"Medicine, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Popov, Jesse"@en . "2012-03-27T16:59:00Z"@en . "2012"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "This body of work describes a novel methodology for discovering and developing new cancer drugs based on therapeutic monoclonal antibodies. Such antibodies generally contain two sites where they bind to their target, but interesting improvements are often observed when the valence (number of target-binding sites) is increased above two. The methodology outlined in this dissertation involves using liposomes to prepare multivalent antibody-lipid nanoparticle formulations of different valence that can be utilized for preclinical drug development. \nAs a proof-of-concept, we applied the methodology to rituximab, a therapeutic antibody used to treat lymphomas and leukemias. For the same dose of rituximab, multivalent rituximab-lipid nanoparticles with valences up to ~250 showed significantly elevated anticancer activity from enhanced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and direct induction of apoptosis. A valence-dependent improvement in apoptosis in lymphoma cells was observed up to levels that were 21-fold higher than those observed after treatment with bivalent rituximab.\nWe subsequently employed the different valences of multivalent rituximab to investigate its poorly defined direct mechanism of action. We uncovered a novel mechanism consisting of upregulation and activation of CD120a which led to ensuing apoptosis. Effector cells of the immune system were capable of hypercrosslinking rituximab on lymphoma cells and reproducing this mechanism, suggesting that it contributes to the in vivo cytotoxicity of regular bivalent rituximab therapy.\nThe methodology described in this dissertation can therefore serve to identify antibodies that are more active as multivalent rather than bivalent molecules, define the optimal valence of such antibodies, and elucidate the mechanism of action of the new multivalent drugs. Furthermore, we illustrate that this information applies to other types of constructs with similar valences, enabling use of the methodology for advancing both liposomal and non-liposomal multivalent antibody formulations to preclinical maturity. \nFinally, this work suggests that every therapeutic antibody may have a different valence where it shows optimal therapeutic activity. For example, antibodies directed against targets that exert therapeutic effects upon clustering may show maximum activity at valences above two. This methodology can easily be applied to other antibodies in an effort to develop superior therapies against nearly any type of cancer."@en . "https://circle.library.ubc.ca/rest/handle/2429/41804?expand=metadata"@en . "MULTIVALENT\u00C2\u00A0MONOCLONAL\u00C2\u00A0ANTIBODIES\u00C2\u00A0AS\u00C2\u00A0IMPROVED\u00C2\u00A0CANCER\u00C2\u00A0THERAPIES:\u00C2\u00A0 PROOF\u00E2\u0080\u0090OF\u00E2\u0080\u0090CONCEPT\u00C2\u00A0AND\u00C2\u00A0MECHANISTIC\u00C2\u00A0STUDIES\u00C2\u00A0USING\u00C2\u00A0RITUXIMAB\u00E2\u0080\u0090LIPID\u00C2\u00A0NANOPARTICLES\u00C2\u00A0 by\u00C2\u00A0 \u00C2\u00A0 Jesse\u00C2\u00A0Popov\u00C2\u00A0 \u00C2\u00A0 B.Sc.,\u00C2\u00A0B.Mus.,\u00C2\u00A0The\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0Western\u00C2\u00A0Ontario,\u00C2\u00A02004\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0THESIS\u00C2\u00A0SUBMITTED\u00C2\u00A0IN\u00C2\u00A0PARTIAL\u00C2\u00A0FULFILLMENT\u00C2\u00A0OF\u00C2\u00A0 THE\u00C2\u00A0REQUIREMENTS\u00C2\u00A0FOR\u00C2\u00A0THE\u00C2\u00A0DEGREE\u00C2\u00A0OF\u00C2\u00A0 \u00C2\u00A0 DOCTOR\u00C2\u00A0OF\u00C2\u00A0PHILOSOPHY\u00C2\u00A0 \u00C2\u00A0 in\u00C2\u00A0 \u00C2\u00A0 THE\u00C2\u00A0FACULTY\u00C2\u00A0OF\u00C2\u00A0GRADUATE\u00C2\u00A0STUDIES\u00C2\u00A0 (Interdisciplinary\u00C2\u00A0Oncology)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 THE\u00C2\u00A0UNIVERSITY\u00C2\u00A0OF\u00C2\u00A0BRITISH\u00C2\u00A0COLUMBIA\u00C2\u00A0 (Vancouver)\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 March\u00C2\u00A02012\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A9\u00C2\u00A0Jesse\u00C2\u00A0Popov,\u00C2\u00A02012\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0ii\u00C2\u00A0\u00C2\u00A0 Abstract\u00C2\u00A0 This\u00C2\u00A0 body\u00C2\u00A0 of\u00C2\u00A0 work\u00C2\u00A0 describes\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 methodology\u00C2\u00A0 for\u00C2\u00A0 discovering\u00C2\u00A0 and\u00C2\u00A0 developing\u00C2\u00A0 new\u00C2\u00A0 cancer\u00C2\u00A0drugs\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0 therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0antibodies.\u00C2\u00A0 \u00C2\u00A0Such\u00C2\u00A0antibodies\u00C2\u00A0generally\u00C2\u00A0contain\u00C2\u00A0 two\u00C2\u00A0 sites\u00C2\u00A0where\u00C2\u00A0 they\u00C2\u00A0bind\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0 target,\u00C2\u00A0but\u00C2\u00A0 interesting\u00C2\u00A0 improvements\u00C2\u00A0are\u00C2\u00A0often\u00C2\u00A0observed\u00C2\u00A0when\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0(number\u00C2\u00A0of\u00C2\u00A0target\u00E2\u0080\u0090binding\u00C2\u00A0sites)\u00C2\u00A0 is\u00C2\u00A0 increased\u00C2\u00A0above\u00C2\u00A0two.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0methodology\u00C2\u00A0outlined\u00C2\u00A0 in\u00C2\u00A0this\u00C2\u00A0 dissertation\u00C2\u00A0 involves\u00C2\u00A0 using\u00C2\u00A0 liposomes\u00C2\u00A0 to\u00C2\u00A0 prepare\u00C2\u00A0 multivalent\u00C2\u00A0 antibody\u00E2\u0080\u0090lipid\u00C2\u00A0 nanoparticle\u00C2\u00A0 formulations\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valence\u00C2\u00A0that\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0utilized\u00C2\u00A0for\u00C2\u00A0preclinical\u00C2\u00A0drug\u00C2\u00A0development.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0 a\u00C2\u00A0proof\u00E2\u0080\u0090of\u00E2\u0080\u0090concept,\u00C2\u00A0we\u00C2\u00A0 applied\u00C2\u00A0 the\u00C2\u00A0methodology\u00C2\u00A0 to\u00C2\u00A0 rituximab,\u00C2\u00A0 a\u00C2\u00A0 therapeutic\u00C2\u00A0 antibody\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 treat\u00C2\u00A0 lymphomas\u00C2\u00A0and\u00C2\u00A0 leukemias.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0 rituximab,\u00C2\u00A0multivalent\u00C2\u00A0 rituximab\u00E2\u0080\u0090 lipid\u00C2\u00A0nanoparticles\u00C2\u00A0with\u00C2\u00A0valences\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0~250\u00C2\u00A0showed\u00C2\u00A0significantly\u00C2\u00A0elevated\u00C2\u00A0anticancer\u00C2\u00A0activity\u00C2\u00A0from\u00C2\u00A0 enhanced\u00C2\u00A0complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity,\u00C2\u00A0antibody\u00E2\u0080\u0090dependent\u00C2\u00A0cell\u00E2\u0080\u0090mediated\u00C2\u00A0cytotoxicity,\u00C2\u00A0and\u00C2\u00A0 direct\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0 improvement\u00C2\u00A0 in\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0 was\u00C2\u00A0 observed\u00C2\u00A0 up\u00C2\u00A0 to\u00C2\u00A0 levels\u00C2\u00A0 that\u00C2\u00A0 were\u00C2\u00A0 21\u00E2\u0080\u0090fold\u00C2\u00A0 higher\u00C2\u00A0 than\u00C2\u00A0 those\u00C2\u00A0 observed\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 bivalent\u00C2\u00A0rituximab.\u00C2\u00A0 We\u00C2\u00A0subsequently\u00C2\u00A0employed\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0rituximab\u00C2\u00A0to\u00C2\u00A0investigate\u00C2\u00A0its\u00C2\u00A0 poorly\u00C2\u00A0 defined\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action.\u00C2\u00A0 \u00C2\u00A0 We\u00C2\u00A0 uncovered\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 mechanism\u00C2\u00A0 consisting\u00C2\u00A0 of\u00C2\u00A0 upregulation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0which\u00C2\u00A0led\u00C2\u00A0to\u00C2\u00A0ensuing\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0Effector\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0 system\u00C2\u00A0 were\u00C2\u00A0 capable\u00C2\u00A0 of\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 rituximab\u00C2\u00A0 on\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 reproducing\u00C2\u00A0 this\u00C2\u00A0 mechanism,\u00C2\u00A0 suggesting\u00C2\u00A0 that\u00C2\u00A0 it\u00C2\u00A0 contributes\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0 cytotoxicity\u00C2\u00A0of\u00C2\u00A0 regular\u00C2\u00A0bivalent\u00C2\u00A0 rituximab\u00C2\u00A0 therapy.\u00C2\u00A0 The\u00C2\u00A0methodology\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0dissertation\u00C2\u00A0 can\u00C2\u00A0 therefore\u00C2\u00A0 serve\u00C2\u00A0 to\u00C2\u00A0 identify\u00C2\u00A0antibodies\u00C2\u00A0 that\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0active\u00C2\u00A0as\u00C2\u00A0multivalent\u00C2\u00A0 rather\u00C2\u00A0 than\u00C2\u00A0bivalent\u00C2\u00A0molecules,\u00C2\u00A0define\u00C2\u00A0 the\u00C2\u00A0optimal\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0 such\u00C2\u00A0antibodies,\u00C2\u00A0and\u00C2\u00A0elucidate\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0new\u00C2\u00A0multivalent\u00C2\u00A0drugs.\u00C2\u00A0\u00C2\u00A0Furthermore,\u00C2\u00A0 we\u00C2\u00A0illustrate\u00C2\u00A0that\u00C2\u00A0this\u00C2\u00A0information\u00C2\u00A0applies\u00C2\u00A0to\u00C2\u00A0other\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0constructs\u00C2\u00A0with\u00C2\u00A0similar\u00C2\u00A0valences,\u00C2\u00A0enabling\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 methodology\u00C2\u00A0 for\u00C2\u00A0 advancing\u00C2\u00A0 both\u00C2\u00A0 liposomal\u00C2\u00A0 and\u00C2\u00A0 non\u00E2\u0080\u0090liposomal\u00C2\u00A0 multivalent\u00C2\u00A0 antibody\u00C2\u00A0 formulations\u00C2\u00A0to\u00C2\u00A0preclinical\u00C2\u00A0maturity.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Finally,\u00C2\u00A0 this\u00C2\u00A0work\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0 every\u00C2\u00A0 therapeutic\u00C2\u00A0 antibody\u00C2\u00A0may\u00C2\u00A0 have\u00C2\u00A0 a\u00C2\u00A0 different\u00C2\u00A0 valence\u00C2\u00A0 where\u00C2\u00A0 it\u00C2\u00A0shows\u00C2\u00A0optimal\u00C2\u00A0therapeutic\u00C2\u00A0activity.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0antibodies\u00C2\u00A0directed\u00C2\u00A0against\u00C2\u00A0targets\u00C2\u00A0that\u00C2\u00A0 exert\u00C2\u00A0therapeutic\u00C2\u00A0effects\u00C2\u00A0upon\u00C2\u00A0clustering\u00C2\u00A0may\u00C2\u00A0show\u00C2\u00A0maximum\u00C2\u00A0activity\u00C2\u00A0at\u00C2\u00A0valences\u00C2\u00A0above\u00C2\u00A0two.\u00C2\u00A0This\u00C2\u00A0 methodology\u00C2\u00A0 can\u00C2\u00A0easily\u00C2\u00A0be\u00C2\u00A0applied\u00C2\u00A0 to\u00C2\u00A0other\u00C2\u00A0antibodies\u00C2\u00A0 in\u00C2\u00A0an\u00C2\u00A0effort\u00C2\u00A0 to\u00C2\u00A0develop\u00C2\u00A0 superior\u00C2\u00A0 therapies\u00C2\u00A0 against\u00C2\u00A0nearly\u00C2\u00A0any\u00C2\u00A0type\u00C2\u00A0of\u00C2\u00A0cancer.\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0iii\u00C2\u00A0\u00C2\u00A0 Preface\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0version\u00C2\u00A0of\u00C2\u00A0Chapter\u00C2\u00A02\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0published\u00C2\u00A0[Popov\u00C2\u00A0J,\u00C2\u00A0Kapanen\u00C2\u00A0AI,\u00C2\u00A0Turner\u00C2\u00A0C,\u00C2\u00A0Ng\u00C2\u00A0R,\u00C2\u00A0Tucker\u00C2\u00A0C,\u00C2\u00A0 Chiu\u00C2\u00A0G,\u00C2\u00A0Klasa\u00C2\u00A0R,\u00C2\u00A0Bally\u00C2\u00A0MB,\u00C2\u00A0Chikh\u00C2\u00A0G.\u00C2\u00A0Multivalent\u00C2\u00A0rituximab\u00C2\u00A0lipid\u00C2\u00A0nanoparticles\u00C2\u00A0as\u00C2\u00A0improved\u00C2\u00A0lymphoma\u00C2\u00A0 therapies:\u00C2\u00A0Indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0activity.\u00C2\u00A0Nanomedicine\u00C2\u00A02011\u00C2\u00A0Nov;6(9):1575\u00E2\u0080\u009091].\u00C2\u00A0\u00C2\u00A0 I\u00C2\u00A0collaborated\u00C2\u00A0primarily\u00C2\u00A0with\u00C2\u00A0Dr.\u00C2\u00A0Ghania\u00C2\u00A0Chikh\u00C2\u00A0for\u00C2\u00A0collection\u00C2\u00A0of\u00C2\u00A0data\u00C2\u00A0 in\u00C2\u00A0this\u00C2\u00A0article,\u00C2\u00A0and\u00C2\u00A0 I\u00C2\u00A0wrote\u00C2\u00A0the\u00C2\u00A0 manuscript\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 preliminary\u00C2\u00A0 draft\u00C2\u00A0 by\u00C2\u00A0 Dr.\u00C2\u00A0 Chikh.\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0 obtained\u00C2\u00A0 the\u00C2\u00A0 content\u00C2\u00A0 in\u00C2\u00A0 Chapters\u00C2\u00A0 3\u00E2\u0080\u00936\u00C2\u00A0 independently;\u00C2\u00A0I\u00C2\u00A0planned\u00C2\u00A0and\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0all\u00C2\u00A0experiments,\u00C2\u00A0performed\u00C2\u00A0data\u00C2\u00A0analysis,\u00C2\u00A0prepared\u00C2\u00A0figures,\u00C2\u00A0 and\u00C2\u00A0wrote\u00C2\u00A0manuscripts\u00C2\u00A0for\u00C2\u00A0future\u00C2\u00A0publication.\u00C2\u00A0 A\u00C2\u00A0 separate\u00C2\u00A0 article\u00C2\u00A0 resulting\u00C2\u00A0 from\u00C2\u00A0 collaboration\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 laboratory\u00C2\u00A0 of\u00C2\u00A0 Dr.\u00C2\u00A0 Ruth\u00C2\u00A0 Signorell\u00C2\u00A0 (Department\u00C2\u00A0of\u00C2\u00A0Chemistry,\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0British\u00C2\u00A0Columbia)\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0published\u00C2\u00A0and\u00C2\u00A0referenced\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0 dissertation\u00C2\u00A0 [Weiss\u00C2\u00A0A,\u00C2\u00A0Preston\u00C2\u00A0TC,\u00C2\u00A0Popov\u00C2\u00A0 J,\u00C2\u00A0 Li\u00C2\u00A0Q,\u00C2\u00A0Wu\u00C2\u00A0 S,\u00C2\u00A0Chou\u00C2\u00A0KC,\u00C2\u00A0Burt\u00C2\u00A0HM,\u00C2\u00A0Bally\u00C2\u00A0MB,\u00C2\u00A0 Signorell\u00C2\u00A0R.\u00C2\u00A0 Selective\u00C2\u00A0recognition\u00C2\u00A0of\u00C2\u00A0rituximab\u00E2\u0080\u0090functionalized\u00C2\u00A0gold\u00C2\u00A0nanoparticles\u00C2\u00A0by\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0studied\u00C2\u00A0with\u00C2\u00A0 3D\u00C2\u00A0imaging.\u00C2\u00A0J\u00C2\u00A0Phys\u00C2\u00A0Chem\u00C2\u00A0C\u00C2\u00A02009\u00C2\u00A0Oct\u00C2\u00A021;113(47):20252\u00E2\u0080\u00908].\u00C2\u00A0 All\u00C2\u00A0experiments\u00C2\u00A0involving\u00C2\u00A0the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0animals\u00C2\u00A0were\u00C2\u00A0completed\u00C2\u00A0in\u00C2\u00A0accordance\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0current\u00C2\u00A0 guidelines\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Canadian\u00C2\u00A0Council\u00C2\u00A0of\u00C2\u00A0Animal\u00C2\u00A0Care.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0British\u00C2\u00A0Columbia\u00C2\u00A0Animal\u00C2\u00A0Care\u00C2\u00A0 Committee\u00C2\u00A0 reviewed\u00C2\u00A0 and\u00C2\u00A0 approved\u00C2\u00A0 certificate\u00C2\u00A0 numbers\u00C2\u00A0 A05\u00E2\u0080\u00901582\u00C2\u00A0 and\u00C2\u00A0 A06\u00E2\u0080\u00901537\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 animal\u00C2\u00A0 studies\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A02\u00C2\u00A0(in\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0rituximab\u00C2\u00A0and\u00C2\u00A0rituximab\u00E2\u0080\u0090lipid\u00C2\u00A0nanoparticles,\u00C2\u00A0and\u00C2\u00A0 pharmacokinetics\u00C2\u00A0of\u00C2\u00A0rituximab\u00E2\u0080\u0090lipid\u00C2\u00A0nanoparticles).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0document\u00C2\u00A0is\u00C2\u00A0written\u00C2\u00A0using\u00C2\u00A0United\u00C2\u00A0States\u00C2\u00A0English\u00C2\u00A0spelling.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0iv\u00C2\u00A0\u00C2\u00A0 Table\u00C2\u00A0of\u00C2\u00A0Contents\u00C2\u00A0 \u00C2\u00A0 Abstract\u00C2\u00A0.........................................................................................................................................\u00C2\u00A0ii\u00C2\u00A0 Preface\u00C2\u00A0..........................................................................................................................................\u00C2\u00A0iii\u00C2\u00A0 Table\u00C2\u00A0of\u00C2\u00A0Contents\u00C2\u00A0..........................................................................................................................\u00C2\u00A0iv\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0Tables\u00C2\u00A0................................................................................................................................\u00C2\u00A0xii\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0Figures\u00C2\u00A0..............................................................................................................................\u00C2\u00A0xiii\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0Abbreviations\u00C2\u00A0.....................................................................................................................\u00C2\u00A0xv\u00C2\u00A0 Acknowledgements\u00C2\u00A0....................................................................................................................\u00C2\u00A0xvii\u00C2\u00A0 Dedication\u00C2\u00A0.................................................................................................................................\u00C2\u00A0xviii\u00C2\u00A0 1\u00C2\u00A0 \u00C2\u00A0 Introduction:\u00C2\u00A0Developing\u00C2\u00A0novel\u00C2\u00A0therapies\u00C2\u00A0for\u00C2\u00A0treating\u00C2\u00A0cancer\u00C2\u00A0.................................................\u00C2\u00A01\u00C2\u00A0 \u00C2\u00A0 1.1\u00C2\u00A0 \u00C2\u00A0 Overview\u00C2\u00A0.................................................................................................................................\u00C2\u00A01\u00C2\u00A0 \u00C2\u00A0 1.2\u00C2\u00A0 \u00C2\u00A0 Liposomal\u00C2\u00A0formulations\u00C2\u00A0..........................................................................................................\u00C2\u00A02\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.2.1\u00C2\u00A0 \u00C2\u00A0 Liposomes\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0treatment\u00C2\u00A0....................................................................................\u00C2\u00A02\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.2.2\u00C2\u00A0 \u00C2\u00A0 What\u00C2\u00A0are\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs?\u00C2\u00A0.......................................................................................................\u00C2\u00A03\u00C2\u00A0 \u00C2\u00A0 1.3\u00C2\u00A0 \u00C2\u00A0 Therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0..................................................................................................\u00C2\u00A06\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.3.1\u00C2\u00A0 \u00C2\u00A0 Rituximab\u00C2\u00A0and\u00C2\u00A0CD20\u00C2\u00A0.....................................................................................................\u00C2\u00A08\u00C2\u00A0 \u00C2\u00A0 1.4\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0...................................................\u00C2\u00A09\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.4.1\u00C2\u00A0 \u00C2\u00A0 Complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0........................................................................\u00C2\u00A010\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.4.2\u00C2\u00A0 \u00C2\u00A0 Ab\u00E2\u0080\u0090dependent\u00C2\u00A0cell\u00E2\u0080\u0090mediated\u00C2\u00A0cytotoxicity\u00C2\u00A0..................................................................\u00C2\u00A010\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.4.3\u00C2\u00A0 \u00C2\u00A0 Direct\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0................................\u00C2\u00A012\u00C2\u00A0 \u00C2\u00A0 1.5\u00C2\u00A0 \u00C2\u00A0 Apoptosis\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0target\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0therapy\u00C2\u00A0...............................................................................\u00C2\u00A014\u00C2\u00A0 \u00C2\u00A0 1.6\u00C2\u00A0 \u00C2\u00A0 Multivalent\u00C2\u00A0Abs\u00C2\u00A0as\u00C2\u00A0improved\u00C2\u00A0therapies\u00C2\u00A0................................................................................\u00C2\u00A015\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.6.1\u00C2\u00A0 \u00C2\u00A0 Characteristics\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0definition\u00C2\u00A0of\u00C2\u00A0valence\u00C2\u00A0..................\u00C2\u00A015\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.6.2\u00C2\u00A0 \u00C2\u00A0 Examples\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0interactions\u00C2\u00A0in\u00C2\u00A0biology\u00C2\u00A0and\u00C2\u00A0their\u00C2\u00A0physical\u00C2\u00A0basis\u00C2\u00A0..................\u00C2\u00A017\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0v\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 1.6.3\u00C2\u00A0 \u00C2\u00A0 Descriptions\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0that\u00C2\u00A0exhibit\u00C2\u00A0superior\u00C2\u00A0efficacies\u00C2\u00A0...............................\u00C2\u00A019\u00C2\u00A0 \u00C2\u00A0 1.7\u00C2\u00A0 \u00C2\u00A0 Multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0dissertation\u00C2\u00A0...................................................\u00C2\u00A020\u00C2\u00A0 2\u00C2\u00A0 \u00C2\u00A0 Indirect\u00C2\u00A0mechanisms\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0rituximab\u00E2\u0080\u0090lipid\u00C2\u00A0nanoparticles\u00C2\u00A0...........22\u00C2\u00A0 \u00C2\u00A0 2.1\u00C2\u00A0 \u00C2\u00A0 Synopsis\u00C2\u00A0................................................................................................................................\u00C2\u00A022\u00C2\u00A0 \u00C2\u00A0 2.2\u00C2\u00A0 \u00C2\u00A0 Background\u00C2\u00A0...........................................................................................................................\u00C2\u00A022\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.2.1\u00C2\u00A0 \u00C2\u00A0 Complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0..............................................................\u00C2\u00A022\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.2.2\u00C2\u00A0 \u00C2\u00A0 Ab\u00E2\u0080\u0090dependent\u00C2\u00A0cell\u00E2\u0080\u0090mediated\u00C2\u00A0cytotoxicity\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0.......................................................\u00C2\u00A023\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.2.3\u00C2\u00A0 \u00C2\u00A0 Does\u00C2\u00A0 the\u00C2\u00A0 creation\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 affect\u00C2\u00A0 the\u00C2\u00A0 indirect\u00C2\u00A0 mechanisms\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 properties\u00C2\u00A0of\u00C2\u00A0Rtx?\u00C2\u00A0.......................................................................................................\u00C2\u00A024\u00C2\u00A0 \u00C2\u00A0 2.3\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0.........................................................................................................\u00C2\u00A024\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.1\u00C2\u00A0 \u00C2\u00A0 Reagents\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0...............................................................................................\u00C2\u00A024\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.2\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0...........................................................................\u00C2\u00A026\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.3\u00C2\u00A0 \u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0cell\u00E2\u0080\u0090surface\u00C2\u00A0protein\u00C2\u00A0expression\u00C2\u00A0levels\u00C2\u00A0............................................\u00C2\u00A028\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.4\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0vivo\u00C2\u00A0xenograft\u00C2\u00A0models\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0Rtx\u00C2\u00A0efficacy\u00C2\u00A0.........................................................\u00C2\u00A028\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.5\u00C2\u00A0 \u00C2\u00A0 Quantification\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0..................................................................\u00C2\u00A028\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.6\u00C2\u00A0 \u00C2\u00A0 Annexin\u00E2\u0080\u0090V/PI\u00C2\u00A0flow\u00C2\u00A0cytometry\u00E2\u0080\u0090based\u00C2\u00A0apoptosis\u00C2\u00A0assay\u00C2\u00A0................................................\u00C2\u00A029\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.7\u00C2\u00A0 \u00C2\u00A0 Assay\u00C2\u00A0for\u00C2\u00A0measuring\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0.........................\u00C2\u00A030\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.8\u00C2\u00A0 \u00C2\u00A0 Assay\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0quantification\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090dependent\u00C2\u00A0cell\u00E2\u0080\u0090mediated\u00C2\u00A0cytotoxicity\u00C2\u00A0................\u00C2\u00A030\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.9\u00C2\u00A0 \u00C2\u00A0 Quantification\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0natural\u00C2\u00A0killer\u00C2\u00A0cells\u00C2\u00A0...........................................................\u00C2\u00A031\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.10\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0vivo\u00C2\u00A0xenograft\u00C2\u00A0models\u00C2\u00A0for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0efficacy\u00C2\u00A0studies\u00C2\u00A0...............................................\u00C2\u00A031\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.3.11\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0vivo\u00C2\u00A0studies\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0..................................................\u00C2\u00A032\u00C2\u00A0 \u00C2\u00A0 2.4\u00C2\u00A0 \u00C2\u00A0 Results\u00C2\u00A0...................................................................................................................................\u00C2\u00A033\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.1\u00C2\u00A0 \u00C2\u00A0 Characterization\u00C2\u00A0of\u00C2\u00A0Z138\u00C2\u00A0and\u00C2\u00A0 JVM2\u00C2\u00A0cell\u00C2\u00A0 lines:\u00C2\u00A0surface\u00C2\u00A0protein\u00C2\u00A0expression\u00C2\u00A0 levels\u00C2\u00A0 and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0treatment\u00C2\u00A0.......................................................................\u00C2\u00A033\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.2\u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0bind\u00C2\u00A0specifically\u00C2\u00A0to\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0..................\u00C2\u00A035\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0vi\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.3\u00C2\u00A0 \u00C2\u00A0 Multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0elicit\u00C2\u00A0superior\u00C2\u00A0complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0and\u00C2\u00A0Ab\u00E2\u0080\u0090 dependent\u00C2\u00A0cell\u00E2\u0080\u0090mediated\u00C2\u00A0cytotoxicity\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0........................................\u00C2\u00A038\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.4\u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0induces\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0natural\u00C2\u00A0killer\u00C2\u00A0cells\u00C2\u00A0.................................................\u00C2\u00A040\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.5\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0.......................................................................\u00C2\u00A041\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.4.6\u00C2\u00A0 \u00C2\u00A0 Pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0........................................................................\u00C2\u00A044\u00C2\u00A0 \u00C2\u00A0 2.5\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0...................................................................................................\u00C2\u00A045\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.5.1\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0...................................................................................................................\u00C2\u00A045\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 2.5.2\u00C2\u00A0 \u00C2\u00A0 Conclusions\u00C2\u00A0.................................................................................................................\u00C2\u00A048\u00C2\u00A0 3\u00C2\u00A0 \u00C2\u00A0 Improved\u00C2\u00A0methodology\u00C2\u00A0for\u00C2\u00A0preparing\u00C2\u00A0multivalent\u00C2\u00A0antibody\u00E2\u0080\u0090lipid\u00C2\u00A0nanoparticles\u00C2\u00A0....................50\u00C2\u00A0 \u00C2\u00A0 3.1\u00C2\u00A0 \u00C2\u00A0 Synopsis\u00C2\u00A0................................................................................................................................\u00C2\u00A050\u00C2\u00A0 \u00C2\u00A0 3.2\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Background\u00C2\u00A0...........................................................................................................................\u00C2\u00A050\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.1\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0need\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0a\u00C2\u00A0new\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0creating\u00C2\u00A0many\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0...........\u00C2\u00A050\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.2.2\u00C2\u00A0 \u00C2\u00A0 How\u00C2\u00A0the\u00C2\u00A0new\u00C2\u00A0methodology\u00C2\u00A0overcomes\u00C2\u00A0previous\u00C2\u00A0limitations\u00C2\u00A0....................................\u00C2\u00A051\u00C2\u00A0 \u00C2\u00A0 3.3\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0.........................................................................................................\u00C2\u00A052\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.1\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0.....................................................................................................................\u00C2\u00A052\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.2\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090micelles\u00C2\u00A0.....................................................................................\u00C2\u00A053\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.3\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0............................................................................................\u00C2\u00A053\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.4\u00C2\u00A0 \u00C2\u00A0 CBQCA\u00C2\u00A0 assay\u00C2\u00A0 for\u00C2\u00A0 measuring\u00C2\u00A0 protein\u00C2\u00A0 concentration\u00C2\u00A0 in\u00C2\u00A0 solution\u00C2\u00A0 or\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 suspension\u00C2\u00A0..................................................................................................................\u00C2\u00A054\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.5\u00C2\u00A0 \u00C2\u00A0 Ammonium\u00C2\u00A0ferrothiocyanate\u00C2\u00A0assay\u00C2\u00A0for\u00C2\u00A0measuring\u00C2\u00A0polyethylene\u00C2\u00A0glycol\u00C2\u00A0content\u00C2\u00A0in\u00C2\u00A0 LNPs\u00C2\u00A0............................................................................................................................\u00C2\u00A054\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.6\u00C2\u00A0 \u00C2\u00A0 Ab\u00C2\u00A0biotinylation\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0nickel\u00C2\u00A0immobilized\u00C2\u00A0metal\u00C2\u00A0affinity\u00C2\u00A0chromatography\u00C2\u00A0column\u00C2\u00A0....\u00C2\u00A055\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.7\u00C2\u00A0 \u00C2\u00A0 Ab\u00C2\u00A0biotinylation\u00C2\u00A0in\u00C2\u00A0solution\u00C2\u00A0........................................................................................\u00C2\u00A056\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.8\u00C2\u00A0 \u00C2\u00A0 HABA/avidin\u00C2\u00A0assay\u00C2\u00A0for\u00C2\u00A0measuring\u00C2\u00A0the\u00C2\u00A0extent\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0biotinylation\u00C2\u00A0..............................\u00C2\u00A056\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.3.9\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0Coupling\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0...............................................\u00C2\u00A057\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0vii\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 3.4\u00C2\u00A0 \u00C2\u00A0 Results\u00C2\u00A0...................................................................................................................................\u00C2\u00A057\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.4.1\u00C2\u00A0 \u00C2\u00A0 Overview\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0improved\u00C2\u00A0methodology\u00C2\u00A0for\u00C2\u00A0creating\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valence\u00C2\u00A0 57\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.4.2\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0measurement\u00C2\u00A0of\u00C2\u00A0protein\u00C2\u00A0content\u00C2\u00A0...............................\u00C2\u00A059\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.4.3\u00C2\u00A0 \u00C2\u00A0 Direct\u00C2\u00A0measurement\u00C2\u00A0of\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0post\u00E2\u0080\u0090inserted\u00C2\u00A0polyethylene\u00C2\u00A0glycol\u00C2\u00A0in\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0.....\u00C2\u00A061\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.4.4\u00C2\u00A0 \u00C2\u00A0 Ab\u00C2\u00A0biotinylation\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0nickel\u00C2\u00A0immobilized\u00C2\u00A0metal\u00C2\u00A0affinity\u00C2\u00A0chromatography\u00C2\u00A0support\u00C2\u00A0...\u00C2\u00A062\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.4.5\u00C2\u00A0 \u00C2\u00A0 Different\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 valences\u00C2\u00A0 are\u00C2\u00A0 achieved\u00C2\u00A0 by\u00C2\u00A0 taking\u00C2\u00A0 advantage\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 slow\u00C2\u00A0 association\u00C2\u00A0dynamics\u00C2\u00A0between\u00C2\u00A0Ab\u00E2\u0080\u0090biotin\u00C2\u00A0and\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0...........................................\u00C2\u00A065\u00C2\u00A0 \u00C2\u00A0 3.5\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0...................................................................................................\u00C2\u00A067\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.5.1\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0...................................................................................................................\u00C2\u00A067\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 3.5.2\u00C2\u00A0 \u00C2\u00A0 Conclusions\u00C2\u00A0.................................................................................................................\u00C2\u00A069\u00C2\u00A0 4\u00C2\u00A0 \u00C2\u00A0 Unique\u00C2\u00A0biological\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0rituximab\u00E2\u0080\u0090lipid\u00C2\u00A0nanoparticles\u00C2\u00A0................................................71\u00C2\u00A0 \u00C2\u00A0 4.1\u00C2\u00A0 \u00C2\u00A0 Synopsis\u00C2\u00A0................................................................................................................................\u00C2\u00A071\u00C2\u00A0 \u00C2\u00A0 4.2\u00C2\u00A0 \u00C2\u00A0 Background\u00C2\u00A0...........................................................................................................................\u00C2\u00A071\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.2.1\u00C2\u00A0 \u00C2\u00A0 Properties\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0Abs\u00C2\u00A0.......................................\u00C2\u00A071\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.2.2\u00C2\u00A0 \u00C2\u00A0 Hypotheses\u00C2\u00A0concerning\u00C2\u00A0the\u00C2\u00A0observed\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0...\u00C2\u00A073\u00C2\u00A0 \u00C2\u00A0 4.3\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0.........................................................................................................\u00C2\u00A074\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.1\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0.....................................................................................................................\u00C2\u00A074\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.2\u00C2\u00A0 \u00C2\u00A0 Cell\u00C2\u00A0lines\u00C2\u00A0......................................................................................................................\u00C2\u00A074\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.3\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0.............................................................................................\u00C2\u00A075\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.4\u00C2\u00A0 \u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0free\u00C2\u00A0CD20\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0.......................................................................................................................\u00C2\u00A075\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.5\u00C2\u00A0 \u00C2\u00A0 Confocal\u00C2\u00A0laser\u00E2\u0080\u0090scanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0of\u00C2\u00A0treated\u00C2\u00A0cells\u00C2\u00A0............................\u00C2\u00A076\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.6\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0Measurement\u00C2\u00A0of\u00C2\u00A0total\u00C2\u00A0CD20\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0treated\u00C2\u00A0cells\u00C2\u00A0....................................................\u00C2\u00A076\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.7\u00C2\u00A0 \u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0mitochondrial\u00C2\u00A0activity\u00C2\u00A0using\u00C2\u00A0AlamarBlue\u00C2\u00A0.......................................\u00C2\u00A077\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.3.8\u00C2\u00A0 \u00C2\u00A0 Annexin\u00E2\u0080\u0090V/PI\u00C2\u00A0apoptosis\u00C2\u00A0assay\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0.................................................\u00C2\u00A077\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0viii\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 4.4\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Results\u00C2\u00A0...................................................................................................................................\u00C2\u00A078\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.4.1\u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0exhibit\u00C2\u00A0unique\u00C2\u00A0binding\u00C2\u00A0properties\u00C2\u00A0to\u00C2\u00A0CD20+\u00C2\u00A0target\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0...........\u00C2\u00A078\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.4.2\u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0domains\u00C2\u00A0are\u00C2\u00A0found\u00C2\u00A0on\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0on\u00C2\u00A0those\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0............................................................................................\u00C2\u00A082\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.4.3\u00C2\u00A0 \u00C2\u00A0 Expression\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0change\u00C2\u00A0substantially\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0....\u00C2\u00A083\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.4.4\u00C2\u00A0 \u00C2\u00A0 Cytotoxicity\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0in\u00C2\u00A0two\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0.................................................\u00C2\u00A085\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.4.5\u00C2\u00A0 \u00C2\u00A0 Time\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0....................\u00C2\u00A086\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.4.6\u00C2\u00A0 \u00C2\u00A0 Levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0...............\u00C2\u00A089\u00C2\u00A0 \u00C2\u00A0 4.5\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0...................................................................................................\u00C2\u00A090\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.5.1\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0...................................................................................................................\u00C2\u00A090\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 4.5.2\u00C2\u00A0 \u00C2\u00A0 Conclusions\u00C2\u00A0.................................................................................................................\u00C2\u00A093\u00C2\u00A0 5\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0novel\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0rituximab\u00C2\u00A0................................................94\u00C2\u00A0 \u00C2\u00A0 5.1\u00C2\u00A0 \u00C2\u00A0 Synopsis\u00C2\u00A0................................................................................................................................\u00C2\u00A094\u00C2\u00A0 \u00C2\u00A0 5.2\u00C2\u00A0 \u00C2\u00A0 Background\u00C2\u00A0...........................................................................................................................\u00C2\u00A094\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.2.1\u00C2\u00A0 \u00C2\u00A0 Extrinsic\u00C2\u00A0and\u00C2\u00A0intrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathways\u00C2\u00A0.................................................................\u00C2\u00A094\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.2.2\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 death\u00E2\u0080\u0090inducing\u00C2\u00A0 signaling\u00C2\u00A0 complex\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 superfamily\u00C2\u00A0.................................................................................................................\u00C2\u00A095\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.2.3\u00C2\u00A0 \u00C2\u00A0 Death\u00C2\u00A0receptors\u00C2\u00A0..........................................................................................................\u00C2\u00A096\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.2.4\u00C2\u00A0 \u00C2\u00A0 Deciphering\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0...............................\u00C2\u00A097\u00C2\u00A0 \u00C2\u00A0 5.3\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0.........................................................................................................\u00C2\u00A098\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.1\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0...............................................................................................\u00C2\u00A098\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.2\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0.............................................................................................\u00C2\u00A098\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.3\u00C2\u00A0 \u00C2\u00A0 Assay\u00C2\u00A0for\u00C2\u00A0quantifying\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0treated\u00C2\u00A0cells\u00C2\u00A0..............................................\u00C2\u00A099\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.4\u00C2\u00A0 \u00C2\u00A0 Profiling\u00C2\u00A0of\u00C2\u00A0caspases\u00C2\u00A0and\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0.....................................................\u00C2\u00A0100\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.5\u00C2\u00A0 \u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0...........................\u00C2\u00A0100\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0ix\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.6\u00C2\u00A0 \u00C2\u00A0 Annexin\u00E2\u0080\u0090V/PI\u00C2\u00A0 apoptosis\u00C2\u00A0 assay\u00C2\u00A0 and\u00C2\u00A0 measurement\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 levels\u00C2\u00A0 within\u00C2\u00A0 apoptotic\u00C2\u00A0subpopulations.........................................................................................\u00C2\u00A0101\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.7\u00C2\u00A0 \u00C2\u00A0 Confocal\u00C2\u00A0laser\u00E2\u0080\u0090scanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0...................................................\u00C2\u00A0102\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.3.8\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0..............................................................................................\u00C2\u00A0103\u00C2\u00A0 \u00C2\u00A0 5.4\u00C2\u00A0 \u00C2\u00A0 Results\u00C2\u00A0.................................................................................................................................\u00C2\u00A0103\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.4.1\u00C2\u00A0 \u00C2\u00A0 Caspase\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis\u00C2\u00A0...............................................\u00C2\u00A0103\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.4.2\u00C2\u00A0 \u00C2\u00A0 Dependence\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0receptor\u00C2\u00A0superfamily\u00C2\u00A0member\u00C2\u00A0expression\u00C2\u00A0 on\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0.............................................................................\u00C2\u00A0106\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.4.3\u00C2\u00A0 \u00C2\u00A0 CD120a\u00C2\u00A0is\u00C2\u00A0equally\u00C2\u00A0upregulated\u00C2\u00A0in\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0 with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0.............................................................................................................\u00C2\u00A0109\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.4.4\u00C2\u00A0 \u00C2\u00A0 Time\u00C2\u00A0course\u00C2\u00A0and\u00C2\u00A0valence\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0..............................\u00C2\u00A0111\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.4.5\u00C2\u00A0 \u00C2\u00A0 Colocalization\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0..................................................................\u00C2\u00A0113\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.4.6\u00C2\u00A0 \u00C2\u00A0 CD120a\u00C2\u00A0 upregulation\u00C2\u00A0 and\u00C2\u00A0 direct\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 result\u00C2\u00A0 from\u00C2\u00A0 Rtx\u00C2\u00A0 multivalency\u00C2\u00A0and\u00C2\u00A0not\u00C2\u00A0the\u00C2\u00A0liposomal\u00C2\u00A0component\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0....................................\u00C2\u00A0115\u00C2\u00A0 \u00C2\u00A0 5.5\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0.................................................................................................\u00C2\u00A0118\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.5.1\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0.................................................................................................................\u00C2\u00A0118\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 5.5.2\u00C2\u00A0 \u00C2\u00A0 Conclusions\u00C2\u00A0...............................................................................................................\u00C2\u00A0121\u00C2\u00A0 6\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0vivo\u00C2\u00A0relevance\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0rituximab\u00C2\u00A0......................\u00C2\u00A0122\u00C2\u00A0 \u00C2\u00A0 6.1\u00C2\u00A0 \u00C2\u00A0 Synopsis\u00C2\u00A0..............................................................................................................................\u00C2\u00A0122\u00C2\u00A0 \u00C2\u00A0 6.2\u00C2\u00A0 \u00C2\u00A0 Background\u00C2\u00A0.........................................................................................................................\u00C2\u00A0122\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.2.1\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0biology\u00C2\u00A0and\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0........................................................................\u00C2\u00A0122\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.2.2\u00C2\u00A0 \u00C2\u00A0 Plasma\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0...........................................................................................\u00C2\u00A0124\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.2.3\u00C2\u00A0 \u00C2\u00A0 Studying\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0............................................................................\u00C2\u00A0126\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.2.3.1\u00C2\u00A0 \u00C2\u00A0 Detergent\u00E2\u0080\u0090resistant\u00C2\u00A0membranes\u00C2\u00A0......................................................................\u00C2\u00A0126\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.2.3.2\u00C2\u00A0 Manipulation\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0cholesterol\u00C2\u00A0content\u00C2\u00A0.................................\u00C2\u00A0127\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0x\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.2.4\u00C2\u00A0 \u00C2\u00A0 Can\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0involve\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0vivo?\u00C2\u00A0.........\u00C2\u00A0128\u00C2\u00A0 \u00C2\u00A0 6.3\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0.......................................................................................................\u00C2\u00A0129\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.1\u00C2\u00A0 \u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0.............................................................................................\u00C2\u00A0129\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.2\u00C2\u00A0 \u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0.........................................................................\u00C2\u00A0130\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.3\u00C2\u00A0 \u00C2\u00A0 Depletion\u00C2\u00A0and\u00C2\u00A0augmentation\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0cholesterol\u00C2\u00A0levels\u00C2\u00A0...................\u00C2\u00A0130\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.4\u00C2\u00A0 \u00C2\u00A0 Use\u00C2\u00A0of\u00C2\u00A0 flow\u00C2\u00A0cytometry\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0or\u00C2\u00A0expression\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 cell\u00E2\u0080\u0090surface\u00C2\u00A0proteins\u00C2\u00A0.................................................................................................\u00C2\u00A0131\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.5\u00C2\u00A0 \u00C2\u00A0 Assay\u00C2\u00A0for\u00C2\u00A0quantifying\u00C2\u00A0the\u00C2\u00A0detergent\u00C2\u00A0resistance\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00E2\u0080\u0090associated\u00C2\u00A0 proteins\u00C2\u00A0....................................................................................................................\u00C2\u00A0131\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.6\u00C2\u00A0 \u00C2\u00A0 Confocal\u00C2\u00A0laser\u00E2\u0080\u0090scanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0...................................................\u00C2\u00A0132\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.7\u00C2\u00A0 \u00C2\u00A0 Experiments\u00C2\u00A0 using\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 pretreated\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00C2\u00A0 or\u00C2\u00A0 A568\u00E2\u0080\u0090Rtx\u00C2\u00A0 followed\u00C2\u00A0 by\u00C2\u00A0 addition\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0or\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0....................................................................................\u00C2\u00A0132\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.3.8\u00C2\u00A0 \u00C2\u00A0 Coculture\u00C2\u00A0of\u00C2\u00A0treated\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0human\u00C2\u00A0peripheral\u00C2\u00A0blood\u00C2\u00A0mononuclear\u00C2\u00A0cells.133\u00C2\u00A0 \u00C2\u00A0 6.4\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Results\u00C2\u00A0.................................................................................................................................\u00C2\u00A0135\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.4.1\u00C2\u00A0 \u00C2\u00A0 Apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0cholesterol\u00E2\u0080\u0090dependent\u00C2\u00A0............................\u00C2\u00A0135\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.4.2\u00C2\u00A0 \u00C2\u00A0 Inhibition\u00C2\u00A0of\u00C2\u00A0cholesterol\u00C2\u00A0synthesis\u00C2\u00A0using\u00C2\u00A0simvastatin\u00C2\u00A0sensitizes\u00C2\u00A0cells\u00C2\u00A0 to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090 induced\u00C2\u00A0apoptosis\u00C2\u00A0....................................................................................................\u00C2\u00A0140\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.4.3\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0model\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0hypercrosslinking\u00C2\u00A0that\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0after\u00C2\u00A0normal\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0......\u00C2\u00A0142\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.4.4\u00C2\u00A0 \u00C2\u00A0 CD120a\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 excluded\u00C2\u00A0 from\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 rafts\u00C2\u00A0 is\u00C2\u00A0 colocalized\u00C2\u00A0 with\u00C2\u00A0 hypercrosslinked\u00C2\u00A0Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0patches\u00C2\u00A0...................................................................\u00C2\u00A0145\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.4.5\u00C2\u00A0 \u00C2\u00A0 Effector\u00C2\u00A0 cells\u00C2\u00A0 induce\u00C2\u00A0 elevated\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 only\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0..............................................................................\u00C2\u00A0149\u00C2\u00A0 \u00C2\u00A0 6.5\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0.................................................................................................\u00C2\u00A0151\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.5.1\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0.................................................................................................................\u00C2\u00A0151\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 6.5.2\u00C2\u00A0 \u00C2\u00A0 Conclusions\u00C2\u00A0...............................................................................................................\u00C2\u00A0155\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xi\u00C2\u00A0\u00C2\u00A0 7\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0 and\u00C2\u00A0 conclusions:\u00C2\u00A0 Antibody\u00E2\u0080\u0090lipid\u00C2\u00A0 nanoparticles\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 promising\u00C2\u00A0 tool\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 drug\u00C2\u00A0development\u00C2\u00A0...............................................................................................................\u00C2\u00A0156\u00C2\u00A0 \u00C2\u00A0 7.1\u00C2\u00A0 \u00C2\u00A0 Recapitulation\u00C2\u00A0.....................................................................................................................\u00C2\u00A0156\u00C2\u00A0 \u00C2\u00A0 7.2\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0concerning\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0...............................\u00C2\u00A0157\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.2.1\u00C2\u00A0 \u00C2\u00A0 Strengths\u00C2\u00A0and\u00C2\u00A0weaknesses\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0tool\u00C2\u00A0for\u00C2\u00A0developing\u00C2\u00A0new\u00C2\u00A0drugs\u00C2\u00A0............\u00C2\u00A0157\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.2.2\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0whole\u00C2\u00A0Abs\u00C2\u00A0in\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0versus\u00C2\u00A0Ab\u00C2\u00A0fragments\u00C2\u00A0...........................................\u00C2\u00A0158\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.2.3\u00C2\u00A0 \u00C2\u00A0 Relevance\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0studied\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0.........................................................................\u00C2\u00A0160\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.2.4\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0interaction\u00C2\u00A0between\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0....\u00C2\u00A0162\u00C2\u00A0 \u00C2\u00A0 7.3\u00C2\u00A0 \u00C2\u00A0 Discussion\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0...................................................................\u00C2\u00A0163\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.3.1\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0issue\u00C2\u00A0of\u00C2\u00A0rapid\u00C2\u00A0clearance\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0remains\u00C2\u00A0unresolved\u00C2\u00A0......................\u00C2\u00A0163\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.3.2\u00C2\u00A0 \u00C2\u00A0 Local\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0and\u00C2\u00A0not\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 Rtx\u00E2\u0080\u0090CD20\u00C2\u00A0 interactions,\u00C2\u00A0determine\u00C2\u00A0the\u00C2\u00A0 level\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00C2\u00A0.............................................................................................................................\u00C2\u00A0165\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.3.3\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0may\u00C2\u00A0not\u00C2\u00A0exclusively\u00C2\u00A0 involve\u00C2\u00A0CD120a,\u00C2\u00A0but\u00C2\u00A0 also\u00C2\u00A0other\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0and\u00C2\u00A0signaling\u00C2\u00A0molecules\u00C2\u00A0................................................\u00C2\u00A0166\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.3.4\u00C2\u00A0 \u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 levels\u00C2\u00A0 or\u00C2\u00A0 mutation\u00C2\u00A0 status\u00C2\u00A0 as\u00C2\u00A0 predictive\u00C2\u00A0 markers\u00C2\u00A0 for\u00C2\u00A0 Rtx\u00C2\u00A0 response\u00C2\u00A0...................................................................................................................\u00C2\u00A0167\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 7.3.5\u00C2\u00A0 \u00C2\u00A0 Statins\u00C2\u00A0in\u00C2\u00A0combination\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0..................................................................\u00C2\u00A0169\u00C2\u00A0 \u00C2\u00A0 7.4\u00C2\u00A0 \u00C2\u00A0 Future\u00C2\u00A0work\u00C2\u00A0........................................................................................................................\u00C2\u00A0171\u00C2\u00A0 \u00C2\u00A0 7.5\u00C2\u00A0 \u00C2\u00A0 Conclusions\u00C2\u00A0.........................................................................................................................\u00C2\u00A0172\u00C2\u00A0 References\u00C2\u00A0.................................................................................................................................\u00C2\u00A0174\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xii\u00C2\u00A0\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0Tables\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A01.1\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Pharmacokinetic\u00C2\u00A0variables\u00C2\u00A0and\u00C2\u00A0tumor\u00C2\u00A0localization\u00C2\u00A0of\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0..........................................\u00C2\u00A06\u00C2\u00A0 Table\u00C2\u00A01.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Selected\u00C2\u00A0therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0approved\u00C2\u00A0or\u00C2\u00A0under\u00C2\u00A0development\u00C2\u00A0in\u00C2\u00A0oncology\u00C2\u00A0.....\u00C2\u00A07\u00C2\u00A0 Table\u00C2\u00A03.1\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Comparison\u00C2\u00A0 of\u00C2\u00A0 Ab\u00C2\u00A0 yields\u00C2\u00A0 between\u00C2\u00A0 current\u00C2\u00A0 and\u00C2\u00A0 previous\u00C2\u00A0 methods\u00C2\u00A0 of\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 production\u00C2\u00A0.......................................................................................................................\u00C2\u00A058\u00C2\u00A0 Table\u00C2\u00A03.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 PEG\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A00.5\u00C2\u00A0mL\u00C2\u00A0fractions\u00C2\u00A0during\u00C2\u00A0purification\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090SUV\u00C2\u00A0..................................\u00C2\u00A062\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xiii\u00C2\u00A0\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0Figures\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A01.1\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0exhibit\u00C2\u00A0enhanced\u00C2\u00A0therapeutic\u00C2\u00A0responses\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0 of\u00C2\u00A0Ab\u00C2\u00A0..................................................................................................................................\u00C2\u00A04\u00C2\u00A0 Figure\u00C2\u00A01.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP\u00C2\u00A0 exhibits\u00C2\u00A0 improved\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 efficacy\u00C2\u00A0 in\u00C2\u00A0 an\u00C2\u00A0 LCC6ErbB2\u00C2\u00A0 breast\u00C2\u00A0 cancer\u00C2\u00A0 model\u00C2\u00A0 compared\u00C2\u00A0to\u00C2\u00A0Trz.................................................................................................................\u00C2\u00A05\u00C2\u00A0 Figure\u00C2\u00A02.1\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0 treatment\u00C2\u00A0.........................................................................................................................\u00C2\u00A034\u00C2\u00A0 Figure\u00C2\u00A02.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Binding\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0............................\u00C2\u00A036\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A02.3\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0vitro\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090mediated\u00C2\u00A0CDC\u00C2\u00A0and\u00C2\u00A0ADCC\u00C2\u00A0are\u00C2\u00A0augmented\u00C2\u00A0when\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0presented\u00C2\u00A0 to\u00C2\u00A0cells\u00C2\u00A0as\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0\u00C2\u00A0.......................................................................................\u00C2\u00A039\u00C2\u00A0 Figure\u00C2\u00A02.4\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 NK\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0upon\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0forms\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0\u00C2\u00A0.................\u00C2\u00A041\u00C2\u00A0 Figure\u00C2\u00A02.5\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Efficacy\u00C2\u00A0study\u00C2\u00A0for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0................................................................................................\u00C2\u00A043\u00C2\u00A0 Figure\u00C2\u00A02.6\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0in\u00C2\u00A0two\u00C2\u00A0mouse\u00C2\u00A0models\u00C2\u00A0.......................................................\u00C2\u00A045\u00C2\u00A0 Figure\u00C2\u00A03.1\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Overview\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0improved\u00C2\u00A0methodology\u00C2\u00A0for\u00C2\u00A0preparing\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0................\u00C2\u00A059\u00C2\u00A0 Figure\u00C2\u00A03.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Control\u00C2\u00A0over\u00C2\u00A0Neut\u00C2\u00A0and\u00C2\u00A0PEG\u00C2\u00A0content\u00C2\u00A0during\u00C2\u00A0the\u00C2\u00A0preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNPs\u00C2\u00A0....................\u00C2\u00A061\u00C2\u00A0 Figure\u00C2\u00A03.3\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Ab\u00C2\u00A0biotinylation\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0NIMAC\u00C2\u00A0column\u00C2\u00A0allows\u00C2\u00A0for\u00C2\u00A0easy\u00C2\u00A0purification,\u00C2\u00A0high\u00C2\u00A0recovery,\u00C2\u00A0and\u00C2\u00A0 precise\u00C2\u00A0degree\u00C2\u00A0of\u00C2\u00A0biotinylation\u00C2\u00A0\u00C2\u00A0.......................................................................................\u00C2\u00A064\u00C2\u00A0 Figure\u00C2\u00A03.4\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Control\u00C2\u00A0over\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0when\u00C2\u00A0coupling\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0.................\u00C2\u00A066\u00C2\u00A0 Figure\u00C2\u00A04.1\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0show\u00C2\u00A0an\u00C2\u00A0inverse\u00C2\u00A0correlation\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0..............................................................\u00C2\u00A079\u00C2\u00A0 Figure\u00C2\u00A04.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Representative\u00C2\u00A0 confocal\u00C2\u00A0 fluorescence\u00C2\u00A0 microscopy\u00C2\u00A0 images\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 distribution\u00C2\u00A0 on\u00C2\u00A0 Ramos\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0.........................................................................\u00C2\u00A083\u00C2\u00A0 Figure\u00C2\u00A04.3\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Time\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0........................................................................................................................\u00C2\u00A085\u00C2\u00A0 Figure\u00C2\u00A04.4\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Particularly\u00C2\u00A0 at\u00C2\u00A0 higher\u00C2\u00A0 doses,\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 are\u00C2\u00A0 significantly\u00C2\u00A0 more\u00C2\u00A0 cytotoxic\u00C2\u00A0 than\u00C2\u00A0 equivalent\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0............................................................................................\u00C2\u00A086\u00C2\u00A0 Figure\u00C2\u00A04.5\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 directly\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 while\u00C2\u00A0 the\u00C2\u00A0 individual\u00C2\u00A0 precursors\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0..........................................................................................\u00C2\u00A088\u00C2\u00A0 Figure\u00C2\u00A04.6\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 level\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 is\u00C2\u00A0 observed\u00C2\u00A0 in\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090 treated\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0all\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0...................\u00C2\u00A089\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xiv\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A05.1\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis\u00C2\u00A0is\u00C2\u00A0dependent\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0....................\u00C2\u00A0104\u00C2\u00A0 Figure\u00C2\u00A05.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 CD120a\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0dramatically\u00C2\u00A0elevated\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0\u00C2\u00A0.........................................................................................................................\u00C2\u00A0107\u00C2\u00A0 Figure\u00C2\u00A05.3\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 CD120a\u00C2\u00A0levels\u00C2\u00A0are\u00C2\u00A0elevated\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0apoptotic\u00C2\u00A0fractions\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090treated\u00C2\u00A0 cells\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0\u00C2\u00A0...............................................................................................\u00C2\u00A0109\u00C2\u00A0 Figure\u00C2\u00A05.4\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 levels\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 are\u00C2\u00A0 highest\u00C2\u00A0 at\u00C2\u00A0 8\u00C2\u00A0 h\u00C2\u00A0 post\u00E2\u0080\u0090 treatment\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0.........................................................................\u00C2\u00A0112\u00C2\u00A0 Figure\u00C2\u00A05.5\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Two\u00C2\u00A0 slices\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 representative\u00C2\u00A0 non\u00E2\u0080\u0090necrotic\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0\u00C2\u00A0..............................................................\u00C2\u00A0114\u00C2\u00A0 Figure\u00C2\u00A05.6\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Apoptosis\u00C2\u00A0via\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0an\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 liposomal\u00C2\u00A0component\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0formulation\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0.......................................................................................\u00C2\u00A0117\u00C2\u00A0 Figure\u00C2\u00A06.1\u00C2\u00A0 The\u00C2\u00A0manipulation\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0Chol\u00C2\u00A0 content\u00C2\u00A0using\u00C2\u00A0MBCD\u00C2\u00A0or\u00C2\u00A0MBCD/Chol\u00C2\u00A0 significantly\u00C2\u00A0impacts\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0.......................\u00C2\u00A0136\u00C2\u00A0 Figure\u00C2\u00A06.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Cells\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 rescued\u00C2\u00A0 from\u00C2\u00A0 apoptosis\u00C2\u00A0 by\u00C2\u00A0 increasing\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 Chol\u00C2\u00A0 content\u00C2\u00A0show\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0raft\u00E2\u0080\u0090associated\u00C2\u00A0CD120a\u00C2\u00A0.............................................\u00C2\u00A0139\u00C2\u00A0 Figure\u00C2\u00A06.3\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Simvastatin\u00C2\u00A0decreases\u00C2\u00A0 the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0sensitizes\u00C2\u00A0cells\u00C2\u00A0 to\u00C2\u00A0apoptosis\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0......................................................................................................................\u00C2\u00A0141\u00C2\u00A0 Figure\u00C2\u00A06.4\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0model\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0that\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0by\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090bearing\u00C2\u00A0effector\u00C2\u00A0 cells\u00C2\u00A0................................................................................................................................\u00C2\u00A0143\u00C2\u00A0 Figure\u00C2\u00A06.5\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Membrane\u00C2\u00A0 raft\u00E2\u0080\u0090associated\u00C2\u00A0 CD120a\u00C2\u00A0 is\u00C2\u00A0 excluded\u00C2\u00A0 from\u00C2\u00A0 regions\u00C2\u00A0 enriched\u00C2\u00A0 in\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 Rtx/CD20,\u00C2\u00A0 and\u00C2\u00A0 non\u00E2\u0080\u0090raft\u00C2\u00A0 CD120a\u00C2\u00A0 is\u00C2\u00A0 colocalized\u00C2\u00A0 with\u00C2\u00A0 hypercrosslinked\u00C2\u00A0Rtx/CD20\u00C2\u00A0...........................................................................................\u00C2\u00A0146\u00C2\u00A0 Figure\u00C2\u00A06.6\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Human\u00C2\u00A0 PBMCs\u00C2\u00A0 induce\u00C2\u00A0 significantly\u00C2\u00A0 higher\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0 plasma\u00E2\u0080\u0090 membrane\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0 non\u00E2\u0080\u0090pretreated\u00C2\u00A0cells\u00C2\u00A0......................................................................................................\u00C2\u00A0150\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xv\u00C2\u00A0\u00C2\u00A0 List\u00C2\u00A0of\u00C2\u00A0Abbreviations\u00C2\u00A0 \u00C2\u00A0 2oAb\u00C2\u00A0 secondary\u00C2\u00A0antibody\u00C2\u00A0 [3H]CHE\u00C2\u00A0 [3H]cholesteryl\u00C2\u00A0hexadecyl\u00C2\u00A0ether\u00C2\u00A0 A###\u00E2\u0080\u0090\u00CF\u0084\u00C2\u00A0 Alexa\u00C2\u00A0Fluor\u00C2\u00A0###\u00E2\u0080\u0090labeled\u00C2\u00A0antibody\u00C2\u00A0against\u00C2\u00A0antigen\u00C2\u00A0\u00CF\u0084\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 e.g.\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0=\u00C2\u00A0Alexa\u00C2\u00A0Fluor\u00C2\u00A0647\u00C2\u00A0labeled\u00C2\u00A0anti\u00E2\u0080\u0090CD120a\u00C2\u00A0antibody\u00C2\u00A0 Ab\u00C2\u00A0 antibody\u00C2\u00A0 ADCC\u00C2\u00A0 antibody\u00E2\u0080\u0090dependent\u00C2\u00A0cell\u00E2\u0080\u0090mediated\u00C2\u00A0cytotoxicity\u00C2\u00A0 AF\u00C2\u00A0 ammonium\u00C2\u00A0ferrothiocyanate\u00C2\u00A0 AFC\u00C2\u00A0\u00C2\u00A0 7\u00E2\u0080\u0090amino\u00E2\u0080\u00904\u00E2\u0080\u0090trifluoromethyl\u00C2\u00A0coumarin\u00C2\u00A0 BB\u00C2\u00A0 binding\u00C2\u00A0buffer\u00C2\u00A0 BCA\u00C2\u00A0 bicinchoninic\u00C2\u00A0acid\u00C2\u00A0 CBQCA\u00C2\u00A0 3\u00E2\u0080\u0090(4\u00E2\u0080\u0090carboxybenzoyl)quinoline\u00E2\u0080\u00902\u00E2\u0080\u0090carboxaldehyde\u00C2\u00A0 CDC\u00C2\u00A0 complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0 CFSE\u00C2\u00A0 carboxyfluorescein\u00C2\u00A0diacetate\u00C2\u00A0succinimidyl\u00C2\u00A0ester\u00C2\u00A0 Chol\u00C2\u00A0 cholesterol\u00C2\u00A0 CLL\u00C2\u00A0 chronic\u00C2\u00A0lymphocytic\u00C2\u00A0leukemia\u00C2\u00A0 CTX\u00C2\u00A0 cholera\u00C2\u00A0toxin\u00C2\u00A0B\u00C2\u00A0subunit\u00C2\u00A0 D649\u00C2\u00A0 DyLight\u00C2\u00A0649\u00C2\u00A0 DISC\u00C2\u00A0 death\u00E2\u0080\u0090inducing\u00C2\u00A0signaling\u00C2\u00A0complex\u00C2\u00A0 DLBCL\u00C2\u00A0\u00C2\u00A0 diffuse\u00C2\u00A0large\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0lymphoma\u00C2\u00A0 DMSO\u00C2\u00A0 dimethyl\u00C2\u00A0sulfoxide\u00C2\u00A0 DR\u00C2\u00A0 detergent\u00C2\u00A0resistance\u00C2\u00A0 DSPC\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphocholine\u00C2\u00A0 DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphoethanolamine\u00E2\u0080\u0090N\u00E2\u0080\u0090[methoxy(polyethylene\u00C2\u00A0\u00C2\u00A0 glycol)\u00E2\u0080\u00902000]\u00C2\u00A0 DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphoethanolamine\u00E2\u0080\u0090N\u00E2\u0080\u0090[biotinyl(polyethylene\u00C2\u00A0glycol)\u00E2\u0080\u0090\u00C2\u00A0 2000]\u00C2\u00A0 DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090Mal\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphoethanolamine\u00E2\u0080\u0090N\u00E2\u0080\u0090[maleimide(polyethylene\u00C2\u00A0\u00C2\u00A0 glycol)\u00E2\u0080\u00902000]\u00C2\u00A0 DTT\u00C2\u00A0 dithiothreitol\u00C2\u00A0 EB\u00C2\u00A0 elution\u00C2\u00A0buffer\u00C2\u00A0 EDTA\u00C2\u00A0 ethylene\u00C2\u00A0diamine\u00C2\u00A0tetraacetic\u00C2\u00A0acid\u00C2\u00A0 ELISA\u00C2\u00A0 enzyme\u00E2\u0080\u0090linked\u00C2\u00A0immunosorbent\u00C2\u00A0assay\u00C2\u00A0 FAM\u00C2\u00A0 carboxyfluorescein\u00C2\u00A0 Fc\u00CE\u00B3R\u00C2\u00A0 IgG\u00C2\u00A0Fc\u00C2\u00A0receptor\u00C2\u00A0 Fc\u00CE\u00B5RI\u00C2\u00A0\u00C2\u00A0 IgE\u00C2\u00A0Fc\u00C2\u00A0receptor\u00C2\u00A0I\u00C2\u00A0 FITC\u00C2\u00A0 fluorescein\u00C2\u00A0isothiocyanate\u00C2\u00A0 FL\u00C2\u00A0 follicular\u00C2\u00A0lymphoma\u00C2\u00A0 FLICA\u00C2\u00A0 fluorescent\u00C2\u00A0inhibitor\u00C2\u00A0of\u00C2\u00A0caspase\u00C2\u00A0 FMK\u00C2\u00A0 fluoromethyl\u00C2\u00A0ketone\u00C2\u00A0 FSC\u00C2\u00A0 forward\u00C2\u00A0scatter\u00C2\u00A0 HABA\u00C2\u00A0 4\u00E2\u0080\u0090hydroxyazobenzene\u00E2\u0080\u00902\u00E2\u0080\u0090carboxylic\u00C2\u00A0acid\u00C2\u00A0 HEPES\u00C2\u00A0 2\u00E2\u0080\u0090[4\u00E2\u0080\u0090(2\u00E2\u0080\u0090hydroxyethyl)piperazin\u00E2\u0080\u00901\u00E2\u0080\u0090yl]ethanesulfonic\u00C2\u00A0acid\u00C2\u00A0 HBS\u00C2\u00A0 HEPES\u00C2\u00A0buffered\u00C2\u00A0saline\u00C2\u00A0 HMG\u00E2\u0080\u0090CoA\u00C2\u00A0\u00C2\u00A0 3\u00E2\u0080\u0090hydroxy\u00E2\u0080\u00903\u00E2\u0080\u0090methylglutaryl\u00C2\u00A0coenzyme\u00C2\u00A0A\u00C2\u00A0 IgE/IgG\u00C2\u00A0 immunoglobulin\u00C2\u00A0E/immunoglobulin\u00C2\u00A0G\u00C2\u00A0 IL\u00C2\u00A0 interleukin\u00C2\u00A0 IP\u00C2\u00A0 intraperitoneally\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xvi\u00C2\u00A0\u00C2\u00A0 IV\u00C2\u00A0 intravenous/intravenously\u00C2\u00A0 LNP\u00C2\u00A0 lipid\u00C2\u00A0nanoparticle\u00C2\u00A0 Luc\u00C2\u00A0 luciferase\u00C2\u00A0 LV\u00C2\u00A0 lentivirus\u00C2\u00A0 Mal\u00C2\u00A0 maleimide\u00C2\u00A0 MCL\u00C2\u00A0 mantle\u00C2\u00A0cell\u00C2\u00A0lymphoma\u00C2\u00A0 MS\u00C2\u00A0 microsphere\u00C2\u00A0 Neut\u00C2\u00A0 NeutrAvidin\u00C2\u00A0 NHL\u00C2\u00A0 non\u00E2\u0080\u0090Hodgkin\u00E2\u0080\u0099s\u00C2\u00A0lymphoma\u00C2\u00A0 NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 N\u00E2\u0080\u0090Hydroxysuccinimide\u00C2\u00A0ester\u00E2\u0080\u0090dPEG4\u00E2\u0080\u0090biotin\u00C2\u00A0 NIMAC\u00C2\u00A0 nickel\u00C2\u00A0immobilized\u00C2\u00A0metal\u00C2\u00A0affinity\u00C2\u00A0chromatography\u00C2\u00A0 NK\u00C2\u00A0 natural\u00C2\u00A0killer\u00C2\u00A0 NP\u00C2\u00A0 nanoparticle\u00C2\u00A0 PBMC\u00C2\u00A0 peripheral\u00C2\u00A0blood\u00C2\u00A0mononuclear\u00C2\u00A0cell\u00C2\u00A0 PBS\u00C2\u00A0 phosphate\u00C2\u00A0buffered\u00C2\u00A0saline\u00C2\u00A0 PE\u00C2\u00A0 phycoerythrin\u00C2\u00A0 PEG\u00C2\u00A0 polyethylene\u00C2\u00A0glycol\u00C2\u00A0 PI\u00C2\u00A0 propidium\u00C2\u00A0iodide\u00C2\u00A0 Prz\u00C2\u00A0 pertuzumab\u00C2\u00A0 QLS\u00C2\u00A0 quasi\u00E2\u0080\u0090elastic\u00C2\u00A0light\u00C2\u00A0scattering\u00C2\u00A0 \u00C2\u00A0 Rtx\u00C2\u00A0 rituximab\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(\u00CE\u00B1)\u00C2\u00A0 rituximab\u00E2\u0080\u0090lipid\u00C2\u00A0nanoparticle\u00C2\u00A0of\u00C2\u00A0valence\u00C2\u00A0\u00CE\u00B1\u00C2\u00A0 ROI\u00C2\u00A0 region\u00C2\u00A0of\u00C2\u00A0interest\u00C2\u00A0 SB\u00C2\u00A0 stripping\u00C2\u00A0buffer\u00C2\u00A0 SC\u00C2\u00A0 subcutaneous/subcutaneously\u00C2\u00A0 SCID\u00C2\u00A0 severe\u00C2\u00A0combined\u00C2\u00A0immunodeficiency\u00C2\u00A0 SD\u00C2\u00A0 standard\u00C2\u00A0deviation\u00C2\u00A0 SPDP\u00C2\u00A0 N\u00E2\u0080\u0090Succinimidyl\u00C2\u00A03\u00E2\u0080\u0090(2\u00E2\u0080\u0090pyridyldithio)propionate\u00C2\u00A0 SSC\u00C2\u00A0 side\u00C2\u00A0scatter\u00C2\u00A0 SUV\u00C2\u00A0 small\u00C2\u00A0unilamellar\u00C2\u00A0vesicle\u00C2\u00A0 TNF\u00CE\u00B1\u00C2\u00A0 tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0\u00CE\u00B1\u00C2\u00A0 TNFR\u00C2\u00A0 tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0receptor\u00C2\u00A0 TRAPS\u00C2\u00A0 TNFR\u00E2\u0080\u0090associated\u00C2\u00A0periodic\u00C2\u00A0syndrome\u00C2\u00A0 Trz\u00C2\u00A0 trastuzumab\u00C2\u00A0 TX100\u00C2\u00A0 Triton\u00C2\u00A0X\u00E2\u0080\u0090100\u00C2\u00A0 wrt\u00C2\u00A0 with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xvii\u00C2\u00A0\u00C2\u00A0 Acknowledgements\u00C2\u00A0 \u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0 like\u00C2\u00A0 to\u00C2\u00A0offer\u00C2\u00A0my\u00C2\u00A0gratitude\u00C2\u00A0 to\u00C2\u00A0my\u00C2\u00A0 supervisor,\u00C2\u00A0Dr.\u00C2\u00A0Marcel\u00C2\u00A0Bally,\u00C2\u00A0 for\u00C2\u00A0his\u00C2\u00A0 support\u00C2\u00A0and\u00C2\u00A0 guidance\u00C2\u00A0in\u00C2\u00A0my\u00C2\u00A0graduate\u00C2\u00A0studies,\u00C2\u00A0and\u00C2\u00A0for\u00C2\u00A0encouraging\u00C2\u00A0my\u00C2\u00A0growth\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0scientist.\u00C2\u00A0\u00C2\u00A0I\u00C2\u00A0would\u00C2\u00A0also\u00C2\u00A0like\u00C2\u00A0to\u00C2\u00A0 acknowledge\u00C2\u00A0 the\u00C2\u00A0other\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0my\u00C2\u00A0 supervisory\u00C2\u00A0committee,\u00C2\u00A0Dr.\u00C2\u00A0Pieter\u00C2\u00A0Cullis\u00C2\u00A0and\u00C2\u00A0Dr.\u00C2\u00A0 I.\u00C2\u00A0Robert\u00C2\u00A0 Nabi\u00C2\u00A0for\u00C2\u00A0their\u00C2\u00A0scientific\u00C2\u00A0direction.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0 like\u00C2\u00A0 to\u00C2\u00A0 give\u00C2\u00A0 special\u00C2\u00A0 thanks\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0members\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 Department\u00C2\u00A0 of\u00C2\u00A0 Experimental\u00C2\u00A0 Therapeutics\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 British\u00C2\u00A0 Columbia\u00C2\u00A0 Cancer\u00C2\u00A0 Research\u00C2\u00A0 Centre,\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 laboratory\u00C2\u00A0 and\u00C2\u00A0 administrative\u00C2\u00A0groups,\u00C2\u00A0who\u00C2\u00A0provided\u00C2\u00A0assistance\u00C2\u00A0and\u00C2\u00A0encouragement\u00C2\u00A0with\u00C2\u00A0my\u00C2\u00A0work.\u00C2\u00A0\u00C2\u00A0I\u00C2\u00A0would\u00C2\u00A0also\u00C2\u00A0like\u00C2\u00A0 to\u00C2\u00A0thank\u00C2\u00A0the\u00C2\u00A0Interdisciplinary\u00C2\u00A0Oncology\u00C2\u00A0Program\u00C2\u00A0(College\u00C2\u00A0for\u00C2\u00A0Interdisciplinary\u00C2\u00A0Studies),\u00C2\u00A0who\u00C2\u00A0provided\u00C2\u00A0 support\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0financial\u00C2\u00A0assistance\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0travel\u00C2\u00A0awards.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0also\u00C2\u00A0like\u00C2\u00A0to\u00C2\u00A0thank\u00C2\u00A0Dr.\u00C2\u00A0Gert\u00C2\u00A0Storm,\u00C2\u00A0my\u00C2\u00A0supervisor\u00C2\u00A0during\u00C2\u00A0a\u00C2\u00A0six\u00E2\u0080\u0090month\u00C2\u00A0work\u00C2\u00A0term\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0 Utrecht\u00C2\u00A0 Institute\u00C2\u00A0 for\u00C2\u00A0Pharmaceutical\u00C2\u00A0Sciences\u00C2\u00A0at\u00C2\u00A0Utrecht\u00C2\u00A0University,\u00C2\u00A0The\u00C2\u00A0Netherlands.\u00C2\u00A0 \u00C2\u00A0Some\u00C2\u00A0data\u00C2\u00A0 found\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A04\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0during\u00C2\u00A0 this\u00C2\u00A0 time,\u00C2\u00A0 including\u00C2\u00A0 confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0 microscopy\u00C2\u00A0 images\u00C2\u00A0 which\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 Center\u00C2\u00A0 for\u00C2\u00A0 Cellular\u00C2\u00A0 Imaging\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 Faculty\u00C2\u00A0 of\u00C2\u00A0 Veterinary\u00C2\u00A0Medicine,\u00C2\u00A0and\u00C2\u00A0I\u00C2\u00A0would\u00C2\u00A0like\u00C2\u00A0to\u00C2\u00A0thank\u00C2\u00A0Dr.\u00C2\u00A0Richard\u00C2\u00A0Wubboltz\u00C2\u00A0and\u00C2\u00A0Esther\u00C2\u00A0van\u00C2\u00A0\u00E2\u0080\u0098t\u00C2\u00A0Veld\u00C2\u00A0for\u00C2\u00A0help\u00C2\u00A0 and\u00C2\u00A0technical\u00C2\u00A0advice.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 I\u00C2\u00A0would\u00C2\u00A0also\u00C2\u00A0like\u00C2\u00A0to\u00C2\u00A0acknowledge\u00C2\u00A0the\u00C2\u00A0generous\u00C2\u00A0financial\u00C2\u00A0support\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0following\u00C2\u00A0sources:\u00C2\u00A0 \u00EF\u0082\u00B7 Canadian\u00C2\u00A0Institutes\u00C2\u00A0of\u00C2\u00A0Health\u00C2\u00A0Research\u00C2\u00A0 \u00EF\u0082\u00B7 Michael\u00C2\u00A0Smith\u00C2\u00A0Foundation\u00C2\u00A0for\u00C2\u00A0Health\u00C2\u00A0Research\u00C2\u00A0 \u00EF\u0082\u00B7 British\u00C2\u00A0Columbia\u00C2\u00A0Innovation\u00C2\u00A0Council\u00C2\u00A0 \u00EF\u0082\u00B7 Natural\u00C2\u00A0Sciences\u00C2\u00A0and\u00C2\u00A0Engineering\u00C2\u00A0Research\u00C2\u00A0Council\u00C2\u00A0 \u00C2\u00A0 Finally,\u00C2\u00A0 a\u00C2\u00A0 heartfelt\u00C2\u00A0 thank\u00C2\u00A0 you\u00C2\u00A0 to\u00C2\u00A0my\u00C2\u00A0 friends,\u00C2\u00A0 family,\u00C2\u00A0 and\u00C2\u00A0 partner\u00C2\u00A0 S\u00C3\u00A9bastien\u00C2\u00A0 for\u00C2\u00A0 their\u00C2\u00A0 love,\u00C2\u00A0 encouragement,\u00C2\u00A0and\u00C2\u00A0enduring\u00C2\u00A0support.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0xviii\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Dedication\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0my\u00C2\u00A0father\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A01\u00C2\u00A0\u00C2\u00A0 1\u00C2\u00A0 Introduction:\u00C2\u00A0Developing\u00C2\u00A0novel\u00C2\u00A0therapies\u00C2\u00A0for\u00C2\u00A0treating\u00C2\u00A0cancer\u00C2\u00A0 1.1\u00C2\u00A0 Overview\u00C2\u00A0 The\u00C2\u00A0goal\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0work\u00C2\u00A0presented\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0dissertation\u00C2\u00A0is\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0new\u00C2\u00A0drug\u00C2\u00A0technologies\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 treatment\u00C2\u00A0 of\u00C2\u00A0 cancer.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 involves\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 methodology\u00C2\u00A0 for\u00C2\u00A0 discovering\u00C2\u00A0 and\u00C2\u00A0 developing\u00C2\u00A0 new\u00C2\u00A0 treatments\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0 antibodies\u00C2\u00A0 (Abs),\u00C2\u00A0 a\u00C2\u00A0 class\u00C2\u00A0 of\u00C2\u00A0 drugs\u00C2\u00A0 that\u00C2\u00A0 hold\u00C2\u00A0 great\u00C2\u00A0 promise\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 treatment1,\u00C2\u00A0 2.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0methodology\u00C2\u00A0makes\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 drug\u00C2\u00A0 formulation\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 liposomes,\u00C2\u00A0 a\u00C2\u00A0 type\u00C2\u00A0of\u00C2\u00A0 lipid\u00C2\u00A0nanoparticle\u00C2\u00A0 (LNP)\u00C2\u00A0 that\u00C2\u00A0normally\u00C2\u00A0 serves\u00C2\u00A0 to\u00C2\u00A0 enhance\u00C2\u00A0 the\u00C2\u00A0delivery\u00C2\u00A0of\u00C2\u00A0 an\u00C2\u00A0 encapsulated\u00C2\u00A0chemotherapeutic\u00C2\u00A0drug\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0disease\u00C2\u00A0and\u00C2\u00A0therefore\u00C2\u00A0increase\u00C2\u00A0its\u00C2\u00A0efficacy3.\u00C2\u00A0\u00C2\u00A0One\u00C2\u00A0 common\u00C2\u00A0 way\u00C2\u00A0 to\u00C2\u00A0 further\u00C2\u00A0 improve\u00C2\u00A0 this\u00C2\u00A0 favorable\u00C2\u00A0 targeting\u00C2\u00A0 effect\u00C2\u00A0 is\u00C2\u00A0 by\u00C2\u00A0 attaching\u00C2\u00A0 tumor\u00E2\u0080\u0090specific\u00C2\u00A0 proteins\u00C2\u00A0or\u00C2\u00A0peptides\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0liposome\u00C2\u00A0surface4\u00E2\u0080\u00906.\u00C2\u00A0\u00C2\u00A0We\u00C2\u00A0generated\u00C2\u00A0such\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0constructs\u00C2\u00A0by\u00C2\u00A0attaching\u00C2\u00A0 several\u00C2\u00A0copies\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0rituximab\u00C2\u00A0(Rtx)\u00C2\u00A0or\u00C2\u00A0trastuzumab\u00C2\u00A0(Trz)\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0exterior\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0liposome,\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0exception\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0liposome\u00C2\u00A0interior\u00C2\u00A0was\u00C2\u00A0devoid\u00C2\u00A0of\u00C2\u00A0chemotherapy.\u00C2\u00A0\u00C2\u00A0Strikingly,\u00C2\u00A0 these\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0exhibited\u00C2\u00A0much\u00C2\u00A0higher\u00C2\u00A0efficacy\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0than\u00C2\u00A0Ab\u00C2\u00A0or\u00C2\u00A0bare\u00C2\u00A0liposomes\u00C2\u00A0alone7.\u00C2\u00A0 Given\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 encapsulated\u00C2\u00A0 drug,\u00C2\u00A0 the\u00C2\u00A0mechanism\u00C2\u00A0 by\u00C2\u00A0 which\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0 exert\u00C2\u00A0 their\u00C2\u00A0 effects\u00C2\u00A0is\u00C2\u00A0distinct\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0changes\u00C2\u00A0in\u00C2\u00A0drug\u00C2\u00A0pharmacokinetics\u00C2\u00A0and\u00C2\u00A0biodistribution\u00C2\u00A0normally\u00C2\u00A0attributed\u00C2\u00A0 to\u00C2\u00A0 liposomal\u00C2\u00A0formulations3,\u00C2\u00A08.\u00C2\u00A0 \u00C2\u00A0Because\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0entirely\u00C2\u00A0abnormal\u00C2\u00A0behavior,\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 promising\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 constructs\u00C2\u00A0was\u00C2\u00A0 completely\u00C2\u00A0 elusive,\u00C2\u00A0 but\u00C2\u00A0 the\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 critical\u00C2\u00A0element\u00C2\u00A0for\u00C2\u00A0further\u00C2\u00A0preclinical\u00C2\u00A0and\u00C2\u00A0clinical\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0such\u00C2\u00A0drug\u00C2\u00A0technologies.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0work\u00C2\u00A0 presented\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 dissertation\u00C2\u00A0 began\u00C2\u00A0with\u00C2\u00A0 efforts\u00C2\u00A0 to\u00C2\u00A0 uncover\u00C2\u00A0 the\u00C2\u00A0 unique\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 constructs.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 outcomes\u00C2\u00A0were\u00C2\u00A0 not\u00C2\u00A0 only\u00C2\u00A0 the\u00C2\u00A0 uncovering\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 explaining\u00C2\u00A0 the\u00C2\u00A0 enhanced\u00C2\u00A0 efficacy\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00C2\u00A0 itself,\u00C2\u00A0 but\u00C2\u00A0 also\u00C2\u00A0 the\u00C2\u00A0 creation\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 methodology\u00C2\u00A0 with\u00C2\u00A0 clear\u00C2\u00A0 potential\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 pharmaceutical\u00C2\u00A0 industry\u00C2\u00A0 for\u00C2\u00A0 discovering,\u00C2\u00A0 studying,\u00C2\u00A0 and\u00C2\u00A0 developing\u00C2\u00A0new\u00C2\u00A0cancer\u00C2\u00A0treatments\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A02\u00C2\u00A0\u00C2\u00A0 1.2\u00C2\u00A0 Liposomal\u00C2\u00A0formulations\u00C2\u00A0 1.2.1\u00C2\u00A0 Liposomes\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0treatment\u00C2\u00A0 Liposomes\u00C2\u00A0are\u00C2\u00A0nanometer\u00E2\u0080\u0090scale\u00C2\u00A0particles\u00C2\u00A0made\u00C2\u00A0up\u00C2\u00A0of\u00C2\u00A0one\u00C2\u00A0or\u00C2\u00A0more\u00C2\u00A0hydrophobic\u00C2\u00A0lipid\u00C2\u00A0bilayers\u00C2\u00A0 that\u00C2\u00A0 enclose\u00C2\u00A0 an\u00C2\u00A0 internal\u00C2\u00A0 aqueous\u00C2\u00A0 compartment.\u00C2\u00A0 \u00C2\u00A0 There\u00C2\u00A0 are\u00C2\u00A0 currently\u00C2\u00A0 four\u00C2\u00A0 approved\u00C2\u00A0 liposomal\u00C2\u00A0 formulations\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0cancer,\u00C2\u00A0and\u00C2\u00A0at\u00C2\u00A0 least\u00C2\u00A014\u00C2\u00A0 in\u00C2\u00A0Phase\u00C2\u00A0 I\u00C2\u00A0clinical\u00C2\u00A0trials\u00C2\u00A0or\u00C2\u00A0 later\u00C2\u00A0stages\u00C2\u00A0of\u00C2\u00A0 development9.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 cancer\u00C2\u00A0 treatment,\u00C2\u00A0 liposomes\u00C2\u00A0 are\u00C2\u00A0 traditionally\u00C2\u00A0 used\u00C2\u00A0 as\u00C2\u00A0 drug\u00C2\u00A0 delivery\u00C2\u00A0 vehicles,\u00C2\u00A0 where\u00C2\u00A0 a\u00C2\u00A0 chemotherapeutic\u00C2\u00A0 agent\u00C2\u00A0 is\u00C2\u00A0 encapsulated\u00C2\u00A0 within\u00C2\u00A0 the\u00C2\u00A0 liposome\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 drug\u00E2\u0080\u0090liposome\u00C2\u00A0 complex\u00C2\u00A0accumulates\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0site8.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0main\u00C2\u00A0mechanism\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0this\u00C2\u00A0occurs\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0enhanced\u00C2\u00A0 permeability\u00C2\u00A0and\u00C2\u00A0retention\u00C2\u00A0effect,\u00C2\u00A0also\u00C2\u00A0referred\u00C2\u00A0to\u00C2\u00A0as\u00C2\u00A0passive\u00C2\u00A0targeting.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0results\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0passage\u00C2\u00A0 of\u00C2\u00A0 liposomes\u00C2\u00A0 through\u00C2\u00A0 tumor\u00C2\u00A0 vasculature,\u00C2\u00A0 which\u00C2\u00A0 unlike\u00C2\u00A0 normal\u00C2\u00A0 vasculature,\u00C2\u00A0 is\u00C2\u00A0 permeable\u00C2\u00A0 to\u00C2\u00A0 liposomes\u00C2\u00A0 and\u00C2\u00A0 macromolecules.\u00C2\u00A0 \u00C2\u00A0 Because\u00C2\u00A0 tumors\u00C2\u00A0 exhibit\u00C2\u00A0 poor\u00C2\u00A0 lymphatic\u00C2\u00A0 drainage,\u00C2\u00A0 liposomes\u00C2\u00A0 subsequently\u00C2\u00A0accumulate\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0tumor10.\u00C2\u00A0\u00C2\u00A0Compared\u00C2\u00A0to\u00C2\u00A0administration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dose\u00C2\u00A0 of\u00C2\u00A0 free\u00C2\u00A0drug,\u00C2\u00A0 concentrations\u00C2\u00A0of\u00C2\u00A0drug\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 site\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A010\u00E2\u0080\u0090fold\u00C2\u00A0or\u00C2\u00A0higher3.\u00C2\u00A0 \u00C2\u00A0Such\u00C2\u00A0enhanced\u00C2\u00A0 delivery\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0disease,\u00C2\u00A0without\u00C2\u00A0an\u00C2\u00A0associated\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0toxicity,\u00C2\u00A0is\u00C2\u00A0 instrumental\u00C2\u00A0in\u00C2\u00A0achieving\u00C2\u00A0therapeutic\u00C2\u00A0improvement\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0encapsulated\u00C2\u00A0drug8.\u00C2\u00A0 As\u00C2\u00A0a\u00C2\u00A0method\u00C2\u00A0to\u00C2\u00A0further\u00C2\u00A0refine\u00C2\u00A0the\u00C2\u00A0specificity\u00C2\u00A0of\u00C2\u00A0targeting,\u00C2\u00A0the\u00C2\u00A0attachment\u00C2\u00A0of\u00C2\u00A0target\u00E2\u0080\u0090specific\u00C2\u00A0 proteins\u00C2\u00A0or\u00C2\u00A0peptides\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0drug\u00C2\u00A0carrier\u00C2\u00A0surface\u00C2\u00A0has\u00C2\u00A0received\u00C2\u00A0considerable\u00C2\u00A0attention4\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0results\u00C2\u00A0 in\u00C2\u00A0active\u00C2\u00A0targeting\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0liposome\u00C2\u00A0and\u00C2\u00A0its\u00C2\u00A0contents\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0site.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0targeting\u00C2\u00A0protein\u00C2\u00A0is\u00C2\u00A0most\u00C2\u00A0 commonly\u00C2\u00A0a\u00C2\u00A0 tumor\u00E2\u0080\u0090specific\u00C2\u00A0Ab,\u00C2\u00A0and\u00C2\u00A0such\u00C2\u00A0constructs\u00C2\u00A0are\u00C2\u00A0therefore\u00C2\u00A0termed\u00C2\u00A0 immunoliposomes.\u00C2\u00A0 \u00C2\u00A0Abs\u00C2\u00A0 are\u00C2\u00A0selected\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0basis\u00C2\u00A0of\u00C2\u00A0their\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0bind\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0molecules\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0disease\u00C2\u00A0site;\u00C2\u00A0when\u00C2\u00A0these\u00C2\u00A0 Abs\u00C2\u00A0are\u00C2\u00A0coupled\u00C2\u00A0to\u00C2\u00A0 liposomes\u00C2\u00A0containing\u00C2\u00A0chemotherapy,\u00C2\u00A0 liposome\u00C2\u00A0targeting\u00C2\u00A0 is\u00C2\u00A0regularly\u00C2\u00A0enhanced6,\u00C2\u00A0 11,\u00C2\u00A0 12.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown,\u00C2\u00A0 however,\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 chemical\u00C2\u00A0modification\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 protein\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 conjugation\u00C2\u00A0 to\u00C2\u00A0 liposomes\u00C2\u00A0 impairs\u00C2\u00A0 the\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Ab13\u00E2\u0080\u009015.\u00C2\u00A0 \u00C2\u00A0With\u00C2\u00A0 the\u00C2\u00A0advent\u00C2\u00A0of\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 Trz\u00C2\u00A0 (Section\u00C2\u00A0 1.3),\u00C2\u00A0 the\u00C2\u00A0 targeting\u00C2\u00A0 Ab\u00C2\u00A0 itself\u00C2\u00A0 can\u00C2\u00A0 exhibit\u00C2\u00A0 a\u00C2\u00A0 therapeutic\u00C2\u00A0 response\u00C2\u00A0 in\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A03\u00C2\u00A0\u00C2\u00A0 addition\u00C2\u00A0to\u00C2\u00A0encapsulated\u00C2\u00A0drug\u00C2\u00A0(Section\u00C2\u00A01.3).\u00C2\u00A0 \u00C2\u00A0Surprisingly,\u00C2\u00A0previous\u00C2\u00A0work\u00C2\u00A0from\u00C2\u00A0our\u00C2\u00A0 laboratory\u00C2\u00A0using\u00C2\u00A0 \u00E2\u0080\u009Cempty\u00C2\u00A0immunoliposomes\u00E2\u0080\u009D\u00C2\u00A0containing\u00C2\u00A0a\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0(rather\u00C2\u00A0than\u00C2\u00A0simply\u00C2\u00A0a\u00C2\u00A0targeting\u00C2\u00A0Ab)\u00C2\u00A0indicated\u00C2\u00A0 that\u00C2\u00A0 coupling\u00C2\u00A0 the\u00C2\u00A0 Ab\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 liposome\u00C2\u00A0 actually\u00C2\u00A0 benefited\u00C2\u00A0 its\u00C2\u00A0 therapeutic\u00C2\u00A0 activity7.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 special\u00C2\u00A0 constructs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0termed\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs.\u00C2\u00A0\u00C2\u00A0 1.2.2\u00C2\u00A0 What\u00C2\u00A0are\u00C2\u00A0Ab\u00C2\u00ADLNPs?\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0consist\u00C2\u00A0of\u00C2\u00A0many\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0tethered\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0outer\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0small\u00C2\u00A0 unilamellar\u00C2\u00A0vesicle\u00C2\u00A0(SUV),\u00C2\u00A0creating\u00C2\u00A0a\u00C2\u00A0construct\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0diameter\u00C2\u00A0of\u00C2\u00A0approximately\u00C2\u00A0130\u00C2\u00A0nm.\u00C2\u00A0\u00C2\u00A0An\u00C2\u00A0SUV\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0 type\u00C2\u00A0of\u00C2\u00A0liposome\u00C2\u00A0that\u00C2\u00A0contains\u00C2\u00A0one\u00C2\u00A0lipid\u00C2\u00A0bilayer\u00C2\u00A0surrounding\u00C2\u00A0an\u00C2\u00A0aqueous\u00C2\u00A0interior,\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0current\u00C2\u00A0 application,\u00C2\u00A0 the\u00C2\u00A0bilayer\u00C2\u00A0 contains\u00C2\u00A0 a\u00C2\u00A0mixture\u00C2\u00A0of\u00C2\u00A0phospholipids,\u00C2\u00A0 cholesterol\u00C2\u00A0 (Chol),\u00C2\u00A0 and\u00C2\u00A0polyethylene\u00C2\u00A0 glycol\u00C2\u00A0 (PEG)\u00C2\u00A0 (see\u00C2\u00A0Section\u00C2\u00A02.3.2\u00C2\u00A0and\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0 for\u00C2\u00A0more\u00C2\u00A0detail)16,\u00C2\u00A017.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0PEG\u00C2\u00A0 serves\u00C2\u00A0 specifically\u00C2\u00A0 to\u00C2\u00A0 block\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0proteins\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs;\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0PEG,\u00C2\u00A0such\u00C2\u00A0binding\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0 uptake\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0reticuloendothelial\u00C2\u00A0system\u00C2\u00A0and\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0significantly\u00C2\u00A0decreased\u00C2\u00A0circulation\u00C2\u00A0lifetime\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0formulation18,\u00C2\u00A019.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0employ\u00C2\u00A0liposomes\u00C2\u00A0strictly\u00C2\u00A0as\u00C2\u00A0scaffolds\u00C2\u00A0to\u00C2\u00A0bridge\u00C2\u00A0many\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0together,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0distinct\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0traditional\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0 liposomes,\u00C2\u00A0which\u00C2\u00A0actively\u00C2\u00A0or\u00C2\u00A0passively\u00C2\u00A0target\u00C2\u00A0an\u00C2\u00A0encapsulated\u00C2\u00A0 drug.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 lack\u00C2\u00A0 of\u00C2\u00A0 encapsulated\u00C2\u00A0 drug,\u00C2\u00A0 the\u00C2\u00A0 advantages\u00C2\u00A0 normally\u00C2\u00A0 associated\u00C2\u00A0 with\u00C2\u00A0 liposomal\u00C2\u00A0 formulations\u00C2\u00A0 discussed\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 1.2.1\u00C2\u00A0 (improvements\u00C2\u00A0 in\u00C2\u00A0 pharmacokinetics\u00C2\u00A0 and\u00C2\u00A0 biodistribution\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0encapsulated\u00C2\u00A0drug)\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0applicable\u00C2\u00A0to\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs3,\u00C2\u00A08.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0emphasize\u00C2\u00A0this\u00C2\u00A0 distinction\u00C2\u00A0 in\u00C2\u00A0 liposome\u00C2\u00A0 function\u00C2\u00A0and\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action,\u00C2\u00A0 the\u00C2\u00A0 term\u00C2\u00A0 \u00E2\u0080\u009CAb\u00E2\u0080\u0090LNP\u00E2\u0080\u009D\u00C2\u00A0 is\u00C2\u00A0employed\u00C2\u00A0 rather\u00C2\u00A0 than\u00C2\u00A0other\u00C2\u00A0terms\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0\u00E2\u0080\u009Cliposomal\u00C2\u00A0antibody\u00E2\u0080\u009D\u00C2\u00A0or\u00C2\u00A0\u00E2\u0080\u009Cempty\u00C2\u00A0immunoliposome.\u00E2\u0080\u009D\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Previous\u00C2\u00A0 studies\u00C2\u00A0 from\u00C2\u00A0 our\u00C2\u00A0 laboratory\u00C2\u00A0made\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0 prepared\u00C2\u00A0with\u00C2\u00A0 two\u00C2\u00A0 different\u00C2\u00A0 therapeutic\u00C2\u00A0Abs,\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Trz7.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A01.1,\u00C2\u00A0these\u00C2\u00A0studies\u00C2\u00A0demonstrated\u00C2\u00A0that\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0given\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Trz,\u00C2\u00A0the\u00C2\u00A0Abs\u00C2\u00A0exhibited\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0activities\u00C2\u00A0against\u00C2\u00A0 different\u00C2\u00A0cancer\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0Abs\u00C2\u00A0were\u00C2\u00A0attached\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0liposome\u00C2\u00A0as\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs.\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A04\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A01.1\u00C2\u00A0\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0exhibit\u00C2\u00A0enhanced\u00C2\u00A0therapeutic\u00C2\u00A0responses\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u00A0.\u00C2\u00A0 (A\u00C2\u00A0&\u00C2\u00A0B)\u00C2\u00A0Two\u00C2\u00A0different\u00C2\u00A0 lymphoma\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0 (Ramos\u00C2\u00A0and\u00C2\u00A0Z138)\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0 for\u00C2\u00A03\u00C2\u00A0days\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 (white\u00C2\u00A0columns),\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 (gray\u00C2\u00A0columns),\u00C2\u00A0or\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0crosslinking\u00C2\u00A0secondary\u00C2\u00A0Ab\u00C2\u00A0 (2oAb)\u00C2\u00A0 (black\u00C2\u00A0columns).\u00C2\u00A0 \u00C2\u00A0 (C\u00C2\u00A0&\u00C2\u00A0D)\u00C2\u00A0Two\u00C2\u00A0different\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0(LCC6ErbB2\u00C2\u00A0and\u00C2\u00A0MCF7ErbB2)\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0for\u00C2\u00A05\u00C2\u00A0days\u00C2\u00A0with\u00C2\u00A0Trz\u00C2\u00A0(white\u00C2\u00A0columns)\u00C2\u00A0or\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0(gray\u00C2\u00A0columns)\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0 of\u00C2\u00A0heregulin.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u009CFraction\u00C2\u00A0affected\u00E2\u0080\u009D\u00C2\u00A0was\u00C2\u00A0 calculated\u00C2\u00A0by\u00C2\u00A0determining\u00C2\u00A0 the\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0 remaining\u00C2\u00A0viable\u00C2\u00A0 cells\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A03\u00E2\u0080\u0090(4,5\u00E2\u0080\u0090 dimethylthiazol\u00E2\u0080\u00902\u00E2\u0080\u0090yl)\u00E2\u0080\u00902,5\u00E2\u0080\u0090diphenyltetrazolium\u00C2\u00A0bromide\u00C2\u00A0assay,\u00C2\u00A0and\u00C2\u00A0subtracting\u00C2\u00A0this\u00C2\u00A0value\u00C2\u00A0from\u00C2\u00A01.\u00C2\u00A0 \u00C2\u00A0Columns:\u00C2\u00A0mean\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0 independent\u00C2\u00A0experiments;\u00C2\u00A0bars:\u00C2\u00A0standard\u00C2\u00A0error;\u00C2\u00A0*p\u00C2\u00A0<\u00C2\u00A00.05,\u00C2\u00A0#p\u00C2\u00A0<\u00C2\u00A00.05,\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0free\u00C2\u00A0Ab\u00C2\u00A0treatment.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0and\u00C2\u00A0pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Trz\u00E2\u0080\u0090LNPs\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0examined\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0 if\u00C2\u00A0 these\u00C2\u00A0favorable\u00C2\u00A0effects\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A01.2,\u00C2\u00A0the\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0 was\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0 than\u00C2\u00A0that\u00C2\u00A0of\u00C2\u00A0Trz.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0 studied\u00C2\u00A0and\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0of\u00C2\u00A0Trz;\u00C2\u00A0as\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A01.1,\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0exhibited\u00C2\u00A0significantly\u00C2\u00A0elevated\u00C2\u00A0 Trz\u00C2\u00A0plasma\u00C2\u00A0 levels\u00C2\u00A0after\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 free\u00C2\u00A0Trz,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0enhanced\u00C2\u00A0 tumor\u00C2\u00A0 localization\u00C2\u00A0of\u00C2\u00A0Trz,\u00C2\u00A0 explaining\u00C2\u00A0the\u00C2\u00A0increased\u00C2\u00A0efficacy\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0observed7.\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A0Adapted\u00C2\u00A0and\u00C2\u00A0reprinted\u00C2\u00A0by\u00C2\u00A0permission\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0American\u00C2\u00A0Association\u00C2\u00A0for\u00C2\u00A0Cancer\u00C2\u00A0Research:\u00C2\u00A0Chiu,\u00C2\u00A0G.N.C.\u00C2\u00A0et\u00C2\u00A0 al.,\u00C2\u00A0 Modulation\u00C2\u00A0 of\u00C2\u00A0 Cancer\u00C2\u00A0 Cell\u00C2\u00A0 Survival\u00C2\u00A0 Pathways\u00C2\u00A0 Using\u00C2\u00A0 Multivalent\u00C2\u00A0 Liposomal\u00C2\u00A0 Therapeutic\u00C2\u00A0 Antibody\u00C2\u00A0 Constructs,\u00C2\u00A0Molecular\u00C2\u00A0Cancer\u00C2\u00A0Therapeutics,\u00C2\u00A02007,\u00C2\u00A0vol.\u00C2\u00A06(3),\u00C2\u00A0844\u00E2\u0080\u009355.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0Ramos\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0B\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0Z138\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0C\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0LCC6ErbB2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0D\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0MCF7ErbB2\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A05\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A01.2\u00C2\u00A0\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0exhibits\u00C2\u00A0improved\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0in\u00C2\u00A0an\u00C2\u00A0LCC6ErbB2\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0model\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0Trz\u00E2\u0080\u00A0.\u00C2\u00A0 (A)\u00C2\u00A0In\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0LCC6ErbB2\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0model.\u00C2\u00A0\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0administered\u00C2\u00A0at\u00C2\u00A00.25\u00C2\u00A0mg/kg\u00C2\u00A0(\u00EF\u0082\u0098),\u00C2\u00A00.5\u00C2\u00A0mg/kg\u00C2\u00A0 (\u00EF\u0081\u00B0),\u00C2\u00A0and\u00C2\u00A01\u00C2\u00A0mg/kg\u00C2\u00A0(\u00EF\u0081\u00B1)\u00C2\u00A0IV\u00C2\u00A0twice\u00C2\u00A0weekly\u00C2\u00A0for\u00C2\u00A05\u00C2\u00A0weeks\u00C2\u00A0starting\u00C2\u00A0on\u00C2\u00A0day\u00C2\u00A018\u00C2\u00A0after\u00C2\u00A0inoculation\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0containing\u00C2\u00A0 an\u00C2\u00A0irrelevant\u00C2\u00A0Ab\u00C2\u00A0(\u00EF\u0081\u00B5)\u00C2\u00A0was\u00C2\u00A0administered\u00C2\u00A0at\u00C2\u00A01\u00C2\u00A0mg/kg\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dosing\u00C2\u00A0schedule,\u00C2\u00A0and\u00C2\u00A0saline\u00C2\u00A0(\u00EF\u0081\u00AE)\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0 vehicle\u00C2\u00A0control.\u00C2\u00A0\u00C2\u00A0(B)\u00C2\u00A0Fold\u00E2\u0080\u0090change\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0volume\u00C2\u00A0after\u00C2\u00A05\u00C2\u00A0weeks\u00C2\u00A0of\u00C2\u00A0various\u00C2\u00A0treatments\u00C2\u00A0(controls\u00C2\u00A0and\u00C2\u00A0Trz\u00E2\u0080\u0090LNP)\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0dosing\u00C2\u00A0schedule.\u00C2\u00A0\u00C2\u00A0Controls:\u00C2\u00A0a,\u00C2\u00A0saline;\u00C2\u00A0b,\u00C2\u00A0irrelevant\u00C2\u00A0Ab;\u00C2\u00A0c,\u00C2\u00A0Trz\u00C2\u00A0(1.0\u00C2\u00A0mg/kg);\u00C2\u00A0d,\u00C2\u00A0irrelevant\u00C2\u00A0Ab\u00E2\u0080\u0090LNP;\u00C2\u00A0e,\u00C2\u00A0bare\u00C2\u00A0liposomes.\u00C2\u00A0\u00C2\u00A0 *p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0Trz.\u00C2\u00A0 \u00C2\u00A0Data\u00C2\u00A0represent\u00C2\u00A0means\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0using\u00C2\u00A0six\u00C2\u00A0mice\u00C2\u00A0 in\u00C2\u00A0each\u00C2\u00A0study\u00C2\u00A0group.\u00C2\u00A0 \u00C2\u00A0Bars:\u00C2\u00A0standard\u00C2\u00A0 error.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0was\u00C2\u00A0 the\u00C2\u00A0 first\u00C2\u00A0 report\u00C2\u00A0describing\u00C2\u00A0enhanced\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0drugs\u00C2\u00A0attached\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0outside\u00C2\u00A0of\u00C2\u00A0 liposomes,\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0liposomes\u00C2\u00A0serve\u00C2\u00A0a\u00C2\u00A0purely\u00C2\u00A0structural\u00C2\u00A0role.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0studies\u00C2\u00A0therefore\u00C2\u00A0established\u00C2\u00A0 that\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0exhibited\u00C2\u00A0novel\u00C2\u00A0 therapeutic\u00C2\u00A0activity\u00C2\u00A0 that\u00C2\u00A0could\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0explained\u00C2\u00A0by\u00C2\u00A0passive\u00C2\u00A0or\u00C2\u00A0active\u00C2\u00A0 targeting\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0encapsulated\u00C2\u00A0drug,\u00C2\u00A0so\u00C2\u00A0 the\u00C2\u00A0 improvements\u00C2\u00A0 in\u00C2\u00A0Ab\u00C2\u00A0efficacy\u00C2\u00A0resulted\u00C2\u00A0 from\u00C2\u00A0an\u00C2\u00A0unknown\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 that\u00C2\u00A0 remained\u00C2\u00A0elusive.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0complications\u00C2\u00A0 in\u00C2\u00A0understanding\u00C2\u00A0 this\u00C2\u00A0mechanistic\u00C2\u00A0 puzzle\u00C2\u00A0 were\u00C2\u00A0 due,\u00C2\u00A0 wholly\u00C2\u00A0 or\u00C2\u00A0 in\u00C2\u00A0 part,\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 therapeutic\u00C2\u00A0 Abs\u00C2\u00A0 themselves\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0clearly\u00C2\u00A0delineated.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 A B Controls\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP\u00C2\u00A0dose\u00C2\u00A0 (mg/kg) 0. 25 0. 50 1. 0a b c d e \u00E2\u0080\u00A2\u00C2\u00A06\u00C2\u00A0\u00C2\u00A0 Table\u00C2\u00A01.1\u00C2\u00A0\u00C2\u00A0Pharmacokinetic\u00C2\u00A0variables\u00C2\u00A0and\u00C2\u00A0tumor\u00C2\u00A0localization\u00C2\u00A0of\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00E2\u0080\u00A0.\u00C2\u00A0 Pharmacokinetic\u00C2\u00A0comparisona:\u00C2\u00A0 \u00C2\u00A0 Trz\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP\u00C2\u00A0 Plasma\u00C2\u00A0levels\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0(\u00CE\u00BCg/mL)\u00C2\u00A0 2.91\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.13\u00C2\u00A0 27.7\u00C2\u00A0\u00C2\u00B1\u00C2\u00A01.3\u00C2\u00A0 AUC0\u00E2\u0080\u009024\u00C2\u00A0h\u00C2\u00A0(\u00CE\u00BCg\u00C2\u00A0\u00E2\u0088\u0099\u00C2\u00A0h\u00C2\u00A0\u00E2\u0088\u0099\u00C2\u00A0mL\u00E2\u0088\u00921)b\u00C2\u00A0 88.7\u00C2\u00A0 883\u00C2\u00A0 Total\u00C2\u00A0body\u00C2\u00A0clearance\u00C2\u00A0(mL\u00C2\u00A0\u00E2\u0088\u0099\u00C2\u00A0h\u00E2\u0080\u00901)\u00C2\u00A0 4.04\u00C2\u00A0 0.386\u00C2\u00A0 Volume\u00C2\u00A0of\u00C2\u00A0distribution\u00C2\u00A0at\u00C2\u00A0steady\u00C2\u00A0state\u00C2\u00A0(mL)\u00C2\u00A0 277\u00C2\u00A0 29.7\u00C2\u00A0 MRTlast\u00C2\u00A0(h)\u00C2\u00A0 9.64\u00C2\u00A0 10.3\u00C2\u00A0 Tumor\u00C2\u00A0localizationa:\u00C2\u00A0 \u00C2\u00A0 Control\u00C2\u00A0liposome\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP\u00C2\u00A0 %\u00C2\u00A0Injected\u00C2\u00A0dose/g\u00C2\u00A0tumor\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0h 3.84\u00C2\u00A0\u00C2\u00B1\u00C2\u00A02.4\u00C2\u00A0 13.9\u00C2\u00A0\u00C2\u00B1\u00C2\u00A03.4\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Abbreviations:\u00C2\u00A0\u00C2\u00A0AUC0\u00E2\u0080\u009024\u00C2\u00A0h:\u00C2\u00A0Area\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0curve\u00C2\u00A0from\u00C2\u00A0time\u00C2\u00A0of\u00C2\u00A0dosing\u00C2\u00A0(t\u00C2\u00A0=\u00C2\u00A00\u00C2\u00A0h)\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0last\u00C2\u00A0measured\u00C2\u00A0plasma\u00C2\u00A0concentration\u00C2\u00A0 (t\u00C2\u00A0=\u00C2\u00A024\u00C2\u00A0h);\u00C2\u00A0MRTlast:\u00C2\u00A0Mean\u00C2\u00A0residence\u00C2\u00A0time,\u00C2\u00A0calculated\u00C2\u00A0using\u00C2\u00A0values\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0last\u00C2\u00A0measured\u00C2\u00A0plasma\u00C2\u00A0concentration\u00C2\u00A0(t\u00C2\u00A0=\u00C2\u00A024\u00C2\u00A0h).\u00C2\u00A0 a\u00C2\u00A0For\u00C2\u00A0plasma\u00C2\u00A0elimination\u00C2\u00A0studies,\u00C2\u00A012\u00C2\u00A0animals\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0study\u00C2\u00A0group,\u00C2\u00A0with\u00C2\u00A0four\u00C2\u00A0animals\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A0of\u00C2\u00A0 blood\u00C2\u00A0collection\u00C2\u00A0(at\u00C2\u00A01,\u00C2\u00A04,\u00C2\u00A0and\u00C2\u00A024\u00C2\u00A0h).\u00C2\u00A0For\u00C2\u00A0tumor\u00C2\u00A0localization\u00C2\u00A0study,\u00C2\u00A0four\u00C2\u00A0animals\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0study\u00C2\u00A0group,\u00C2\u00A0and\u00C2\u00A0tumors\u00C2\u00A0 were\u00C2\u00A0harvested\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0 b\u00C2\u00A0Noncompartmental\u00C2\u00A0analysis\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 linear\u00C2\u00A0trapezoidal\u00C2\u00A0rule\u00C2\u00A0was\u00C2\u00A0done\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0software\u00C2\u00A0WinNonlin\u00C2\u00A0version\u00C2\u00A01.5\u00C2\u00A0to\u00C2\u00A0 estimate\u00C2\u00A0the\u00C2\u00A0values\u00C2\u00A0of\u00C2\u00A0various\u00C2\u00A0pharmacokinetic\u00C2\u00A0variables.\u00C2\u00A0 \u00C2\u00A0 1.3\u00C2\u00A0 Therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0 Therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0a\u00C2\u00A0class\u00C2\u00A0of\u00C2\u00A0drugs\u00C2\u00A0that\u00C2\u00A0hold\u00C2\u00A0great\u00C2\u00A0promise\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0treatment\u00C2\u00A0 due\u00C2\u00A0to\u00C2\u00A0their\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0and\u00C2\u00A0kill\u00C2\u00A0cancer\u00C2\u00A0cells\u00C2\u00A0while\u00C2\u00A0leaving\u00C2\u00A0healthy\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0body\u00C2\u00A0unaffected.\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 concept\u00C2\u00A0 of\u00C2\u00A0 using\u00C2\u00A0 \u00E2\u0080\u009Cmagic\u00C2\u00A0 bullets\u00E2\u0080\u009D\u00C2\u00A0 to\u00C2\u00A0 treat\u00C2\u00A0 disease\u00C2\u00A0 was\u00C2\u00A0 proposed\u00C2\u00A0 as\u00C2\u00A0 early\u00C2\u00A0 as\u00C2\u00A0 1897\u00C2\u00A0 by\u00C2\u00A0 Paul\u00C2\u00A0 Ehrlich20.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0until\u00C2\u00A01975,\u00C2\u00A0however,\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0as\u00C2\u00A0targeted\u00C2\u00A0drugs\u00C2\u00A0 became\u00C2\u00A0 possible\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 invention\u00C2\u00A0 of\u00C2\u00A0 hybridoma\u00C2\u00A0 technology,\u00C2\u00A0 allowing\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 production\u00C2\u00A0 of\u00C2\u00A0 monoclonal\u00C2\u00A0 Abs\u00C2\u00A0 of\u00C2\u00A0 predefined\u00C2\u00A0 antigen\u00C2\u00A0 specificity21.\u00C2\u00A0 \u00C2\u00A0Monoclonal\u00C2\u00A0 Abs\u00C2\u00A0 were\u00C2\u00A0 thought\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 ideal\u00C2\u00A0 candidates\u00C2\u00A0 for\u00C2\u00A0 administration\u00C2\u00A0 in\u00C2\u00A0 humans\u00C2\u00A0 because\u00C2\u00A0 of\u00C2\u00A0 their\u00C2\u00A0 specificity\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 single\u00C2\u00A0 epitope\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 corresponding\u00C2\u00A0antigen,\u00C2\u00A0but\u00C2\u00A0early\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0were\u00C2\u00A0antigenic\u00C2\u00A0 in\u00C2\u00A0humans\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0their\u00C2\u00A0origins\u00C2\u00A0 in\u00C2\u00A0 mice,\u00C2\u00A0limiting\u00C2\u00A0their\u00C2\u00A0clinical\u00C2\u00A0potential22,\u00C2\u00A023.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Starting\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 early\u00C2\u00A0 1980s,\u00C2\u00A0 protein\u00C2\u00A0 engineering\u00C2\u00A0 techniques\u00C2\u00A0 allowed\u00C2\u00A0 for\u00C2\u00A0 replacement\u00C2\u00A0 of\u00C2\u00A0 mouse\u00C2\u00A0 regions\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 Ab\u00C2\u00A0molecules\u00C2\u00A0 with\u00C2\u00A0 corresponding\u00C2\u00A0 regions\u00C2\u00A0 of\u00C2\u00A0 human\u00C2\u00A0 origin,\u00C2\u00A0 reducing\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A07\u00C2\u00A0\u00C2\u00A0 mouse\u00E2\u0080\u0090specific\u00C2\u00A0 immunogenic\u00C2\u00A0 response\u00C2\u00A0when\u00C2\u00A0administered\u00C2\u00A0 in\u00C2\u00A0humans.\u00C2\u00A0 \u00C2\u00A0Chimeric\u00C2\u00A0Abs\u00C2\u00A0 refer\u00C2\u00A0 to\u00C2\u00A0Abs\u00C2\u00A0 with\u00C2\u00A0 antigen\u00E2\u0080\u0090binding\u00C2\u00A0 regions\u00C2\u00A0 coded\u00C2\u00A0 by\u00C2\u00A0 genes\u00C2\u00A0 of\u00C2\u00A0mouse\u00C2\u00A0 origin\u00C2\u00A0 and\u00C2\u00A0 constant\u00C2\u00A0 regions\u00C2\u00A0 from\u00C2\u00A0 human\u00C2\u00A0 genes.\u00C2\u00A0\u00C2\u00A0Humanized\u00C2\u00A0Abs\u00C2\u00A0refer\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0genetically\u00C2\u00A0engineered\u00C2\u00A0mouse\u00C2\u00A0Ab\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0protein\u00C2\u00A0sequence\u00C2\u00A0has\u00C2\u00A0 been\u00C2\u00A0modified\u00C2\u00A0 to\u00C2\u00A0 increase\u00C2\u00A0 the\u00C2\u00A0 similarity\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Ab\u00C2\u00A0 to\u00C2\u00A0human\u00C2\u00A0Abs22,\u00C2\u00A023.\u00C2\u00A0 \u00C2\u00A0 Subsequent\u00C2\u00A0 technological\u00C2\u00A0 advances\u00C2\u00A0have\u00C2\u00A0allowed\u00C2\u00A0fully\u00C2\u00A0human\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0also\u00C2\u00A0be\u00C2\u00A0produced24\u00E2\u0080\u009026.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0first\u00C2\u00A0monoclonal\u00C2\u00A0Ab\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0approved\u00C2\u00A0for\u00C2\u00A0use\u00C2\u00A0 in\u00C2\u00A0oncology\u00C2\u00A0was\u00C2\u00A0Rtx\u00C2\u00A0 in\u00C2\u00A0199727,\u00C2\u00A028,\u00C2\u00A0and\u00C2\u00A0over\u00C2\u00A0 the\u00C2\u00A0past\u00C2\u00A0decade,\u00C2\u00A0the\u00C2\u00A0market\u00C2\u00A0for\u00C2\u00A0therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0has\u00C2\u00A0grown\u00C2\u00A0exponentially29\u00E2\u0080\u009031.\u00C2\u00A0\u00C2\u00A0Over\u00C2\u00A030\u00C2\u00A0 Abs\u00C2\u00A0 and\u00C2\u00A0 their\u00C2\u00A0 derivatives\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 approved\u00C2\u00A0 for\u00C2\u00A0 use\u00C2\u00A0 in\u00C2\u00A0 various\u00C2\u00A0 indications,\u00C2\u00A0 including\u00C2\u00A0 10\u00C2\u00A0 in\u00C2\u00A0 oncology,\u00C2\u00A0and\u00C2\u00A0there\u00C2\u00A0are\u00C2\u00A0hundreds\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0late\u00C2\u00A0stages\u00C2\u00A0of\u00C2\u00A0preclinical\u00C2\u00A0and\u00C2\u00A0clinical\u00C2\u00A0development31.\u00C2\u00A0\u00C2\u00A0Table\u00C2\u00A0 1.2\u00C2\u00A0shows\u00C2\u00A0a\u00C2\u00A0selection\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0currently\u00C2\u00A0approved\u00C2\u00A0for\u00C2\u00A0use\u00C2\u00A0in\u00C2\u00A0treating\u00C2\u00A0 cancer,\u00C2\u00A0or\u00C2\u00A0at\u00C2\u00A0late\u00C2\u00A0stages\u00C2\u00A0of\u00C2\u00A0clinical\u00C2\u00A0trials.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A01.2\u00C2\u00A0\u00C2\u00A0Selected\u00C2\u00A0therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0approved\u00C2\u00A0or\u00C2\u00A0under\u00C2\u00A0development\u00C2\u00A0in\u00C2\u00A0oncology.\u00C2\u00A0 \u00C2\u00A0 Generic\u00C2\u00A0name\u00C2\u00A0 Trade\u00C2\u00A0name\u00C2\u00A0 Target\u00C2\u00A0 Ab\u00C2\u00A0format\u00C2\u00A0 Cancer\u00C2\u00A0indication\u00C2\u00A0 Status\u00C2\u00A0 Refs.\u00C2\u00A0 Rituximab\u00C2\u00A0(Rtx)\u00C2\u00A0 Rituxan/Mabthera\u00C2\u00A0 CD20\u00C2\u00A0 Chimeric\u00C2\u00A0IgG1\u00C2\u00A0 NHL\u00C2\u00A0 Approved\u00C2\u00A0 27,\u00C2\u00A028\u00C2\u00A0 Trastuzumab\u00C2\u00A0(Trz)\u00C2\u00A0 Herceptin\u00C2\u00A0 ErbB2\u00C2\u00A0 Humanized\u00C2\u00A0IgG1\u00C2\u00A0 Breast\u00C2\u00A0 Approved\u00C2\u00A0 32,\u00C2\u00A033\u00C2\u00A0 Alemtuzumab\u00C2\u00A0 Campath/MabCampath CD52\u00C2\u00A0 Humanized\u00C2\u00A0IgG1\u00C2\u00A0 CLL\u00C2\u00A0 Approved\u00C2\u00A0 34\u00C2\u00A0 Cetuximab\u00C2\u00A0 Erbitux\u00C2\u00A0 EGFR\u00C2\u00A0 Chimeric\u00C2\u00A0IgG1\u00C2\u00A0 Colorectal\u00C2\u00A0 Approved\u00C2\u00A0 35\u00C2\u00A0 Bevacizumab\u00C2\u00A0 Avastin\u00C2\u00A0 VEGFA Humanized\u00C2\u00A0IgG1\u00C2\u00A0 Colorectal,\u00C2\u00A0breast,\u00C2\u00A0lung\u00C2\u00A0 Approved\u00C2\u00A0 36\u00C2\u00A0 Panitumumab\u00C2\u00A0 Vectibix\u00C2\u00A0 EGFR\u00C2\u00A0 Human\u00C2\u00A0IgG2\u00C2\u00A0 Colorectal\u00C2\u00A0 Approved\u00C2\u00A0 24\u00C2\u00A0 Ofatumumab\u00C2\u00A0 Arzerra\u00C2\u00A0 CD20\u00C2\u00A0 Human\u00C2\u00A0IgG1\u00C2\u00A0 CLL\u00C2\u00A0 Approved\u00C2\u00A0 25,\u00C2\u00A037\u00C2\u00A0 Pertuzumab\u00C2\u00A0(Prz)\u00C2\u00A0 Omnitarg\u00C2\u00A0 ErbB2\u00C2\u00A0 Humanized\u00C2\u00A0IgG1\u00C2\u00A0 Breast,\u00C2\u00A0ovarian\u00C2\u00A0 Phase\u00C2\u00A0III\u00C2\u00A0 38,\u00C2\u00A039\u00C2\u00A0 Veltuzumab\u00C2\u00A0 N/A\u00C2\u00A0\u00C2\u00A0 CD20\u00C2\u00A0 Humanized\u00C2\u00A0IgG1\u00C2\u00A0 NHL,\u00C2\u00A0CLL\u00C2\u00A0 Phase\u00C2\u00A0III\u00C2\u00A0 40,\u00C2\u00A041\u00C2\u00A0 \u00C2\u00A0 Abbreviations:\u00C2\u00A0\u00C2\u00A0CLL:\u00C2\u00A0Chronic\u00C2\u00A0lymphocytic\u00C2\u00A0leukemia;\u00C2\u00A0N/A:\u00C2\u00A0Not\u00C2\u00A0applicable;\u00C2\u00A0NHL:\u00C2\u00A0Non\u00E2\u0080\u0090Hodgkin\u00C2\u00A0lymphoma.\u00C2\u00A0 \u00C2\u00A0 Even\u00C2\u00A0though\u00C2\u00A0in\u00C2\u00A0humans\u00C2\u00A0there\u00C2\u00A0are\u00C2\u00A0five\u00C2\u00A0classes\u00C2\u00A0of\u00C2\u00A0immunoglobulin\u00C2\u00A0(IgM,\u00C2\u00A0IgD,\u00C2\u00A0IgG,\u00C2\u00A0IgA,\u00C2\u00A0and\u00C2\u00A0IgE)\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0four\u00C2\u00A0 IgG\u00C2\u00A0subclasses\u00C2\u00A0 (IgG1,\u00C2\u00A0 IgG2,\u00C2\u00A0 IgG3,\u00C2\u00A0and\u00C2\u00A0 IgG4),\u00C2\u00A0by\u00C2\u00A0far\u00C2\u00A0most\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 IgG1\u00C2\u00A0 subtype\u00C2\u00A0 (Table\u00C2\u00A01.2).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 is\u00C2\u00A0because\u00C2\u00A0 IgG1\u00C2\u00A0exhibits\u00C2\u00A0a\u00C2\u00A0 long\u00C2\u00A0half\u00E2\u0080\u0090life\u00C2\u00A0 in\u00C2\u00A0blood\u00C2\u00A0 (~21\u00C2\u00A0days)\u00C2\u00A0and\u00C2\u00A0 it\u00C2\u00A0 elicits\u00C2\u00A0more\u00C2\u00A0 favorable\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 (see\u00C2\u00A0Sections\u00C2\u00A01.4.1\u00C2\u00A0&\u00C2\u00A01.4.2)\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A08\u00C2\u00A0\u00C2\u00A0 other\u00C2\u00A0 Ig\u00C2\u00A0 classes\u00C2\u00A0and\u00C2\u00A0 subclasses42.\u00C2\u00A0 \u00C2\u00A0Cancer\u00C2\u00A0patients\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 therapeutic\u00C2\u00A0Abs\u00C2\u00A0 typically\u00C2\u00A0need\u00C2\u00A0 to\u00C2\u00A0 receive\u00C2\u00A0weekly\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0 several\u00C2\u00A0hundred\u00C2\u00A0milligrams\u00C2\u00A0over\u00C2\u00A0 several\u00C2\u00A0months\u00C2\u00A0 to\u00C2\u00A0maintain\u00C2\u00A0an\u00C2\u00A0effective\u00C2\u00A0 serum\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0over\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg/mL43.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0150\u00C2\u00A0kDa\u00C2\u00A0Y\u00E2\u0080\u0090shaped\u00C2\u00A0structure\u00C2\u00A0of\u00C2\u00A0 IgGs\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0divided\u00C2\u00A0 into\u00C2\u00A0two\u00C2\u00A0distinct\u00C2\u00A0functional\u00C2\u00A0units:\u00C2\u00A0the\u00C2\u00A0fragment\u00C2\u00A0of\u00C2\u00A0antigen\u00C2\u00A0binding\u00C2\u00A0(Fab;\u00C2\u00A0the\u00C2\u00A0v\u00E2\u0080\u0090shaped\u00C2\u00A0upper\u00C2\u00A0part\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0molecule)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0constant\u00C2\u00A0fragment\u00C2\u00A0(Fc),\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0connected\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0site\u00C2\u00A0called\u00C2\u00A0the\u00C2\u00A0hinge\u00C2\u00A0region.\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0Fab\u00C2\u00A0region\u00C2\u00A0contains\u00C2\u00A0two\u00C2\u00A0identical\u00C2\u00A0sites\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0(drug)\u00C2\u00A0binds\u00C2\u00A0its\u00C2\u00A0antigen\u00C2\u00A0(target),\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0 Fc\u00C2\u00A0region\u00C2\u00A0interacts\u00C2\u00A0with\u00C2\u00A0specific\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system1.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0Fab\u00C2\u00A0and\u00C2\u00A0Fc\u00C2\u00A0regions\u00C2\u00A0each\u00C2\u00A0 are\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0different\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs,\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A01.4.\u00C2\u00A0\u00C2\u00A0 1.3.1\u00C2\u00A0 Rituximab\u00C2\u00A0and\u00C2\u00A0CD20\u00C2\u00A0 Rtx\u00C2\u00A0 is\u00C2\u00A0 often\u00C2\u00A0 heralded\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 \u00E2\u0080\u009Cblockbuster\u00E2\u0080\u009D\u00C2\u00A0 Ab\u00C2\u00A0 given\u00C2\u00A0 its\u00C2\u00A0widespread\u00C2\u00A0 success\u00C2\u00A0 in\u00C2\u00A0 significantly\u00C2\u00A0 improving\u00C2\u00A0 the\u00C2\u00A0 survival\u00C2\u00A0 statistics\u00C2\u00A0 for\u00C2\u00A0 B\u00E2\u0080\u0090cell\u00C2\u00A0 non\u00E2\u0080\u0090Hodgkin\u00C2\u00A0 lymphomas\u00C2\u00A0 (NHLs),\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 other\u00C2\u00A0 malignancies\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 chronic\u00C2\u00A0 lymphocytic\u00C2\u00A0 leukemia\u00C2\u00A0 (CLL)\u00C2\u00A0 and\u00C2\u00A0 autoimmune\u00C2\u00A0 disorders\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 rheumatoid\u00C2\u00A0arthritis31,\u00C2\u00A044.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0opposed\u00C2\u00A0to\u00C2\u00A0more\u00C2\u00A0recently\u00C2\u00A0developed\u00C2\u00A0Abs,\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0often\u00C2\u00A0humanized\u00C2\u00A0 or\u00C2\u00A0 fully\u00C2\u00A0human\u00C2\u00A0molecules,\u00C2\u00A0Rtx\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0 chimeric\u00C2\u00A0Ab\u00C2\u00A0 containing\u00C2\u00A0a\u00C2\u00A0murine\u00C2\u00A0variable\u00C2\u00A0 region\u00C2\u00A0attached\u00C2\u00A0 to\u00C2\u00A0a\u00C2\u00A0 human\u00C2\u00A0IgG1\u00C2\u00A0constant\u00C2\u00A0region.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0target\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0CD20,\u00C2\u00A0a\u00C2\u00A033\u00C2\u00A0to\u00C2\u00A037\u00C2\u00A0kD\u00C2\u00A0membrane\u00E2\u0080\u0090associated\u00C2\u00A0phosphoprotein\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0highly\u00C2\u00A0 expressed\u00C2\u00A0on\u00C2\u00A0all\u00C2\u00A0normal\u00C2\u00A0and\u00C2\u00A0most\u00C2\u00A0malignant\u00C2\u00A0B\u00C2\u00A0cells,\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0other\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0body,\u00C2\u00A0making\u00C2\u00A0 it\u00C2\u00A0an\u00C2\u00A0 attractive\u00C2\u00A0target\u00C2\u00A0for\u00C2\u00A0B\u00C2\u00A0cell\u00E2\u0080\u0090based\u00C2\u00A0therapy.\u00C2\u00A0 \u00C2\u00A0CD20\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0detected\u00C2\u00A0on\u00C2\u00A0embryonic\u00C2\u00A0stem\u00C2\u00A0or\u00C2\u00A0pro\u00E2\u0080\u0090B\u00C2\u00A0cells,\u00C2\u00A0 but\u00C2\u00A0is\u00C2\u00A0expressed\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0mature\u00C2\u00A0B\u00C2\u00A0cells;\u00C2\u00A0expression\u00C2\u00A0ceases\u00C2\u00A0upon\u00C2\u00A0differentiation\u00C2\u00A0into\u00C2\u00A0plasma\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0protein\u00C2\u00A0spans\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0four\u00C2\u00A0times\u00C2\u00A0and\u00C2\u00A0there\u00C2\u00A0 is\u00C2\u00A0some\u00C2\u00A0evidence\u00C2\u00A0that\u00C2\u00A0 it\u00C2\u00A0may\u00C2\u00A0 function\u00C2\u00A0in\u00C2\u00A0calcium\u00C2\u00A0entry45,\u00C2\u00A046.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0tetramers\u00C2\u00A0which\u00C2\u00A0associate\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0B\u00C2\u00A0cell\u00C2\u00A0 antigen\u00C2\u00A0receptor\u00C2\u00A0in\u00C2\u00A0unstimulated\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0upon\u00C2\u00A0antigen\u00C2\u00A0binding,\u00C2\u00A0CD20\u00C2\u00A0is\u00C2\u00A0released\u00C2\u00A0and\u00C2\u00A0it\u00C2\u00A0associates\u00C2\u00A0 transiently\u00C2\u00A0with\u00C2\u00A0phosphoproteins\u00C2\u00A0and\u00C2\u00A0calmodulin\u00E2\u0080\u0090binding\u00C2\u00A0proteins,\u00C2\u00A0which\u00C2\u00A0regulate\u00C2\u00A0calcium\u00C2\u00A0entry47.\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A09\u00C2\u00A0\u00C2\u00A0 Most\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 therapeutic\u00C2\u00A0 Abs\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 internalize\u00C2\u00A0 upon\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 CD2040.\u00C2\u00A0 \u00C2\u00A0 Despite\u00C2\u00A0 its\u00C2\u00A0 proven\u00C2\u00A0 success\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0target\u00C2\u00A0for\u00C2\u00A0Ab\u00C2\u00A0therapy,\u00C2\u00A0the\u00C2\u00A0precise\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0is\u00C2\u00A0still\u00C2\u00A0elusive.\u00C2\u00A0\u00C2\u00A0 1.4\u00C2\u00A0 The\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0monoclonal\u00C2\u00A0Abs\u00C2\u00A0 In\u00C2\u00A0general,\u00C2\u00A0 the\u00C2\u00A0precise\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0 therapeutic\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0completely\u00C2\u00A0clear,\u00C2\u00A0 and\u00C2\u00A0they\u00C2\u00A0vary\u00C2\u00A0from\u00C2\u00A0Ab\u00C2\u00A0to\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0given\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0is\u00C2\u00A0commonly\u00C2\u00A0viewed\u00C2\u00A0as\u00C2\u00A0resulting\u00C2\u00A0 from\u00C2\u00A0a\u00C2\u00A0combination\u00C2\u00A0of\u00C2\u00A0 indirect\u00C2\u00A0and\u00C2\u00A0direct\u00C2\u00A0effects\u00C2\u00A0that\u00C2\u00A0occur\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0binds\u00C2\u00A0to\u00C2\u00A0 its\u00C2\u00A0target44.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 indirect\u00C2\u00A0 and\u00C2\u00A0 direct\u00C2\u00A0 mechanisms\u00C2\u00A0 are\u00C2\u00A0 mediated\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 Fc\u00C2\u00A0 and\u00C2\u00A0 Fab\u00C2\u00A0 regions\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 Ab\u00C2\u00A0 molecule,\u00C2\u00A0 respectively.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0are\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system\u00C2\u00A0on\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0 are\u00C2\u00A0 coated\u00C2\u00A0with\u00C2\u00A0 therapeutic\u00C2\u00A0 Ab.\u00C2\u00A0 \u00C2\u00A0 They\u00C2\u00A0 involve\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0 of\u00C2\u00A0 specific\u00C2\u00A0 cell\u00E2\u0080\u0090surface\u00C2\u00A0 or\u00C2\u00A0 plasma\u00C2\u00A0 proteins\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0molecule;\u00C2\u00A0since\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0is\u00C2\u00A0common\u00C2\u00A0to\u00C2\u00A0most\u00C2\u00A0 therapeutic\u00C2\u00A0Abs,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0surprising\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0are\u00C2\u00A0common\u00C2\u00A0to\u00C2\u00A0all\u00C2\u00A0therapeutic\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0 Specifically,\u00C2\u00A0 the\u00C2\u00A0 two\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0are\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0 (CDC;\u00C2\u00A0 Section\u00C2\u00A01.4.1)\u00C2\u00A0 and\u00C2\u00A0Ab\u00E2\u0080\u0090dependent\u00C2\u00A0 cell\u00E2\u0080\u0090mediated\u00C2\u00A0 cytotoxicity\u00C2\u00A0 (ADCC;\u00C2\u00A0 Section\u00C2\u00A0 1.4.2).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 Fc\u00C2\u00A0 region\u00C2\u00A0 possesses\u00C2\u00A0 interaction\u00C2\u00A0 sites\u00C2\u00A0 for\u00C2\u00A0 ligands\u00C2\u00A0 which\u00C2\u00A0 can\u00C2\u00A0 induce\u00C2\u00A0 effector\u00C2\u00A0 functions,\u00C2\u00A0 including\u00C2\u00A0 three\u00C2\u00A0 structurally\u00C2\u00A0 homologous\u00C2\u00A0 cellular\u00C2\u00A0 IgG\u00C2\u00A0 Fc\u00C2\u00A0 receptor\u00C2\u00A0 types\u00C2\u00A0 (Fc\u00CE\u00B3RI,\u00C2\u00A0 Fc\u00CE\u00B3RII,\u00C2\u00A0 Fc\u00CE\u00B3RIII)\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 C1q\u00C2\u00A0 component\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 complement48.\u00C2\u00A0 \u00C2\u00A0 Even\u00C2\u00A0 though\u00C2\u00A0 every\u00C2\u00A0 therapeutic\u00C2\u00A0 Ab\u00C2\u00A0 elicits\u00C2\u00A0 CDC\u00C2\u00A0 and\u00C2\u00A0 ADCC,\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0contributions\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0two\u00C2\u00A0mechanisms\u00C2\u00A0vary\u00C2\u00A0from\u00C2\u00A0one\u00C2\u00A0Ab\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0next44,\u00C2\u00A049,\u00C2\u00A050.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0opposed\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00E2\u0080\u0090mediated\u00C2\u00A0indirect\u00C2\u00A0mechanisms,\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 of\u00C2\u00A0a\u00C2\u00A0 therapeutic\u00C2\u00A0Ab\u00C2\u00A0 results\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0physical\u00C2\u00A0 interaction\u00C2\u00A0of\u00C2\u00A0 its\u00C2\u00A0 two\u00C2\u00A0 identical\u00C2\u00A0Fab\u00C2\u00A0 regions\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 target\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface48\u00C2\u00A0(Section\u00C2\u00A01.4.3).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0is\u00C2\u00A0therefore\u00C2\u00A0Ab\u00E2\u0080\u0090specific,\u00C2\u00A0and\u00C2\u00A0highly\u00C2\u00A0 variable\u00C2\u00A0 between\u00C2\u00A0 different\u00C2\u00A0 Abs;\u00C2\u00A0 for\u00C2\u00A0 example,\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 can\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 therapeutic\u00C2\u00A0effects\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0growth\u00C2\u00A0inhibition,\u00C2\u00A0chemosensitization,\u00C2\u00A0or\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis51.\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A010\u00C2\u00A0\u00C2\u00A0 1.4.1\u00C2\u00A0 Complement\u00C2\u00ADdependent\u00C2\u00A0cytotoxicity\u00C2\u00A0 As\u00C2\u00A0part\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 innate\u00C2\u00A0 immune\u00C2\u00A0system,\u00C2\u00A0complement\u00C2\u00A0 is\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0main\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090 mediated\u00C2\u00A0immunity\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0protecting\u00C2\u00A0the\u00C2\u00A0host\u00C2\u00A0from\u00C2\u00A0attack\u00C2\u00A0by\u00C2\u00A0intruding\u00C2\u00A0pathogens.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0 was\u00C2\u00A0first\u00C2\u00A0identified\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0serum\u00C2\u00A0component\u00C2\u00A0that\u00C2\u00A0\u00E2\u0080\u009Ccomplemented\u00E2\u0080\u009D\u00C2\u00A0Abs\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0kill\u00C2\u00A0bacteria,\u00C2\u00A0but\u00C2\u00A0we\u00C2\u00A0 now\u00C2\u00A0know\u00C2\u00A0today\u00C2\u00A0that\u00C2\u00A0it\u00C2\u00A0consists\u00C2\u00A0of\u00C2\u00A0more\u00C2\u00A0than\u00C2\u00A030\u00C2\u00A0proteins\u00C2\u00A0found\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0and\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surfaces\u00C2\u00A0of\u00C2\u00A0 cells52,\u00C2\u00A0 53.\u00C2\u00A0 \u00C2\u00A0 Activation\u00C2\u00A0 of\u00C2\u00A0 complement\u00C2\u00A0 occurs\u00C2\u00A0 after\u00C2\u00A0 distinct\u00C2\u00A0 events\u00C2\u00A0 that\u00C2\u00A0 trigger\u00C2\u00A0 different\u00C2\u00A0 protease\u00C2\u00A0 cascades\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0classical,\u00C2\u00A0mannose\u00E2\u0080\u0090binding\u00C2\u00A0 lectin,\u00C2\u00A0and\u00C2\u00A0alternative\u00C2\u00A0pathways.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0 the\u00C2\u00A0classical\u00C2\u00A0pathway\u00C2\u00A0is\u00C2\u00A0activated\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0complement\u00C2\u00A0protein\u00C2\u00A0C1q\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Ab\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0invading\u00C2\u00A0pathogen\u00C2\u00A0or\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0cancer\u00C2\u00A0cell\u00C2\u00A0coated\u00C2\u00A0with\u00C2\u00A0therapeutic\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0 final\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0 three\u00C2\u00A0complement\u00C2\u00A0pathways\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0assembly\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A05\u00E2\u0080\u009310\u00C2\u00A0nm\u00C2\u00A0diameter\u00C2\u00A0membrane\u00E2\u0080\u0090 attack\u00C2\u00A0complex\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0foreign\u00C2\u00A0cell,\u00C2\u00A0which\u00C2\u00A0causes\u00C2\u00A0cell\u00C2\u00A0death\u00C2\u00A0by\u00C2\u00A0disrupting\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane53,\u00C2\u00A054.\u00C2\u00A0\u00C2\u00A0 Complement\u00C2\u00A0 is\u00C2\u00A0 finely\u00C2\u00A0regulated\u00C2\u00A0so\u00C2\u00A0 that\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0activated\u00C2\u00A0on\u00C2\u00A0 foreign\u00C2\u00A0Ab\u00E2\u0080\u0090coated\u00C2\u00A0cells\u00C2\u00A0 (resulting\u00C2\u00A0 in\u00C2\u00A0their\u00C2\u00A0 elimination)\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0on\u00C2\u00A0normal\u00C2\u00A0cells52.\u00C2\u00A0 For\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 strong\u00C2\u00A0 CDC\u00C2\u00A0 activity\u00C2\u00A0 during\u00C2\u00A0 monoclonal\u00C2\u00A0 Ab\u00C2\u00A0 therapy,\u00C2\u00A0 relatively\u00C2\u00A0 high\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0target\u00C2\u00A0antigen\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0attain\u00C2\u00A0sufficiently\u00C2\u00A0high\u00C2\u00A0Ab\u00C2\u00A0levels\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0tumor\u00E2\u0080\u0090cell\u00C2\u00A0 surface\u00C2\u00A0to\u00C2\u00A0initiate\u00C2\u00A0CDC.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0is\u00C2\u00A0also\u00C2\u00A0clear\u00C2\u00A0that\u00C2\u00A0higher\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0C1q\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0 correlate\u00C2\u00A0with\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CDC43,\u00C2\u00A054.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0given\u00C2\u00A0the\u00C2\u00A0importance\u00C2\u00A0of\u00C2\u00A0CDC\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0 therapeutic\u00C2\u00A0Abs,\u00C2\u00A0 several\u00C2\u00A0 strategies\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0employed\u00C2\u00A0 to\u00C2\u00A0enhance\u00C2\u00A0CDC\u00C2\u00A0 in\u00C2\u00A0new\u00C2\u00A0Abs\u00C2\u00A0 under\u00C2\u00A0 development,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 amino\u00C2\u00A0 acid\u00C2\u00A0 mutations\u00C2\u00A0 that\u00C2\u00A0 enhance\u00C2\u00A0 CDC\u00C2\u00A0 through\u00C2\u00A0 improved\u00C2\u00A0 C1q\u00C2\u00A0 binding55,\u00C2\u00A056.\u00C2\u00A0 \u00C2\u00A0 Another\u00C2\u00A0 strategy\u00C2\u00A0 involves\u00C2\u00A0 shuffling\u00C2\u00A0 IgG1\u00C2\u00A0 and\u00C2\u00A0 IgG3\u00C2\u00A0 sequences\u00C2\u00A0within\u00C2\u00A0 a\u00C2\u00A0 heavy\u00C2\u00A0 chain\u00C2\u00A0 constant\u00C2\u00A0 region,\u00C2\u00A0since\u00C2\u00A0 IgG3\u00C2\u00A0exhibits\u00C2\u00A0superior\u00C2\u00A0CDC\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0other\u00C2\u00A0 IgG\u00C2\u00A0 isotypes,\u00C2\u00A0while\u00C2\u00A0 IgG1\u00C2\u00A0 exhibits\u00C2\u00A0 higher\u00C2\u00A0 ADCC,\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 principal\u00C2\u00A0 indirect\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action57.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 discussion\u00C2\u00A0 of\u00C2\u00A0 CDC\u00C2\u00A0 relating\u00C2\u00A0specifically\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0provided\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.2.1.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A011\u00C2\u00A0\u00C2\u00A0 1.4.2\u00C2\u00A0 Ab\u00C2\u00ADdependent\u00C2\u00A0cell\u00C2\u00ADmediated\u00C2\u00A0cytotoxicity\u00C2\u00A0 ADCC\u00C2\u00A0 results\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0 between\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A0 foreign\u00C2\u00A0cell\u00C2\u00A0coated\u00C2\u00A0with\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0Effector\u00C2\u00A0cells\u00C2\u00A0contain\u00C2\u00A0IgG\u00C2\u00A0Fc\u00C2\u00A0receptors\u00C2\u00A0(Fc\u00CE\u00B3Rs)\u00C2\u00A0that\u00C2\u00A0bind\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0 of\u00C2\u00A0IgG\u00C2\u00A0molecules,\u00C2\u00A0and\u00C2\u00A0they\u00C2\u00A0consist\u00C2\u00A0of\u00C2\u00A0macrophages,\u00C2\u00A0natural\u00C2\u00A0killer\u00C2\u00A0(NK)\u00C2\u00A0cells,\u00C2\u00A0granulocytes,\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 leukocytes43,\u00C2\u00A058.\u00C2\u00A0 \u00C2\u00A0 Fc\u00CE\u00B3Rs\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 classified\u00C2\u00A0 as\u00C2\u00A0 activating\u00C2\u00A0 or\u00C2\u00A0 inhibitory;\u00C2\u00A0 for\u00C2\u00A0 example,\u00C2\u00A0 Fc\u00CE\u00B3RIa,\u00C2\u00A0 Fc\u00CE\u00B3RIIa,\u00C2\u00A0 Fc\u00CE\u00B3RIIc\u00C2\u00A0and\u00C2\u00A0 Fc\u00CE\u00B3RIIIa,\u00C2\u00A0are\u00C2\u00A0activating\u00C2\u00A0 receptors,\u00C2\u00A0while\u00C2\u00A0 Fc\u00CE\u00B3RIIb\u00C2\u00A0 is\u00C2\u00A0an\u00C2\u00A0 inhibitory\u00C2\u00A0 receptor.\u00C2\u00A0 \u00C2\u00A0NK\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 granulocytes\u00C2\u00A0 contain\u00C2\u00A0 only\u00C2\u00A0 activating\u00C2\u00A0 Fc\u00CE\u00B3Rs,\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 therefore\u00C2\u00A0 better\u00C2\u00A0 inducers\u00C2\u00A0 of\u00C2\u00A0 ADCC\u00C2\u00A0 compared\u00C2\u00A0to\u00C2\u00A0macrophages,\u00C2\u00A0which\u00C2\u00A0contain\u00C2\u00A0both\u00C2\u00A0activating\u00C2\u00A0and\u00C2\u00A0 inhibitory\u00C2\u00A0Fc\u00CE\u00B3Rs54.\u00C2\u00A0 \u00C2\u00A0When\u00C2\u00A0activating\u00C2\u00A0 Fc\u00CE\u00B3Rs\u00C2\u00A0 on\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 crosslinked\u00C2\u00A0 by\u00C2\u00A0 IgG\u00C2\u00A0 bound\u00C2\u00A0 to\u00C2\u00A0 its\u00C2\u00A0 target,\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 effector\u00C2\u00A0 cell\u00C2\u00A0 occurs\u00C2\u00A0 and\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0phagocytosis\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 target\u00C2\u00A0 cell\u00C2\u00A0or\u00C2\u00A0 cell\u00C2\u00A0 lysis\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 release\u00C2\u00A0of\u00C2\u00A0 cytotoxic\u00C2\u00A0 granules\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0effector\u00C2\u00A0cell43,\u00C2\u00A054,\u00C2\u00A058.\u00C2\u00A0 When\u00C2\u00A0studied\u00C2\u00A0in\u00C2\u00A0vitro,\u00C2\u00A0ADCC\u00C2\u00A0resulting\u00C2\u00A0from\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0treatment\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0achieved\u00C2\u00A0at\u00C2\u00A0Ab\u00C2\u00A0 concentrations\u00C2\u00A0below\u00C2\u00A010\u00C2\u00A0ng/mL,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 several\u00C2\u00A0orders\u00C2\u00A0of\u00C2\u00A0magnitude\u00C2\u00A0 lower\u00C2\u00A0 than\u00C2\u00A0 the\u00C2\u00A0 required\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 serum\u00C2\u00A0 concentrations\u00C2\u00A0 mentioned\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 1.3\u00C2\u00A0 (ref.\u00C2\u00A0 43).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 discrepancy\u00C2\u00A0 results\u00C2\u00A0 from\u00C2\u00A0 competition\u00C2\u00A0between\u00C2\u00A0endogenous\u00C2\u00A0human\u00C2\u00A0serum\u00C2\u00A0 IgG\u00C2\u00A0and\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0for\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0activating\u00C2\u00A0 Fc\u00CE\u00B3R\u00C2\u00A0on\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0(such\u00C2\u00A0as\u00C2\u00A0Fc\u00CE\u00B3RIIIa\u00C2\u00A0on\u00C2\u00A0NK\u00C2\u00A0cells),\u00C2\u00A0which\u00C2\u00A0leads\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0requirement\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0 amount\u00C2\u00A0of\u00C2\u00A0drug,\u00C2\u00A0contributing\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0very\u00C2\u00A0high\u00C2\u00A0costs\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0such\u00C2\u00A0therapies58.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Different\u00C2\u00A0Abs\u00C2\u00A0against\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0target\u00C2\u00A0show\u00C2\u00A0variable\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0ADCC,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0magnitude\u00C2\u00A0of\u00C2\u00A0 induced\u00C2\u00A0ADCC\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0correlate\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0density\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface,\u00C2\u00A0underscoring\u00C2\u00A0the\u00C2\u00A0 complexity\u00C2\u00A0of\u00C2\u00A0 this\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action59.\u00C2\u00A0 \u00C2\u00A0Since\u00C2\u00A0 increased\u00C2\u00A0ADCC\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 to\u00C2\u00A0enhance\u00C2\u00A0 the\u00C2\u00A0 efficacy\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs,\u00C2\u00A0strategies\u00C2\u00A0for\u00C2\u00A0 improving\u00C2\u00A0ADCC\u00C2\u00A0activity\u00C2\u00A0occupy\u00C2\u00A0an\u00C2\u00A0 important\u00C2\u00A0role\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 development\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0next\u00C2\u00A0generation\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs58.\u00C2\u00A0 \u00C2\u00A0Some\u00C2\u00A0approaches\u00C2\u00A0 include\u00C2\u00A0modifications\u00C2\u00A0 of\u00C2\u00A0glycosylation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0that\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0enhanced\u00C2\u00A0ADCC58,\u00C2\u00A060,\u00C2\u00A061\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0amino\u00C2\u00A0acid\u00C2\u00A0mutations\u00C2\u00A0that\u00C2\u00A0 result\u00C2\u00A0in\u00C2\u00A0enhanced\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0Fc\u00CE\u00B3RIIIa62\u00E2\u0080\u009064.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0Properties\u00C2\u00A0relating\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0ADCC\u00C2\u00A0 are\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0further\u00C2\u00A0detail\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.2.2.\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A012\u00C2\u00A0\u00C2\u00A0 1.4.3\u00C2\u00A0 Direct\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0 Because\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 therapeutic\u00C2\u00A0Ab\u00C2\u00A0 is\u00C2\u00A0 highly\u00C2\u00A0Ab\u00E2\u0080\u0090specific,\u00C2\u00A0 it\u00C2\u00A0 depends\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 multitude\u00C2\u00A0of\u00C2\u00A0factors.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0include\u00C2\u00A0the\u00C2\u00A0molecular\u00C2\u00A0biology\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target,\u00C2\u00A0the\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0interaction\u00C2\u00A0 between\u00C2\u00A0Ab\u00C2\u00A0and\u00C2\u00A0target,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0specific\u00C2\u00A0characteristics\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0cell,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0mutation\u00C2\u00A0status,\u00C2\u00A0 target\u00C2\u00A0 expression\u00C2\u00A0 levels,\u00C2\u00A0 and\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0proteins\u00C2\u00A0 that\u00C2\u00A0 regulate\u00C2\u00A0 activation\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 target51.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 direct\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0therefore\u00C2\u00A0far\u00C2\u00A0less\u00C2\u00A0well\u00E2\u0080\u0090defined\u00C2\u00A0and\u00C2\u00A0more\u00C2\u00A0variable\u00C2\u00A0than\u00C2\u00A0the\u00C2\u00A0 indirect\u00C2\u00A0ones,\u00C2\u00A0even\u00C2\u00A0when\u00C2\u00A0 looking\u00C2\u00A0at\u00C2\u00A0direct\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0Abs\u00C2\u00A0directed\u00C2\u00A0against\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0 target.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 One\u00C2\u00A0such\u00C2\u00A0example\u00C2\u00A0 is\u00C2\u00A0 two\u00C2\u00A0 therapeutic\u00C2\u00A0Abs\u00C2\u00A0used\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 treatment\u00C2\u00A0of\u00C2\u00A0breast\u00C2\u00A0cancer,\u00C2\u00A0Trz\u00C2\u00A0and\u00C2\u00A0 pertuzumab\u00C2\u00A0(Prz)32,\u00C2\u00A065.\u00C2\u00A0\u00C2\u00A0Both\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0directed\u00C2\u00A0against\u00C2\u00A0ErbB2\u00C2\u00A0(also\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0Her2\u00C2\u00A0or\u00C2\u00A0EGFR2),\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0overexpressed\u00C2\u00A0 in\u00C2\u00A015\u00E2\u0080\u009030%\u00C2\u00A0of\u00C2\u00A0 invasive\u00C2\u00A0breast\u00C2\u00A0 carcinomas,\u00C2\u00A0 and\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0predictive\u00C2\u00A0of\u00C2\u00A0poor\u00C2\u00A0 prognosis66.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0ErbB\u00C2\u00A0 family\u00C2\u00A0 includes\u00C2\u00A0 four\u00C2\u00A0 receptor\u00C2\u00A0 tyrosine\u00C2\u00A0kinases\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0common\u00C2\u00A0extracellular\u00C2\u00A0 ligand\u00E2\u0080\u0090binding\u00C2\u00A0 domain.\u00C2\u00A0 \u00C2\u00A0Upon\u00C2\u00A0 ligand\u00C2\u00A0 binding,\u00C2\u00A0 various\u00C2\u00A0 homodimers\u00C2\u00A0 and\u00C2\u00A0 heterodimers\u00C2\u00A0 are\u00C2\u00A0 formed\u00C2\u00A0 among\u00C2\u00A0 the\u00C2\u00A0 four\u00C2\u00A0 family\u00C2\u00A0members,\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0 intracellular\u00C2\u00A0pathways\u00C2\u00A0 that\u00C2\u00A0control\u00C2\u00A0 survival\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0cycle\u00C2\u00A0progression.\u00C2\u00A0 \u00C2\u00A0Dysregulation\u00C2\u00A0of\u00C2\u00A0 this\u00C2\u00A0kinase\u00C2\u00A0pathway,\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0constitutive\u00C2\u00A0 activation,\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0regulate\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0cancers67.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Trz\u00C2\u00A0 acts\u00C2\u00A0 by\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 extracellular\u00C2\u00A0 juxtamembrane\u00C2\u00A0 domain\u00C2\u00A0 of\u00C2\u00A0 ErbB2.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 causes\u00C2\u00A0 dimerization\u00C2\u00A0of\u00C2\u00A0ErbB2\u00C2\u00A0and\u00C2\u00A0 subsequent\u00C2\u00A0 internalization\u00C2\u00A0and\u00C2\u00A0downmodulation\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0protein\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 surface.\u00C2\u00A0 \u00C2\u00A0 Downstream\u00C2\u00A0 signaling\u00C2\u00A0 from\u00C2\u00A0 ErbB2\u00C2\u00A0 overexpression\u00C2\u00A0 therefore\u00C2\u00A0 ceases,\u00C2\u00A0 resulting\u00C2\u00A0 disruption\u00C2\u00A0of\u00C2\u00A0cell\u00E2\u0080\u0090cycle\u00C2\u00A0progression\u00C2\u00A0and\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0growth\u00C2\u00A0of\u00C2\u00A0metastases68\u00E2\u0080\u009070.\u00C2\u00A0\u00C2\u00A0Prz,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0 hand,\u00C2\u00A0binds\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0epitope\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0extracellular\u00C2\u00A0domain\u00C2\u00A0of\u00C2\u00A0ErbB2\u00C2\u00A0that\u00C2\u00A0sterically\u00C2\u00A0inhibits\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 ErbB2\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0ligand\u00E2\u0080\u0090activated\u00C2\u00A0dimer\u00C2\u00A0complexes.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0downmodulation\u00C2\u00A0like\u00C2\u00A0with\u00C2\u00A0 Trz,\u00C2\u00A0but\u00C2\u00A0instead\u00C2\u00A0it\u00C2\u00A0causes\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0ErbB\u00C2\u00A0signaling\u00C2\u00A0by\u00C2\u00A0disrupting\u00C2\u00A0the\u00C2\u00A0vast\u00C2\u00A0array\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0ligand\u00E2\u0080\u0090 dependent\u00C2\u00A0receptor\u00C2\u00A0combinations\u00C2\u00A0that\u00C2\u00A0exist\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0ErbB\u00C2\u00A0kinase\u00C2\u00A0pathway71.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A013\u00C2\u00A0\u00C2\u00A0 Because\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0differences\u00C2\u00A0 in\u00C2\u00A0direct\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action,\u00C2\u00A0Trz\u00C2\u00A0 requires\u00C2\u00A0overexpression\u00C2\u00A0of\u00C2\u00A0 ErbB2\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0have\u00C2\u00A0therapeutic\u00C2\u00A0activity,\u00C2\u00A0and\u00C2\u00A0it\u00C2\u00A0shows\u00C2\u00A0low\u00C2\u00A0activity\u00C2\u00A0when\u00C2\u00A0ErbB2\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0overexpressed.\u00C2\u00A0 \u00C2\u00A0Prz,\u00C2\u00A0however,\u00C2\u00A0 is\u00C2\u00A0highly\u00C2\u00A0effective\u00C2\u00A0at\u00C2\u00A0 inhibiting\u00C2\u00A0 tumor\u00C2\u00A0growth\u00C2\u00A0when\u00C2\u00A0ErbB2\u00C2\u00A0 is\u00C2\u00A0 overexpressed\u00C2\u00A0or\u00C2\u00A0when\u00C2\u00A0ErbB2\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0low71.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0the\u00C2\u00A0two\u00C2\u00A0Abs\u00C2\u00A0together\u00C2\u00A0have\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 show\u00C2\u00A0 synergistic\u00C2\u00A0effects\u00C2\u00A0 against\u00C2\u00A0breast\u00C2\u00A0 cancer\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 vitro72\u00C2\u00A0 and\u00C2\u00A0 against\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0breast\u00C2\u00A0 cancer\u00C2\u00A0 xenografts73,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 combination\u00C2\u00A0exhibited\u00C2\u00A0 favorable\u00C2\u00A0 response\u00C2\u00A0 rates\u00C2\u00A0 in\u00C2\u00A0patients\u00C2\u00A0who\u00C2\u00A0had\u00C2\u00A0 progressed\u00C2\u00A0following\u00C2\u00A0previous\u00C2\u00A0Trz\u00C2\u00A0therapy38.\u00C2\u00A0 The\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0involves\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0mechanism:\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0consist\u00C2\u00A0 of\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0 target\u00C2\u00A0cells.\u00C2\u00A0 \u00C2\u00A0Generally,\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0and\u00C2\u00A0CDC\u00C2\u00A0 in\u00C2\u00A0vitro,\u00C2\u00A0Rtx\u00C2\u00A0 exhibits\u00C2\u00A0low\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0cytotoxicity\u00C2\u00A0in\u00C2\u00A0neoplastic\u00C2\u00A0cells\u00C2\u00A0unless\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0crosslinked\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0reagent\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0an\u00C2\u00A0 anti\u00E2\u0080\u0090human\u00E2\u0080\u0090IgG\u00C2\u00A0secondary\u00C2\u00A0Ab\u00C2\u00A0(2oAb).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0demonstrated\u00C2\u00A0with\u00C2\u00A0established\u00C2\u00A0cell\u00C2\u00A0 lines7,\u00C2\u00A074,\u00C2\u00A075\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0primary\u00C2\u00A0cells76.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0direct\u00C2\u00A0effect\u00C2\u00A0is\u00C2\u00A0generally\u00C2\u00A0thought\u00C2\u00A0to\u00C2\u00A0result\u00C2\u00A0from\u00C2\u00A0clustering\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0and\u00C2\u00A0since\u00C2\u00A0 it\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0occur\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0or\u00C2\u00A0CDC,\u00C2\u00A0the\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0also\u00C2\u00A0plays\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0overall\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0postulated\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0vivo,\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0may\u00C2\u00A0occur\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090 mediated\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0by\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0effector\u00C2\u00A0cells,\u00C2\u00A0since\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0known\u00C2\u00A0that\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0 other\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0mediate\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0crosslinking\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0ADCC43,\u00C2\u00A054,\u00C2\u00A058.\u00C2\u00A0\u00C2\u00A0Such\u00C2\u00A0direct\u00C2\u00A0effects\u00C2\u00A0 may\u00C2\u00A0be\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0a\u00C2\u00A0substantial\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cytotoxic\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0therapy,\u00C2\u00A0although\u00C2\u00A0this\u00C2\u00A0 has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0definitively\u00C2\u00A0shown77.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0a\u00C2\u00A0direct\u00C2\u00A0mechanistic\u00C2\u00A0link\u00C2\u00A0between\u00C2\u00A0Rtx\u00C2\u00A0crosslinking\u00C2\u00A0and\u00C2\u00A0 apoptosis\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0established,\u00C2\u00A0so\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0understood\u00C2\u00A0how\u00C2\u00A0crosslinking\u00C2\u00A0can\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0crosslinking\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0is\u00C2\u00A0further\u00C2\u00A0discussed\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A01.6.\u00C2\u00A0 Taken\u00C2\u00A0together,\u00C2\u00A0the\u00C2\u00A0complex\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0makes\u00C2\u00A0it\u00C2\u00A0 difficult\u00C2\u00A0 to\u00C2\u00A0define\u00C2\u00A0 features\u00C2\u00A0of\u00C2\u00A0 specific\u00C2\u00A0Abs\u00C2\u00A0 that,\u00C2\u00A0upon\u00C2\u00A0modulation,\u00C2\u00A0would\u00C2\u00A0 increase\u00C2\u00A0or\u00C2\u00A0decrease\u00C2\u00A0 the\u00C2\u00A0 efficacy\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0Several\u00C2\u00A0strategies\u00C2\u00A0for\u00C2\u00A0improving\u00C2\u00A0the\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0by\u00C2\u00A0increasing\u00C2\u00A0CDC\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A014\u00C2\u00A0\u00C2\u00A0 or\u00C2\u00A0ADCC\u00C2\u00A0were\u00C2\u00A0mentioned\u00C2\u00A0above,\u00C2\u00A0and\u00C2\u00A0equivalent\u00C2\u00A0strategies\u00C2\u00A0can\u00C2\u00A0also\u00C2\u00A0be\u00C2\u00A0employed\u00C2\u00A0for\u00C2\u00A0enhancing\u00C2\u00A0the\u00C2\u00A0 Ab\u00E2\u0080\u0090specific\u00C2\u00A0direct\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0case\u00C2\u00A0of\u00C2\u00A0Abs\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0Rtx\u00C2\u00A0whose\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0 consists\u00C2\u00A0of\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis,\u00C2\u00A0methods\u00C2\u00A0to\u00C2\u00A0enhance\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0would\u00C2\u00A0have\u00C2\u00A0clear\u00C2\u00A0benefit\u00C2\u00A0 in\u00C2\u00A0improving\u00C2\u00A0these\u00C2\u00A0drugs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 1.5\u00C2\u00A0 Apoptosis\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0target\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0therapy\u00C2\u00A0 Evasion\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0is\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0hallmarks\u00C2\u00A0of\u00C2\u00A0cancer78,\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0large\u00C2\u00A0and\u00C2\u00A0promising\u00C2\u00A0focus\u00C2\u00A0in\u00C2\u00A0 cancer\u00C2\u00A0 research\u00C2\u00A0 involves\u00C2\u00A0 inducing\u00C2\u00A0apoptosis\u00C2\u00A0 specifically\u00C2\u00A0 in\u00C2\u00A0cancer\u00C2\u00A0cells\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0 in\u00C2\u00A0normal\u00C2\u00A0cells79\u00E2\u0080\u009082.\u00C2\u00A0\u00C2\u00A0 Apoptosis\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 process\u00C2\u00A0 of\u00C2\u00A0 programmed\u00C2\u00A0 cell\u00C2\u00A0 death,\u00C2\u00A0 and\u00C2\u00A0 is\u00C2\u00A0 conserved\u00C2\u00A0 across\u00C2\u00A0 all\u00C2\u00A0metazoans;\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 necessary\u00C2\u00A0 under\u00C2\u00A0 normal\u00C2\u00A0 physiological\u00C2\u00A0 conditions\u00C2\u00A0 at\u00C2\u00A0 all\u00C2\u00A0 stages\u00C2\u00A0 of\u00C2\u00A0 life.\u00C2\u00A0 \u00C2\u00A0 During\u00C2\u00A0 embryonic\u00C2\u00A0 development,\u00C2\u00A0 apoptosis\u00C2\u00A0 is\u00C2\u00A0 essential\u00C2\u00A0 for\u00C2\u00A0organogenesis\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0of\u00C2\u00A0multicellular\u00C2\u00A0 tissues,\u00C2\u00A0 where\u00C2\u00A0 it\u00C2\u00A0 ensures\u00C2\u00A0 the\u00C2\u00A0 proper\u00C2\u00A0 balance\u00C2\u00A0 of\u00C2\u00A0 each\u00C2\u00A0 differentiated\u00C2\u00A0 cell\u00C2\u00A0 lineage,\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 adult\u00C2\u00A0 organisms,\u00C2\u00A0 apoptosis\u00C2\u00A0maintains\u00C2\u00A0normal\u00C2\u00A0cellular\u00C2\u00A0homeostasis83.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0multicellular\u00C2\u00A0organisms,\u00C2\u00A0the\u00C2\u00A0total\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0balance\u00C2\u00A0between\u00C2\u00A0new\u00C2\u00A0cells\u00C2\u00A0created\u00C2\u00A0 through\u00C2\u00A0 mitosis\u00C2\u00A0 and\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0 die\u00C2\u00A0 through\u00C2\u00A0 apoptosis80.\u00C2\u00A0 \u00C2\u00A0 Different\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0 disruptions\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 balance\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 altered\u00C2\u00A0 apoptotic\u00C2\u00A0 signaling\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 primary\u00C2\u00A0 pathogenic\u00C2\u00A0 events\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0disease.\u00C2\u00A0Accelerated\u00C2\u00A0 cell\u00C2\u00A0death\u00C2\u00A0 is\u00C2\u00A0evident\u00C2\u00A0 in\u00C2\u00A0acute\u00C2\u00A0and\u00C2\u00A0chronic\u00C2\u00A0degenerative\u00C2\u00A0diseases,\u00C2\u00A0 immunodeficiency,\u00C2\u00A0 and\u00C2\u00A0 infertility,\u00C2\u00A0 while\u00C2\u00A0 insufficient\u00C2\u00A0 apoptosis\u00C2\u00A0 can\u00C2\u00A0manifest\u00C2\u00A0 as\u00C2\u00A0 autoimmunity\u00C2\u00A0 or\u00C2\u00A0 cancer80,\u00C2\u00A083.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Apoptosis\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 contrasted\u00C2\u00A0 to\u00C2\u00A0 necrosis,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 traumatic\u00C2\u00A0 cell\u00C2\u00A0 death\u00C2\u00A0 following\u00C2\u00A0 acute\u00C2\u00A0 cellular\u00C2\u00A0 injury\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0 rupture\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0and\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0an\u00C2\u00A0 inflammatory\u00C2\u00A0 response.\u00C2\u00A0\u00C2\u00A0Apoptosis,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0controlled\u00C2\u00A0process\u00C2\u00A0that\u00C2\u00A0leads\u00C2\u00A0to\u00C2\u00A0characteristic\u00C2\u00A0changes\u00C2\u00A0 in\u00C2\u00A0biochemistry\u00C2\u00A0and\u00C2\u00A0morphology\u00C2\u00A0before\u00C2\u00A0death.\u00C2\u00A0\u00C2\u00A0Morphological\u00C2\u00A0changes\u00C2\u00A0include\u00C2\u00A0condensation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 chromatin\u00C2\u00A0and\u00C2\u00A0DNA\u00C2\u00A0 fragmentation,\u00C2\u00A0 reduction\u00C2\u00A0of\u00C2\u00A0 cell\u00C2\u00A0 volume,\u00C2\u00A0 cytoskeletal\u00C2\u00A0 rearrangement,\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0blebbing,\u00C2\u00A0and\u00C2\u00A0eventual\u00C2\u00A0 formation\u00C2\u00A0of\u00C2\u00A0 small\u00C2\u00A0vesicles\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0apoptotic\u00C2\u00A0bodies.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A015\u00C2\u00A0\u00C2\u00A0 apoptotic\u00C2\u00A0bodies\u00C2\u00A0are\u00C2\u00A0rapidly\u00C2\u00A0phagocytosed\u00C2\u00A0by\u00C2\u00A0macrophages\u00C2\u00A0so\u00C2\u00A0the\u00C2\u00A0dead\u00C2\u00A0cell\u00C2\u00A0is\u00C2\u00A0removed\u00C2\u00A0without\u00C2\u00A0any\u00C2\u00A0 inflammatory\u00C2\u00A0response81.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 One\u00C2\u00A0early\u00C2\u00A0biochemical\u00C2\u00A0change\u00C2\u00A0in\u00C2\u00A0apoptosis\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0loss\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0lipid\u00C2\u00A0asymmetry.\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 exposure\u00C2\u00A0of\u00C2\u00A0 phosphatidylserine\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0outer\u00C2\u00A0 leaflet\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0 which\u00C2\u00A0under\u00C2\u00A0normal\u00C2\u00A0conditions\u00C2\u00A0is\u00C2\u00A0present\u00C2\u00A0almost\u00C2\u00A0exclusively\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0inner\u00C2\u00A0leaflet84.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0appearance\u00C2\u00A0 of\u00C2\u00A0phosphatidylserine\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0extracellular\u00C2\u00A0leaflet\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0using\u00C2\u00A0 fluorescently\u00E2\u0080\u0090labeled\u00C2\u00A0conjugates\u00C2\u00A0of\u00C2\u00A0Annexin\u00E2\u0080\u0090V,\u00C2\u00A0a\u00C2\u00A0protein\u00C2\u00A0that\u00C2\u00A0interacts\u00C2\u00A0strongly\u00C2\u00A0and\u00C2\u00A0specifically\u00C2\u00A0with\u00C2\u00A0 phosphatidylserine.\u00C2\u00A0\u00C2\u00A0Using\u00C2\u00A0appropriate\u00C2\u00A0protocols,\u00C2\u00A0labeled\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00C2\u00A0conjugates\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0employed\u00C2\u00A0in\u00C2\u00A0 applications\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0and\u00C2\u00A0fluorescence\u00C2\u00A0microscopy85.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Apoptosis\u00C2\u00A0signaling\u00C2\u00A0most\u00C2\u00A0often\u00C2\u00A0results\u00C2\u00A0from\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0caspases,\u00C2\u00A0a\u00C2\u00A0family\u00C2\u00A0of\u00C2\u00A0cysteine\u00C2\u00A0 proteases\u00C2\u00A0that\u00C2\u00A0also\u00C2\u00A0plays\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0inflammation.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0date,\u00C2\u00A012\u00C2\u00A0human\u00C2\u00A0caspases\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0discovered,\u00C2\u00A0 and\u00C2\u00A0each\u00C2\u00A0is\u00C2\u00A0activated\u00C2\u00A0by\u00C2\u00A0cleavage\u00C2\u00A0of\u00C2\u00A0its\u00C2\u00A0procaspase\u00C2\u00A0precursor.\u00C2\u00A0\u00C2\u00A0Cleavage\u00C2\u00A0is\u00C2\u00A0often\u00C2\u00A0performed\u00C2\u00A0by\u00C2\u00A0other\u00C2\u00A0 activated\u00C2\u00A0caspases,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0proteolytic\u00C2\u00A0cascades82,\u00C2\u00A086,\u00C2\u00A087.\u00C2\u00A0\u00C2\u00A0When\u00C2\u00A0a\u00C2\u00A0cell\u00C2\u00A0receives\u00C2\u00A0a\u00C2\u00A0stimulus\u00C2\u00A0 to\u00C2\u00A0die,\u00C2\u00A0 caspase\u00C2\u00A0 signaling\u00C2\u00A0begins\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 cleavage\u00C2\u00A0of\u00C2\u00A0one\u00C2\u00A0or\u00C2\u00A0more\u00C2\u00A0 specific\u00C2\u00A0 initiator\u00C2\u00A0 caspases;\u00C2\u00A0 this\u00C2\u00A0 early\u00C2\u00A0 event\u00C2\u00A0 corresponds\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 loss\u00C2\u00A0 of\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 asymmetry\u00C2\u00A0 and\u00C2\u00A0 appearance\u00C2\u00A0 of\u00C2\u00A0 phosphatidylserine\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 outer\u00C2\u00A0 leaflet\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane84,\u00C2\u00A0 85.\u00C2\u00A0 \u00C2\u00A0 Specific\u00C2\u00A0 molecular\u00C2\u00A0 mechanisms\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0are\u00C2\u00A0discussed\u00C2\u00A0in\u00C2\u00A0greater\u00C2\u00A0 detail\u00C2\u00A0in\u00C2\u00A0Sections\u00C2\u00A05.2\u00C2\u00A0and\u00C2\u00A06.2.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 1.6\u00C2\u00A0 Multivalent\u00C2\u00A0Abs\u00C2\u00A0as\u00C2\u00A0improved\u00C2\u00A0therapies\u00C2\u00A0\u00C2\u00A0 1.6.1\u00C2\u00A0 Characteristics\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0definition\u00C2\u00A0of\u00C2\u00A0valence\u00C2\u00A0 As\u00C2\u00A0mentioned\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A01.4.3,\u00C2\u00A0Rtx\u00C2\u00A0induces\u00C2\u00A0low\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0unless\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0 crosslinked\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A02oAb7,\u00C2\u00A074\u00E2\u0080\u009076.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 importance\u00C2\u00A0of\u00C2\u00A0 this\u00C2\u00A0mechanism\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0was\u00C2\u00A0provided\u00C2\u00A0by\u00C2\u00A0studies\u00C2\u00A0 showing\u00C2\u00A0 that\u00C2\u00A0cross\u00E2\u0080\u0090linked\u00C2\u00A0Rtx\u00C2\u00A0 induced\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0dose\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 time\u00E2\u0080\u0090dependent\u00C2\u00A0manner\u00C2\u00A0 in\u00C2\u00A0 freshly\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A016\u00C2\u00A0\u00C2\u00A0 isolated\u00C2\u00A0clinical\u00C2\u00A0samples\u00C2\u00A0of\u00C2\u00A0B\u00E2\u0080\u0090CLL\u00C2\u00A0cells,\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0and\u00C2\u00A0CDC76.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0make\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0effect\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0challenge\u00C2\u00A0because\u00C2\u00A0clearly,\u00C2\u00A0administration\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A02oAb\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0feasible.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0different\u00C2\u00A0method\u00C2\u00A0 that\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 to\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0constructs75,\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 88\u00E2\u0080\u009090.\u00C2\u00A0 The\u00C2\u00A0 valence\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0multivalent\u00C2\u00A0 ligand\u00C2\u00A0 is\u00C2\u00A0 defined\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 target\u00E2\u0080\u0090binding\u00C2\u00A0 sites\u00C2\u00A0 it\u00C2\u00A0 contains.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0normal\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0bivalent\u00C2\u00A0molecules\u00C2\u00A0because\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0IgGs\u00C2\u00A0(Table\u00C2\u00A0 1.2),\u00C2\u00A0which\u00C2\u00A0contain\u00C2\u00A0 two\u00C2\u00A0 identical\u00C2\u00A0sites\u00C2\u00A0where\u00C2\u00A0 they\u00C2\u00A0bind\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0 target.\u00C2\u00A0 \u00C2\u00A0Multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0 possess\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0or\u00C2\u00A0higher,\u00C2\u00A0and\u00C2\u00A0consist\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0or\u00C2\u00A0more\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0and/or\u00C2\u00A0Fab\u00C2\u00A0fragments\u00C2\u00A0 bound\u00C2\u00A0together\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0stable\u00C2\u00A0configuration.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0general,\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0prepared\u00C2\u00A0using\u00C2\u00A0 whole\u00C2\u00A0 IgG\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0equal\u00C2\u00A0 to\u00C2\u00A0 twice\u00C2\u00A0 the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 IgG\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0construct;\u00C2\u00A0 for\u00C2\u00A0example,\u00C2\u00A0an\u00C2\u00A0 IgG\u00C2\u00A0 dimer\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0chemically\u00C2\u00A0crosslinking\u00C2\u00A0two\u00C2\u00A0IgG\u00C2\u00A0molecules\u00C2\u00A0has\u00C2\u00A0a\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0four.\u00C2\u00A0\u00C2\u00A0Rtx,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 variety\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0Abs,\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0some\u00C2\u00A0cases\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0efficacious\u00C2\u00A0as\u00C2\u00A0multivalent\u00C2\u00A0rather\u00C2\u00A0 than\u00C2\u00A0bivalent\u00C2\u00A0constructs\u00C2\u00A0(see\u00C2\u00A0Section\u00C2\u00A01.6.3).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Multivalent\u00C2\u00A0Abs\u00C2\u00A0of\u00C2\u00A0 low\u00C2\u00A0valence\u00C2\u00A0 (<10)\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0Ab\u00C2\u00A0or\u00C2\u00A0Ab\u00E2\u0080\u0090like\u00C2\u00A0molecules\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0protein\u00C2\u00A0 engineering\u00C2\u00A0and/or\u00C2\u00A0recombination\u00C2\u00A0techniques89,\u00C2\u00A091,\u00C2\u00A0or\u00C2\u00A0can\u00C2\u00A0involve\u00C2\u00A0coupling\u00C2\u00A0Ab\u00C2\u00A0or\u00C2\u00A0fragments\u00C2\u00A0together\u00C2\u00A0 using\u00C2\u00A0crosslinking\u00C2\u00A0chemistry\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0molecular\u00C2\u00A0scaffolds92.\u00C2\u00A0\u00C2\u00A0Higher\u00C2\u00A0valences\u00C2\u00A0can\u00C2\u00A0achieved\u00C2\u00A0by\u00C2\u00A0 coupling\u00C2\u00A0 Abs\u00C2\u00A0 to\u00C2\u00A0 nanostructures\u00C2\u00A0 consisting\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 polymers93,\u00C2\u00A0 dendrimers94,\u00C2\u00A0 95,\u00C2\u00A0 rotaxanes92,\u00C2\u00A0or\u00C2\u00A0gold\u00C2\u00A0nanoparticles96.\u00C2\u00A0\u00C2\u00A0Regardless\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0type\u00C2\u00A0of\u00C2\u00A0construct,\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0 crosslink\u00C2\u00A0their\u00C2\u00A0target\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0without\u00C2\u00A0the\u00C2\u00A0need\u00C2\u00A0for\u00C2\u00A0a\u00C2\u00A0crosslinking\u00C2\u00A02oAb.\u00C2\u00A0\u00C2\u00A0When\u00C2\u00A0the\u00C2\u00A0 crosslinking\u00C2\u00A0 involves\u00C2\u00A0 more\u00C2\u00A0 than\u00C2\u00A0 two\u00C2\u00A0 proteins\u00C2\u00A0 being\u00C2\u00A0 bridged,\u00C2\u00A0 creating\u00C2\u00A0 an\u00C2\u00A0 extensive\u00C2\u00A0 network\u00C2\u00A0 of\u00C2\u00A0 crosslinked\u00C2\u00A0 target\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 numerous\u00C2\u00A0 binding\u00C2\u00A0 sites\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 multivalent\u00C2\u00A0 Ab,\u00C2\u00A0 the\u00C2\u00A0 term\u00C2\u00A0 \u00E2\u0080\u009Chypercrosslinking\u00E2\u0080\u009D\u00C2\u00A0is\u00C2\u00A0employed.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 increased\u00C2\u00A0 efficacy\u00C2\u00A0 of\u00C2\u00A0many\u00C2\u00A0multivalent\u00C2\u00A0 Abs\u00C2\u00A0 is\u00C2\u00A0 related\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 they\u00C2\u00A0 exhibit\u00C2\u00A0 different\u00C2\u00A0properties\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 their\u00C2\u00A0bivalent\u00C2\u00A0 counterparts.\u00C2\u00A0 \u00C2\u00A0Conversion\u00C2\u00A0of\u00C2\u00A0Abs\u00C2\u00A0 into\u00C2\u00A0multivalent\u00C2\u00A0 formats\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 increased\u00C2\u00A0 functional\u00C2\u00A0 affinity\u00C2\u00A0 toward\u00C2\u00A0 the\u00C2\u00A0 cell\u00E2\u0080\u0090surface\u00C2\u00A0 target,\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A017\u00C2\u00A0\u00C2\u00A0 decreased\u00C2\u00A0 dissociation\u00C2\u00A0 rates,\u00C2\u00A0 enhanced\u00C2\u00A0 biodistribution,\u00C2\u00A0 and\u00C2\u00A0 longer\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 half\u00E2\u0080\u0090life97,\u00C2\u00A0 98.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 characteristics\u00C2\u00A0provide\u00C2\u00A0 the\u00C2\u00A0basis\u00C2\u00A0 for\u00C2\u00A0mechanisms\u00C2\u00A0 of\u00C2\u00A0 enhancements\u00C2\u00A0 (or\u00C2\u00A0 inhibitions)\u00C2\u00A0of\u00C2\u00A0 therapeutic\u00C2\u00A0 activity\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0fundamentally\u00C2\u00A0different\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0case\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0bivalent\u00C2\u00A0one99.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 1.6.2\u00C2\u00A0 Examples\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0interactions\u00C2\u00A0in\u00C2\u00A0biology\u00C2\u00A0and\u00C2\u00A0their\u00C2\u00A0physical\u00C2\u00A0basis\u00C2\u00A0 Multivalent\u00C2\u00A0interactions\u00C2\u00A0are\u00C2\u00A0found\u00C2\u00A0throughout\u00C2\u00A0biology\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0necessary\u00C2\u00A0for\u00C2\u00A0processes\u00C2\u00A0such\u00C2\u00A0 as\u00C2\u00A0adhesion\u00C2\u00A0of\u00C2\u00A0viruses\u00C2\u00A0or\u00C2\u00A0bacteria\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0cells,\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0transcription\u00C2\u00A0factors\u00C2\u00A0to\u00C2\u00A0multiple\u00C2\u00A0 sites\u00C2\u00A0on\u00C2\u00A0DNA,\u00C2\u00A0and\u00C2\u00A0Abs\u00C2\u00A0interacting\u00C2\u00A0with\u00C2\u00A0macrophages\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0immune\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0through\u00C2\u00A0their\u00C2\u00A0 Fc\u00C2\u00A0 regions99.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 nature,\u00C2\u00A0 examples\u00C2\u00A0 of\u00C2\u00A0 biological\u00C2\u00A0molecules\u00C2\u00A0with\u00C2\u00A0 clustered\u00C2\u00A0 and\u00C2\u00A0 repeated\u00C2\u00A0 epitopes\u00C2\u00A0 include\u00C2\u00A0 biopolymers\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 polysaccharides,\u00C2\u00A0 proteoglycans,\u00C2\u00A0 filamentous\u00C2\u00A0 proteins,\u00C2\u00A0 and\u00C2\u00A0 DNA100.\u00C2\u00A0 Besides\u00C2\u00A0 Abs,\u00C2\u00A0 other\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0 ligands\u00C2\u00A0 include\u00C2\u00A0 carbohydrates,\u00C2\u00A0 peptides,\u00C2\u00A0 and\u00C2\u00A0 small\u00C2\u00A0 molecules,\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0demonstrate\u00C2\u00A0enhanced\u00C2\u00A0binding\u00C2\u00A0affinity\u00C2\u00A0to\u00C2\u00A0targeted\u00C2\u00A0tissue15.\u00C2\u00A0 A\u00C2\u00A0multivalent\u00C2\u00A0 interaction\u00C2\u00A0 consists,\u00C2\u00A0 in\u00C2\u00A0 theory,\u00C2\u00A0of\u00C2\u00A0 a\u00C2\u00A0 complex\u00C2\u00A0 created\u00C2\u00A0by\u00C2\u00A0 an\u00C2\u00A0 initial\u00C2\u00A0univalent\u00C2\u00A0 interaction\u00C2\u00A0 where\u00C2\u00A0 the\u00C2\u00A0 local\u00C2\u00A0 concentrations\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 remaining\u00C2\u00A0 free\u00C2\u00A0 binding\u00C2\u00A0 sites\u00C2\u00A0 on\u00C2\u00A0 both\u00C2\u00A0 species\u00C2\u00A0 become\u00C2\u00A0greatly\u00C2\u00A0enhanced\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0their\u00C2\u00A0close\u00C2\u00A0proximity15.\u00C2\u00A0\u00C2\u00A0Due\u00C2\u00A0to\u00C2\u00A0this\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0effective\u00C2\u00A0ligand\u00C2\u00A0and\u00C2\u00A0 target\u00C2\u00A0 concentrations,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 significantly\u00C2\u00A0 higher\u00C2\u00A0 probability\u00C2\u00A0 that\u00C2\u00A0 subsequent\u00C2\u00A0 ligand\u00E2\u0080\u0090target\u00C2\u00A0 interactions\u00C2\u00A0will\u00C2\u00A0result\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0univalent\u00C2\u00A0interaction.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0increased\u00C2\u00A0probability\u00C2\u00A0translates\u00C2\u00A0into\u00C2\u00A0 a\u00C2\u00A0higher\u00C2\u00A0avidity\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0ligand.\u00C2\u00A0\u00C2\u00A0Avidity\u00C2\u00A0refers\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0association\u00C2\u00A0constant\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0polyvalent\u00C2\u00A0 interaction,\u00C2\u00A0 as\u00C2\u00A0 opposed\u00C2\u00A0 to\u00C2\u00A0 affinity,\u00C2\u00A0 which\u00C2\u00A0 refers\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 association\u00C2\u00A0 constant\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 univalent\u00C2\u00A0 interaction;\u00C2\u00A0avidity\u00C2\u00A0is\u00C2\u00A0usually\u00C2\u00A0greater\u00C2\u00A0than\u00C2\u00A0the\u00C2\u00A0sum\u00C2\u00A0of\u00C2\u00A0constituent\u00C2\u00A0affinities\u00C2\u00A0for\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0ligand99,\u00C2\u00A0 100.\u00C2\u00A0 Kinetic\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0 enhanced\u00C2\u00A0 avidity\u00C2\u00A0 results\u00C2\u00A0 from\u00C2\u00A0 decreases\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 rate\u00C2\u00A0 of\u00C2\u00A0 dissociation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0ligand\u00C2\u00A0from\u00C2\u00A0its\u00C2\u00A0target,\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0association;\u00C2\u00A0 a\u00C2\u00A0greater\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0univalent\u00C2\u00A0interactions\u00C2\u00A0must\u00C2\u00A0be\u00C2\u00A0broken99.\u00C2\u00A0\u00C2\u00A0While\u00C2\u00A0some\u00C2\u00A0are\u00C2\u00A0being\u00C2\u00A0broken,\u00C2\u00A0others\u00C2\u00A0 continue\u00C2\u00A0to\u00C2\u00A0 form,\u00C2\u00A0resulting\u00C2\u00A0 in\u00C2\u00A0recapture\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 ligand\u00C2\u00A0before\u00C2\u00A0the\u00C2\u00A0 ligand\u00E2\u0080\u0090target\u00C2\u00A0complex\u00C2\u00A0dissociates\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A018\u00C2\u00A0\u00C2\u00A0 completely100.\u00C2\u00A0\u00C2\u00A0From\u00C2\u00A0a\u00C2\u00A0molecular\u00C2\u00A0point\u00C2\u00A0of\u00C2\u00A0view,\u00C2\u00A0the\u00C2\u00A0avidity\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0interaction\u00C2\u00A0results\u00C2\u00A0from\u00C2\u00A0 the\u00C2\u00A0 length\u00C2\u00A0 and\u00C2\u00A0 flexibility\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 linker(s),\u00C2\u00A0 the\u00C2\u00A0 strength\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 noncovalent\u00C2\u00A0 forces\u00C2\u00A0 involved,\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0of\u00C2\u00A0binding\u00C2\u00A0elements,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0conformational\u00C2\u00A0freedom\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0construct101.\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0 top\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0enormous\u00C2\u00A0diversity\u00C2\u00A0of\u00C2\u00A0constructs\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0materials\u00C2\u00A0employed,\u00C2\u00A0significant\u00C2\u00A0work\u00C2\u00A0has\u00C2\u00A0 also\u00C2\u00A0been\u00C2\u00A0described\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0configuration,\u00C2\u00A0architecture,\u00C2\u00A0and\u00C2\u00A0spacing\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target\u00E2\u0080\u0090binding\u00C2\u00A0sites102,\u00C2\u00A0103.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 multivalency\u00C2\u00A0 can\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 many\u00C2\u00A0 biological\u00C2\u00A0 consequences;\u00C2\u00A0 for\u00C2\u00A0 example,\u00C2\u00A0 multivalent\u00C2\u00A0 ligands\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0strong\u00C2\u00A0 inhibitors,\u00C2\u00A0particularly\u00C2\u00A0 if\u00C2\u00A0they\u00C2\u00A0bind\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0receptor\u00C2\u00A0whose\u00C2\u00A0agonistic\u00C2\u00A0 ligand\u00C2\u00A0is\u00C2\u00A0monovalent;\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0significant\u00C2\u00A0therapeutic\u00C2\u00A0interest\u00C2\u00A0in\u00C2\u00A0creating\u00C2\u00A0such\u00C2\u00A0multivalent\u00C2\u00A0ligands104.\u00C2\u00A0\u00C2\u00A0 Multivalent\u00C2\u00A0interactions\u00C2\u00A0can\u00C2\u00A0also\u00C2\u00A0serve\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0specific\u00C2\u00A0geometric\u00C2\u00A0shapes\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0clathrin\u00E2\u0080\u0090coated\u00C2\u00A0pits99.\u00C2\u00A0 \u00C2\u00A0They\u00C2\u00A0may\u00C2\u00A0also\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0grading\u00C2\u00A0of\u00C2\u00A0biological\u00C2\u00A0responses;\u00C2\u00A0for\u00C2\u00A0 example,\u00C2\u00A0macrophages\u00C2\u00A0cannot\u00C2\u00A0ingest\u00C2\u00A0a\u00C2\u00A0pathogen\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0recognizing\u00C2\u00A0a\u00C2\u00A0single\u00C2\u00A0Ab\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0with\u00C2\u00A0its\u00C2\u00A0 Fc\u00CE\u00B3Rs,\u00C2\u00A0 but\u00C2\u00A0 more\u00C2\u00A0 Fc\u00E2\u0080\u0090Fc\u00CE\u00B3R\u00C2\u00A0 associations\u00C2\u00A0 strengthen\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0 between\u00C2\u00A0 pathogen\u00C2\u00A0 and\u00C2\u00A0 macrophage,\u00C2\u00A0increasing\u00C2\u00A0the\u00C2\u00A0likelihood\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0pathogen\u00C2\u00A0will\u00C2\u00A0be\u00C2\u00A0cleared99.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Interactions\u00C2\u00A0between\u00C2\u00A0Fc\u00C2\u00A0 regions\u00C2\u00A0on\u00C2\u00A0antibody\u00E2\u0080\u0090coated\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0Fc\u00CE\u00B3Rs\u00C2\u00A0 in\u00C2\u00A0general\u00C2\u00A0constitute\u00C2\u00A0 multivalent\u00C2\u00A0interactions;\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0crosslinking,\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0interaction,\u00C2\u00A0is\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0initiation\u00C2\u00A0of\u00C2\u00A0 ADCC\u00C2\u00A0 by\u00C2\u00A0 NK\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 other\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 (Section\u00C2\u00A0 1.4.2).\u00C2\u00A0 \u00C2\u00A0Moreover,\u00C2\u00A0 initiation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 classical\u00C2\u00A0 complement\u00C2\u00A0 cascade\u00C2\u00A0 requires\u00C2\u00A0 a\u00C2\u00A0multivalent\u00C2\u00A0 interaction;\u00C2\u00A0 C1q\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 hexavalent\u00C2\u00A0 ligand\u00C2\u00A0 and\u00C2\u00A0 requires\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0many\u00C2\u00A0Fc\u00C2\u00A0regions\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0pathogen\u00C2\u00A0or\u00C2\u00A0cancer\u00C2\u00A0cell\u00C2\u00A0in\u00C2\u00A0 order\u00C2\u00A0 for\u00C2\u00A0 CDC\u00C2\u00A0 to\u00C2\u00A0 result105,\u00C2\u00A0106\u00C2\u00A0 (Section\u00C2\u00A0 1.4.1).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 cell\u00C2\u00A0 signaling\u00C2\u00A0 processes,\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 innumerable\u00C2\u00A0 examples\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0ligands\u00C2\u00A0that\u00C2\u00A0cause\u00C2\u00A0receptor\u00C2\u00A0activation\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0oligomerization.\u00C2\u00A0\u00C2\u00A0Clustering\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 receptor\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 signal\u00C2\u00A0amplification\u00C2\u00A0because\u00C2\u00A0many\u00C2\u00A0 receptors\u00C2\u00A0become\u00C2\u00A0activated\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0 single\u00C2\u00A0 ligand,\u00C2\u00A0which\u00C2\u00A0creates\u00C2\u00A0high\u00C2\u00A0local\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0receptor\u00C2\u00A0and\u00C2\u00A0which\u00C2\u00A0also\u00C2\u00A0causes\u00C2\u00A0exclusion\u00C2\u00A0 of\u00C2\u00A0negative\u00C2\u00A0modulators107,\u00C2\u00A0108.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0\u00CE\u00B1\u00C2\u00A0 (TNF\u00E2\u0080\u0090\u00CE\u00B1),\u00C2\u00A0the\u00C2\u00A0 ligand\u00C2\u00A0to\u00C2\u00A0CD120a\u00C2\u00A0 (Tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 1\u00C2\u00A0 (TNFR1)),\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 trivalent\u00C2\u00A0 ligand\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 capable\u00C2\u00A0 of\u00C2\u00A0 binding\u00C2\u00A0 and\u00C2\u00A0 crosslinking\u00C2\u00A0 three\u00C2\u00A0CD120a\u00C2\u00A0molecules;\u00C2\u00A0 receptor\u00C2\u00A0 clustering\u00C2\u00A0 is\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A019\u00C2\u00A0\u00C2\u00A0 and\u00C2\u00A0the\u00C2\u00A0subsequent\u00C2\u00A0initiation\u00C2\u00A0of\u00C2\u00A0diverse\u00C2\u00A0signaling\u00C2\u00A0processes\u00C2\u00A0ranging\u00C2\u00A0from\u00C2\u00A0proliferation\u00C2\u00A0to\u00C2\u00A0apoptosis\u00C2\u00A0 (see\u00C2\u00A0Section\u00C2\u00A06.2.1).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Taken\u00C2\u00A0together,\u00C2\u00A0these\u00C2\u00A0descriptions\u00C2\u00A0highlight\u00C2\u00A0that\u00C2\u00A0multivalent\u00C2\u00A0 interactions\u00C2\u00A0are\u00C2\u00A0ubiquitous\u00C2\u00A0 in\u00C2\u00A0 nature.\u00C2\u00A0\u00C2\u00A0They\u00C2\u00A0also\u00C2\u00A0illustrate\u00C2\u00A0that\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0a\u00C2\u00A0univalent\u00C2\u00A0and\u00C2\u00A0multivalent\u00C2\u00A0ligand\u00C2\u00A0may\u00C2\u00A0share\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0 target\u00E2\u0080\u0090binding\u00C2\u00A0site,\u00C2\u00A0they\u00C2\u00A0exhibit\u00C2\u00A0distinct\u00C2\u00A0physiochemical\u00C2\u00A0and\u00C2\u00A0biochemical\u00C2\u00A0properties\u00C2\u00A0that\u00C2\u00A0can\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0 remarkably\u00C2\u00A0different\u00C2\u00A0biological\u00C2\u00A0responses.\u00C2\u00A0 1.6.3\u00C2\u00A0 Descriptions\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0that\u00C2\u00A0exhibit\u00C2\u00A0superior\u00C2\u00A0efficacies\u00C2\u00A0 Although\u00C2\u00A0 all\u00C2\u00A0 therapeutic\u00C2\u00A0Abs\u00C2\u00A0differ\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0of\u00C2\u00A0 their\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0 (Section\u00C2\u00A0 1.4.3),\u00C2\u00A0 the\u00C2\u00A0 multimerization\u00C2\u00A0 of\u00C2\u00A0 many\u00C2\u00A0 bivalent\u00C2\u00A0 therapeutic\u00C2\u00A0 Abs\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 approved\u00C2\u00A0 or\u00C2\u00A0 under\u00C2\u00A0 development\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 enhance\u00C2\u00A0 their\u00C2\u00A0 associated\u00C2\u00A0 biological\u00C2\u00A0 responses7,\u00C2\u00A074,\u00C2\u00A089,\u00C2\u00A0109\u00E2\u0080\u0090113.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0 example,\u00C2\u00A0using\u00C2\u00A0two\u00C2\u00A0different\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0cell\u00C2\u00A0 lines,\u00C2\u00A0one\u00C2\u00A0that\u00C2\u00A0responds\u00C2\u00A0to\u00C2\u00A0Trz\u00C2\u00A0and\u00C2\u00A0one\u00C2\u00A0that\u00C2\u00A0 is\u00C2\u00A0Trz\u00E2\u0080\u0090 resistant,\u00C2\u00A0 it\u00C2\u00A0 was\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0 creating\u00C2\u00A0 multivalent\u00C2\u00A0 Trz\u00C2\u00A0 by\u00C2\u00A0 coupling\u00C2\u00A0 it\u00C2\u00A0 to\u00C2\u00A0 magnetic\u00C2\u00A0 microspheres\u00C2\u00A0 resulted\u00C2\u00A0in\u00C2\u00A0therapeutic\u00C2\u00A0effects\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0including\u00C2\u00A0the\u00C2\u00A0Trz\u00E2\u0080\u0090resistant\u00C2\u00A0one110.\u00C2\u00A0\u00C2\u00A0Another\u00C2\u00A0example\u00C2\u00A0 illustrated\u00C2\u00A0that\u00C2\u00A0multivalent\u00C2\u00A0Trz\u00C2\u00A0constructs\u00C2\u00A0with\u00C2\u00A0valences\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0four\u00C2\u00A0showed\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0growth\u00C2\u00A0 inhibition\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0Trz,\u00C2\u00A0and\u00C2\u00A0similarly\u00C2\u00A0constructed\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0anti\u00E2\u0080\u0090death\u00C2\u00A0 receptor\u00C2\u00A05\u00C2\u00A0(DR5)\u00C2\u00A0Abs\u00C2\u00A0showed\u00C2\u00A0equivalent\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0to\u00C2\u00A0bivalent\u00C2\u00A0Ab\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0~100\u00E2\u0080\u0090 fold\u00C2\u00A0lower89.\u00C2\u00A0 Consistent\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 requirement\u00C2\u00A0of\u00C2\u00A0crosslinking\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0apoptosis,\u00C2\u00A0many\u00C2\u00A0 many\u00C2\u00A0multivalent\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0designs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0described,\u00C2\u00A0 including\u00C2\u00A0 tetravalent\u00C2\u00A0Rtx\u00C2\u00A0dimers88,\u00C2\u00A0Rtx\u00E2\u0080\u0090 dextran\u00C2\u00A0polymers\u00C2\u00A0 (valence\u00C2\u00A0~10)\u00C2\u00A0and\u00C2\u00A0nanoparticles\u00C2\u00A0 (NPs)\u00C2\u00A0prepared\u00C2\u00A0by\u00C2\u00A0coupling\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0Dynabeads75,\u00C2\u00A0 trivalent\u00C2\u00A0and\u00C2\u00A0tetravalent\u00C2\u00A0Rtx\u00C2\u00A0constructs\u00C2\u00A0obtained\u00C2\u00A0through\u00C2\u00A0protein\u00C2\u00A0engineering89,\u00C2\u00A0Rtx\u00C2\u00A0coupled\u00C2\u00A0to\u00C2\u00A0gold\u00C2\u00A0 nanoparticles96,\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0multivalent\u00C2\u00A0branched\u00C2\u00A0copolymer\u00E2\u0080\u0090Fab\u00C2\u00A0conjugates15,\u00C2\u00A0and\u00C2\u00A0hexavalent\u00C2\u00A0anti\u00E2\u0080\u0090 CD20\u00C2\u00A0created\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A0Dock\u00E2\u0080\u0090and\u00E2\u0080\u0090Lock\u00C2\u00A0method90.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0general,\u00C2\u00A0 the\u00C2\u00A0multivalent\u00C2\u00A0constructs\u00C2\u00A0exhibited\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A020\u00C2\u00A0\u00C2\u00A0 superior\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0along\u00C2\u00A0with\u00C2\u00A0elevated\u00C2\u00A0responses\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0vivo;\u00C2\u00A0these\u00C2\u00A0responses\u00C2\u00A0 are\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0greater\u00C2\u00A0detail\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A04.2.\u00C2\u00A0 One\u00C2\u00A0 type\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0not\u00C2\u00A0 listed\u00C2\u00A0 above\u00C2\u00A0 is\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0before\u00C2\u00A0 the\u00C2\u00A0experiments\u00C2\u00A0 described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A01.2.2\u00C2\u00A0(ref.\u00C2\u00A07)\u00C2\u00A0were\u00C2\u00A0carried\u00C2\u00A0out,\u00C2\u00A0the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0liposome\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0nanoscale\u00C2\u00A0scaffold\u00C2\u00A0for\u00C2\u00A0 creating\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0had\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0considered.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0surprising\u00C2\u00A0in\u00C2\u00A0light\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 interest\u00C2\u00A0 surrounding\u00C2\u00A0 immunoliposomes,\u00C2\u00A0 but\u00C2\u00A0 the\u00C2\u00A0 focus\u00C2\u00A0 of\u00C2\u00A0 such\u00C2\u00A0 work\u00C2\u00A0 had\u00C2\u00A0 always\u00C2\u00A0 been\u00C2\u00A0 to\u00C2\u00A0 target\u00C2\u00A0 encapsulated\u00C2\u00A0 chemotherapy;\u00C2\u00A0 the\u00C2\u00A0Abs\u00C2\u00A0employed\u00C2\u00A0 therefore\u00C2\u00A0possessed\u00C2\u00A0 targeting\u00C2\u00A0properties\u00C2\u00A0and\u00C2\u00A0not\u00C2\u00A0 necessarily\u00C2\u00A0 therapeutic\u00C2\u00A0activity\u00C2\u00A0 (Section\u00C2\u00A01.2).\u00C2\u00A0 \u00C2\u00A0Since\u00C2\u00A0 the\u00C2\u00A0work\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0dissertation\u00C2\u00A0began,\u00C2\u00A0only\u00C2\u00A0one\u00C2\u00A0 report\u00C2\u00A0describing\u00C2\u00A0a\u00C2\u00A0 similar\u00C2\u00A0application\u00C2\u00A0of\u00C2\u00A0 liposomes\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0published;\u00C2\u00A0Oliveira\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0describe\u00C2\u00A0a\u00C2\u00A0 situation\u00C2\u00A0where\u00C2\u00A0anti\u00E2\u0080\u0090EGFR\u00C2\u00A0nanobodies\u00C2\u00A0coupled\u00C2\u00A0to\u00C2\u00A0empty\u00C2\u00A0liposomes\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0enhanced\u00C2\u00A0anticancer\u00C2\u00A0 activity\u00C2\u00A0through\u00C2\u00A0downregulation\u00C2\u00A0of\u00C2\u00A0EGFR114.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0work\u00C2\u00A0presented\u00C2\u00A0herein\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0provide\u00C2\u00A0 a\u00C2\u00A0 convenient,\u00C2\u00A0 useful,\u00C2\u00A0 and\u00C2\u00A0 promising\u00C2\u00A0 means\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 preclinical\u00C2\u00A0 discovery\u00C2\u00A0 and\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 liposomal\u00C2\u00A0and\u00C2\u00A0non\u00E2\u0080\u0090liposomal\u00C2\u00A0therapeutic\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 1.7\u00C2\u00A0 Multivalent\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0dissertation\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0work\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0dissertation\u00C2\u00A0makes\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 therapeutic\u00C2\u00A0Ab\u00C2\u00A0Rtx\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0prove\u00C2\u00A0 the\u00C2\u00A0 concept\u00C2\u00A0that\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0are\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0with\u00C2\u00A0enhanced\u00C2\u00A0activity\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0 bivalent\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0also\u00C2\u00A0sets\u00C2\u00A0the\u00C2\u00A0groundwork\u00C2\u00A0for\u00C2\u00A0developing\u00C2\u00A0improved\u00C2\u00A0multivalent\u00C2\u00A0drugs\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0other\u00C2\u00A0 therapeutic\u00C2\u00A0Abs.\u00C2\u00A0 \u00C2\u00A0Section\u00C2\u00A01.2.2\u00C2\u00A0 illustrated\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 (induction\u00C2\u00A0of\u00C2\u00A0 apoptosis)\u00C2\u00A0is\u00C2\u00A0enhanced\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0instead\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Chapter\u00C2\u00A02\u00C2\u00A0 serves\u00C2\u00A0to\u00C2\u00A0examine\u00C2\u00A0how\u00C2\u00A0the\u00C2\u00A0coupling\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0to\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0affects\u00C2\u00A0the\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0(CDC\u00C2\u00A0 and\u00C2\u00A0ADCC),\u00C2\u00A0since\u00C2\u00A0the\u00C2\u00A0improved\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0should\u00C2\u00A0not\u00C2\u00A0occur\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0cost\u00C2\u00A0of\u00C2\u00A0decreased\u00C2\u00A0indirect\u00C2\u00A0 mechanisms.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 chapter\u00C2\u00A0 also\u00C2\u00A0 examines\u00C2\u00A0 the\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 efficacy\u00C2\u00A0 and\u00C2\u00A0 pharmacokinetics\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0 Next,\u00C2\u00A0 to\u00C2\u00A0 study\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 it\u00C2\u00A0 was\u00C2\u00A0 necessary\u00C2\u00A0 to\u00C2\u00A0 prepare\u00C2\u00A0 many\u00C2\u00A0 different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0available\u00C2\u00A0methods\u00C2\u00A0for\u00C2\u00A0coupling\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0liposomes\u00C2\u00A0were\u00C2\u00A0found\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A021\u00C2\u00A0\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 unsuitable.\u00C2\u00A0 \u00C2\u00A0 Chapter\u00C2\u00A0 3\u00C2\u00A0 describes\u00C2\u00A0 a\u00C2\u00A0 superior\u00C2\u00A0methodology\u00C2\u00A0 that\u00C2\u00A0was\u00C2\u00A0 developed\u00C2\u00A0 for\u00C2\u00A0 creating\u00C2\u00A0 numerous\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 constructs\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 valence\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 Ab.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0methodology\u00C2\u00A0 shows\u00C2\u00A0 significant\u00C2\u00A0improvements\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0yield\u00C2\u00A0of\u00C2\u00A0coupled\u00C2\u00A0Ab,\u00C2\u00A0time\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0many\u00C2\u00A0valences,\u00C2\u00A0 and\u00C2\u00A0reproducibility.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Chapter\u00C2\u00A04\u00C2\u00A0makes\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0new\u00C2\u00A0methodology\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0many\u00C2\u00A0different\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0valences\u00C2\u00A0in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 examine\u00C2\u00A0 their\u00C2\u00A0 novel\u00C2\u00A0 physiochemical\u00C2\u00A0 and\u00C2\u00A0 biological\u00C2\u00A0 properties.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 include\u00C2\u00A0 binding\u00C2\u00A0 characteristics\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0 improvements\u00C2\u00A0 in\u00C2\u00A0their\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0 in\u00C2\u00A0CD20+\u00C2\u00A0 lymphoma\u00C2\u00A0cells.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 chapter\u00C2\u00A0establishes\u00C2\u00A0a\u00C2\u00A0 relationship\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0construct\u00C2\u00A0and\u00C2\u00A0 its\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0work\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A05\u00C2\u00A0employs\u00C2\u00A0 the\u00C2\u00A0different\u00C2\u00A0 valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 to\u00C2\u00A0 uncover\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 molecular\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 that\u00C2\u00A0 explains\u00C2\u00A0 its\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 extended\u00C2\u00A0 into\u00C2\u00A0 Chapter\u00C2\u00A0 6,\u00C2\u00A0 where\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0 conditions\u00C2\u00A0 involving\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0of\u00C2\u00A0 immune\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0coated\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0give\u00C2\u00A0rise\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0 same\u00C2\u00A0mechanism,\u00C2\u00A0 suggesting\u00C2\u00A0 that\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 also\u00C2\u00A0 a\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 under\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 conditions.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0provides\u00C2\u00A0compelling\u00C2\u00A0evidence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0defined\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0which\u00C2\u00A0 so\u00C2\u00A0far\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0described.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Overall,\u00C2\u00A0 this\u00C2\u00A0 proof\u00E2\u0080\u0090of\u00E2\u0080\u0090concept\u00C2\u00A0 serves\u00C2\u00A0 to\u00C2\u00A0 illustrate\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0methodology\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 dissertation\u00C2\u00A0 would\u00C2\u00A0 be\u00C2\u00A0 invaluable\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0 therapeutic\u00C2\u00A0 Abs.\u00C2\u00A0 \u00C2\u00A0 Because\u00C2\u00A0 multivalent\u00C2\u00A0Abs\u00C2\u00A0show\u00C2\u00A0enhanced\u00C2\u00A0efficacies\u00C2\u00A0over\u00C2\u00A0bivalent\u00C2\u00A0ones,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0possible\u00C2\u00A0 that\u00C2\u00A0some\u00C2\u00A0\u00E2\u0080\u009Cdiscarded\u00E2\u0080\u009D\u00C2\u00A0 Abs\u00C2\u00A0under\u00C2\u00A0development\u00C2\u00A0(due\u00C2\u00A0to\u00C2\u00A0 low\u00C2\u00A0activity\u00C2\u00A0as\u00C2\u00A0bivalent\u00C2\u00A0molecules)\u00C2\u00A0may\u00C2\u00A0actually\u00C2\u00A0show\u00C2\u00A0significantly\u00C2\u00A0 improved\u00C2\u00A0activity\u00C2\u00A0as\u00C2\u00A0multivalent\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0methodology\u00C2\u00A0can\u00C2\u00A0therefore\u00C2\u00A0be\u00C2\u00A0employed\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0context\u00C2\u00A0of\u00C2\u00A0 drug\u00C2\u00A0discovery\u00C2\u00A0to\u00C2\u00A0progress\u00C2\u00A0promising\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0formulations\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0advanced\u00C2\u00A0stage\u00C2\u00A0 of\u00C2\u00A0preclinical\u00C2\u00A0development.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A022\u00C2\u00A0\u00C2\u00A0 2\u00C2\u00A0 Indirect\u00C2\u00A0 mechanisms\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0 rituximab\u00C2\u00ADlipid\u00C2\u00A0nanoparticles\u00E2\u0080\u00A1\u00C2\u00A0 2.1\u00C2\u00A0 Synopsis\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0creating\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0enhance\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0 (Section\u00C2\u00A01.2.2),\u00C2\u00A0 the\u00C2\u00A0effects\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0are\u00C2\u00A0unclear.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 indirect\u00C2\u00A0 mechanisms\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0consist\u00C2\u00A0of\u00C2\u00A0complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0(CDC;\u00C2\u00A0Section\u00C2\u00A01.4.1)\u00C2\u00A0 and\u00C2\u00A0 antibody\u00E2\u0080\u0090dependent\u00C2\u00A0 cell\u00E2\u0080\u0090mediated\u00C2\u00A0 cytotoxicity\u00C2\u00A0 (ADCC;\u00C2\u00A0 Section\u00C2\u00A01.4.2).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 chapter\u00C2\u00A0examines\u00C2\u00A0 how\u00C2\u00A0the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0are\u00C2\u00A0affected\u00C2\u00A0by\u00C2\u00A0creating\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs,\u00C2\u00A0and\u00C2\u00A0 it\u00C2\u00A0also\u00C2\u00A0examines\u00C2\u00A0the\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0efficacy\u00C2\u00A0and\u00C2\u00A0pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP. 2.2\u00C2\u00A0 Background 2.2.1\u00C2\u00A0 Complement\u00C2\u00ADdependent\u00C2\u00A0cytotoxicity\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 Rtx\u00C2\u00A0activates\u00C2\u00A0the\u00C2\u00A0classical\u00C2\u00A0complement\u00C2\u00A0pathway\u00C2\u00A0by\u00C2\u00A0binding\u00C2\u00A0C1q115,\u00C2\u00A0 leading\u00C2\u00A0to\u00C2\u00A0generation\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0membrane\u00C2\u00A0attack\u00C2\u00A0complex\u00C2\u00A0and\u00C2\u00A0ensuing\u00C2\u00A0tumor\u00C2\u00A0cell\u00C2\u00A0lysis116.\u00C2\u00A0\u00C2\u00A0Rtx\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0CDC\u00C2\u00A0in\u00C2\u00A0 many\u00C2\u00A0different\u00C2\u00A0 lymphoma\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0 in\u00C2\u00A0vitro115,\u00C2\u00A0117\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0effective\u00C2\u00A0only\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0 mouse\u00C2\u00A0model\u00C2\u00A0expressing\u00C2\u00A0a\u00C2\u00A0 full\u00C2\u00A0complement\u00C2\u00A0system,\u00C2\u00A0while\u00C2\u00A0 its\u00C2\u00A0activity\u00C2\u00A0was\u00C2\u00A0ablated\u00C2\u00A0 in\u00C2\u00A0C1q\u00E2\u0080\u0090deficient\u00C2\u00A0 mice118.\u00C2\u00A0 \u00C2\u00A0 Infusion\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 in\u00C2\u00A0CLL\u00C2\u00A0patients\u00C2\u00A0has\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 to\u00C2\u00A0deplete\u00C2\u00A0complement,\u00C2\u00A0and\u00C2\u00A0pre\u00E2\u0080\u0090Rtx\u00C2\u00A0 complement\u00C2\u00A0 levels\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 return\u00C2\u00A0 until\u00C2\u00A0 several\u00C2\u00A0 weeks\u00C2\u00A0 after\u00C2\u00A0 completion\u00C2\u00A0 of\u00C2\u00A0 therapy119.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 complement\u00C2\u00A0cascade\u00C2\u00A0leading\u00C2\u00A0to\u00C2\u00A0CDC\u00C2\u00A0is\u00C2\u00A0regulated\u00C2\u00A0by\u00C2\u00A0proteins\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0 CD55\u00C2\u00A0and\u00C2\u00A0CD59.\u00C2\u00A0 \u00C2\u00A0Blocking\u00C2\u00A0CD55\u00C2\u00A0and/or\u00C2\u00A0CD59\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0 increase\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CDC\u00C2\u00A0 in\u00C2\u00A0NHL\u00C2\u00A0cell\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A1\u00C2\u00A0A\u00C2\u00A0 version\u00C2\u00A0 of\u00C2\u00A0 Chapter\u00C2\u00A0 2\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 published16.\u00C2\u00A0 \u00C2\u00A0 Adapted\u00C2\u00A0 and\u00C2\u00A0 reprinted\u00C2\u00A0 by\u00C2\u00A0 permission\u00C2\u00A0 from\u00C2\u00A0 Future\u00C2\u00A0 Medicine\u00C2\u00A0Ltd.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A023\u00C2\u00A0\u00C2\u00A0 lines\u00C2\u00A0 and\u00C2\u00A0 primary\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 Rtx,\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0 two\u00C2\u00A0 proteins\u00C2\u00A0 function\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0as\u00C2\u00A0important\u00C2\u00A0regulators\u00C2\u00A0of\u00C2\u00A0CDC46,\u00C2\u00A0120.\u00C2\u00A0 2.2.2\u00C2\u00A0 Ab\u00C2\u00ADdependent\u00C2\u00A0cell\u00C2\u00ADmediated\u00C2\u00A0cytotoxicity\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 ADCC\u00C2\u00A0 is\u00C2\u00A0also\u00C2\u00A0an\u00C2\u00A0established\u00C2\u00A0contributor\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0therapeutic\u00C2\u00A0 Abs\u00C2\u00A0 in\u00C2\u00A0 general.\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0Ab\u00E2\u0080\u0090coated\u00C2\u00A0 tumor\u00C2\u00A0 cells,\u00C2\u00A0 it\u00C2\u00A0occurs\u00C2\u00A0upon\u00C2\u00A0 binding\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 Fc\u00C2\u00A0 region\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Ab\u00C2\u00A0 to\u00C2\u00A0 activating\u00C2\u00A0Fc\u00EF\u0081\u00A7\u00C2\u00A0receptors\u00C2\u00A0 (Fc\u00EF\u0081\u00A7R)\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0Fc\u00EF\u0081\u00A7RIIIa\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0present\u00C2\u00A0on\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0natural\u00C2\u00A0 killer\u00C2\u00A0 (NK)\u00C2\u00A0 cells,\u00C2\u00A0 granulocytes,\u00C2\u00A0 and\u00C2\u00A0macrophages.\u00C2\u00A0 \u00C2\u00A0 Rtx\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 ADCC\u00C2\u00A0 in\u00C2\u00A0many\u00C2\u00A0 human\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0lines117\u00C2\u00A0and\u00C2\u00A0ADCC\u00C2\u00A0was\u00C2\u00A0abolished\u00C2\u00A0in\u00C2\u00A0nude\u00C2\u00A0mice\u00C2\u00A0deficient\u00C2\u00A0in\u00C2\u00A0activating\u00C2\u00A0Fc\u00EF\u0081\u00A7R\u00C2\u00A0yet\u00C2\u00A0 present\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0wild\u00E2\u0080\u0090type\u00C2\u00A0mice121,\u00C2\u00A0establishing\u00C2\u00A0a\u00C2\u00A0central\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 importance\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090induced\u00C2\u00A0 ADCC\u00C2\u00A0 in\u00C2\u00A0 humans\u00C2\u00A0was\u00C2\u00A0 illustrated\u00C2\u00A0 in\u00C2\u00A0 studies\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 Fc\u00EF\u0081\u00A7RIIIa\u00C2\u00A0 dimorphism\u00C2\u00A0 that\u00C2\u00A0 influences\u00C2\u00A0 the\u00C2\u00A0 binding\u00C2\u00A0 affinity\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 receptor\u00C2\u00A0 to\u00C2\u00A0 Rtx,\u00C2\u00A0where\u00C2\u00A0 enhanced\u00C2\u00A0 binding\u00C2\u00A0 corresponds\u00C2\u00A0 to\u00C2\u00A0 elevated\u00C2\u00A0 ADCC.\u00C2\u00A0 \u00C2\u00A0 Patients\u00C2\u00A0 homozygous\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 high\u00E2\u0080\u0090affinity\u00C2\u00A0 dimorphism\u00C2\u00A0 showed\u00C2\u00A0 enhanced\u00C2\u00A0clinical\u00C2\u00A0and\u00C2\u00A0molecular\u00C2\u00A0responses\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0patients122,\u00C2\u00A0123.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0ADCC\u00E2\u0080\u0090 regulating\u00C2\u00A0molecules\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0themselves,\u00C2\u00A0expression\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0ligands\u00C2\u00A0to\u00C2\u00A0NKG2D,\u00C2\u00A0a\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0 receptor,\u00C2\u00A0 influence\u00C2\u00A0 the\u00C2\u00A0 susceptibility\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 cell\u00C2\u00A0 to\u00C2\u00A0Rtx\u00E2\u0080\u0090induced\u00C2\u00A0ADCC124.\u00C2\u00A0 \u00C2\u00A0Other\u00C2\u00A0molecules\u00C2\u00A0 present\u00C2\u00A0on\u00C2\u00A0B\u00C2\u00A0cells\u00C2\u00A0which\u00C2\u00A0trigger\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0include\u00C2\u00A0CD40125,\u00C2\u00A0CD80,\u00C2\u00A0and\u00C2\u00A0CD86126,\u00C2\u00A0but\u00C2\u00A0in\u00C2\u00A0general,\u00C2\u00A0 the\u00C2\u00A0molecular\u00C2\u00A0determinants\u00C2\u00A0of\u00C2\u00A0whether\u00C2\u00A0a\u00C2\u00A0tumor\u00C2\u00A0cell\u00C2\u00A0will\u00C2\u00A0elicit\u00C2\u00A0ADCC\u00C2\u00A0upon\u00C2\u00A0Rtx\u00C2\u00A0binding\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0well\u00C2\u00A0 understood.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0the\u00C2\u00A0relative\u00C2\u00A0contributions\u00C2\u00A0of\u00C2\u00A0CDC\u00C2\u00A0and\u00C2\u00A0ADCC\u00C2\u00A0that\u00C2\u00A0occur\u00C2\u00A0after\u00C2\u00A0Rtx\u00C2\u00A0treatment,\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0contribution\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0 (Section\u00C2\u00A01.4.3),\u00C2\u00A0are\u00C2\u00A0unclear\u00C2\u00A0and\u00C2\u00A0have\u00C2\u00A0 been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0origin\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0cell44,\u00C2\u00A049,\u00C2\u00A050.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A024\u00C2\u00A0\u00C2\u00A0 2.2.3\u00C2\u00A0 Does\u00C2\u00A0 the\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNPs\u00C2\u00A0affect\u00C2\u00A0 the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0 properties\u00C2\u00A0of\u00C2\u00A0Rtx?\u00C2\u00A0 We\u00C2\u00A0examined\u00C2\u00A0how\u00C2\u00A0the\u00C2\u00A0known\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0are\u00C2\u00A0affected\u00C2\u00A0by\u00C2\u00A0creating\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0 \u00C2\u00A0We\u00C2\u00A0 have\u00C2\u00A0 previously\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 exhibit\u00C2\u00A0 increased\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0 those\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0 free\u00C2\u00A0Rtx\u00C2\u00A0 (Section\u00C2\u00A01.2.2)7.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0studies\u00C2\u00A0 established\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0exhibits\u00C2\u00A0an\u00C2\u00A0 improved\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 objective\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0current\u00C2\u00A0study\u00C2\u00A0was\u00C2\u00A0to\u00C2\u00A0establish\u00C2\u00A0whether\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0and\u00C2\u00A0CDC\u00C2\u00A0are\u00C2\u00A0preserved,\u00C2\u00A0since\u00C2\u00A0 under\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 conditions,\u00C2\u00A0 the\u00C2\u00A0 favorable\u00C2\u00A0 direct\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 should\u00C2\u00A0 not\u00C2\u00A0 occur\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 cost\u00C2\u00A0 of\u00C2\u00A0 decreased\u00C2\u00A0indirect\u00C2\u00A0contributions\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0efficacy.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0examine\u00C2\u00A0differences\u00C2\u00A0between\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0their\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0 action,\u00C2\u00A0we\u00C2\u00A0employed\u00C2\u00A0two\u00C2\u00A0mantle\u00C2\u00A0cell\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0lines,\u00C2\u00A0Z138\u00C2\u00A0and\u00C2\u00A0JVM2,\u00C2\u00A0which\u00C2\u00A0exhibited\u00C2\u00A0different\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0sensitivities\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0along\u00C2\u00A0with\u00C2\u00A0variable\u00C2\u00A0expression\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0cell\u00E2\u0080\u0090surface\u00C2\u00A0proteins\u00C2\u00A0that\u00C2\u00A0regulate\u00C2\u00A0 ADCC\u00C2\u00A0 and\u00C2\u00A0 CDC.\u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 were\u00C2\u00A0 prepared\u00C2\u00A0 which\u00C2\u00A0 showed\u00C2\u00A0 enhanced\u00C2\u00A0 binding\u00C2\u00A0 and\u00C2\u00A0 which\u00C2\u00A0 induced\u00C2\u00A0 apoptosis\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0lines.\u00C2\u00A0\u00C2\u00A0We\u00C2\u00A0demonstrate\u00C2\u00A0that\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0exhibited\u00C2\u00A0increased\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0 CDC\u00C2\u00A0and\u00C2\u00A0ADCC\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0Rtx,\u00C2\u00A0alongside\u00C2\u00A0an\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0levels.\u00C2\u00A0\u00C2\u00A0Finally,\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0 understand\u00C2\u00A0the\u00C2\u00A0manifestations\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0in\u00C2\u00A0vivo,\u00C2\u00A0we\u00C2\u00A0examined\u00C2\u00A0the\u00C2\u00A0efficacy\u00C2\u00A0and\u00C2\u00A0 pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 2.3\u00C2\u00A0\u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0 2.3.1\u00C2\u00A0 Reagents\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0 The\u00C2\u00A0 following\u00C2\u00A0 lipids\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 Avanti\u00C2\u00A0 Polar\u00C2\u00A0 Lipids\u00C2\u00A0 (Alabaster\u00C2\u00A0 AL,\u00C2\u00A0 USA):\u00C2\u00A0 1,2\u00E2\u0080\u0090 distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphocholine\u00C2\u00A0 (DSPC),\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphoethanolamine\u00E2\u0080\u0090 N\u00E2\u0080\u0090[methoxy(polyethylene\u00C2\u00A0 glycol)\u00E2\u0080\u00902000]\u00C2\u00A0 (DSPE\u00E2\u0080\u0090PEG),\u00C2\u00A0 and\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090 phosphoethanolamine\u00E2\u0080\u0090N\u00E2\u0080\u0090[maleimide(polyethylene\u00C2\u00A0 glycol)\u00E2\u0080\u00902000]\u00C2\u00A0 (DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090Mal).\u00C2\u00A0 \u00C2\u00A0 [3H]cholesteryl\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A025\u00C2\u00A0\u00C2\u00A0 hexadecyl\u00C2\u00A0 ether\u00C2\u00A0 ([3H]CHE)\u00C2\u00A0 was\u00C2\u00A0 from\u00C2\u00A0 Perkin\u00E2\u0080\u0090Elmer\u00C2\u00A0 Life\u00C2\u00A0 Sciences\u00C2\u00A0 (Woodbridge\u00C2\u00A0 ON,\u00C2\u00A0 Canada).\u00C2\u00A0\u00C2\u00A0 Cholesterol\u00C2\u00A0(Chol)\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0Sigma\u00E2\u0080\u0090Aldrich\u00C2\u00A0(St.\u00C2\u00A0Louis\u00C2\u00A0MO,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0Pico\u00E2\u0080\u0090Fluor\u00C2\u00A015\u00C2\u00A0and\u00C2\u00A0Pico\u00E2\u0080\u0090 Fluor\u00C2\u00A040\u00C2\u00A0scintillation\u00C2\u00A0cocktails\u00C2\u00A0were\u00C2\u00A0bought\u00C2\u00A0from\u00C2\u00A0PerkinElmer\u00C2\u00A0BioSignal\u00C2\u00A0(Montreal\u00C2\u00A0QC,\u00C2\u00A0Canada).\u00C2\u00A0 \u00C2\u00A0N\u00E2\u0080\u0090 Succinimidyl\u00C2\u00A0 3\u00E2\u0080\u0090(2\u00E2\u0080\u0090pyridyldithio)propionate\u00C2\u00A0 (SPDP)\u00C2\u00A0 and\u00C2\u00A0 dithiothreitol\u00C2\u00A0 (DTT)\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 Pierce/Thermo\u00C2\u00A0 Fisher\u00C2\u00A0 Scientific\u00C2\u00A0 (Rockford\u00C2\u00A0 IL,\u00C2\u00A0 USA).\u00C2\u00A0 \u00C2\u00A0 Rituximab\u00C2\u00A0 (Rtx)\u00C2\u00A0 and\u00C2\u00A0 trastuzumab\u00C2\u00A0 (Trz)\u00C2\u00A0were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0BC\u00C2\u00A0Cancer\u00C2\u00A0Agency\u00C2\u00A0pharmacy\u00C2\u00A0 (Vancouver\u00C2\u00A0BC,\u00C2\u00A0Canada).\u00C2\u00A0 \u00C2\u00A0Matrigel\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0 from\u00C2\u00A0 Collaborative\u00C2\u00A0 Biomedical\u00C2\u00A0 Products\u00C2\u00A0 Inc.\u00C2\u00A0 (Chicago\u00C2\u00A0 IL,\u00C2\u00A0 USA).\u00C2\u00A0 \u00C2\u00A0 Carboxyfluorescein\u00C2\u00A0 diacetate\u00C2\u00A0 succinimidyl\u00C2\u00A0ester\u00C2\u00A0(CFSE)\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Molecular\u00C2\u00A0Probes\u00C2\u00A0(Eugene\u00C2\u00A0OR,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0Anti\u00E2\u0080\u0090human\u00C2\u00A0CD3\u00E2\u0080\u0090 FITC,\u00C2\u00A0 CD56\u00E2\u0080\u0090PE,\u00C2\u00A0 CD54\u00E2\u0080\u0090APC,\u00C2\u00A0 and\u00C2\u00A0 Fc\u00CE\u00B3RIII/II\u00C2\u00A0 (CD16/32)\u00C2\u00A0 Abs\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 BD\u00C2\u00A0 Biosciences\u00C2\u00A0 (Mississauga\u00C2\u00A0ON,\u00C2\u00A0Canada).\u00C2\u00A0\u00C2\u00A0Anti\u00E2\u0080\u0090Rtx\u00E2\u0080\u0090FITC\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0Serotec\u00C2\u00A0(Oxford,\u00C2\u00A0UK).\u00C2\u00A0\u00C2\u00A0Rabbit\u00C2\u00A0anti\u00E2\u0080\u0090 human\u00C2\u00A0IgG\u00C2\u00A0Fc\u00C2\u00A0fragment\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0MP\u00C2\u00A0Biomedicals\u00C2\u00A0(Irvine\u00C2\u00A0CA,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0Lympholyte\u00E2\u0080\u0090Mammal\u00C2\u00A0 was\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Cedarlane\u00C2\u00A0(Burlington\u00C2\u00A0ON,\u00C2\u00A0Canada).\u00C2\u00A0\u00C2\u00A0Unless\u00C2\u00A0otherwise\u00C2\u00A0noted,\u00C2\u00A0all\u00C2\u00A0other\u00C2\u00A0reagents\u00C2\u00A0 were\u00C2\u00A0from\u00C2\u00A0Sigma\u00E2\u0080\u0090Aldrich\u00C2\u00A0(St.\u00C2\u00A0Louis\u00C2\u00A0MO,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Mantle\u00C2\u00A0cell\u00C2\u00A0lymphoma\u00C2\u00A0(MCL)\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0Z138\u00C2\u00A0and\u00C2\u00A0JVM2\u00C2\u00A0were\u00C2\u00A0generously\u00C2\u00A0provided\u00C2\u00A0by\u00C2\u00A0Dr.\u00C2\u00A0Zeev\u00C2\u00A0 Estrov\u00C2\u00A0 (University\u00C2\u00A0of\u00C2\u00A0Texas)\u00C2\u00A0and\u00C2\u00A0previously\u00C2\u00A0 characterized127.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0maintained\u00C2\u00A0 in\u00C2\u00A0RPMI\u00C2\u00A01640\u00C2\u00A0 medium\u00C2\u00A0 (Stem\u00C2\u00A0 Cell\u00C2\u00A0 Technologies)\u00C2\u00A0 supplemented\u00C2\u00A0 with\u00C2\u00A0 2\u00C2\u00A0 mM\u00C2\u00A0 L\u00E2\u0080\u0090glutamine,\u00C2\u00A0 10%\u00C2\u00A0 FBS,\u00C2\u00A0 and\u00C2\u00A0 1%\u00C2\u00A0 penicillin/streptomycin.\u00C2\u00A0 \u00C2\u00A0All\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0maintained\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0 \u00C2\u00B0C\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0humidified\u00C2\u00A0atmosphere\u00C2\u00A0containing\u00C2\u00A0 5%\u00C2\u00A0CO2.\u00C2\u00A0\u00C2\u00A0 For\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 imaging\u00C2\u00A0 studies,\u00C2\u00A0 Z138\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 transfected\u00C2\u00A0 to\u00C2\u00A0 express\u00C2\u00A0 luciferase\u00C2\u00A0 (Luc).\u00C2\u00A0 Constructs\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0lentivirus\u00C2\u00A0(LV)\u00C2\u00A0vector\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0Luc\u00C2\u00A0genes\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Dr.\u00C2\u00A0Alice\u00C2\u00A0Mui\u00C2\u00A0 (Jack\u00C2\u00A0Bell\u00C2\u00A0Research\u00C2\u00A0Centre,\u00C2\u00A0Vancouver\u00C2\u00A0General\u00C2\u00A0Hospital,\u00C2\u00A0Vancouver\u00C2\u00A0BC,\u00C2\u00A0Canada)\u00C2\u00A0who\u00C2\u00A0also\u00C2\u00A0assisted\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0transfection\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0line.\u00C2\u00A0\u00C2\u00A0Briefly,\u00C2\u00A0the\u00C2\u00A0Luc\u00C2\u00A0coding\u00C2\u00A0sequence\u00C2\u00A0was\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0pGL\u00E2\u0080\u00903\u00C2\u00A0vector\u00C2\u00A0 (Promega,\u00C2\u00A0Madison,\u00C2\u00A0WI,\u00C2\u00A0USA)\u00C2\u00A0and\u00C2\u00A0cloned\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0 lentiviral\u00C2\u00A0vector\u00C2\u00A0FG9\u00C2\u00A0behind\u00C2\u00A0the\u00C2\u00A0CMV\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0LTR\u00C2\u00A0and\u00C2\u00A0 UBiC\u00C2\u00A0promoters.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0generate\u00C2\u00A0Luc\u00E2\u0080\u0090expressing\u00C2\u00A0lentivirus\u00C2\u00A0(LV\u00E2\u0080\u0090Luc),\u00C2\u00A0this\u00C2\u00A0vector\u00C2\u00A0was\u00C2\u00A0cotransfected\u00C2\u00A0using\u00C2\u00A0 calcium\u00C2\u00A0 phosphate\u00C2\u00A0with\u00C2\u00A0 packaging\u00C2\u00A0 constructs\u00C2\u00A0 pRSVREV,\u00C2\u00A0 pMDLg/pRRE,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 VSV\u00E2\u0080\u0090G\u00C2\u00A0 expression\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A026\u00C2\u00A0\u00C2\u00A0 plasmid\u00C2\u00A0 pHCMVG\u00C2\u00A0 into\u00C2\u00A0HEK\u00E2\u0080\u0090293T\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0 Five\u00C2\u00A0million\u00C2\u00A0 293T\u00C2\u00A0HEK\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 plated\u00C2\u00A0 on\u00C2\u00A0 poly\u00E2\u0080\u0090L\u00E2\u0080\u0090lysine\u00E2\u0080\u0090 coated\u00C2\u00A0 tissue\u00C2\u00A0 culture\u00C2\u00A0plates\u00C2\u00A0 allowed\u00C2\u00A0 to\u00C2\u00A0 adhere\u00C2\u00A0 for\u00C2\u00A0 24\u00C2\u00A0 hours.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 following\u00C2\u00A0 day,\u00C2\u00A0 10\u00C2\u00A0 \u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 transducing\u00C2\u00A0vector,\u00C2\u00A07.5\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0packaging\u00C2\u00A0vector,\u00C2\u00A0and\u00C2\u00A02.5\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0VSV\u00C2\u00A0envelope\u00C2\u00A0pMD.G\u00C2\u00A0were\u00C2\u00A0co\u00E2\u0080\u0090 transfected\u00C2\u00A0 by\u00C2\u00A0 LipofectAMINE\u00C2\u00A0 2000\u00C2\u00A0 (Invitrogen,\u00C2\u00A0 Burlington\u00C2\u00A0 ON,\u00C2\u00A0 Canada),\u00C2\u00A0 according\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 manufacturer's\u00C2\u00A0 instructions.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0fresh\u00C2\u00A0medium\u00C2\u00A0was\u00C2\u00A0applied\u00C2\u00A0to\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0cultured\u00C2\u00A0 for\u00C2\u00A0 another\u00C2\u00A0 24\u00C2\u00A0 h.\u00C2\u00A0 \u00C2\u00A0 Conditioned\u00C2\u00A0medium\u00C2\u00A0was\u00C2\u00A0 then\u00C2\u00A0 collected\u00C2\u00A0 and\u00C2\u00A0cleared\u00C2\u00A0 of\u00C2\u00A0 debris\u00C2\u00A0 by\u00C2\u00A0 low\u00C2\u00A0 speed\u00C2\u00A0 centrifugation,\u00C2\u00A0filtered,\u00C2\u00A0and\u00C2\u00A0stored\u00C2\u00A0at\u00C2\u00A0\u00E2\u0080\u009070\u00C2\u00A0\u00C2\u00B0C.\u00C2\u00A0\u00C2\u00A0Supernatant\u00C2\u00A0was\u00C2\u00A0collected\u00C2\u00A0daily\u00C2\u00A0for\u00C2\u00A04\u00C2\u00A0days,\u00C2\u00A0pooled\u00C2\u00A0and\u00C2\u00A0 ultracentrifuged.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0pellet\u00C2\u00A0was\u00C2\u00A0re\u00E2\u0080\u0090suspended\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0medium,\u00C2\u00A0and\u00C2\u00A0aliquots\u00C2\u00A0were\u00C2\u00A0stored\u00C2\u00A0at\u00C2\u00A0\u00E2\u0080\u009070\u00C2\u00A0 \u00C2\u00B0C.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0infected\u00C2\u00A0with\u00C2\u00A0LV\u00E2\u0080\u0090Luc\u00C2\u00A0(25\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0viral\u00C2\u00A0supernatant/mL\u00C2\u00A0medium).\u00C2\u00A0\u00C2\u00A0Briefly,\u00C2\u00A0one\u00C2\u00A0 million\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0add\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A012\u00E2\u0080\u0090well\u00C2\u00A0plate\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0complete\u00C2\u00A0medium\u00C2\u00A0and\u00C2\u00A0these\u00C2\u00A0were\u00C2\u00A0 then\u00C2\u00A0cultured\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0Subsequently,\u00C2\u00A0LV\u00E2\u0080\u0090vector\u00C2\u00A0or\u00C2\u00A0LV\u00E2\u0080\u0090Luc\u00C2\u00A0constructs\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0medium\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0Polybrene\u00C2\u00A0(8\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0medium).\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0approximately\u00C2\u00A05\u00C2\u00A0h\u00C2\u00A0of\u00C2\u00A0incubation,\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 washed\u00C2\u00A0with\u00C2\u00A0phosphate\u00E2\u0080\u0090buffered\u00C2\u00A0 saline\u00C2\u00A0 (PBS,\u00C2\u00A0pH\u00C2\u00A07.4),\u00C2\u00A0 fresh\u00C2\u00A0 culture\u00C2\u00A0medium\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0and\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A06\u00C2\u00A0days.\u00C2\u00A0To\u00C2\u00A0enrich\u00C2\u00A0for\u00C2\u00A0Luc\u00E2\u0080\u0090positive\u00C2\u00A0cells,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0sorted\u00C2\u00A0by\u00C2\u00A0FACS\u00C2\u00A0for\u00C2\u00A0GFP\u00C2\u00A0 expression\u00C2\u00A0(cells\u00C2\u00A0were\u00C2\u00A0cotransfected\u00C2\u00A0with\u00C2\u00A0LV\u00E2\u0080\u0090GFP\u00C2\u00A0constructs).\u00C2\u00A0GFP\u00E2\u0080\u0090positive\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0considered\u00C2\u00A0to\u00C2\u00A0 be\u00C2\u00A0positive\u00C2\u00A0for\u00C2\u00A0Luc\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0was\u00C2\u00A0confirmed\u00C2\u00A0by\u00C2\u00A0culturing\u00C2\u00A0the\u00C2\u00A0sorted\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0low\u00C2\u00A0concentrations\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 wells\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plate.\u00C2\u00A0 \u00C2\u00A0Luciferin\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0and\u00C2\u00A0plates\u00C2\u00A0were\u00C2\u00A0 imaged\u00C2\u00A0using\u00C2\u00A0 IVIS\u00C2\u00A0 (see\u00C2\u00A0 below)\u00C2\u00A0 to\u00C2\u00A0 confirm\u00C2\u00A0 Luc\u00C2\u00A0expression.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0Z138\u00E2\u0080\u0090Luc\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0expanded\u00C2\u00A0and\u00C2\u00A0used\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 studies\u00C2\u00A0described\u00C2\u00A0below.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 2.3.2\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0 Small\u00C2\u00A0 unilamellar\u00C2\u00A0 vesicles\u00C2\u00A0 composed\u00C2\u00A0 of\u00C2\u00A0 DSPC/Chol/DSPE\u00E2\u0080\u0090PEG/DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090Mal\u00C2\u00A0 (mole\u00C2\u00A0 ratio\u00C2\u00A0 48:45:5:2)\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0extrusion\u00C2\u00A0procedure128,\u00C2\u00A0129.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A0200\u00C2\u00A0\u00C2\u00B5mol\u00C2\u00A0lipid\u00C2\u00A0were\u00C2\u00A0dissolved\u00C2\u00A0 in\u00C2\u00A0CHCl3\u00C2\u00A0 and\u00C2\u00A0 an\u00C2\u00A0 aliquot\u00C2\u00A0of\u00C2\u00A0 [3H]CHE\u00C2\u00A0 (0.009\u00C2\u00A0\u00C2\u00B5Ci/\u00C2\u00B5mol\u00C2\u00A0 liposomal\u00C2\u00A0 lipid)\u00C2\u00A0was\u00C2\u00A0 added.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0 lipid\u00C2\u00A0 film\u00C2\u00A0was\u00C2\u00A0 formed\u00C2\u00A0by\u00C2\u00A0drying\u00C2\u00A0 the\u00C2\u00A0solution\u00C2\u00A0 first\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0stream\u00C2\u00A0of\u00C2\u00A0N2\u00C2\u00A0 then\u00C2\u00A0under\u00C2\u00A0vacuum\u00C2\u00A0 for\u00C2\u00A03\u00C2\u00A0h.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 film\u00C2\u00A0was\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A027\u00C2\u00A0\u00C2\u00A0 hydrated\u00C2\u00A0in\u00C2\u00A02.0\u00C2\u00A0mL\u00C2\u00A0HEPES\u00E2\u0080\u0090buffered\u00C2\u00A0saline\u00C2\u00A0(HBS)\u00C2\u00A0at\u00C2\u00A065\u00C2\u00A0oC\u00C2\u00A0for\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0with\u00C2\u00A0stirring,\u00C2\u00A0then\u00C2\u00A0was\u00C2\u00A0subjected\u00C2\u00A0to\u00C2\u00A0 five\u00C2\u00A0freeze\u00E2\u0080\u0090thaw\u00C2\u00A0cycles\u00C2\u00A0each\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0five\u00E2\u0080\u0090minute\u00C2\u00A0treatments\u00C2\u00A0in\u00C2\u00A0liquid\u00C2\u00A0N2\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A065\u00C2\u00A0oC\u00C2\u00A0water\u00C2\u00A0bath,\u00C2\u00A0 respectively.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 suspension\u00C2\u00A0was\u00C2\u00A0 then\u00C2\u00A0 extruded\u00C2\u00A0 ten\u00C2\u00A0 times\u00C2\u00A0 through\u00C2\u00A0 two\u00C2\u00A0 stacked\u00C2\u00A0 polycarbonate\u00C2\u00A0 filters\u00C2\u00A0(100\u00C2\u00A0nm\u00C2\u00A0and\u00C2\u00A080\u00C2\u00A0nm\u00C2\u00A0pore\u00C2\u00A0size)\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0Lipex\u00C2\u00A0extruder\u00C2\u00A0(Northern\u00C2\u00A0Lipids,\u00C2\u00A0Burnaby\u00C2\u00A0BC,\u00C2\u00A0Canada).\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0resulting\u00C2\u00A0mean\u00C2\u00A0vesicle\u00C2\u00A0diameter\u00C2\u00A0was\u00C2\u00A095\u00E2\u0080\u0093110\u00C2\u00A0nm\u00C2\u00A0as\u00C2\u00A0determined\u00C2\u00A0by\u00C2\u00A0quasielastic\u00C2\u00A0 light\u00C2\u00A0scattering\u00C2\u00A0 (QLS)\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0Nicomp\u00C2\u00A0 submicron\u00C2\u00A0 particle\u00C2\u00A0 sizer\u00C2\u00A0 (model\u00C2\u00A0 370/270).\u00C2\u00A0 \u00C2\u00A0 Liposomal\u00C2\u00A0 lipid\u00C2\u00A0 concentrations\u00C2\u00A0 were\u00C2\u00A0measured\u00C2\u00A0by\u00C2\u00A0liquid\u00C2\u00A0scintillation\u00C2\u00A0counting\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0Packard\u00C2\u00A0scintillation\u00C2\u00A0counter\u00C2\u00A0(model\u00C2\u00A01900\u00C2\u00A0TR)\u00C2\u00A0 using\u00C2\u00A0aliquots\u00C2\u00A0mixed\u00C2\u00A0with\u00C2\u00A05.0\u00C2\u00A0mL\u00C2\u00A0Pico\u00E2\u0080\u0090Fluor\u00C2\u00A015\u00C2\u00A0scintillation\u00C2\u00A0fluid.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Rtx\u00C2\u00A0was\u00C2\u00A0coupled\u00C2\u00A0to\u00C2\u00A0 liposomes\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0established\u00C2\u00A0procedure130.\u00C2\u00A0 \u00C2\u00A0Briefly,\u00C2\u00A080\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0 12.5\u00C2\u00A0mM\u00C2\u00A0solution\u00C2\u00A0of\u00C2\u00A0SPDP\u00C2\u00A0in\u00C2\u00A0ethanol\u00C2\u00A0were\u00C2\u00A0diluted\u00C2\u00A0with\u00C2\u00A0920\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0HBS\u00C2\u00A0(25\u00C2\u00A0mM\u00C2\u00A0HEPES,\u00C2\u00A0150\u00C2\u00A0mM\u00C2\u00A0NaCl,\u00C2\u00A0pH\u00C2\u00A0 7.4)\u00C2\u00A0 to\u00C2\u00A0give\u00C2\u00A0a\u00C2\u00A0 final\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A01.0\u00C2\u00A0nmol/\u00C2\u00B5L.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0Ab\u00C2\u00A0 (8\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A09\u00C2\u00A0mg)\u00C2\u00A0was\u00C2\u00A0 reacted\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0 five\u00E2\u0080\u0090fold\u00C2\u00A0 molar\u00C2\u00A0 excess\u00C2\u00A0 of\u00C2\u00A0 SPDP\u00C2\u00A0 for\u00C2\u00A0 25\u00C2\u00A0min\u00C2\u00A0 at\u00C2\u00A0 room\u00C2\u00A0 temperature,\u00C2\u00A0 and\u00C2\u00A0was\u00C2\u00A0 subsequently\u00C2\u00A0 eluted\u00C2\u00A0 by\u00C2\u00A0 gravity\u00C2\u00A0 through\u00C2\u00A0a\u00C2\u00A0Sephadex\u00C2\u00A0G\u00E2\u0080\u009050\u00C2\u00A0column\u00C2\u00A0equilibrated\u00C2\u00A0with\u00C2\u00A0sodium\u00C2\u00A0acetate\u00C2\u00A0buffer\u00C2\u00A0(100\u00C2\u00A0mM\u00C2\u00A0sodium\u00C2\u00A0acetate,\u00C2\u00A0 150\u00C2\u00A0mM\u00C2\u00A0NaCl,\u00C2\u00A0pH\u00C2\u00A04.5).\u00C2\u00A0\u00C2\u00A0Fractions\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0were\u00C2\u00A0determined\u00C2\u00A0by\u00C2\u00A0diluting\u00C2\u00A0aliquots\u00C2\u00A050\u00E2\u0080\u0090fold\u00C2\u00A0in\u00C2\u00A0 HBS\u00C2\u00A0and\u00C2\u00A0by\u00C2\u00A0measuring\u00C2\u00A0the\u00C2\u00A0absorbance\u00C2\u00A0at\u00C2\u00A0280\u00C2\u00A0nm.\u00C2\u00A0\u00C2\u00A0Ab\u00E2\u0080\u0090containing\u00C2\u00A0fractions\u00C2\u00A0were\u00C2\u00A0pooled\u00C2\u00A0and\u00C2\u00A0added\u00C2\u00A0 to\u00C2\u00A0 solid\u00C2\u00A0DTT\u00C2\u00A0 to\u00C2\u00A0 give\u00C2\u00A0 a\u00C2\u00A0 final\u00C2\u00A0 concentration\u00C2\u00A0 of\u00C2\u00A0 25\u00C2\u00A0mM\u00C2\u00A0DTT.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 solution\u00C2\u00A0was\u00C2\u00A0 incubated\u00C2\u00A0 at\u00C2\u00A0 room\u00C2\u00A0 temperature\u00C2\u00A0 for\u00C2\u00A025\u00C2\u00A0min\u00C2\u00A0with\u00C2\u00A0 stirring.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 thiolated\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0 then\u00C2\u00A0 isolated\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0Sephadex\u00C2\u00A0G\u00E2\u0080\u009050\u00C2\u00A0 column\u00C2\u00A0 equilibrated\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0 and\u00C2\u00A0was\u00C2\u00A0 immediately\u00C2\u00A0 added\u00C2\u00A0 to\u00C2\u00A0 liposomes\u00C2\u00A0 (10\u00C2\u00A0mM\u00C2\u00A0 final\u00C2\u00A0 liposomal\u00C2\u00A0 lipid\u00C2\u00A0concentration).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0mixture\u00C2\u00A0was\u00C2\u00A0incubated\u00C2\u00A0at\u00C2\u00A0room\u00C2\u00A0temperature\u00C2\u00A0for\u00C2\u00A018\u00C2\u00A0h\u00C2\u00A0with\u00C2\u00A0gentle\u00C2\u00A0mixing.\u00C2\u00A0\u00C2\u00A0 At\u00C2\u00A0the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0reaction,\u00C2\u00A0the\u00C2\u00A0mixture\u00C2\u00A0was\u00C2\u00A0eluted\u00C2\u00A0through\u00C2\u00A0a\u00C2\u00A0Sepharose\u00C2\u00A0CL\u00E2\u0080\u00904B\u00C2\u00A0column\u00C2\u00A0equilibrated\u00C2\u00A0 with\u00C2\u00A0HBS\u00C2\u00A0to\u00C2\u00A0separate\u00C2\u00A0the\u00C2\u00A0unreacted\u00C2\u00A0Ab\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0diameter\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0was\u00C2\u00A0~130\u00C2\u00A0 nm,\u00C2\u00A0as\u00C2\u00A0determined\u00C2\u00A0using\u00C2\u00A0QLS.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0conjugated\u00C2\u00A0to\u00C2\u00A0liposomes\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0 Pierce\u00C2\u00A0Micro\u00C2\u00A0bicinchoninic\u00C2\u00A0acid\u00C2\u00A0(BCA)\u00C2\u00A0protein\u00C2\u00A0assay\u00C2\u00A0kit.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0quantitation\u00C2\u00A0was\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0according\u00C2\u00A0 to\u00C2\u00A0the\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions\u00C2\u00A0with\u00C2\u00A00.5%\u00C2\u00A0Triton\u00C2\u00A0X\u00E2\u0080\u0090100\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0reagent\u00C2\u00A0solution\u00C2\u00A0and\u00C2\u00A0using\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A028\u00C2\u00A0\u00C2\u00A0 Ab\u00C2\u00A0solutions\u00C2\u00A0of\u00C2\u00A0known\u00C2\u00A0concentration\u00C2\u00A0as\u00C2\u00A0standards.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0liposome\u00C2\u00A0was\u00C2\u00A0 calculated\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0previously7\u00C2\u00A0and\u00C2\u00A0was\u00C2\u00A0found\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0between\u00C2\u00A038\u00C2\u00A0and\u00C2\u00A047.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 2.3.3\u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0cell\u00C2\u00ADsurface\u00C2\u00A0protein\u00C2\u00A0expression\u00C2\u00A0levels\u00C2\u00A0 A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A01\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0Z138\u00C2\u00A0or\u00C2\u00A0 JVM2\u00C2\u00A0MCL\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0 \u00C2\u00A0 the\u00C2\u00A0exponential\u00C2\u00A0growth\u00C2\u00A0phase\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0 with\u00C2\u00A0cold\u00C2\u00A0PBS\u00C2\u00A0containing\u00C2\u00A01%\u00C2\u00A0FBS,\u00C2\u00A0then\u00C2\u00A0incubated\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0oC\u00C2\u00A0\u00C2\u00A0for\u00C2\u00A010\u00C2\u00A0min\u00C2\u00A0with\u00C2\u00A0anti\u00E2\u0080\u0090Fc\u00CE\u00B3RIII/II\u00C2\u00A0Ab\u00C2\u00A0followed\u00C2\u00A0 by 20\u00C2\u00A0min\u00C2\u00A0with\u00C2\u00A0saturating\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0FITC\u00E2\u0080\u0090labeled\u00C2\u00A0anti\u00E2\u0080\u0090human\u00C2\u00A0CD20,\u00C2\u00A0CD40,\u00C2\u00A0CD80,\u00C2\u00A0CD86,\u00C2\u00A0CD95\u00C2\u00A0(Fas\u00C2\u00A0 receptor),\u00C2\u00A0CD54,\u00C2\u00A0CD55\u00C2\u00A0or\u00C2\u00A0CD59\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0Samples\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0washed\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0FACSCalibur\u00C2\u00A0flow\u00C2\u00A0 cytometer\u00C2\u00A0 (Becton\u00C2\u00A0Dickinson,\u00C2\u00A0 San\u00C2\u00A0 Jose,\u00C2\u00A0CA)\u00C2\u00A0 collecting\u00C2\u00A0 10,000\u00C2\u00A0 events\u00C2\u00A0 for\u00C2\u00A0 each\u00C2\u00A0 sample.\u00C2\u00A0 \u00C2\u00A0Data\u00C2\u00A0was\u00C2\u00A0 analyzed\u00C2\u00A0 using\u00C2\u00A0 BD\u00C2\u00A0 CellQuest\u00C2\u00A0 software.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 this\u00C2\u00A0 and\u00C2\u00A0 all\u00C2\u00A0 subsequent\u00C2\u00A0 experiments,\u00C2\u00A0 data\u00C2\u00A0 values\u00C2\u00A0 are\u00C2\u00A0 reported\u00C2\u00A0 as\u00C2\u00A0mean\u00C2\u00A0 \u00C2\u00B1\u00C2\u00A0 standard\u00C2\u00A0 deviation\u00C2\u00A0 (SD)\u00C2\u00A0 unless\u00C2\u00A0 indicated\u00C2\u00A0 otherwise.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 all\u00C2\u00A0 cases,\u00C2\u00A0 two\u00E2\u0080\u0090tailed\u00C2\u00A0 unpaired\u00C2\u00A0Student\u00E2\u0080\u0099s\u00C2\u00A0t\u00E2\u0080\u0090tests\u00C2\u00A0were\u00C2\u00A0applied\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0significance\u00C2\u00A0of\u00C2\u00A00.05.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 2.3.4\u00C2\u00A0 In\u00C2\u00A0vivo\u00C2\u00A0xenograft\u00C2\u00A0models\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0Rtx\u00C2\u00A0efficacy\u00C2\u00A0 All\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0studies\u00C2\u00A0were\u00C2\u00A0completed\u00C2\u00A0using\u00C2\u00A0protocols\u00C2\u00A0approved\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0Animal\u00C2\u00A0Care\u00C2\u00A0Committee\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0University\u00C2\u00A0of\u00C2\u00A0British\u00C2\u00A0Columbia,\u00C2\u00A0 and\u00C2\u00A0were\u00C2\u00A0 in\u00C2\u00A0 accordance\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 current\u00C2\u00A0 guidelines\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 Canadian\u00C2\u00A0Council\u00C2\u00A0of\u00C2\u00A0Animal\u00C2\u00A0Care.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0assess\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0treatment,\u00C2\u00A05\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0Z138\u00C2\u00A0or\u00C2\u00A0JVM2\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0exponential\u00C2\u00A0growth\u00C2\u00A0phase\u00C2\u00A0were\u00C2\u00A0mixed\u00C2\u00A0with\u00C2\u00A0Matrigel\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0final\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0\u00C2\u00B5L,\u00C2\u00A0then\u00C2\u00A0 injected\u00C2\u00A0 subcutaneously\u00C2\u00A0(SC)\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0flank\u00C2\u00A0of\u00C2\u00A0male\u00C2\u00A0Rag\u00E2\u0080\u00902M\u00C2\u00A0mice.\u00C2\u00A0\u00C2\u00A0Treatments\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0control\u00C2\u00A0(PBS)\u00C2\u00A0were\u00C2\u00A0 initiated\u00C2\u00A0 when\u00C2\u00A0 tumors\u00C2\u00A0 were\u00C2\u00A0 palpable\u00C2\u00A0 (0.5\u00C2\u00A0 \u00C3\u0097\u00C2\u00A0 0.5\u00C2\u00A0 \u00C3\u0097\u00C2\u00A0 0.5\u00C2\u00A0 mm),\u00C2\u00A0 typically\u00C2\u00A0 25\u00C2\u00A0 to\u00C2\u00A0 28\u00C2\u00A0 days\u00C2\u00A0 after\u00C2\u00A0 cell\u00C2\u00A0 inoculation.\u00C2\u00A0 \u00C2\u00A0Groups\u00C2\u00A0of\u00C2\u00A06\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0(2.5\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg/kg)\u00C2\u00A0given\u00C2\u00A0 intraperitoneally\u00C2\u00A0(IP)\u00C2\u00A0 every\u00C2\u00A03\u00C2\u00A0days\u00C2\u00A0 for\u00C2\u00A0a\u00C2\u00A0 total\u00C2\u00A0of\u00C2\u00A06\u00C2\u00A0 treatments\u00C2\u00A0 (Q3D\u00C2\u00A0\u00C3\u0097\u00C2\u00A06).\u00C2\u00A0 \u00C2\u00A0Tumor\u00C2\u00A0 size\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0calibrated\u00C2\u00A0 caliper\u00C2\u00A0every\u00C2\u00A02\u00E2\u0080\u00933\u00C2\u00A0days.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A029\u00C2\u00A0\u00C2\u00A0 2.3.5\u00C2\u00A0 Quantification\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0 Z138\u00C2\u00A0or\u00C2\u00A0JVM2\u00C2\u00A0cells\u00C2\u00A0(1\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106)\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0for\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0\u00C2\u00B0C\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0or\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0at\u00C2\u00A0 different\u00C2\u00A0doses\u00C2\u00A0as\u00C2\u00A0 indicated.\u00C2\u00A0 \u00C2\u00A0All\u00C2\u00A0treatments\u00C2\u00A0were\u00C2\u00A0done\u00C2\u00A0 in\u00C2\u00A0triplicate.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 incubation\u00C2\u00A0 time,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0cold\u00C2\u00A0PBS\u00C2\u00A0containing\u00C2\u00A01%\u00C2\u00A0FBS\u00C2\u00A0and\u00C2\u00A0labeled\u00C2\u00A0with\u00C2\u00A0anti\u00E2\u0080\u0090Rtx\u00E2\u0080\u0090FITC\u00C2\u00A0for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0 at\u00C2\u00A0 4\u00C2\u00A0 \u00C2\u00B0C,\u00C2\u00A0 then\u00C2\u00A0 analyzed\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 FACSCalibur\u00C2\u00A0 flow\u00C2\u00A0 cytometer\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 above\u00C2\u00A0 (10,000\u00C2\u00A0 events\u00C2\u00A0 per\u00C2\u00A0 sample).\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0parallel\u00C2\u00A0study\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0to\u00C2\u00A0measure\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0through\u00C2\u00A0quantification\u00C2\u00A0of\u00C2\u00A0 [3H]CHE\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0liposomes\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0the\u00C2\u00A0incubation,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0centrifuged\u00C2\u00A0at\u00C2\u00A0300g\u00C2\u00A0for\u00C2\u00A0 5\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0\u00EF\u0082\u00B0C,\u00C2\u00A0then\u00C2\u00A0washed\u00C2\u00A0three\u00C2\u00A0times\u00C2\u00A0with\u00C2\u00A01\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0PBS\u00C2\u00A0to\u00C2\u00A0eliminate\u00C2\u00A0unbound\u00C2\u00A0Ab\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0Washed\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0resuspended\u00C2\u00A0and\u00C2\u00A0solubilized\u00C2\u00A0with\u00C2\u00A01\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A00.9%\u00C2\u00A0Triton\u00E2\u0080\u0090X\u00C2\u00A0100\u00C2\u00A0in\u00C2\u00A0PBS,\u00C2\u00A0and\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0 [3H]CHE\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0by\u00C2\u00A0liquid\u00C2\u00A0scintillation\u00C2\u00A0counting\u00C2\u00A0using\u00C2\u00A0Pico\u00E2\u0080\u0090Fluor\u00C2\u00A040\u00C2\u00A0scintillation\u00C2\u00A0cocktail\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0 Canberra\u00E2\u0080\u0090Packard\u00C2\u00A0Scintillation\u00C2\u00A0\u00CE\u00B2\u00C2\u00A0counter\u00C2\u00A0(1900\u00C2\u00A0TR\u00C2\u00A0Tri\u00C2\u00A0Carb).\u00C2\u00A0 2.3.6\u00C2\u00A0 Annexin\u00C2\u00ADV/PI\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00ADbased\u00C2\u00A0apoptosis\u00C2\u00A0assay\u00C2\u00A0 Cells\u00C2\u00A0were\u00C2\u00A0seeded\u00C2\u00A0 in\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plates\u00C2\u00A0 (20,000\u00C2\u00A0cells/well)\u00C2\u00A0and\u00C2\u00A0 treatments\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0day\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 seeded.\u00C2\u00A0 \u00C2\u00A0Control\u00C2\u00A0 treatments\u00C2\u00A0consisted\u00C2\u00A0of\u00C2\u00A0HBS\u00C2\u00A0and\u00C2\u00A0uncoupled\u00C2\u00A0 liposomes,\u00C2\u00A0 negative\u00C2\u00A0controls\u00C2\u00A0employed\u00C2\u00A0Trz\u00C2\u00A0 instead\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0positive\u00C2\u00A0controls\u00C2\u00A0 included\u00C2\u00A0campothecin\u00E2\u0080\u0090treated\u00C2\u00A0 samples.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0analysis\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0 later,\u00C2\u00A0samples\u00C2\u00A0were\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A01.5\u00C2\u00A0mL\u00C2\u00A0Eppendorf\u00C2\u00A0tubes\u00C2\u00A0and\u00C2\u00A0spun\u00C2\u00A0at\u00C2\u00A0 7000\u00C2\u00A0rpm\u00C2\u00A0for\u00C2\u00A015\u00C2\u00A0s.\u00C2\u00A0 \u00C2\u00A0Supernatants\u00C2\u00A0were\u00C2\u00A0removed\u00C2\u00A0and\u00C2\u00A0pellets\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0cold\u00C2\u00A0Hanks\u00E2\u0080\u0099\u00C2\u00A0 balanced\u00C2\u00A0 salt\u00C2\u00A0 solution.\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0 were\u00C2\u00A0 spun\u00C2\u00A0 again\u00C2\u00A0 at\u00C2\u00A0 7000\u00C2\u00A0 rpm\u00C2\u00A0 /\u00C2\u00A0 15\u00C2\u00A0 s\u00C2\u00A0 and\u00C2\u00A0 supernatants\u00C2\u00A0 were\u00C2\u00A0 removed.\u00C2\u00A0 \u00C2\u00A0 Positive\u00C2\u00A0 control\u00C2\u00A0 samples\u00C2\u00A0 were\u00C2\u00A0 unstained,\u00C2\u00A0 stained\u00C2\u00A0 with\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC,\u00C2\u00A0 stained\u00C2\u00A0 with\u00C2\u00A0 propidium\u00C2\u00A0 iodide\u00C2\u00A0 (PI),\u00C2\u00A0 or\u00C2\u00A0 double\u00E2\u0080\u0090stained\u00C2\u00A0with\u00C2\u00A0 both;\u00C2\u00A0 all\u00C2\u00A0 other\u00C2\u00A0 samples\u00C2\u00A0were\u00C2\u00A0 double\u00E2\u0080\u0090stained.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0 staining,\u00C2\u00A0pellets\u00C2\u00A0were\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0mixture\u00C2\u00A0of\u00C2\u00A05\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0and\u00C2\u00A040\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00C2\u00A0binding\u00C2\u00A0buffer\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0at\u00C2\u00A0room\u00C2\u00A0temperature\u00C2\u00A0for\u00C2\u00A020\u00C2\u00A0min.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0PI\u00C2\u00A0staining,\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0 cold\u00C2\u00A00.5\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0solution\u00C2\u00A0of\u00C2\u00A0PI\u00C2\u00A0 in\u00C2\u00A0Hanks\u00E2\u0080\u0099\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0samples.\u00C2\u00A0 \u00C2\u00A0Analysis\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A030\u00C2\u00A0\u00C2\u00A0 Becton\u00C2\u00A0Dickinson\u00C2\u00A0FACSCalibur\u00C2\u00A0flow\u00C2\u00A0cytometer.\u00C2\u00A0\u00C2\u00A0Compensation\u00C2\u00A0values\u00C2\u00A0were\u00C2\u00A0set\u00C2\u00A0with\u00C2\u00A0positive\u00C2\u00A0controls\u00C2\u00A0 and\u00C2\u00A0 quadrant\u00C2\u00A0 placement\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 plot\u00C2\u00A0 of\u00C2\u00A0 PI\u00C2\u00A0 fluorescence\u00C2\u00A0 intensity\u00C2\u00A0 (FL3)\u00C2\u00A0 versus\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0 fluorescence\u00C2\u00A0 intensity\u00C2\u00A0 (FL1)\u00C2\u00A0 in\u00C2\u00A0 each\u00C2\u00A0 case\u00C2\u00A0 was\u00C2\u00A0 decided\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 basis\u00C2\u00A0 of\u00C2\u00A0 data\u00C2\u00A0 obtained\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 negative\u00C2\u00A0 controls.\u00C2\u00A0 \u00C2\u00A0Early\u00C2\u00A0apoptotic\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0defined\u00C2\u00A0as\u00C2\u00A0PI\u00C2\u00A0negative\u00C2\u00A0and\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0positive,\u00C2\u00A0 necrotic\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0positive\u00C2\u00A0for\u00C2\u00A0both\u00C2\u00A0fluorophores,\u00C2\u00A0and\u00C2\u00A0viable\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0negative\u00C2\u00A0for\u00C2\u00A0both.\u00C2\u00A0 2.3.7\u00C2\u00A0 Assay\u00C2\u00A0for\u00C2\u00A0measuring\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0complement\u00C2\u00ADdependent\u00C2\u00A0cytotoxicity\u00C2\u00A0 MCL\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0 (1\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0cells/mL)\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0or\u00C2\u00A0controls\u00C2\u00A0 (including\u00C2\u00A0 HBS,\u00C2\u00A0uncoupled\u00C2\u00A0liposomes,\u00C2\u00A0and\u00C2\u00A0Trz\u00E2\u0080\u0090NP)\u00C2\u00A0for\u00C2\u00A015\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0\u00C2\u00B0C,\u00C2\u00A0then\u00C2\u00A0a\u00C2\u00A025%\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0human\u00C2\u00A0serum\u00C2\u00A0(50\u00C2\u00A0 \u00C2\u00B5L)\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0sample\u00C2\u00A0was\u00C2\u00A0further\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A04\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0Informed\u00C2\u00A0consent\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0 volunteers\u00C2\u00A0who\u00C2\u00A0provided\u00C2\u00A0the\u00C2\u00A0serum.\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0and\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0PI,\u00C2\u00A0then\u00C2\u00A0analyzed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0 cytometry\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0above.\u00C2\u00A0\u00C2\u00A0Cell\u00C2\u00A0lysis\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0CDC\u00C2\u00A0was\u00C2\u00A0expressed\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0percentage\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0 following:\u00C2\u00A0%\u00C2\u00A0 cell\u00C2\u00A0 lysis\u00C2\u00A0=\u00C2\u00A0[1\u00C2\u00A0\u00E2\u0088\u0092\u00C2\u00A0(fraction\u00C2\u00A0 of\u00C2\u00A0 viable\u00C2\u00A0 treated\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 human\u00C2\u00A0 serum)\u00C2\u00A0 /\u00C2\u00A0 (fraction\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0treated\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0human\u00C2\u00A0serum)]\u00C2\u00A0\u00C3\u0097\u00C2\u00A0100%.\u00C2\u00A0 2.3.8\u00C2\u00A0 Assay\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0quantification\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00ADdependent\u00C2\u00A0cell\u00C2\u00ADmediated\u00C2\u00A0cytotoxicity\u00C2\u00A0 A\u00C2\u00A0fluorometric\u00C2\u00A0method\u00C2\u00A0described\u00C2\u00A0by\u00C2\u00A0Gomez\u00E2\u0080\u0090Roman\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0was\u00C2\u00A0used131.\u00C2\u00A0\u00C2\u00A0Briefly,\u00C2\u00A01\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0Z138\u00C2\u00A0 or\u00C2\u00A0JVM2\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0double\u00E2\u0080\u0090stained\u00C2\u00A0with\u00C2\u00A02.5\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0PKH26\u00C2\u00A0(a\u00C2\u00A0membrane\u00C2\u00A0dye)\u00C2\u00A0and\u00C2\u00A02.5\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0CFSE\u00C2\u00A0(a\u00C2\u00A0 viability\u00C2\u00A0dye).\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0 then\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 controls\u00C2\u00A0 (as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0CDC\u00C2\u00A0assay\u00C2\u00A0above)\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0 \u00C2\u00B5g/mL\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 (either\u00C2\u00A0as\u00C2\u00A0 free\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP)\u00C2\u00A0 for\u00C2\u00A030\u00C2\u00A0min.\u00C2\u00A0 \u00C2\u00A0Next,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0with\u00C2\u00A0mouse\u00C2\u00A0 splenocytes\u00C2\u00A0(at\u00C2\u00A05:1,\u00C2\u00A050:1,\u00C2\u00A0and\u00C2\u00A0100:1\u00C2\u00A0ratios\u00C2\u00A0of\u00C2\u00A0effector\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0cells)\u00C2\u00A0previously\u00C2\u00A0stimulated\u00C2\u00A0overnight\u00C2\u00A0 with\u00C2\u00A0 25\u00C2\u00A0 ng/mL\u00C2\u00A0of\u00C2\u00A0 interleukin\u00E2\u0080\u009012\u00C2\u00A0 (IL\u00E2\u0080\u009012).\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0 a\u00C2\u00A0 24\u00C2\u00A0h\u00C2\u00A0 incubation,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0 in\u00C2\u00A0 cold\u00C2\u00A0 PBS\u00C2\u00A0 containing\u00C2\u00A01%\u00C2\u00A0FBS\u00C2\u00A0and\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0buffer\u00C2\u00A0before\u00C2\u00A0analysis\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry,\u00C2\u00A0acquiring\u00C2\u00A0 10,000\u00C2\u00A0nongated\u00C2\u00A0events.\u00C2\u00A0 \u00C2\u00A0Flow\u00C2\u00A0 cytometry\u00C2\u00A0data\u00C2\u00A0was\u00C2\u00A0acquired\u00C2\u00A0by\u00C2\u00A0 setting\u00C2\u00A0FL1\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0CFSE\u00C2\u00A0emission\u00C2\u00A0 channel\u00C2\u00A0and\u00C2\u00A0FL2\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0PKH26\u00C2\u00A0emission\u00C2\u00A0channel.\u00C2\u00A0Percent\u00C2\u00A0cell\u00C2\u00A0kill\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0by\u00C2\u00A0back\u00E2\u0080\u0090gating\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A031\u00C2\u00A0\u00C2\u00A0 PKH26bright\u00C2\u00A0population\u00C2\u00A0of\u00C2\u00A0targets\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0reported\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0membrane\u00E2\u0080\u0090labeled\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0 having\u00C2\u00A0 lost\u00C2\u00A0the\u00C2\u00A0viability\u00C2\u00A0dye,\u00C2\u00A0 i.e.\u00C2\u00A0%\u00C2\u00A0CFSE\u00E2\u0080\u0093\u00C2\u00A0within\u00C2\u00A0PKH26bright.\u00C2\u00A0 \u00C2\u00A0Non\u00E2\u0080\u0090stained\u00C2\u00A0and\u00C2\u00A0single\u00E2\u0080\u0090stained\u00C2\u00A0targets\u00C2\u00A0 were\u00C2\u00A0included\u00C2\u00A0in\u00C2\u00A0every\u00C2\u00A0experiment\u00C2\u00A0to\u00C2\u00A0compensate\u00C2\u00A0for\u00C2\u00A0single\u00E2\u0080\u0090stained\u00C2\u00A0CFSE\u00C2\u00A0and\u00C2\u00A0PKH26\u00C2\u00A0emissions,\u00C2\u00A0and\u00C2\u00A0 double\u00E2\u0080\u0090stained\u00C2\u00A0 targets\u00C2\u00A0 incubated\u00C2\u00A0with\u00C2\u00A0Ab\u00C2\u00A0without\u00C2\u00A0effectors\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0 to\u00C2\u00A0define\u00C2\u00A0spontaneous\u00C2\u00A0cell\u00C2\u00A0 death\u00C2\u00A0 to\u00C2\u00A0 exposure\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0media\u00C2\u00A0 and\u00C2\u00A0 Ab\u00C2\u00A0 alone.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 percentage\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 undergoing\u00C2\u00A0 ADCC\u00C2\u00A0was\u00C2\u00A0 quantified\u00C2\u00A0 as\u00C2\u00A0 follows:\u00C2\u00A0%\u00C2\u00A0 undergoing\u00C2\u00A0 ADCC\u00C2\u00A0=\u00C2\u00A0[1\u00C2\u00A0\u00E2\u0088\u0092\u00C2\u00A0(fraction\u00C2\u00A0 of\u00C2\u00A0 viable\u00C2\u00A0 double\u00E2\u0080\u0090stained\u00C2\u00A0 target\u00C2\u00A0 cells)\u00C2\u00A0 /\u00C2\u00A0 (fraction\u00C2\u00A0of\u00C2\u00A0double\u00E2\u0080\u0090stained\u00C2\u00A0untreated\u00C2\u00A0control\u00C2\u00A0cells)]\u00C2\u00A0\u00C3\u0097\u00C2\u00A0100%.\u00C2\u00A0\u00C2\u00A0 2.3.9\u00C2\u00A0 Quantification\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0natural\u00C2\u00A0killer\u00C2\u00A0cells\u00C2\u00A0 Fresh\u00C2\u00A0 peripheral\u00C2\u00A0 blood\u00C2\u00A0 mononuclear\u00C2\u00A0 cells\u00C2\u00A0 (PBMCs;\u00C2\u00A0 effector\u00C2\u00A0 cells)\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 volunteers\u00C2\u00A0who\u00C2\u00A0provided\u00C2\u00A0 informed\u00C2\u00A0consent.\u00C2\u00A0 \u00C2\u00A0PBMCs\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Lympholyte\u00E2\u0080\u0090Mammal,\u00C2\u00A0then\u00C2\u00A0 incubated\u00C2\u00A0with\u00C2\u00A0Z138\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A01:1\u00C2\u00A0ratio,\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0final\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A01\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0effector\u00C2\u00A0cells/mL\u00C2\u00A0 and\u00C2\u00A01\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0target\u00C2\u00A0cells/mL.\u00C2\u00A0\u00C2\u00A0Cultures\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0either\u00C2\u00A0as:\u00C2\u00A0free\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 a\u00C2\u00A0mixture\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0unconjugated\u00C2\u00A0liposomes,\u00C2\u00A0or\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0(negative\u00C2\u00A0control).\u00C2\u00A0\u00C2\u00A0Treated\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 for\u00C2\u00A0 20\u00C2\u00A0 h\u00C2\u00A0 at\u00C2\u00A0 37\u00C2\u00A0 \u00EF\u0082\u00B0C\u00C2\u00A0 in\u00C2\u00A0 RPMI\u00C2\u00A0 1640\u00C2\u00A0 complete\u00C2\u00A0 medium\u00C2\u00A0 supplemented\u00C2\u00A0 with\u00C2\u00A0 50\u00C2\u00A0 \u00C2\u00B5M\u00EF\u0080\u00A0\u00EF\u0081\u00A2\u00E2\u0080\u0090 mercaptoethanol.\u00C2\u00A0\u00C2\u00A0Several\u00C2\u00A0control\u00C2\u00A0samples\u00C2\u00A0containing\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0only\u00C2\u00A0(without\u00C2\u00A0target\u00C2\u00A0cells)\u00C2\u00A0were\u00C2\u00A0 also\u00C2\u00A0included.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0incubation,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0and\u00C2\u00A0labeled\u00C2\u00A0with\u00C2\u00A0anti\u00E2\u0080\u0090human\u00C2\u00A0CD3\u00E2\u0080\u0090FITC,\u00C2\u00A0CD56\u00E2\u0080\u0090PE\u00C2\u00A0 and\u00C2\u00A0CD54\u00E2\u0080\u0090APC\u00C2\u00A0Abs\u00C2\u00A0 for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0 \u00C2\u00B0C\u00C2\u00A0before\u00C2\u00A0analysis\u00C2\u00A0by\u00C2\u00A0 flow\u00C2\u00A0cytometry.\u00C2\u00A0 \u00C2\u00A0Natural\u00C2\u00A0killer\u00C2\u00A0 (NK)\u00C2\u00A0cells\u00C2\u00A0 were\u00C2\u00A0identified\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0CD3\u00E2\u0080\u0093/CD56+\u00C2\u00A0population,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0CD54bright\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0 within\u00C2\u00A0this\u00C2\u00A0population.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A050,000\u00C2\u00A0events\u00C2\u00A0were\u00C2\u00A0collected\u00C2\u00A0per\u00C2\u00A0sample.\u00C2\u00A0\u00C2\u00A0 2.3.10\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0vivo\u00C2\u00A0xenograft\u00C2\u00A0models\u00C2\u00A0for\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0efficacy\u00C2\u00A0studies\u00C2\u00A0 For\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 efficacy\u00C2\u00A0 studies,\u00C2\u00A0 female\u00C2\u00A0C.B\u00E2\u0080\u009017\u00C2\u00A0mice\u00C2\u00A0with\u00C2\u00A0 severe\u00C2\u00A0 combined\u00C2\u00A0 immunodeficiency\u00C2\u00A0 (SCID)\u00C2\u00A0weighing\u00C2\u00A020\u00E2\u0080\u009025\u00C2\u00A0g\u00C2\u00A0were\u00C2\u00A0 inoculated\u00C2\u00A0 intravenously\u00C2\u00A0(IV)\u00C2\u00A0with\u00C2\u00A05\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0Z138\u00E2\u0080\u0090Luc\u00C2\u00A0cells\u00C2\u00A0(see\u00C2\u00A0Section\u00C2\u00A0 2.3.1).\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0day\u00C2\u00A07\u00C2\u00A0after\u00C2\u00A0cell\u00C2\u00A0 inoculation,\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0tumors\u00C2\u00A0was\u00C2\u00A0assessed\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0 IVIS\u00C2\u00A0optical\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A032\u00C2\u00A0\u00C2\u00A0 imaging\u00C2\u00A0 system\u00C2\u00A0 200\u00C2\u00A0 Series\u00C2\u00A0 (Xenogen)\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0 10\u00C2\u00A0min\u00C2\u00A0 before\u00C2\u00A0 imaging,\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0 injected\u00C2\u00A0 IP\u00C2\u00A0with\u00C2\u00A0200\u00C2\u00A0\u00C2\u00B5l\u00C2\u00A0of\u00C2\u00A015\u00C2\u00A0mg/mL\u00C2\u00A0 firefly\u00C2\u00A0D\u00E2\u0080\u0090luciferin\u00C2\u00A0potassium\u00C2\u00A0salt,\u00C2\u00A0which\u00C2\u00A0emits\u00C2\u00A0photons\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 oxygen,\u00C2\u00A0 adenosine\u00C2\u00A0 triphosphate,\u00C2\u00A0 and\u00C2\u00A0 Luc.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 animals\u00C2\u00A0were\u00C2\u00A0 imaged\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0 photon\u00C2\u00A0 counts\u00C2\u00A0 from\u00C2\u00A0 captured\u00C2\u00A0 images\u00C2\u00A0were\u00C2\u00A0 quantified\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 Living\u00C2\u00A0 Image\u00C2\u00A0 software\u00C2\u00A0 package\u00C2\u00A0 (version\u00C2\u00A0 2.50;\u00C2\u00A0 Xenogen).\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 photograph\u00C2\u00A0 was\u00C2\u00A0 first\u00C2\u00A0 obtained\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 imaging\u00C2\u00A0 chamber\u00C2\u00A0 under\u00C2\u00A0 dim\u00C2\u00A0 illumination,\u00C2\u00A0 then\u00C2\u00A0 a\u00C2\u00A0 luminescence\u00C2\u00A0 image\u00C2\u00A0was\u00C2\u00A0 acquired.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0overlay\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0pseudocolour\u00C2\u00A0 images\u00C2\u00A0 represents\u00C2\u00A0the\u00C2\u00A0spatial\u00C2\u00A0distribution\u00C2\u00A0of\u00C2\u00A0photon\u00C2\u00A0counts\u00C2\u00A0produced\u00C2\u00A0by\u00C2\u00A0active\u00C2\u00A0Luc,\u00C2\u00A0with\u00C2\u00A0red\u00C2\u00A0representing\u00C2\u00A0 the\u00C2\u00A0most\u00C2\u00A0 intense\u00C2\u00A0 (saturated)\u00C2\u00A0 luminescence\u00C2\u00A0and\u00C2\u00A0blue\u00C2\u00A0 representing\u00C2\u00A0 the\u00C2\u00A0 least\u00C2\u00A0 intense\u00C2\u00A0 luminescence.\u00C2\u00A0\u00C2\u00A0 Captured\u00C2\u00A0 images\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0quantified\u00C2\u00A0using\u00C2\u00A0Living\u00C2\u00A0Image.\u00C2\u00A0Bioluminescent\u00C2\u00A0signals\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0regions\u00C2\u00A0 of\u00C2\u00A0 interest\u00C2\u00A0 (ROI)\u00C2\u00A0 expressed\u00C2\u00A0 in\u00C2\u00A0 pseudocolour\u00C2\u00A0 are\u00C2\u00A0 presented\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 cumulative\u00C2\u00A0 photon\u00C2\u00A0 counts\u00C2\u00A0 collected\u00C2\u00A0within\u00C2\u00A0each\u00C2\u00A0ROI.\u00C2\u00A0\u00C2\u00A0Uninfected\u00C2\u00A0animals\u00C2\u00A0were\u00C2\u00A0imaged\u00C2\u00A0to\u00C2\u00A0detect\u00C2\u00A0background\u00C2\u00A0signals.\u00C2\u00A0\u00C2\u00A0Starting\u00C2\u00A0 on\u00C2\u00A0 day\u00C2\u00A0 7\u00C2\u00A0 after\u00C2\u00A0 Z138\u00C2\u00A0 cell\u00C2\u00A0 inoculation,\u00C2\u00A0 treatments\u00C2\u00A0 consisting\u00C2\u00A0 of\u00C2\u00A0 either\u00C2\u00A0 free\u00C2\u00A0 Rtx\u00C2\u00A0 or\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 were\u00C2\u00A0 administered\u00C2\u00A0Q3D\u00C2\u00A0\u00C3\u0097\u00C2\u00A06.\u00C2\u00A0\u00C2\u00A0Tumor\u00C2\u00A0measurements\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0IVIS\u00C2\u00A0optical\u00C2\u00A0imaging\u00C2\u00A0system\u00C2\u00A0were\u00C2\u00A0made\u00C2\u00A0twice\u00C2\u00A0 (early\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0study)\u00C2\u00A0or\u00C2\u00A0once\u00C2\u00A0per\u00C2\u00A0week.\u00C2\u00A0 \u00C2\u00A0Mice\u00C2\u00A0were\u00C2\u00A0monitored\u00C2\u00A0daily\u00C2\u00A0for\u00C2\u00A0extended\u00C2\u00A0time\u00C2\u00A0frames\u00C2\u00A0using\u00C2\u00A0 both\u00C2\u00A0qualitative\u00C2\u00A0and\u00C2\u00A0quantitative\u00C2\u00A0health\u00C2\u00A0status\u00C2\u00A0indicators.\u00C2\u00A0\u00C2\u00A0When\u00C2\u00A0the\u00C2\u00A0health\u00C2\u00A0status\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0animal\u00C2\u00A0was\u00C2\u00A0 considered\u00C2\u00A0poor\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0predefined\u00C2\u00A0 scale,\u00C2\u00A0 the\u00C2\u00A0animals\u00C2\u00A0were\u00C2\u00A0killed\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 following\u00C2\u00A0day\u00C2\u00A0was\u00C2\u00A0 recorded\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0time\u00C2\u00A0of\u00C2\u00A0death\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0generate\u00C2\u00A0Kaplan\u00E2\u0080\u0090Meier\u00C2\u00A0survival\u00C2\u00A0curves.\u00C2\u00A0 2.3.11\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0vivo\u00C2\u00A0studies\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 For\u00C2\u00A0studies\u00C2\u00A0of\u00C2\u00A0liposome\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0clearance\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0plasma,\u00C2\u00A0BALB/c\u00C2\u00A0mice\u00C2\u00A0(17\u00E2\u0080\u009019\u00C2\u00A0g)\u00C2\u00A0and\u00C2\u00A0C.B\u00E2\u0080\u0090 17\u00C2\u00A0SCID\u00C2\u00A0mice\u00C2\u00A0(19\u00E2\u0080\u009022\u00C2\u00A0g)\u00C2\u00A0were\u00C2\u00A0injected\u00C2\u00A0IV\u00C2\u00A0with\u00C2\u00A05\u00C2\u00A0mg/kg\u00C2\u00A0(in\u00C2\u00A0200\u00C2\u00A0\u00C2\u00B5L)\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0various\u00C2\u00A0time\u00C2\u00A0 points\u00C2\u00A0 post\u00E2\u0080\u0090injection,\u00C2\u00A0 blood\u00C2\u00A0 was\u00C2\u00A0 collected\u00C2\u00A0 by\u00C2\u00A0 cardiac\u00C2\u00A0 puncture\u00C2\u00A0 and\u00C2\u00A0 placed\u00C2\u00A0 into\u00C2\u00A0 EDTA\u00E2\u0080\u0090coated\u00C2\u00A0 microtainer\u00C2\u00A0tubes.\u00C2\u00A0\u00C2\u00A0Plasma\u00C2\u00A0was\u00C2\u00A0isolated\u00C2\u00A0from\u00C2\u00A0blood\u00C2\u00A0samples\u00C2\u00A0by\u00C2\u00A0centrifugation\u00C2\u00A0at\u00C2\u00A01000g\u00C2\u00A0for\u00C2\u00A015\u00C2\u00A0min.\u00C2\u00A0\u00C2\u00A0 Aliquots\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 were\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 Rtx\u00C2\u00A0 levels\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 colorimetric\u00C2\u00A0 enzyme\u00E2\u0080\u0090linked\u00C2\u00A0 immunosorbent\u00C2\u00A0assay\u00C2\u00A0(ELISA).\u00C2\u00A0\u00C2\u00A0Briefly,\u00C2\u00A0a\u00C2\u00A0rabbit\u00C2\u00A0anti\u00E2\u0080\u0090human\u00C2\u00A0IgG\u00C2\u00A0Fc\u00C2\u00A0fragment\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0coating\u00C2\u00A0and\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A033\u00C2\u00A0\u00C2\u00A0 capturing\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 horseradish\u00C2\u00A0 peroxidase\u00E2\u0080\u0090conjugated\u00C2\u00A0 rabbit\u00C2\u00A0 anti\u00E2\u0080\u0090human\u00C2\u00A0whole\u00C2\u00A0 IgG\u00C2\u00A0was\u00C2\u00A0 used\u00C2\u00A0 for\u00C2\u00A0 detection,\u00C2\u00A0 with\u00C2\u00A0 orthophenylenediamine\u00C2\u00A0 added\u00C2\u00A0 as\u00C2\u00A0 substrate.\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 determine\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 in\u00C2\u00A0 plasma,\u00C2\u00A0 absorbance\u00C2\u00A0 at\u00C2\u00A0 405\u00C2\u00A0 nm\u00C2\u00A0 was\u00C2\u00A0measured\u00C2\u00A0 and\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 values\u00C2\u00A0 from\u00C2\u00A0 a\u00C2\u00A0 standard\u00C2\u00A0 curve\u00C2\u00A0 constructed\u00C2\u00A0 from\u00C2\u00A0 known\u00C2\u00A0 amounts\u00C2\u00A0 of\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0 liposomal\u00C2\u00A0 lipid\u00C2\u00A0 concentrations,\u00C2\u00A0 aliquots\u00C2\u00A0 of\u00C2\u00A0 plasma\u00C2\u00A0 were\u00C2\u00A0counted\u00C2\u00A0in\u00C2\u00A05\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0Pico\u00E2\u0080\u0090Fluor\u00C2\u00A040\u00C2\u00A0scintillation\u00C2\u00A0fluid.\u00C2\u00A0 2.4\u00C2\u00A0 Results\u00C2\u00A0 2.4.1\u00C2\u00A0 Characterization\u00C2\u00A0of\u00C2\u00A0Z138\u00C2\u00A0 and\u00C2\u00A0 JVM2\u00C2\u00A0 cell\u00C2\u00A0 lines:\u00C2\u00A0 surface\u00C2\u00A0protein\u00C2\u00A0 expression\u00C2\u00A0 levels\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0treatment\u00C2\u00A0 Two\u00C2\u00A0 human\u00C2\u00A0 MCL\u00C2\u00A0 cell\u00C2\u00A0 lines,\u00C2\u00A0 JVM2\u00C2\u00A0 and\u00C2\u00A0 Z138,\u00C2\u00A0 were\u00C2\u00A0 used\u00C2\u00A0 in\u00C2\u00A0 our\u00C2\u00A0 studies\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 indirect\u00C2\u00A0 mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0were\u00C2\u00A0selected\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0previous\u00C2\u00A0studies\u00C2\u00A0 that\u00C2\u00A0characterized\u00C2\u00A0several\u00C2\u00A0MCL\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0their\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0classic\u00C2\u00A0features\u00C2\u00A0of\u00C2\u00A0MCL,\u00C2\u00A0 namely\u00C2\u00A0 the\u00C2\u00A0 t(11;14)(q13;q32)\u00C2\u00A0 translocation\u00C2\u00A0and\u00C2\u00A0overexpression\u00C2\u00A0of\u00C2\u00A0cyclin\u00C2\u00A0D1127.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0Z138\u00C2\u00A0 line\u00C2\u00A0was\u00C2\u00A0 found\u00C2\u00A0to\u00C2\u00A0possess\u00C2\u00A0both\u00C2\u00A0traits\u00C2\u00A0of\u00C2\u00A0classic\u00C2\u00A0MCL,\u00C2\u00A0and\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0JVM2\u00C2\u00A0cells\u00C2\u00A0retained\u00C2\u00A0the\u00C2\u00A0translocation,\u00C2\u00A0they\u00C2\u00A0 expressed\u00C2\u00A0cyclin\u00C2\u00A0D2\u00C2\u00A0and\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0overexpress\u00C2\u00A0cyclin\u00C2\u00A0D1,\u00C2\u00A0therefore\u00C2\u00A0representing\u00C2\u00A0a\u00C2\u00A0variant\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 disease132,\u00C2\u00A0133.\u00C2\u00A0 Given\u00C2\u00A0 these\u00C2\u00A0phenotypic\u00C2\u00A0differences,\u00C2\u00A0 the\u00C2\u00A0 two\u00C2\u00A0 cell\u00C2\u00A0 lines\u00C2\u00A0were\u00C2\u00A0 further\u00C2\u00A0 characterized\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0of\u00C2\u00A0 select\u00C2\u00A0cell\u00E2\u0080\u0090surface\u00C2\u00A0proteins\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0and\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0 molecules\u00C2\u00A0consist\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0CD20,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0proteins\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0regulate\u00C2\u00A0CDC\u00C2\u00A0 and\u00C2\u00A0ADCC.\u00C2\u00A0\u00C2\u00A0Figure\u00C2\u00A02.1A\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0while\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0similar\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0lines,\u00C2\u00A0the\u00C2\u00A0JVM2\u00C2\u00A0line\u00C2\u00A0 expresses\u00C2\u00A0 significantly\u00C2\u00A0 higher\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 (TNFR)\u00C2\u00A0 receptor\u00C2\u00A0 family\u00C2\u00A0 receptors\u00C2\u00A0 (CD95\u00C2\u00A0 and\u00C2\u00A0 CD40),\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 costimulatory\u00C2\u00A0molecules\u00C2\u00A0 CD80\u00C2\u00A0 and\u00C2\u00A0 CD86\u00C2\u00A0 which\u00C2\u00A0 are\u00C2\u00A0 necessary\u00C2\u00A0 for\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 activation\u00C2\u00A0 and\u00C2\u00A0 survival.\u00C2\u00A0 \u00C2\u00A0 NK\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 also\u00C2\u00A0 activated\u00C2\u00A0 by\u00C2\u00A0 CD40125,\u00C2\u00A0 CD80,\u00C2\u00A0 and\u00C2\u00A0 CD86126.\u00C2\u00A0\u00C2\u00A0Levels\u00C2\u00A0of\u00C2\u00A0CD55\u00C2\u00A0are\u00C2\u00A0elevated\u00C2\u00A0in\u00C2\u00A0Z138\u00C2\u00A0cells,\u00C2\u00A0whereas\u00C2\u00A0CD59\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0relatively\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0in\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A034\u00C2\u00A0\u00C2\u00A0 both\u00C2\u00A0 lines;\u00C2\u00A0 these\u00C2\u00A0 proteins\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 inhibit\u00C2\u00A0 Rtx\u00E2\u0080\u0090induced\u00C2\u00A0 CDC46,\u00C2\u00A0 120.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 intercellular\u00C2\u00A0 adhesion\u00C2\u00A0molecule\u00C2\u00A0CD54\u00C2\u00A0also\u00C2\u00A0shows\u00C2\u00A0similar\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0lines.\u00C2\u00A0\u00C2\u00A0Overall,\u00C2\u00A0the\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0 phenotype\u00C2\u00A0observed\u00C2\u00A0suggest\u00C2\u00A0 that\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0and\u00C2\u00A0CDC\u00C2\u00A0may\u00C2\u00A0vary\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0 two\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP46,\u00C2\u00A049.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A02.1\u00C2\u00A0\u00C2\u00A0Phenotypic\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0treatment.\u00C2\u00A0 (A)\u00C2\u00A0 Cell\u00E2\u0080\u0090surface\u00C2\u00A0 marker\u00C2\u00A0 expression\u00C2\u00A0 on\u00C2\u00A0 JVM2\u00C2\u00A0 and\u00C2\u00A0 Z138\u00C2\u00A0 cells,\u00C2\u00A0 assessed\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0 cytometry.\u00C2\u00A0 \u00C2\u00A0 *p\u00C2\u00A0 <\u00C2\u00A0 0.05.\u00C2\u00A0 \u00C2\u00A0 MFI:\u00C2\u00A0 mean\u00C2\u00A0 fluorescence\u00C2\u00A0intensity.\u00C2\u00A0\u00C2\u00A0(B\u00C2\u00A0&\u00C2\u00A0C)\u00C2\u00A0To\u00C2\u00A0assess\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0responses\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A05\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0JVM2\u00C2\u00A0(B)\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0(C)\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 injected\u00C2\u00A0SC\u00C2\u00A0 into\u00C2\u00A0Rag\u00E2\u0080\u00902M\u00C2\u00A0mice.\u00C2\u00A0 \u00C2\u00A0Once\u00C2\u00A0 the\u00C2\u00A0 tumors\u00C2\u00A0 reached\u00C2\u00A0~200\u00C2\u00A0mm3,\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0 twice\u00C2\u00A0weekly\u00C2\u00A0 for\u00C2\u00A03\u00C2\u00A0weeks\u00C2\u00A0with\u00C2\u00A0 either\u00C2\u00A02.5\u00C2\u00A0mg/kg\u00C2\u00A0(\u00EF\u0081\u00AE)\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg/kg\u00C2\u00A0(\u00EF\u0081\u00B0)\u00C2\u00A0Rtx.\u00C2\u00A0Control\u00C2\u00A0mice\u00C2\u00A0(\u00EF\u0081\u00B5)\u00C2\u00A0were\u00C2\u00A0injected\u00C2\u00A0with\u00C2\u00A0PBS.\u00C2\u00A0\u00C2\u00A0Treatments\u00C2\u00A0are\u00C2\u00A0indicated\u00C2\u00A0by\u00C2\u00A0arrows.\u00C2\u00A0\u00C2\u00A0 Data\u00C2\u00A0correspond\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0average\u00C2\u00A0metastasis\u00C2\u00A0from\u00C2\u00A06\u00C2\u00A0mice\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SD\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0separate\u00C2\u00A0 experiments.\u00C2\u00A0 0 0.3 0.6 0.9 1.2 25 30 35 40 45 50 Tu m or vo lu m e\u00C2\u00A0/ \u00C2\u00A0m m 3 Days\u00C2\u00A0postinoculation JVM2B 0 0.3 0.6 0.9 1.2 10 20 30 40 50 Tu m or vo lu m e\u00C2\u00A0/ \u00C2\u00A0m m 3 Days\u00C2\u00A0postinoculation Z138C \u00E2\u0080\u00A2\u00C2\u00A035\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0responses\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0Rag\u00E2\u0080\u00902M\u00C2\u00A0mice\u00C2\u00A0inoculated\u00C2\u00A0subcutaneously\u00C2\u00A0(SC)\u00C2\u00A0with\u00C2\u00A0JVM2\u00C2\u00A0 and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0determined.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A02.1B,\u00C2\u00A0JVM2\u00C2\u00A0tumors\u00C2\u00A0 in\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0 Rtx\u00C2\u00A0 exhibited\u00C2\u00A0 an\u00C2\u00A0 initial\u00C2\u00A0 growth\u00C2\u00A0 delay\u00C2\u00A0 (after\u00C2\u00A0 day\u00C2\u00A0 35\u00C2\u00A0 postinoculation),\u00C2\u00A0 but\u00C2\u00A0 eventually\u00C2\u00A0 the\u00C2\u00A0 tumors\u00C2\u00A0 progressed\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0manner\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0comparable\u00C2\u00A0to\u00C2\u00A0controls.\u00C2\u00A0\u00C2\u00A0On\u00C2\u00A0day\u00C2\u00A045\u00C2\u00A0after\u00C2\u00A0JVM2\u00C2\u00A0cell\u00C2\u00A0inoculation,\u00C2\u00A0the\u00C2\u00A0 control\u00C2\u00A0animals\u00C2\u00A0possessed\u00C2\u00A0tumors\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0approximately\u00C2\u00A0600\u00C2\u00A0mg\u00C2\u00A0in\u00C2\u00A0size.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0animals\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0 2.5\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg/kg\u00C2\u00A0Rtx\u00C2\u00A0exhibited\u00C2\u00A0 slightly\u00C2\u00A0 smaller\u00C2\u00A0 tumors,\u00C2\u00A0albeit\u00C2\u00A0 the\u00C2\u00A0differences\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0 significant.\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A02.1C\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0mice\u00C2\u00A0bearing\u00C2\u00A0established\u00C2\u00A0Z138\u00C2\u00A0tumors\u00C2\u00A0were\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0 Control\u00C2\u00A0animals\u00C2\u00A0exhibited\u00C2\u00A0 tumors\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0approximately\u00C2\u00A0500\u00C2\u00A0mg\u00C2\u00A0 in\u00C2\u00A0 size\u00C2\u00A040\u00C2\u00A0days\u00C2\u00A0after\u00C2\u00A0Z138\u00C2\u00A0cell\u00C2\u00A0 inoculation,\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0tumors\u00C2\u00A0in\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0(2.5\u00C2\u00A0or\u00C2\u00A010\u00C2\u00A0mg/kg)\u00C2\u00A0were\u00C2\u00A050%\u00C2\u00A0smaller\u00C2\u00A0then\u00C2\u00A0the\u00C2\u00A0 tumors\u00C2\u00A0measured\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0 initiation\u00C2\u00A0 of\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 Based\u00C2\u00A0 on\u00C2\u00A0 these\u00C2\u00A0 data,\u00C2\u00A0 the\u00C2\u00A0 JVM2\u00C2\u00A0 tumors\u00C2\u00A0were\u00C2\u00A0 considered\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0Rtx\u00E2\u0080\u0090insensitive\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0Z138\u00C2\u00A0tumors\u00C2\u00A0were\u00C2\u00A0considered\u00C2\u00A0Rtx\u00E2\u0080\u0090sensitive.\u00C2\u00A0 2.4.2\u00C2\u00A0 Rtx\u00C2\u00ADLNPs\u00C2\u00A0bind\u00C2\u00A0specifically\u00C2\u00A0to\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0 Multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0were\u00C2\u00A0 prepared\u00C2\u00A0 from\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 2.3.2.\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 examine\u00C2\u00A0 the\u00C2\u00A0behavior\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0constructs\u00C2\u00A0when\u00C2\u00A0 tested\u00C2\u00A0against\u00C2\u00A0 the\u00C2\u00A0Z138\u00C2\u00A0and\u00C2\u00A0 JVM2\u00C2\u00A0cell\u00C2\u00A0 lines,\u00C2\u00A0cells\u00C2\u00A0 were\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 bound\u00C2\u00A0 Rtx\u00C2\u00A0 were\u00C2\u00A0 determined\u00C2\u00A0 using\u00C2\u00A0 flow\u00C2\u00A0 cytometry.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0results,\u00C2\u00A0summarized\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A02.2A\u00C2\u00A0&\u00C2\u00A0B,\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A04.9\u00C2\u00A0and\u00C2\u00A03.2\u00C2\u00A0 times\u00C2\u00A0more\u00C2\u00A0Rtx\u00C2\u00A0 became\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells,\u00C2\u00A0respectively,\u00C2\u00A0when\u00C2\u00A0added\u00C2\u00A0as\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0compared\u00C2\u00A0 with\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0\u00C2\u00B5g/mL.\u00C2\u00A0\u00C2\u00A0Comparable\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0binding\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0observed\u00C2\u00A0at\u00C2\u00A0lower\u00C2\u00A0 Rtx\u00C2\u00A0doses,\u00C2\u00A0and\u00C2\u00A0 these\u00C2\u00A0data\u00C2\u00A0agree\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 general\u00C2\u00A0observation\u00C2\u00A0 that\u00C2\u00A0multivalent\u00C2\u00A0 constructs\u00C2\u00A0exhibit\u00C2\u00A0 increased\u00C2\u00A0functional\u00C2\u00A0affinity\u00C2\u00A0and\u00C2\u00A0decreased\u00C2\u00A0dissociation\u00C2\u00A0rates\u00C2\u00A0when\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0cell\u00E2\u0080\u0090surface\u00C2\u00A0antigens97.\u00C2\u00A0\u00C2\u00A0 Surprisingly,\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0had\u00C2\u00A0similar\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0 (Figure\u00C2\u00A02.1A),\u00C2\u00A0 JVM2\u00C2\u00A0 cells\u00C2\u00A0had\u00C2\u00A0 the\u00C2\u00A0ability\u00C2\u00A0 to\u00C2\u00A0bind\u00C2\u00A0more\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A025\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0and\u00C2\u00A0above,\u00C2\u00A0 the\u00C2\u00A0mean\u00C2\u00A0 fluorescence\u00C2\u00A0intensities\u00C2\u00A0for\u00C2\u00A0both\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.2A\u00C2\u00A0are\u00C2\u00A0between\u00C2\u00A01.8\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A02.5\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0 than\u00C2\u00A0 those\u00C2\u00A0 of\u00C2\u00A0 Figure\u00C2\u00A0 2.2B\u00C2\u00A0 (p\u00C2\u00A0 <\u00C2\u00A0 0.05\u00C2\u00A0 for\u00C2\u00A0 all\u00C2\u00A0 equivalent\u00C2\u00A0 treatments).\u00C2\u00A0 \u00C2\u00A0 Using\u00C2\u00A0 non\u00E2\u0080\u0090exchangeable\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A036\u00C2\u00A0\u00C2\u00A0 [3H]cholesteryl\u00C2\u00A0hexadecyl\u00C2\u00A0ether\u00C2\u00A0([3H]CHE)\u00C2\u00A0 incorporated\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0liposomes\u00C2\u00A0as\u00C2\u00A0an\u00C2\u00A0alternative\u00C2\u00A0means\u00C2\u00A0to\u00C2\u00A0 assess\u00C2\u00A0cell\u00C2\u00A0binding,\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0lipid\u00C2\u00A0following\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0approximately\u00C2\u00A0two\u00E2\u0080\u0090 fold\u00C2\u00A0greater\u00C2\u00A0for\u00C2\u00A0JVM2\u00C2\u00A0cells\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05;\u00C2\u00A0data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0JVM2\u00E2\u0080\u0090derived\u00C2\u00A0tumors\u00C2\u00A0 were\u00C2\u00A0less\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0treatment\u00C2\u00A0although\u00C2\u00A0they\u00C2\u00A0bound\u00C2\u00A0more\u00C2\u00A0Ab\u00C2\u00A0than\u00C2\u00A0Z138\u00C2\u00A0cells,\u00C2\u00A0again\u00C2\u00A0suggesting\u00C2\u00A0 that\u00C2\u00A0the\u00C2\u00A0JVM2\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0resistant\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0therapy.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A02.2\u00C2\u00A0\u00C2\u00A0Binding\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 (A\u00C2\u00A0&\u00C2\u00A0B)\u00C2\u00A0Rtx\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0JVM2\u00C2\u00A0(A)\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0(B)\u00C2\u00A0MCL\u00C2\u00A0cells\u00C2\u00A0when\u00C2\u00A0incubated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(\u00EF\u0081\u00AE),\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0(\u00EF\u0081\u00B0),\u00C2\u00A0or\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0(\u00C3\u0097),\u00C2\u00A0as\u00C2\u00A0 assessed\u00C2\u00A0 by\u00C2\u00A0 flow\u00C2\u00A0 cytometry.\u00C2\u00A0 \u00C2\u00A0 Error\u00C2\u00A0 bars\u00C2\u00A0 are\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0 size\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0 points.\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0 are\u00C2\u00A0 the\u00C2\u00A0means\u00C2\u00A0 of\u00C2\u00A0 experiments\u00C2\u00A0 performed\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 \u00C2\u00A0 MFI:\u00C2\u00A0 mean\u00C2\u00A0 fluorescence\u00C2\u00A0 intensity.\u00C2\u00A0 (C\u00C2\u00A0 &\u00C2\u00A0 D)\u00C2\u00A0 Fraction\u00C2\u00A0 of\u00C2\u00A0 viable\u00C2\u00A0 cells\u00C2\u00A0 remaining\u00C2\u00A0 after\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 0 100 200 300 400 0 25 50 75 100 M FI [Rtx]\u00C2\u00A0\u00C2\u00A0/\u00C2\u00A0\u00C2\u00A0\u00CE\u00BCg/mL JVM2A 0 100 200 300 400 0 25 50 75 100 M FI [Rtx]\u00C2\u00A0\u00C2\u00A0/\u00C2\u00A0\u00C2\u00A0\u00CE\u00BCg/mL Z138B \u00E2\u0080\u00A2\u00C2\u00A037\u00C2\u00A0\u00C2\u00A0 treatment\u00C2\u00A0of\u00C2\u00A0JVM2\u00C2\u00A0(C)\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0(D)\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC\u00C2\u00A0for\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A0as\u00C2\u00A0assessed\u00C2\u00A0by\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0after\u00C2\u00A0Annexin\u00E2\u0080\u0090 V\u00E2\u0080\u0090FITC\u00C2\u00A0and\u00C2\u00A0PI\u00C2\u00A0staining.\u00C2\u00A0\u00C2\u00A0Double\u00E2\u0080\u0090negatively\u00C2\u00A0stained\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0considered\u00C2\u00A0viable.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0different\u00C2\u00A0treatments\u00C2\u00A0are\u00C2\u00A0indicated\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0legend.\u00C2\u00A0\u00C2\u00A0Data\u00C2\u00A0correspond\u00C2\u00A0to\u00C2\u00A0means\u00C2\u00A0of\u00C2\u00A0triplicates,\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0separate\u00C2\u00A0experiments.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0responses\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0 assessed.\u00C2\u00A0\u00C2\u00A0Previous\u00C2\u00A0work\u00C2\u00A0has\u00C2\u00A0demonstrated\u00C2\u00A0that\u00C2\u00A0Rtx,\u00C2\u00A0on\u00C2\u00A0its\u00C2\u00A0own,\u00C2\u00A0has\u00C2\u00A0little\u00C2\u00A0therapeutic\u00C2\u00A0effect\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0 unless\u00C2\u00A0a\u00C2\u00A0crosslinking\u00C2\u00A0Ab\u00C2\u00A0 is\u00C2\u00A0added7,\u00C2\u00A074\u00E2\u0080\u009076.\u00C2\u00A0 \u00C2\u00A0Using\u00C2\u00A0 the\u00C2\u00A0AlamarBlue\u00C2\u00A0assay,\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0previously\u00C2\u00A0observed\u00C2\u00A0 that\u00C2\u00A0 both\u00C2\u00A0 crosslinked\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 decrease\u00C2\u00A0 Z138\u00C2\u00A0 cell\u00C2\u00A0 proliferation\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 to\u00C2\u00A0 an\u00C2\u00A0 equivalent\u00C2\u00A0 extent7.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 this\u00C2\u00A0 study,\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0 was\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 measure\u00C2\u00A0 the\u00C2\u00A0 fraction\u00C2\u00A0 of\u00C2\u00A0 viable\u00C2\u00A0 (nonapoptotic)\u00C2\u00A0 JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0 remaining\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 free\u00C2\u00A0Rtx,\u00C2\u00A0crosslinked\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0The\u00C2\u00A0results,\u00C2\u00A0summarized\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A02.2C\u00C2\u00A0&\u00C2\u00A0D,\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0when\u00C2\u00A0using\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00C2\u00A0and\u00C2\u00A0PI\u00C2\u00A0to\u00C2\u00A0 identify\u00C2\u00A0nonviable\u00C2\u00A0 cells,\u00C2\u00A0 free\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0 the\u00C2\u00A0 least\u00C2\u00A0 effective\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 treatments\u00C2\u00A0 (dark\u00C2\u00A0 gray\u00C2\u00A0 bars),\u00C2\u00A0with\u00C2\u00A0 minimum\u00C2\u00A0cell\u00C2\u00A0viabilities\u00C2\u00A0of\u00C2\u00A080%\u00C2\u00A0in\u00C2\u00A0JVM2\u00C2\u00A0cells\u00C2\u00A0(at\u00C2\u00A01.5\u00C2\u00A0\u00C2\u00B5g/mL)\u00C2\u00A0and\u00C2\u00A069%\u00C2\u00A0in\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0(0.15\u00C2\u00A0\u00C2\u00B5g/mL).\u00C2\u00A0The\u00C2\u00A0 percentages\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0untreated\u00C2\u00A0control\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0cases.\u00C2\u00A0\u00C2\u00A0 Compared\u00C2\u00A0to\u00C2\u00A0free\u00C2\u00A0Rtx,\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0engenders\u00C2\u00A0significant\u00C2\u00A0therapeutic\u00C2\u00A0effects\u00C2\u00A0 on\u00C2\u00A0both\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0a\u00C2\u00A0treatment\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A015\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A042%\u00C2\u00A0and\u00C2\u00A062%\u00C2\u00A0of\u00C2\u00A0JVM2\u00C2\u00A0 and\u00C2\u00A0Z138\u00C2\u00A0cells,\u00C2\u00A0 respectively,\u00C2\u00A0were\u00C2\u00A0considered\u00C2\u00A0non\u00E2\u0080\u0090viable\u00C2\u00A0 (Figure\u00C2\u00A02.2C\u00C2\u00A0&\u00C2\u00A0D).\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0 results\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 Z138\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0more\u00C2\u00A0 sensitive\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 than\u00C2\u00A0 JVM2\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 vitro.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 populations\u00C2\u00A0after\u00C2\u00A0Rtx\u00C2\u00A0treatment\u00C2\u00A0were\u00C2\u00A0equal\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0or\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0bare\u00C2\u00A0uncoupled\u00C2\u00A0liposomes\u00C2\u00A0 (hatched\u00C2\u00A0 bars),\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 enhanced\u00C2\u00A0 effects\u00C2\u00A0 observed\u00C2\u00A0 for\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 result\u00C2\u00A0 from\u00C2\u00A0 Rtx\u00C2\u00A0 being\u00C2\u00A0 bound\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0LNPs.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0an\u00C2\u00A0additional\u00C2\u00A0control,\u00C2\u00A0LNPs\u00C2\u00A0formulated\u00C2\u00A0with\u00C2\u00A0Trz\u00C2\u00A0(an\u00C2\u00A0anti\u00E2\u0080\u0090ErbB2\u00C2\u00A0therapeutic\u00C2\u00A0 monoclonal\u00C2\u00A0 Ab)\u00C2\u00A0 instead\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 showed\u00C2\u00A0 minimal\u00C2\u00A0 activity\u00C2\u00A0 (light\u00C2\u00A0 gray\u00C2\u00A0 bars),\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 therapeutic\u00C2\u00A0effects\u00C2\u00A0are\u00C2\u00A0specific\u00C2\u00A0to\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0were\u00C2\u00A0comparable\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05)\u00C2\u00A0to\u00C2\u00A02oAb\u00E2\u0080\u0090 crosslinked\u00C2\u00A0Rtx\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0used\u00C2\u00A0was\u00C2\u00A015\u00C2\u00A0\u00C2\u00B5g/mL.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A038\u00C2\u00A0\u00C2\u00A0 2.4.3\u00C2\u00A0 Multivalent\u00C2\u00A0 Rtx\u00C2\u00ADLNPs\u00C2\u00A0 elicit\u00C2\u00A0 superior\u00C2\u00A0 complement\u00C2\u00ADdependent\u00C2\u00A0 cytotoxicity\u00C2\u00A0 and\u00C2\u00A0Ab\u00C2\u00ADdependent\u00C2\u00A0cell\u00C2\u00ADmediated\u00C2\u00A0cytotoxicity\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 Taken\u00C2\u00A0together,\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0summarized\u00C2\u00A0above\u00C2\u00A0 indicate\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0exhibit\u00C2\u00A0 enhanced\u00C2\u00A0activity\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0when\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0effects\u00C2\u00A0achieved\u00C2\u00A0with\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0 these\u00C2\u00A0data\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0previous\u00C2\u00A0results7.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A0 2.2\u00C2\u00A0occurred\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0direct\u00C2\u00A0effects\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cells,\u00C2\u00A0yet\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0understood\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0 Rtx\u00C2\u00A0is\u00C2\u00A0largely\u00C2\u00A0dependent\u00C2\u00A0on\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0involving\u00C2\u00A0CDC\u00C2\u00A0and\u00C2\u00A0ADCC115\u00E2\u0080\u0090119,\u00C2\u00A0121,\u00C2\u00A0123.\u00C2\u00A0\u00C2\u00A0Thus\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0 important\u00C2\u00A0 to\u00C2\u00A0 assess\u00C2\u00A0how\u00C2\u00A0 the\u00C2\u00A0CDC\u00C2\u00A0 and\u00C2\u00A0ADCC\u00C2\u00A0 responses\u00C2\u00A0 are\u00C2\u00A0 affected\u00C2\u00A0when\u00C2\u00A0 tumor\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0 To\u00C2\u00A0measure\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CDC,\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0given\u00C2\u00A0equivalent\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP,\u00C2\u00A0then\u00C2\u00A0human\u00C2\u00A0serum\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0lysis\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0later\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0(see\u00C2\u00A0 Section\u00C2\u00A02.3.7).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0summarized\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.3A\u00C2\u00A0&\u00C2\u00A0B.\u00C2\u00A0\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0treatment\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0 more\u00C2\u00A0significant\u00C2\u00A0cell\u00C2\u00A0 lysis\u00C2\u00A0 than\u00C2\u00A0Rtx\u00C2\u00A0 treatment\u00C2\u00A0 in\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0 lines,\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 is\u00C2\u00A0better\u00C2\u00A0at\u00C2\u00A0 inducing\u00C2\u00A0CDC\u00C2\u00A0than\u00C2\u00A0free\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0When\u00C2\u00A0comparing\u00C2\u00A0results\u00C2\u00A0obtained\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0two\u00C2\u00A0cell\u00C2\u00A0lines,\u00C2\u00A0CDC\u00C2\u00A0activity\u00C2\u00A0was\u00C2\u00A0 higher\u00C2\u00A0in\u00C2\u00A0JVM2\u00C2\u00A0cells,\u00C2\u00A0where\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A01\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0induced\u00C2\u00A0a\u00C2\u00A03.4\u00E2\u0080\u0090fold\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0 CDC\u00C2\u00A0activity\u00C2\u00A0 (relative\u00C2\u00A0 to\u00C2\u00A0control),\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A02.0\u00E2\u0080\u0090fold\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0Z138\u00C2\u00A0cells.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0effect\u00C2\u00A0was\u00C2\u00A0 even\u00C2\u00A0more\u00C2\u00A0pronounced\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0dose\u00C2\u00A0 increased\u00C2\u00A0to\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g/mL,\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0values\u00C2\u00A0 increased\u00C2\u00A0to\u00C2\u00A05.3\u00E2\u0080\u0090 fold\u00C2\u00A0(JVM2)\u00C2\u00A0and\u00C2\u00A03.9\u00E2\u0080\u0090fold\u00C2\u00A0(Z138).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 level\u00C2\u00A0of\u00C2\u00A0CDC\u00C2\u00A0observed\u00C2\u00A0following\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0 comparable\u00C2\u00A0 to\u00C2\u00A0 controls,\u00C2\u00A0which\u00C2\u00A0 included\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0mixture\u00C2\u00A0 of\u00C2\u00A0 free\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 uncoupled\u00C2\u00A0 liposomes\u00C2\u00A0 (Rtx\u00C2\u00A0 +\u00C2\u00A0 lip)\u00C2\u00A0 (Figure\u00C2\u00A0 2.3A\u00C2\u00A0&\u00C2\u00A0 B).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 level\u00C2\u00A0 of\u00C2\u00A0 cell\u00C2\u00A0 lysis\u00C2\u00A0 obtained\u00C2\u00A0 for\u00C2\u00A0 Rtx\u00C2\u00A0 plus\u00C2\u00A0 crosslinking\u00C2\u00A0 secondary\u00C2\u00A0Ab\u00C2\u00A0(2oAb)\u00C2\u00A0was\u00C2\u00A0identical\u00C2\u00A0to\u00C2\u00A0that\u00C2\u00A0obtained\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0alone\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0 suggest\u00C2\u00A0that\u00C2\u00A0indirect\u00C2\u00A0CDC\u00E2\u0080\u0090based\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0activity\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0enhanced\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0when\u00C2\u00A0using\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP,\u00C2\u00A0even\u00C2\u00A0when\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0situations\u00C2\u00A0where\u00C2\u00A0Rtx\u00C2\u00A0crosslinking\u00C2\u00A0is\u00C2\u00A0achieved\u00C2\u00A0by\u00C2\u00A0an\u00C2\u00A0anti\u00E2\u0080\u0090Rtx\u00C2\u00A02oAb.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A039\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A02.3\u00C2\u00A0 \u00C2\u00A0In\u00C2\u00A0vitro\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090mediated\u00C2\u00A0CDC\u00C2\u00A0and\u00C2\u00A0ADCC\u00C2\u00A0are\u00C2\u00A0augmented\u00C2\u00A0when\u00C2\u00A0Rtx\u00C2\u00A0 is\u00C2\u00A0presented\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0as\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 (A\u00C2\u00A0&\u00C2\u00A0B)\u00C2\u00A0 For\u00C2\u00A0measuring\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CDC,\u00C2\u00A0 JVM2\u00C2\u00A0or\u00C2\u00A0 Z138\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0untreated\u00C2\u00A0 (U)\u00C2\u00A0or\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP,\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00C2\u00A0 +\u00C2\u00A0bare\u00C2\u00A0 liposomes\u00C2\u00A0(Rtx\u00C2\u00A0+\u00C2\u00A0lip),\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0All\u00C2\u00A0treatments\u00C2\u00A0consisted\u00C2\u00A0of\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0Ab\u00C2\u00A0except\u00C2\u00A0for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(lo),\u00C2\u00A0which\u00C2\u00A0consisted\u00C2\u00A0of\u00C2\u00A01\u00C2\u00A0\u00C2\u00B5g.\u00C2\u00A0\u00C2\u00A0(C\u00C2\u00A0&\u00C2\u00A0 D)\u00C2\u00A0For\u00C2\u00A0ADCC\u00C2\u00A0experiments,\u00C2\u00A0JVM2\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0 (\u00EF\u0081\u00B0),\u00C2\u00A0Rtx\u00C2\u00A0+\u00C2\u00A0bare\u00C2\u00A0liposomes\u00C2\u00A0(\u00E1\u0087\u009E),\u00C2\u00A0Rtx\u00C2\u00A0+\u00C2\u00A0crosslinking\u00C2\u00A02oAb\u00C2\u00A0(\u00EF\u0081\u00B5),\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0(\u00E2\u0097\u0087),\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(\u00EF\u0081\u00AE).\u00C2\u00A0\u00C2\u00A0Stimulated\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0(mouse\u00C2\u00A0 splenocytes)\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0added\u00C2\u00A0in\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0at\u00C2\u00A0effector\u00E2\u0080\u0090to\u00E2\u0080\u0090target\u00C2\u00A0cell\u00C2\u00A0ratios\u00C2\u00A0of\u00C2\u00A05:1,\u00C2\u00A050:1,\u00C2\u00A0and\u00C2\u00A0100:1.\u00C2\u00A0After\u00C2\u00A0a\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0 incubation,\u00C2\u00A0 ADCC\u00C2\u00A0 was\u00C2\u00A0 measured\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 2.3.8.\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0 represent\u00C2\u00A0 the\u00C2\u00A0 means\u00C2\u00A0 of\u00C2\u00A0 triplicates,\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 representative\u00C2\u00A0of\u00C2\u00A0three\u00C2\u00A0separate\u00C2\u00A0experiments.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0assess\u00C2\u00A0ADCC,\u00C2\u00A0a\u00C2\u00A0fluorometric\u00C2\u00A0method\u00C2\u00A0described\u00C2\u00A0by\u00C2\u00A0Gomez\u00E2\u0080\u0090Roman\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0was\u00C2\u00A0used131.\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0 were\u00C2\u00A0 first\u00C2\u00A0 dual\u00E2\u0080\u0090labeled\u00C2\u00A0 with\u00C2\u00A0 PKH26,\u00C2\u00A0 a\u00C2\u00A0 membrane\u00E2\u0080\u0090associating\u00C2\u00A0 lipid\u00C2\u00A0 dye,\u00C2\u00A0 and\u00C2\u00A0 carboxyfluorescein\u00C2\u00A0 diacetate\u00C2\u00A0succinimidyl\u00C2\u00A0ester\u00C2\u00A0(CFSE),\u00C2\u00A0which\u00C2\u00A0stains\u00C2\u00A0viable\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0incubated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 or\u00C2\u00A0other\u00C2\u00A0treatments\u00C2\u00A0for\u00C2\u00A030\u00C2\u00A0min,\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0mouse\u00C2\u00A0splenocytes\u00C2\u00A0(effector\u00C2\u00A0cells).\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0 24\u00C2\u00A0h,\u00C2\u00A0the\u00C2\u00A0JVM2\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0lysed\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0retained\u00C2\u00A0the\u00C2\u00A0PKH26\u00C2\u00A0signal\u00C2\u00A0 but\u00C2\u00A0lost\u00C2\u00A0that\u00C2\u00A0of\u00C2\u00A0CFSE.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0lysed\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0therefore\u00C2\u00A0determined\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A040\u00C2\u00A0\u00C2\u00A0 as\u00C2\u00A0the\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0CFSE\u00E2\u0080\u0090negative\u00C2\u00A0cells\u00C2\u00A0within\u00C2\u00A0the\u00C2\u00A0PKH26bright\u00C2\u00A0population.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0results,\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A0 2.3C\u00C2\u00A0&\u00C2\u00A0D,\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 free\u00C2\u00A0Rtx\u00C2\u00A0 (filled\u00C2\u00A0 triangles)\u00C2\u00A0elicits\u00C2\u00A0an\u00C2\u00A0ADCC\u00C2\u00A0 response\u00C2\u00A0at\u00C2\u00A0 levels\u00C2\u00A0comparable\u00C2\u00A0 to\u00C2\u00A0 those\u00C2\u00A0observed\u00C2\u00A0when\u00C2\u00A0using\u00C2\u00A0secondary\u00E2\u0080\u0090antibody\u00C2\u00A0crosslinked\u00C2\u00A0Rtx\u00C2\u00A0(filled\u00C2\u00A0diamonds)\u00C2\u00A0or\u00C2\u00A0a\u00C2\u00A0mixture\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 and\u00C2\u00A0uncoupled\u00C2\u00A0 liposomes\u00C2\u00A0 (open\u00C2\u00A0 triangles).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0extent\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0 resulting\u00C2\u00A0 from\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 treatment\u00C2\u00A0 (filled\u00C2\u00A0 squares)\u00C2\u00A0was\u00C2\u00A0 significantly\u00C2\u00A0enhanced\u00C2\u00A0 in\u00C2\u00A0both\u00C2\u00A0 cell\u00C2\u00A0 lines,\u00C2\u00A0but\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0particularly\u00C2\u00A0notable\u00C2\u00A0when\u00C2\u00A0 using\u00C2\u00A0Z138\u00C2\u00A0cells,\u00C2\u00A0where\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0100\u00E2\u0080\u0090fold\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0effector\u00C2\u00A0cells,\u00C2\u00A081%\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0undergoing\u00C2\u00A0ADCC\u00C2\u00A0 after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A026%\u00C2\u00A0for\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0at\u00C2\u00A0all\u00C2\u00A0target/effector\u00C2\u00A0cell\u00C2\u00A0ratios\u00C2\u00A0 tested).\u00C2\u00A0\u00C2\u00A0Overall\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0were\u00C2\u00A0significantly\u00C2\u00A0lower\u00C2\u00A0in\u00C2\u00A0JVM2\u00C2\u00A0cells,\u00C2\u00A0where\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0 effector\u00C2\u00A0 cells,\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 treatment\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 an\u00C2\u00A0 ADCC\u00C2\u00A0 response\u00C2\u00A0 of\u00C2\u00A0 14%,\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 5%\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0only\u00C2\u00A0at\u00C2\u00A0this\u00C2\u00A0target\u00C2\u00A0cell/effector\u00C2\u00A0cell\u00C2\u00A0ratio).\u00C2\u00A0 2.4.4\u00C2\u00A0 Rtx\u00C2\u00ADLNP\u00C2\u00A0induces\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0natural\u00C2\u00A0killer\u00C2\u00A0cells\u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0corroborate\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0elicits\u00C2\u00A0a\u00C2\u00A0superior\u00C2\u00A0ADCC\u00C2\u00A0response\u00C2\u00A0 compared\u00C2\u00A0with\u00C2\u00A0free\u00C2\u00A0Rtx,\u00C2\u00A0the\u00C2\u00A0extent\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0activated\u00C2\u00A0after\u00C2\u00A0exposure\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00C2\u00A0was\u00C2\u00A0determined.\u00C2\u00A0 \u00C2\u00A0Bowles\u00C2\u00A0and\u00C2\u00A0Weiner\u00C2\u00A0demonstrated\u00C2\u00A0a\u00C2\u00A0correlation\u00C2\u00A0between\u00C2\u00A0ADCC\u00C2\u00A0activity\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 CD54\u00C2\u00A0 upregulation\u00C2\u00A0 on\u00C2\u00A0 NK\u00C2\u00A0 cells134,\u00C2\u00A0 and\u00C2\u00A0 therefore\u00C2\u00A0 the\u00C2\u00A0 activation\u00C2\u00A0 state\u00C2\u00A0 of\u00C2\u00A0 human\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0quantified\u00C2\u00A0here\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0their\u00C2\u00A0CD54\u00C2\u00A0expression\u00C2\u00A0levels.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0used\u00C2\u00A0were\u00C2\u00A0 a\u00C2\u00A0 subpopulation\u00C2\u00A0within\u00C2\u00A0 a\u00C2\u00A0 sample\u00C2\u00A0 of\u00C2\u00A0 human\u00C2\u00A0 peripheral\u00C2\u00A0 blood\u00C2\u00A0mononuclear\u00C2\u00A0 cells\u00C2\u00A0 (PBMCs).\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0mixed\u00C2\u00A0with\u00C2\u00A0Z138\u00C2\u00A0 cells\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0 target\u00C2\u00A0 cells;\u00C2\u00A0only\u00C2\u00A0Z138\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0used\u00C2\u00A0 since\u00C2\u00A0 the\u00C2\u00A0 ADCC\u00C2\u00A0response\u00C2\u00A0was\u00C2\u00A0most\u00C2\u00A0robust\u00C2\u00A0with\u00C2\u00A0these\u00C2\u00A0cells,\u00C2\u00A0as\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.3D.\u00C2\u00A0\u00C2\u00A0Samples\u00C2\u00A0of\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0 or\u00C2\u00A0combined\u00C2\u00A0effector\u00C2\u00A0and\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0and\u00C2\u00A0controls\u00C2\u00A0(U\u00C2\u00A0or\u00C2\u00A0T\u00E2\u0080\u0090NP).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0 results,\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.4,\u00C2\u00A0compare\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0state\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0 CD54bright\u00C2\u00A0 cells\u00C2\u00A0 contained\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0NK\u00C2\u00A0 cell\u00C2\u00A0population\u00C2\u00A0 (CD3\u00E2\u0080\u0093/CD56+).\u00C2\u00A0 \u00C2\u00A0Open\u00C2\u00A0bars\u00C2\u00A0 indicate\u00C2\u00A0 control\u00C2\u00A0 samples\u00C2\u00A0that\u00C2\u00A0contained\u00C2\u00A0only\u00C2\u00A0effector\u00C2\u00A0cells,\u00C2\u00A0while\u00C2\u00A0black\u00C2\u00A0bars\u00C2\u00A0correspond\u00C2\u00A0to\u00C2\u00A0samples\u00C2\u00A0containing\u00C2\u00A0both\u00C2\u00A0 effector\u00C2\u00A0 and\u00C2\u00A0 target\u00C2\u00A0 (Z138)\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 general,\u00C2\u00A0 a\u00C2\u00A0 greater\u00C2\u00A0 fraction\u00C2\u00A0 of\u00C2\u00A0 NK\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 activated\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A041\u00C2\u00A0\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 Z138\u00C2\u00A0 target\u00C2\u00A0 cells,\u00C2\u00A0 as\u00C2\u00A0 illustrated\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 elevated\u00C2\u00A0 CD54\u00C2\u00A0 expression\u00C2\u00A0 levels\u00C2\u00A0 (black\u00C2\u00A0 bars\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0white\u00C2\u00A0 ones).\u00C2\u00A0 \u00C2\u00A0NK\u00C2\u00A0 cell\u00C2\u00A0 activation\u00C2\u00A0was\u00C2\u00A0 greatest\u00C2\u00A0when\u00C2\u00A0 the\u00C2\u00A0 target\u00C2\u00A0 Z138\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0 level\u00C2\u00A0of\u00C2\u00A0CD54\u00C2\u00A0expression\u00C2\u00A0found\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(rightmost\u00C2\u00A0black\u00C2\u00A0bar).\u00C2\u00A0 \u00C2\u00A0Among\u00C2\u00A0the\u00C2\u00A0samples\u00C2\u00A0containing\u00C2\u00A0both\u00C2\u00A0effector\u00C2\u00A0and\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0(black\u00C2\u00A0 bars),\u00C2\u00A0the\u00C2\u00A0only\u00C2\u00A0significant\u00C2\u00A0difference\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05)\u00C2\u00A0occurred\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0untreated\u00C2\u00A0(34%\u00C2\u00A0activated)\u00C2\u00A0and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0treated\u00C2\u00A0(62%)\u00C2\u00A0samples.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0study\u00C2\u00A0confirms\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0resulting\u00C2\u00A0from\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0treatment\u00C2\u00A0are\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0enhanced\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0activation.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A02.4\u00C2\u00A0\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0activation\u00C2\u00A0upon\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0forms\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Human\u00C2\u00A0 PBMCs\u00C2\u00A0 (effector\u00C2\u00A0 cells)\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 overnight\u00C2\u00A0with\u00C2\u00A0 or\u00C2\u00A0without\u00C2\u00A0 Z138\u00C2\u00A0 target\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0 untreated\u00C2\u00A0 (U)\u00C2\u00A0 or\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Trz\u00E2\u0080\u0090LNP,\u00C2\u00A0Rtx,\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0 labeled\u00C2\u00A0with\u00C2\u00A0anti\u00E2\u0080\u0090human\u00E2\u0080\u0090CD3,\u00C2\u00A0CD56\u00C2\u00A0and\u00C2\u00A0CD54\u00C2\u00A0Abs\u00C2\u00A0conjugated\u00C2\u00A0to\u00C2\u00A0 different\u00C2\u00A0 fluorophores.\u00C2\u00A0 \u00C2\u00A0 Flow\u00C2\u00A0 cytometric\u00C2\u00A0 analysis\u00C2\u00A0 was\u00C2\u00A0 performed\u00C2\u00A0 and\u00C2\u00A0 NK\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 identified\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 CD3\u00E2\u0080\u0093/CD56+\u00C2\u00A0 population.\u00C2\u00A0 \u00C2\u00A0Within\u00C2\u00A0 this\u00C2\u00A0population,\u00C2\u00A0 the\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0CD54bright\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0determined,\u00C2\u00A0corresponding\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0 activated\u00C2\u00A0NK\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0*p\u00C2\u00A0<\u00C2\u00A00.05.\u00C2\u00A0 \u00C2\u00A0 2.4.5\u00C2\u00A0 In\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 The\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0ADCC\u00C2\u00A0and\u00C2\u00A0CDC\u00C2\u00A0 is\u00C2\u00A0enhanced\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0 that\u00C2\u00A0of\u00C2\u00A0 free\u00C2\u00A0Rtx,\u00C2\u00A0 and\u00C2\u00A0when\u00C2\u00A0this\u00C2\u00A0is\u00C2\u00A0considered\u00C2\u00A0alongside\u00C2\u00A0the\u00C2\u00A0increased\u00C2\u00A0direct\u00C2\u00A0activity\u00C2\u00A0leading\u00C2\u00A0to\u00C2\u00A0cell\u00C2\u00A0death,\u00C2\u00A0one\u00C2\u00A0would\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A042\u00C2\u00A0\u00C2\u00A0 expect\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0would\u00C2\u00A0exhibit\u00C2\u00A0significantly\u00C2\u00A0improved\u00C2\u00A0therapeutic\u00C2\u00A0effects\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0 when\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0\u00C2\u00A0We\u00C2\u00A0therefore\u00C2\u00A0completed\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0studies\u00C2\u00A0on\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0in\u00C2\u00A0C.B\u00E2\u0080\u009017\u00C2\u00A0SCID\u00C2\u00A0mice.\u00C2\u00A0\u00C2\u00A0 Although\u00C2\u00A0these\u00C2\u00A0mice\u00C2\u00A0lack\u00C2\u00A0functional\u00C2\u00A0B\u00C2\u00A0and\u00C2\u00A0T\u00C2\u00A0cells\u00C2\u00A0(similar\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Rag\u00E2\u0080\u00902M\u00C2\u00A0mice\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0generate\u00C2\u00A0the\u00C2\u00A0 data\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.1),\u00C2\u00A0they\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0well\u00E2\u0080\u0090defined\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0population\u00C2\u00A0and\u00C2\u00A0should\u00C2\u00A0therefore\u00C2\u00A0be\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0 eliciting\u00C2\u00A0ADCC\u00C2\u00A0 responses135.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 Z138\u00C2\u00A0 cell\u00C2\u00A0 line\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0 for\u00C2\u00A0 these\u00C2\u00A0 studies\u00C2\u00A0 since\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0 the\u00C2\u00A0most\u00C2\u00A0 responsive\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00C2\u00A0 treatment,\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A0 systemic\u00C2\u00A0 tumor\u00C2\u00A0model\u00C2\u00A0was\u00C2\u00A0 employed,\u00C2\u00A0where\u00C2\u00A0 the\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 inoculated\u00C2\u00A0 IV.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0model\u00C2\u00A0 is\u00C2\u00A0 considered\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0more\u00C2\u00A0 aggressive\u00C2\u00A0 and\u00C2\u00A0 less\u00C2\u00A0 sensitive\u00C2\u00A0 to\u00C2\u00A0 treatment\u00C2\u00A0 compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0SC\u00C2\u00A0model.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 follow\u00C2\u00A0 disease\u00C2\u00A0 development\u00C2\u00A0 non\u00E2\u0080\u0090invasively,\u00C2\u00A0 Z138\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 transfected\u00C2\u00A0 to\u00C2\u00A0 express\u00C2\u00A0Luc,\u00C2\u00A0and\u00C2\u00A0tumor\u00E2\u0080\u0090bearing\u00C2\u00A0mice\u00C2\u00A0 (as\u00C2\u00A0confirmed\u00C2\u00A0by\u00C2\u00A0bioluminescent\u00C2\u00A0 imaging)\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0 HBS,\u00C2\u00A0Rtx,\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0specified\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0start\u00C2\u00A0of\u00C2\u00A0treatment,\u00C2\u00A0the\u00C2\u00A0animals\u00C2\u00A0were\u00C2\u00A0imaged\u00C2\u00A0 and\u00C2\u00A0representative\u00C2\u00A0images\u00C2\u00A0are\u00C2\u00A0provided\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.5A.\u00C2\u00A0\u00C2\u00A0Mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0exhibited\u00C2\u00A0 a\u00C2\u00A0significantly\u00C2\u00A0 lower\u00C2\u00A0 tumor\u00C2\u00A0burden\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0control\u00C2\u00A0animals\u00C2\u00A0and\u00C2\u00A0 this\u00C2\u00A0was\u00C2\u00A0 readily\u00C2\u00A0apparent\u00C2\u00A014\u00C2\u00A0 days\u00C2\u00A0after\u00C2\u00A0 initiation\u00C2\u00A0of\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 imaging\u00C2\u00A0data\u00C2\u00A0also\u00C2\u00A0highlighted\u00C2\u00A0 that\u00C2\u00A0 following\u00C2\u00A0 IV\u00C2\u00A0 injection\u00C2\u00A0of\u00C2\u00A0 Z138\u00E2\u0080\u0090Luc\u00C2\u00A0 cells,\u00C2\u00A0 disease\u00C2\u00A0 development\u00C2\u00A0 was\u00C2\u00A0 throughout\u00C2\u00A0 the\u00C2\u00A0 body.\u00C2\u00A0 \u00C2\u00A0 Notably,\u00C2\u00A0 there\u00C2\u00A0 was\u00C2\u00A0 significant\u00C2\u00A0 disease\u00C2\u00A0burden\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0brain,\u00C2\u00A0which\u00C2\u00A0would\u00C2\u00A0partially\u00C2\u00A0explain\u00C2\u00A0why\u00C2\u00A0this\u00C2\u00A0model\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0considered\u00C2\u00A0more\u00C2\u00A0 aggressive.\u00C2\u00A0As\u00C2\u00A0highlighted\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0examples,\u00C2\u00A0disease\u00C2\u00A0development\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0brain\u00C2\u00A0was\u00C2\u00A0 unaffected\u00C2\u00A0by\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0Tumor\u00C2\u00A0burden\u00C2\u00A0was\u00C2\u00A0quantified\u00C2\u00A0by\u00C2\u00A0measuring\u00C2\u00A0the\u00C2\u00A0total\u00C2\u00A0 number\u00C2\u00A0of\u00C2\u00A0emitted\u00C2\u00A0photons\u00C2\u00A0from\u00C2\u00A0each\u00C2\u00A0mouse,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0average\u00C2\u00A0photon\u00C2\u00A0counts\u00C2\u00A0determined\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0 treatment\u00C2\u00A0are\u00C2\u00A0summarized\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.5B.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0burden\u00C2\u00A0in\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 significantly\u00C2\u00A0 different,\u00C2\u00A0 but\u00C2\u00A0 both\u00C2\u00A0 treatment\u00C2\u00A0 groups\u00C2\u00A0 exhibited\u00C2\u00A0 significantly\u00C2\u00A0lower\u00C2\u00A0tumor\u00C2\u00A0burden\u00C2\u00A0when\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0control\u00C2\u00A0(HBS\u00E2\u0080\u0090treated)\u00C2\u00A0mice.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A043\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A02.5\u00C2\u00A0\u00C2\u00A0Efficacy\u00C2\u00A0study\u00C2\u00A0for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0 C.B\u00E2\u0080\u009017\u00C2\u00A0SCID\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0injected\u00C2\u00A0with\u00C2\u00A0Z138\u00E2\u0080\u0090Luc\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0monitored\u00C2\u00A0for\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0IVIS\u00C2\u00A0200\u00C2\u00A0optical\u00C2\u00A0imaging\u00C2\u00A0 system.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A010\u00C2\u00A0days\u00C2\u00A0after\u00C2\u00A0visible\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0appeared,\u00C2\u00A0mice\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0twice\u00C2\u00A0per\u00C2\u00A0week\u00C2\u00A0for\u00C2\u00A03\u00C2\u00A0weeks\u00C2\u00A0with\u00C2\u00A0either\u00C2\u00A0 HBS\u00C2\u00A0or\u00C2\u00A01\u00C2\u00A0mg/kg\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(R\u00E2\u0080\u0090NP).\u00C2\u00A0Tumors\u00C2\u00A0were\u00C2\u00A0scanned\u00C2\u00A0once\u00C2\u00A0or\u00C2\u00A0twice\u00C2\u00A0per\u00C2\u00A0week.\u00C2\u00A0 \u00C2\u00A0(A)\u00C2\u00A0 Images\u00C2\u00A0of\u00C2\u00A0 three\u00C2\u00A0out\u00C2\u00A0of\u00C2\u00A0five\u00C2\u00A0mice\u00C2\u00A0are\u00C2\u00A0presented.\u00C2\u00A0\u00C2\u00A0Red\u00C2\u00A0and\u00C2\u00A0blue\u00C2\u00A0regions\u00C2\u00A0represent\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0and\u00C2\u00A0lowest\u00C2\u00A0luminescence,\u00C2\u00A0respectively.\u00C2\u00A0 (B)\u00C2\u00A0Assessment\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0growth,\u00C2\u00A0as\u00C2\u00A0measured\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0total\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0light\u00C2\u00A0emitted\u00C2\u00A0by\u00C2\u00A0Luc\u00E2\u0080\u0090expressing\u00C2\u00A0tumor\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 entire\u00C2\u00A0body\u00C2\u00A0of\u00C2\u00A0control\u00C2\u00A0mice\u00C2\u00A0(\u00EF\u0081\u00B5),\u00C2\u00A0and\u00C2\u00A0mice\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 (\u00EF\u0081\u00AE)\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(\u00EF\u0081\u00B0).\u00C2\u00A0 \u00C2\u00A0(C)\u00C2\u00A0Kaplan\u00E2\u0080\u0090Meier\u00C2\u00A0survival\u00C2\u00A0curve\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 mice\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0study;\u00C2\u00A0symbols\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0(B).\u00C2\u00A0\u00C2\u00A0Inset:\u00C2\u00A0Increase\u00C2\u00A0in\u00C2\u00A0lifespan\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0control.\u00C2\u00A0\u00C2\u00A0Data\u00C2\u00A0is\u00C2\u00A0representative\u00C2\u00A0 of\u00C2\u00A0two\u00C2\u00A0separate\u00C2\u00A0studies.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Kaplan\u00E2\u0080\u0090Meier\u00C2\u00A0 survival\u00C2\u00A0 curves\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0generated\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A02.3.10,\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0 provided\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.5C.\u00C2\u00A0\u00C2\u00A0They\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0imaging\u00C2\u00A0data\u00C2\u00A0and\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0provided\u00C2\u00A0equivalent\u00C2\u00A0survival\u00C2\u00A0benefits.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0median\u00C2\u00A0survival\u00C2\u00A0time\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00C2\u00A0 treated\u00C2\u00A0animals\u00C2\u00A0was\u00C2\u00A056\u00C2\u00A0and\u00C2\u00A061\u00C2\u00A0days,\u00C2\u00A0 respectively,\u00C2\u00A0while\u00C2\u00A0 the\u00C2\u00A0median\u00C2\u00A0 survival\u00C2\u00A0 time\u00C2\u00A0 for\u00C2\u00A0control\u00C2\u00A0 animals\u00C2\u00A0was\u00C2\u00A035\u00C2\u00A0days.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0were\u00C2\u00A0somewhat\u00C2\u00A0surprising\u00C2\u00A0given\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0studies\u00C2\u00A0suggested\u00C2\u00A0 that\u00C2\u00A0the\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0would\u00C2\u00A0exhibit\u00C2\u00A0improved\u00C2\u00A0treatment\u00C2\u00A0effects\u00C2\u00A0when\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0an\u00C2\u00A0effort\u00C2\u00A0to\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A044\u00C2\u00A0\u00C2\u00A0 understand\u00C2\u00A0why\u00C2\u00A0these\u00C2\u00A0expectations\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0realized,\u00C2\u00A0pharmacokinetic\u00C2\u00A0studies\u00C2\u00A0were\u00C2\u00A0completed\u00C2\u00A0to\u00C2\u00A0 compare\u00C2\u00A0 the\u00C2\u00A0Rtx\u00C2\u00A0 levels\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0compartment\u00C2\u00A0over\u00C2\u00A0 time\u00C2\u00A0 following\u00C2\u00A0 IV\u00C2\u00A0 injection\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 2.4.6\u00C2\u00A0 Pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 Pharmacokinetic\u00C2\u00A0 studies\u00C2\u00A0were\u00C2\u00A0 completed\u00C2\u00A0 in\u00C2\u00A0 immunocompromised\u00C2\u00A0mice\u00C2\u00A0 (C.B\u00E2\u0080\u009017\u00C2\u00A0 SCID)\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0as\u00C2\u00A0immunocompetent\u00C2\u00A0BALB/c\u00C2\u00A0mice,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0obtained\u00C2\u00A0are\u00C2\u00A0summarized\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.6.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0 black\u00C2\u00A0symbols\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.6A\u00C2\u00A0(C.B\u00E2\u0080\u009017\u00C2\u00A0SCID\u00C2\u00A0mice)\u00C2\u00A0and\u00C2\u00A0Figure\u00C2\u00A02.6C\u00C2\u00A0(BALB/c\u00C2\u00A0mice)\u00C2\u00A0represent\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0 concentration\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0mouse\u00C2\u00A0strains,\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0general,\u00C2\u00A0the\u00C2\u00A0elimination\u00C2\u00A0rate\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0 comparable\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0strains.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0initial\u00C2\u00A0Rtx\u00C2\u00A0dose\u00C2\u00A0remaining\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0was\u00C2\u00A027%\u00C2\u00A0for\u00C2\u00A0 C.B\u00E2\u0080\u009017\u00C2\u00A0 SCID\u00C2\u00A0 mice\u00C2\u00A0 and\u00C2\u00A0 24%\u00C2\u00A0 for\u00C2\u00A0 BALB/c\u00C2\u00A0 mice.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 values\u00C2\u00A0 are\u00C2\u00A0 comparable\u00C2\u00A0 to\u00C2\u00A0 those\u00C2\u00A0 reported\u00C2\u00A0 elsewhere136\u00C2\u00A0 and\u00C2\u00A0 highlight\u00C2\u00A0 that\u00C2\u00A0 Rtx\u00C2\u00A0 is\u00C2\u00A0 slowly\u00C2\u00A0 eliminated\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 circulation\u00C2\u00A0 following\u00C2\u00A0 IV\u00C2\u00A0 administration\u00C2\u00A0in\u00C2\u00A0mice.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0open\u00C2\u00A0symbols\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.6A\u00C2\u00A0&\u00C2\u00A0C\u00C2\u00A0represent\u00C2\u00A0plasma\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 following\u00C2\u00A0 IV\u00C2\u00A0 injection\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0and\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0obvious\u00C2\u00A0 that\u00C2\u00A0 these\u00C2\u00A0values\u00C2\u00A0are\u00C2\u00A0considerably\u00C2\u00A0reduced.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 both\u00C2\u00A0mouse\u00C2\u00A0strains,\u00C2\u00A0 the\u00C2\u00A0 injected\u00C2\u00A0dose\u00C2\u00A0was\u00C2\u00A0 reduced\u00C2\u00A0by\u00C2\u00A0as\u00C2\u00A0much\u00C2\u00A0as\u00C2\u00A080%\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0 first\u00C2\u00A0hour\u00C2\u00A0after\u00C2\u00A0 administration,\u00C2\u00A0and\u00C2\u00A0at\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0postinjection,\u00C2\u00A0the\u00C2\u00A0remaining\u00C2\u00A0percentages\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 initial\u00C2\u00A0dose\u00C2\u00A0were\u00C2\u00A00.3%\u00C2\u00A0 (C.B\u00E2\u0080\u009017\u00C2\u00A0SCID)\u00C2\u00A0and\u00C2\u00A02.8%\u00C2\u00A0(BALB/c).\u00C2\u00A0 The\u00C2\u00A0plasma\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0liposomal\u00C2\u00A0lipids\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0monitored\u00C2\u00A0after\u00C2\u00A0administration\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0highlighted\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.6B\u00C2\u00A0&\u00C2\u00A0D,\u00C2\u00A0the\u00C2\u00A0time\u00E2\u0080\u0090dependent\u00C2\u00A0LNP\u00C2\u00A0concentration\u00C2\u00A0curves\u00C2\u00A0mirror\u00C2\u00A0those\u00C2\u00A0 of\u00C2\u00A0Rtx\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0treatments;\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A00.7%\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0initial\u00C2\u00A0dose\u00C2\u00A0remained\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0C.B\u00E2\u0080\u009017\u00C2\u00A0SCID\u00C2\u00A0mice,\u00C2\u00A0 while\u00C2\u00A00.5%\u00C2\u00A0remained\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0BALB/c\u00C2\u00A0animals.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0observation\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 and\u00C2\u00A0LNPs\u00C2\u00A0decrease\u00C2\u00A0at\u00C2\u00A0approximately\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0rate\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0Rtx\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0dissociate\u00C2\u00A0from\u00C2\u00A0LNPs\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 circulation\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 both\u00C2\u00A0 are\u00C2\u00A0 cleared\u00C2\u00A0 together.\u00C2\u00A0 \u00C2\u00A0 Since\u00C2\u00A0 the\u00C2\u00A0 detected\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 are\u00C2\u00A0 similar\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0mouse\u00C2\u00A0strains,\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0also\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0cleared\u00C2\u00A0via\u00C2\u00A0an\u00C2\u00A0immunogenic\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A045\u00C2\u00A0\u00C2\u00A0 mechanism.\u00C2\u00A0\u00C2\u00A0Nevertheless,\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0are\u00C2\u00A0so\u00C2\u00A0low\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0that\u00C2\u00A0its\u00C2\u00A0full\u00C2\u00A0 therapeutic\u00C2\u00A0potential,\u00C2\u00A0as\u00C2\u00A0illustrated\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A02\u00E2\u0080\u00904,\u00C2\u00A0is\u00C2\u00A0compromised\u00C2\u00A0in\u00C2\u00A0mice\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A02.6\u00C2\u00A0\u00C2\u00A0Pharmacokinetics\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0in\u00C2\u00A0two\u00C2\u00A0mouse\u00C2\u00A0models.\u00C2\u00A0\u00C2\u00A0 C.B\u00E2\u0080\u009017\u00C2\u00A0SCID\u00C2\u00A0mice\u00C2\u00A0(A\u00C2\u00A0&\u00C2\u00A0B)\u00C2\u00A0and\u00C2\u00A0BALB/c\u00C2\u00A0mice\u00C2\u00A0(C\u00C2\u00A0&\u00C2\u00A0D)\u00C2\u00A0were\u00C2\u00A0injected\u00C2\u00A0IV\u00C2\u00A0with\u00C2\u00A05\u00C2\u00A0mg/kg\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0free\u00C2\u00A0form\u00C2\u00A0(black\u00C2\u00A0symbols)\u00C2\u00A0or\u00C2\u00A0in\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(open\u00C2\u00A0symbols).\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0of\u00C2\u00A00.5,\u00C2\u00A04,\u00C2\u00A024\u00C2\u00A0and\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0injection,\u00C2\u00A0circulating\u00C2\u00A0Rtx\u00C2\u00A0(A\u00C2\u00A0&\u00C2\u00A0C)\u00C2\u00A0and\u00C2\u00A0lipid\u00C2\u00A0(B\u00C2\u00A0&\u00C2\u00A0D)\u00C2\u00A0 were\u00C2\u00A0 quantified\u00C2\u00A0 by\u00C2\u00A0 ELISA\u00C2\u00A0 and\u00C2\u00A0 liquid\u00C2\u00A0 scintillation\u00C2\u00A0 counting,\u00C2\u00A0 respectively.\u00C2\u00A0 \u00C2\u00A0 Data\u00C2\u00A0 is\u00C2\u00A0 representative\u00C2\u00A0 of\u00C2\u00A0 two\u00C2\u00A0 separate\u00C2\u00A0 experiments.\u00C2\u00A0 \u00C2\u00A0 2.5\u00C2\u00A0\u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0 2.5.1\u00C2\u00A0 Discussion\u00C2\u00A0 In\u00C2\u00A0 spite\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0nearly\u00C2\u00A0all\u00C2\u00A0 therapeutic\u00C2\u00A0Abs\u00C2\u00A0are\u00C2\u00A0bivalent\u00C2\u00A0 IgG\u00C2\u00A0molecules,\u00C2\u00A0 it\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 demonstrated\u00C2\u00A0 in\u00C2\u00A0numerous\u00C2\u00A0cases\u00C2\u00A0 that\u00C2\u00A0equivalent\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0have\u00C2\u00A0more\u00C2\u00A0 therapeutic\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A046\u00C2\u00A0\u00C2\u00A0 potency.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0designs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0which\u00C2\u00A0exhibit\u00C2\u00A0enhanced\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 target\u00C2\u00A0 cells15,\u00C2\u00A096\u00C2\u00A0 and/or\u00C2\u00A0elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 cell\u00C2\u00A0 kill\u00C2\u00A0 in\u00C2\u00A0 vitro7,\u00C2\u00A075,\u00C2\u00A088\u00E2\u0080\u009090\u00C2\u00A0 and\u00C2\u00A0enhanced\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 efficacy75,\u00C2\u00A090.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0studied\u00C2\u00A0here\u00C2\u00A0represent\u00C2\u00A0 the\u00C2\u00A0only\u00C2\u00A0 liposomal\u00C2\u00A0 formulation\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0 therapeutic\u00C2\u00A0IgG\u00C2\u00A0that\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0described137.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0constructs\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0~90,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0much\u00C2\u00A0 higher\u00C2\u00A0 than\u00C2\u00A0 the\u00C2\u00A0other\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0 reported\u00C2\u00A0multivalent\u00C2\u00A0 constructs,\u00C2\u00A0whose\u00C2\u00A0 valences\u00C2\u00A0 generally\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0 exceed\u00C2\u00A010.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 favorable\u00C2\u00A0effects\u00C2\u00A0 related\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 following\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs7\u00C2\u00A0were\u00C2\u00A0confirmed\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0experiments\u00C2\u00A0described\u00C2\u00A0above.\u00C2\u00A0\u00C2\u00A0Figure\u00C2\u00A02.2\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0Z138\u00C2\u00A0cell\u00C2\u00A0 line\u00C2\u00A0was\u00C2\u00A0substantially\u00C2\u00A0more\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0spite\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0that\u00C2\u00A0it\u00C2\u00A0bound\u00C2\u00A0 only\u00C2\u00A0half\u00C2\u00A0as\u00C2\u00A0much\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0higher\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0 result\u00C2\u00A0 from\u00C2\u00A0more\u00C2\u00A0Rtx\u00C2\u00A0 being\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0explained,\u00C2\u00A0in\u00C2\u00A0part,\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0JVM2\u00C2\u00A0line\u00C2\u00A0(and\u00C2\u00A0 not\u00C2\u00A0the\u00C2\u00A0Z138\u00C2\u00A0 line)\u00C2\u00A0 is\u00C2\u00A0Epstein\u00E2\u0080\u0090Barr\u00C2\u00A0virus\u00C2\u00A0positive,\u00C2\u00A0and\u00C2\u00A0 latently\u00C2\u00A0 infected\u00C2\u00A0Epstein\u00E2\u0080\u0090Barr\u00C2\u00A0virus\u00E2\u0080\u0090positive\u00C2\u00A0B\u00E2\u0080\u0090 cell\u00C2\u00A0 lymphomas\u00C2\u00A0are\u00C2\u00A0often\u00C2\u00A0protected\u00C2\u00A0from\u00C2\u00A0death\u00E2\u0080\u0090receptor\u00C2\u00A0 induced\u00C2\u00A0apoptosis\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0that\u00C2\u00A0triggered\u00C2\u00A0 by\u00C2\u00A0CD95\u00C2\u00A0(Fas\u00C2\u00A0receptor)138.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0may\u00C2\u00A0 justify\u00C2\u00A0why\u00C2\u00A0the\u00C2\u00A0high\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD95\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0JVM2\u00C2\u00A0 line\u00C2\u00A0 (Figure\u00C2\u00A02.1)\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0correlate\u00C2\u00A0with\u00C2\u00A0lower\u00C2\u00A0cell\u00C2\u00A0viability\u00C2\u00A0levels\u00C2\u00A0following\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0crosslinked\u00C2\u00A0Rtx\u00C2\u00A0 or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(Figure\u00C2\u00A02.2C\u00C2\u00A0&\u00C2\u00A0D).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 There\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0a\u00C2\u00A0lack\u00C2\u00A0of\u00C2\u00A0studies\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0 (ADCC\u00C2\u00A0 and\u00C2\u00A0CDC),\u00C2\u00A0 even\u00C2\u00A0 though\u00C2\u00A0 the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0 are\u00C2\u00A0 known\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 essential\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 efficacy\u00C2\u00A0of\u00C2\u00A0Rtx118,\u00C2\u00A0119,\u00C2\u00A0121,\u00C2\u00A0123\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0 information\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0critical\u00C2\u00A0for\u00C2\u00A0developing\u00C2\u00A0such\u00C2\u00A0constructs\u00C2\u00A0for\u00C2\u00A0 clinical\u00C2\u00A0use.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0current\u00C2\u00A0study,\u00C2\u00A0 the\u00C2\u00A0 JVM2\u00C2\u00A0cell\u00C2\u00A0 line\u00C2\u00A0was\u00C2\u00A0expected\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0higher\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CDC\u00C2\u00A0 than\u00C2\u00A0 the\u00C2\u00A0 Z138\u00C2\u00A0 line\u00C2\u00A0 since,\u00C2\u00A0 as\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 2.1,\u00C2\u00A0 it\u00C2\u00A0 exhibited\u00C2\u00A0 lower\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 CD55,\u00C2\u00A0 a\u00C2\u00A0 CDC\u00C2\u00A0 inhibitor46.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 was\u00C2\u00A0 also\u00C2\u00A0 expected\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 JVM2\u00C2\u00A0 line\u00C2\u00A0 would\u00C2\u00A0 exhibit\u00C2\u00A0 enhanced\u00C2\u00A0 ADCC\u00C2\u00A0 since\u00C2\u00A0 its\u00C2\u00A0 expression\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD40,\u00C2\u00A0CD80,\u00C2\u00A0and\u00C2\u00A0CD86\u00C2\u00A0were\u00C2\u00A0elevated,\u00C2\u00A0and\u00C2\u00A0these\u00C2\u00A0proteins\u00C2\u00A0activate\u00C2\u00A0NK\u00C2\u00A0cells125,\u00C2\u00A0126.\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0 expected,\u00C2\u00A0 the\u00C2\u00A0 JVM2\u00C2\u00A0 cell\u00C2\u00A0 line\u00C2\u00A0 induced\u00C2\u00A0 a\u00C2\u00A0 more\u00C2\u00A0 robust\u00C2\u00A0 CDC\u00C2\u00A0 response,\u00C2\u00A0 but\u00C2\u00A0 Z138\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 substantially\u00C2\u00A0better\u00C2\u00A0at\u00C2\u00A0 inducing\u00C2\u00A0ADCC\u00C2\u00A0 (Figure\u00C2\u00A02.3).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 illustrates\u00C2\u00A0 the\u00C2\u00A0poor\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0how\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A047\u00C2\u00A0\u00C2\u00A0 different\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0 induce\u00C2\u00A0ADCC\u00C2\u00A0 to\u00C2\u00A0various\u00C2\u00A0extents49.\u00C2\u00A0 \u00C2\u00A0Together\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0apoptosis\u00C2\u00A0 data\u00C2\u00A0from\u00C2\u00A0Figure\u00C2\u00A02.2C\u00C2\u00A0&\u00C2\u00A0D,\u00C2\u00A0however,\u00C2\u00A0the\u00C2\u00A0observations\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.3\u00C2\u00A0support\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.1B\u00C2\u00A0 &\u00C2\u00A0 C,\u00C2\u00A0which\u00C2\u00A0 show\u00C2\u00A0 that\u00C2\u00A0 animals\u00C2\u00A0 bearing\u00C2\u00A0 established\u00C2\u00A0 Z138\u00C2\u00A0 tumors\u00C2\u00A0 are\u00C2\u00A0 Rtx\u00E2\u0080\u0090sensitive\u00C2\u00A0whereas\u00C2\u00A0 those\u00C2\u00A0 bearing\u00C2\u00A0 JVM2\u00C2\u00A0 tumors\u00C2\u00A0 are\u00C2\u00A0 not.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 of\u00C2\u00A0 particular\u00C2\u00A0 interest\u00C2\u00A0 since\u00C2\u00A0 these\u00C2\u00A0models\u00C2\u00A0may\u00C2\u00A0 provide\u00C2\u00A0 a\u00C2\u00A0 framework\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 future\u00C2\u00A0elucidation\u00C2\u00A0of\u00C2\u00A0molecular\u00C2\u00A0markers\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 resistance\u00C2\u00A0or\u00C2\u00A0 response\u00C2\u00A0 in\u00C2\u00A0MCL,\u00C2\u00A0 which\u00C2\u00A0are\u00C2\u00A0currently\u00C2\u00A0poorly\u00C2\u00A0defined139,\u00C2\u00A0140.\u00C2\u00A0 Figure\u00C2\u00A0 2.3\u00C2\u00A0 also\u00C2\u00A0 demonstrates\u00C2\u00A0 that\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 elicits\u00C2\u00A0 superior\u00C2\u00A0 CDC\u00C2\u00A0 and\u00C2\u00A0 ADCC\u00C2\u00A0 responses\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 induce\u00C2\u00A0specific\u00C2\u00A0CDC\u00C2\u00A0activity\u00C2\u00A0 in\u00C2\u00A0 vitro.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0multivalent\u00C2\u00A0 interaction\u00C2\u00A0 consists,\u00C2\u00A0 in\u00C2\u00A0 theory,\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 complex\u00C2\u00A0 created\u00C2\u00A0 by\u00C2\u00A0 an\u00C2\u00A0 initial\u00C2\u00A0 univalent\u00C2\u00A0 interaction\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0 local\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0remaining\u00C2\u00A0 free\u00C2\u00A0binding\u00C2\u00A0sites\u00C2\u00A0on\u00C2\u00A0both\u00C2\u00A0species\u00C2\u00A0are\u00C2\u00A0 greatly\u00C2\u00A0enhanced15.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0may\u00C2\u00A0help\u00C2\u00A0explain\u00C2\u00A0the\u00C2\u00A0increased\u00C2\u00A0CDC\u00C2\u00A0and\u00C2\u00A0ADCC\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090treated\u00C2\u00A0 cells.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0elevated\u00C2\u00A0CDC\u00C2\u00A0may\u00C2\u00A0result\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0that\u00C2\u00A0C1q\u00C2\u00A0is\u00C2\u00A0itself\u00C2\u00A0a\u00C2\u00A0hexavalent\u00C2\u00A0ligand\u00C2\u00A0that\u00C2\u00A0requires\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 several\u00C2\u00A0 IgG\u00C2\u00A0 Fc\u00C2\u00A0 regions\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0activate\u00C2\u00A0a\u00C2\u00A0CDC\u00C2\u00A0 response105,\u00C2\u00A0106.\u00C2\u00A0 \u00C2\u00A0Multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 provides\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0local\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0exposed\u00C2\u00A0Fc\u00C2\u00A0available\u00C2\u00A0to\u00C2\u00A0bind\u00C2\u00A0C1q\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0initial\u00C2\u00A0 univalent\u00C2\u00A0Fc\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0C1q\u00C2\u00A0 interaction.\u00C2\u00A0 \u00C2\u00A0Similarly,\u00C2\u00A0ADCC\u00C2\u00A0requires\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0on\u00C2\u00A0 effector\u00C2\u00A0cells\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0NK\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0Following\u00C2\u00A0the\u00C2\u00A0initial\u00C2\u00A0interaction\u00C2\u00A0between\u00C2\u00A0an\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0on\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0 a\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0NK\u00C2\u00A0cell,\u00C2\u00A0the\u00C2\u00A0remaining\u00C2\u00A0Fc\u00C2\u00A0and\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0are\u00C2\u00A0at\u00C2\u00A0significantly\u00C2\u00A0elevated\u00C2\u00A0local\u00C2\u00A0concentrations\u00C2\u00A0with\u00C2\u00A0 respect\u00C2\u00A0to\u00C2\u00A0one\u00C2\u00A0another.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0facilitates\u00C2\u00A0subsequent\u00C2\u00A0Fc\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0interactions\u00C2\u00A0leading\u00C2\u00A0to\u00C2\u00A0ADCC,\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0 effect\u00C2\u00A0is\u00C2\u00A0absent\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx.\u00C2\u00A0 Despite\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 appear\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 therapeutically\u00C2\u00A0 superior\u00C2\u00A0 in\u00C2\u00A0 vitro,\u00C2\u00A0 we\u00C2\u00A0 unexpectedly\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0exhibited\u00C2\u00A0equivalent\u00C2\u00A0activity\u00C2\u00A0when\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0treat\u00C2\u00A0a\u00C2\u00A0systemic\u00C2\u00A0 Z138\u00C2\u00A0 tumor\u00C2\u00A0 model\u00C2\u00A0 (Figure\u00C2\u00A0 2.5).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 explained\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 rapid\u00C2\u00A0 elimination\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 following\u00C2\u00A0 IV\u00C2\u00A0 injection\u00C2\u00A0 (Figure\u00C2\u00A0 2.6).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 observed\u00C2\u00A0 removal\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 circulation\u00C2\u00A0 was\u00C2\u00A0 surprising\u00C2\u00A0 in\u00C2\u00A0 light\u00C2\u00A0 of\u00C2\u00A0 previous\u00C2\u00A0 studies\u00C2\u00A0 from\u00C2\u00A0 our\u00C2\u00A0 laboratory\u00C2\u00A0 on\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP\u00C2\u00A0 (Section\u00C2\u00A0 1.2.2),\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 identical\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 in\u00C2\u00A0 every\u00C2\u00A0way\u00C2\u00A0 except\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 Ab\u00C2\u00A0 employed.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 studies\u00C2\u00A0 showed\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A048\u00C2\u00A0\u00C2\u00A0 pharmacokinetics\u00C2\u00A0(plasma\u00C2\u00A0concentrations\u00C2\u00A0and\u00C2\u00A0area\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0curve)\u00C2\u00A0and\u00C2\u00A0efficacy\u00C2\u00A0(decrease\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0 volume)\u00C2\u00A0 of\u00C2\u00A0 Trz\u00E2\u0080\u0090LNP\u00C2\u00A0 were\u00C2\u00A0 both\u00C2\u00A0 significantly\u00C2\u00A0 enhanced\u00C2\u00A0 with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0 free\u00C2\u00A0 Trz\u00C2\u00A0 in\u00C2\u00A0 breast\u00C2\u00A0 tumor\u00C2\u00A0 xenograft\u00C2\u00A0mouse\u00C2\u00A0models7.\u00C2\u00A0\u00C2\u00A0Thus,\u00C2\u00A0the\u00C2\u00A0rapid\u00C2\u00A0clearance\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0appear\u00C2\u00A0to\u00C2\u00A0result\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 binding\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0proteins\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0albumin\u00C2\u00A0or\u00C2\u00A0lipoproteins\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0LNPs,\u00C2\u00A0which\u00C2\u00A0would\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0uptake\u00C2\u00A0 by\u00C2\u00A0the\u00C2\u00A0reticuloendothelial\u00C2\u00A0system18,\u00C2\u00A019.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0expected\u00C2\u00A0since\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0contains\u00C2\u00A0PEG\u00C2\u00A0for\u00C2\u00A0this\u00C2\u00A0purpose,\u00C2\u00A0 and\u00C2\u00A0Trz\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0interfere\u00C2\u00A0with\u00C2\u00A0PEG\u00C2\u00A0function\u00C2\u00A0in\u00C2\u00A0Trz\u00E2\u0080\u0090LNP7.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.6,\u00C2\u00A0the\u00C2\u00A0rapid\u00C2\u00A0elimination\u00C2\u00A0 of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 involved\u00C2\u00A0neither\u00C2\u00A0dissociation\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 liposomes\u00C2\u00A0nor\u00C2\u00A0an\u00C2\u00A0 immunogenic\u00C2\u00A0 reaction,\u00C2\u00A0 but\u00C2\u00A0when\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0Trz\u00E2\u0080\u0090LNP,\u00C2\u00A0the\u00C2\u00A0pharmacokinetic\u00C2\u00A0behavior\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0appears\u00C2\u00A0to\u00C2\u00A0arise\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0Ab\u00C2\u00A0used\u00C2\u00A0and\u00C2\u00A0not\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0liposomal\u00C2\u00A0component\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0formulation.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0issue\u00C2\u00A0of\u00C2\u00A0rapid\u00C2\u00A0clearance\u00C2\u00A0 is\u00C2\u00A0discussed\u00C2\u00A0further\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A07.3.1.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 2.5.2\u00C2\u00A0 Conclusions\u00C2\u00A0 The\u00C2\u00A0studies\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0chapter\u00C2\u00A0demonstrate\u00C2\u00A0 that\u00C2\u00A0multivalency\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0an\u00C2\u00A0effective\u00C2\u00A0strategy\u00C2\u00A0 for\u00C2\u00A0 improving\u00C2\u00A0 the\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0 therapeutic\u00C2\u00A0Abs.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0greatest\u00C2\u00A0barrier\u00C2\u00A0 to\u00C2\u00A0developing\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0 for\u00C2\u00A0 clinical\u00C2\u00A0use\u00C2\u00A0is\u00C2\u00A0their\u00C2\u00A0poorly\u00C2\u00A0understood\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action,\u00C2\u00A0but\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0 and\u00C2\u00A0indirect\u00C2\u00A0modes\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0are\u00C2\u00A0significantly\u00C2\u00A0improved\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0chapter\u00C2\u00A0focused\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0enhanced\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0(CDC\u00C2\u00A0and\u00C2\u00A0ADCC),\u00C2\u00A0and\u00C2\u00A0we\u00C2\u00A0further\u00C2\u00A0showed\u00C2\u00A0 that\u00C2\u00A0elevated\u00C2\u00A0ADCC\u00C2\u00A0occurred\u00C2\u00A0alongside\u00C2\u00A0higher\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0NK\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0spite\u00C2\u00A0of\u00C2\u00A0more\u00C2\u00A0favorable\u00C2\u00A0 direct\u00C2\u00A0 and\u00C2\u00A0 indirect\u00C2\u00A0 cytotoxicity,\u00C2\u00A0 we\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 efficacy\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 were\u00C2\u00A0 equivalent,\u00C2\u00A0and\u00C2\u00A0pharmacokinetic\u00C2\u00A0 studies\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0very\u00C2\u00A0 rapidly\u00C2\u00A0 cleared\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 circulation\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0 indicates\u00C2\u00A0the\u00C2\u00A0need\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0constructs\u00C2\u00A0 with\u00C2\u00A0more\u00C2\u00A0extended\u00C2\u00A0circulation\u00C2\u00A0lifetimes\u00C2\u00A0(using\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0type\u00C2\u00A0of\u00C2\u00A0NP\u00C2\u00A0formulation\u00C2\u00A0and/or\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 Ab)\u00C2\u00A0 if\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 developed\u00C2\u00A0 for\u00C2\u00A0 clinical\u00C2\u00A0 use,\u00C2\u00A0 but\u00C2\u00A0 it\u00C2\u00A0 also\u00C2\u00A0 highlights\u00C2\u00A0 the\u00C2\u00A0 therapeutic\u00C2\u00A0 potency\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A049\u00C2\u00A0\u00C2\u00A0 Part\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0potency\u00C2\u00A0results\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action,\u00C2\u00A0which\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0case\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0is\u00C2\u00A0 undefined\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0molecular\u00C2\u00A0point\u00C2\u00A0of\u00C2\u00A0view.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0next\u00C2\u00A0chapter\u00C2\u00A0describes\u00C2\u00A0an\u00C2\u00A0alternative,\u00C2\u00A0more\u00C2\u00A0versatile\u00C2\u00A0 methodology\u00C2\u00A0that\u00C2\u00A0we\u00C2\u00A0developed\u00C2\u00A0for\u00C2\u00A0producing\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0differing\u00C2\u00A0valence\u00C2\u00A0for\u00C2\u00A0use\u00C2\u00A0in\u00C2\u00A0studies\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 direct\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs51.\u00C2\u00A0\u00C2\u00A0Applying\u00C2\u00A0this\u00C2\u00A0methodology\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0will\u00C2\u00A0add\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0current\u00C2\u00A0results\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0indirect\u00C2\u00A0factors\u00C2\u00A0and\u00C2\u00A0will\u00C2\u00A0help\u00C2\u00A0to\u00C2\u00A0delineate\u00C2\u00A0the\u00C2\u00A0overall\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 of\u00C2\u00A0multivalent\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0understanding\u00C2\u00A0will\u00C2\u00A0further\u00C2\u00A0define\u00C2\u00A0cases\u00C2\u00A0where\u00C2\u00A0it\u00C2\u00A0might\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0efficacious\u00C2\u00A0 to\u00C2\u00A0employ\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0a\u00C2\u00A0bivalent\u00C2\u00A0IgG\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0treat\u00C2\u00A0cancer.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A050\u00C2\u00A0\u00C2\u00A0 3\u00C2\u00A0 Improved\u00C2\u00A0methodology\u00C2\u00A0for\u00C2\u00A0preparing\u00C2\u00A0multivalent\u00C2\u00A0antibody\u00C2\u00ADlipid\u00C2\u00A0 nanoparticles\u00C2\u00A0 3.1\u00C2\u00A0 Synopsis\u00C2\u00A0 Previous\u00C2\u00A0methods\u00C2\u00A0of\u00C2\u00A0coupling\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0liposomes\u00C2\u00A0were\u00C2\u00A0unsuitable\u00C2\u00A0for\u00C2\u00A0creating\u00C2\u00A0many\u00C2\u00A0different\u00C2\u00A0 valences\u00C2\u00A0of\u00C2\u00A0 (Ab\u00E2\u0080\u0090LNPs).\u00C2\u00A0 \u00C2\u00A0An\u00C2\u00A0 improved\u00C2\u00A0methodology\u00C2\u00A0developed\u00C2\u00A0 for\u00C2\u00A0 this\u00C2\u00A0purpose\u00C2\u00A0 is\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 chapter.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 exhibits\u00C2\u00A0 significant\u00C2\u00A0 improvements\u00C2\u00A0 over\u00C2\u00A0 previous\u00C2\u00A0methods\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 reproducibility,\u00C2\u00A0 length\u00C2\u00A0of\u00C2\u00A0time\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0many\u00C2\u00A0valences,\u00C2\u00A0and\u00C2\u00A0yields\u00C2\u00A0of\u00C2\u00A0coupled\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 3.2\u00C2\u00A0\u00C2\u00A0 Background\u00C2\u00A0 3.2.1\u00C2\u00A0 The\u00C2\u00A0need\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0a\u00C2\u00A0new\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0creating\u00C2\u00A0many\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0 An\u00C2\u00A0 updated\u00C2\u00A0 methodology\u00C2\u00A0 was\u00C2\u00A0 required\u00C2\u00A0 to\u00C2\u00A0 produce\u00C2\u00A0 many\u00C2\u00A0 different\u00C2\u00A0 valences\u00C2\u00A0 of\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 because\u00C2\u00A0previous\u00C2\u00A0methods,\u00C2\u00A0which\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0described\u00C2\u00A0 largely\u00C2\u00A0 in\u00C2\u00A0 light\u00C2\u00A0of\u00C2\u00A0 immunoliposomes,\u00C2\u00A0were\u00C2\u00A0 inadequate\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 reproducibility,\u00C2\u00A0 time\u00C2\u00A0 required,\u00C2\u00A0 and\u00C2\u00A0 overall\u00C2\u00A0 yields\u00C2\u00A0 of\u00C2\u00A0 coupled\u00C2\u00A0 Ab.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 problems\u00C2\u00A0stem\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0chemical\u00C2\u00A0coupling,\u00C2\u00A0which\u00C2\u00A0normally\u00C2\u00A0involves\u00C2\u00A0thiolation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0 followed\u00C2\u00A0 by\u00C2\u00A0 reaction\u00C2\u00A0with\u00C2\u00A0maleimide\u00C2\u00A0 (Mal)\u00E2\u0080\u0090containing\u00C2\u00A0 liposomes\u00C2\u00A0 or\u00C2\u00A0micelles3,\u00C2\u00A05,\u00C2\u00A0141,\u00C2\u00A0142.\u00C2\u00A0 \u00C2\u00A0 Between\u00C2\u00A0 three\u00C2\u00A0and\u00C2\u00A0seven\u00C2\u00A0 thiol\u00C2\u00A0groups\u00C2\u00A0are\u00C2\u00A0required\u00C2\u00A0per\u00C2\u00A0Ab\u00C2\u00A0molecule7,\u00C2\u00A0143,\u00C2\u00A0resulting\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0very\u00C2\u00A0heterogeneous\u00C2\u00A0 reaction\u00C2\u00A0product\u00C2\u00A0 that\u00C2\u00A0 varies\u00C2\u00A0 from\u00C2\u00A0preparation\u00C2\u00A0 to\u00C2\u00A0preparation.\u00C2\u00A0 \u00C2\u00A0 Such\u00C2\u00A0heavy\u00C2\u00A0 chemical\u00C2\u00A0modification\u00C2\u00A0 may\u00C2\u00A0 also\u00C2\u00A0 pose\u00C2\u00A0 a\u00C2\u00A0 problem\u00C2\u00A0 particularly\u00C2\u00A0 for\u00C2\u00A0 therapeutic\u00C2\u00A0 Abs,\u00C2\u00A0 which\u00C2\u00A0 after\u00C2\u00A0 thiolation\u00C2\u00A0 have\u00C2\u00A0 shown\u00C2\u00A0 decreased\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0target144\u00C2\u00A0and\u00C2\u00A0whose\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0native\u00C2\u00A0Fc\u00C2\u00A0fragment44.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 One\u00C2\u00A0common\u00C2\u00A0coupling\u00C2\u00A0strategy,\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.2,\u00C2\u00A0involves\u00C2\u00A0the\u00C2\u00A0preparation\u00C2\u00A0of\u00C2\u00A0small\u00C2\u00A0 unilamellar\u00C2\u00A0 vesicles\u00C2\u00A0 (SUVs)\u00C2\u00A0 containing\u00C2\u00A0 surface\u00C2\u00A0 Mal\u00C2\u00A0 moieties,\u00C2\u00A0 followed\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 addition\u00C2\u00A0 of\u00C2\u00A0 thiol\u00E2\u0080\u0090 modified\u00C2\u00A0 Ab7,\u00C2\u00A0 144.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0 current\u00C2\u00A0 application,\u00C2\u00A0 where\u00C2\u00A0 smaller\u00C2\u00A0 amounts\u00C2\u00A0 of\u00C2\u00A0 several\u00C2\u00A0 different\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A051\u00C2\u00A0\u00C2\u00A0 formulations\u00C2\u00A0are\u00C2\u00A0required,\u00C2\u00A0this\u00C2\u00A0method\u00C2\u00A0is\u00C2\u00A0impractical\u00C2\u00A0since\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0SUV\u00C2\u00A0composition\u00C2\u00A0would\u00C2\u00A0need\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0prepared,\u00C2\u00A0by\u00C2\u00A0extrusion\u00C2\u00A0or\u00C2\u00A0other\u00C2\u00A0means,\u00C2\u00A0for\u00C2\u00A0every\u00C2\u00A0desired\u00C2\u00A0valence.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0more\u00C2\u00A0recent\u00C2\u00A0variation\u00C2\u00A0 is\u00C2\u00A0the\u00C2\u00A0coupling\u00C2\u00A0of\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0micelles\u00C2\u00A0using\u00C2\u00A0thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0 chemistry,\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0heating\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0SUVs,\u00C2\u00A0enabling\u00C2\u00A0the\u00C2\u00A0\u00E2\u0080\u009Cpost\u00E2\u0080\u0090insertion\u00E2\u0080\u009D\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0micellar\u00C2\u00A0 lipids\u00C2\u00A0and\u00C2\u00A0Ab\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0preformed\u00C2\u00A0SUVs142,\u00C2\u00A0145.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0Although\u00C2\u00A0 this\u00C2\u00A0method\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0useful\u00C2\u00A0 for\u00C2\u00A0preparing\u00C2\u00A0 immunoliposomes,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0unsuitable\u00C2\u00A0for\u00C2\u00A0preparing\u00C2\u00A0many\u00C2\u00A0precise\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0because\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0 undergoes\u00C2\u00A0 substantial\u00C2\u00A0 heterogeneous\u00C2\u00A0 chemical\u00C2\u00A0modification\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 above.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 variable\u00C2\u00A0 valences\u00C2\u00A0 from\u00C2\u00A0 one\u00C2\u00A0 preparation\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 next\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 potentially\u00C2\u00A0 reduced\u00C2\u00A0 therapeutic\u00C2\u00A0 activity\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0low\u00C2\u00A0yields\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0final\u00C2\u00A0product\u00C2\u00A0are\u00C2\u00A0obtained\u00C2\u00A0(59%)\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0initially\u00C2\u00A0modified.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0particularly\u00C2\u00A0a\u00C2\u00A0problem\u00C2\u00A0when\u00C2\u00A0using\u00C2\u00A0therapeutic\u00C2\u00A0 Abs\u00C2\u00A0 since\u00C2\u00A0 they\u00C2\u00A0 are\u00C2\u00A0 generally\u00C2\u00A0 available\u00C2\u00A0 in\u00C2\u00A0 limited\u00C2\u00A0 quantities,\u00C2\u00A0 especially\u00C2\u00A0 novel\u00C2\u00A0 drugs\u00C2\u00A0 under\u00C2\u00A0 development.\u00C2\u00A0 3.2.2\u00C2\u00A0 How\u00C2\u00A0the\u00C2\u00A0new\u00C2\u00A0methodology\u00C2\u00A0overcomes\u00C2\u00A0previous\u00C2\u00A0limitations\u00C2\u00A0 The\u00C2\u00A0 improved\u00C2\u00A0methodology\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 chapter\u00C2\u00A0overcomes\u00C2\u00A0 the\u00C2\u00A0above\u00C2\u00A0 limitations\u00C2\u00A0by\u00C2\u00A0 drawing\u00C2\u00A0on\u00C2\u00A0desirable\u00C2\u00A0 features\u00C2\u00A0 from\u00C2\u00A0earlier\u00C2\u00A0methods,\u00C2\u00A0 including\u00C2\u00A0 the\u00C2\u00A0post\u00E2\u0080\u0090insertion\u00C2\u00A0 technique142,\u00C2\u00A0145,\u00C2\u00A0 while\u00C2\u00A0 replacing\u00C2\u00A0 thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0 chemistry\u00C2\u00A0 with\u00C2\u00A0 high\u00E2\u0080\u0090affinity\u00C2\u00A0 biotin\u00E2\u0080\u0090NeutrAvidin\u00C2\u00A0 (Neut)\u00C2\u00A0 interactions146,\u00C2\u00A0147.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0doing\u00C2\u00A0so,\u00C2\u00A0a\u00C2\u00A0novel\u00C2\u00A0feature\u00C2\u00A0concerning\u00C2\u00A0the\u00C2\u00A0kinetics\u00C2\u00A0of\u00C2\u00A0biotin\u00E2\u0080\u0090Neut\u00C2\u00A0interactions\u00C2\u00A0on\u00C2\u00A0 liposomes\u00C2\u00A0was\u00C2\u00A0discovered\u00C2\u00A0and\u00C2\u00A0incorporated\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0methodology.\u00C2\u00A0\u00C2\u00A0Specifically,\u00C2\u00A0it\u00C2\u00A0involves\u00C2\u00A0surprisingly\u00C2\u00A0 slow\u00C2\u00A0kinetics\u00C2\u00A0of\u00C2\u00A0association\u00C2\u00A0between\u00C2\u00A0biotinylated\u00C2\u00A0protein\u00C2\u00A0and\u00C2\u00A0Neut\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0PEG\u00E2\u0080\u0090containing\u00C2\u00A0 liposomes,\u00C2\u00A0attributed\u00C2\u00A0 to\u00C2\u00A0a\u00C2\u00A0 steric\u00C2\u00A0blocking\u00C2\u00A0effect\u00C2\u00A0by\u00C2\u00A0PEG148.\u00C2\u00A0 \u00C2\u00A0Since\u00C2\u00A0Ab\u00C2\u00A0addition\u00C2\u00A0 is\u00C2\u00A0 slow\u00C2\u00A0 (coupled\u00C2\u00A0Ab\u00C2\u00A0 levels\u00C2\u00A0continued\u00C2\u00A0to\u00C2\u00A0increase\u00C2\u00A0over\u00C2\u00A072\u00C2\u00A0h),\u00C2\u00A0to\u00C2\u00A0make\u00C2\u00A0a\u00C2\u00A0series\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valences,\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0only\u00C2\u00A0necessary\u00C2\u00A0 to\u00C2\u00A0stop\u00C2\u00A0fractions\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0coupling\u00C2\u00A0reaction\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0times.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0improve\u00C2\u00A0yields,\u00C2\u00A0a\u00C2\u00A0solid\u00E2\u0080\u0090support\u00C2\u00A0 Ab\u00C2\u00A0 biotinylation\u00C2\u00A0 technique\u00C2\u00A0was\u00C2\u00A0 employed149\u00C2\u00A0 that\u00C2\u00A0 provides\u00C2\u00A0 high\u00C2\u00A0 recovery\u00C2\u00A0 (96%),\u00C2\u00A0 and\u00C2\u00A0 to\u00C2\u00A0 aid\u00C2\u00A0with\u00C2\u00A0 reproducibility,\u00C2\u00A0 relevant\u00C2\u00A0 analytical\u00C2\u00A0 assays\u00C2\u00A0were\u00C2\u00A0developed\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 characterization\u00C2\u00A0of\u00C2\u00A0preparation\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A052\u00C2\u00A0\u00C2\u00A0 intermediates,\u00C2\u00A0including\u00C2\u00A0fluorescence\u00E2\u0080\u0090based\u00C2\u00A0protein\u00C2\u00A0quantitation150\u00C2\u00A0and\u00C2\u00A0an\u00C2\u00A0assay\u00C2\u00A0for\u00C2\u00A0PEG\u00C2\u00A0content\u00C2\u00A0in\u00C2\u00A0 liposomes151.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0chapter\u00C2\u00A0details\u00C2\u00A0this\u00C2\u00A0new\u00C2\u00A0methodology\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0significant\u00C2\u00A0improvements\u00C2\u00A0it\u00C2\u00A0brings\u00C2\u00A0 to\u00C2\u00A0the\u00C2\u00A0preparation\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valence.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 3.3\u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0 3.3.1\u00C2\u00A0 Materials\u00C2\u00A0 The\u00C2\u00A0 following\u00C2\u00A0 lipids\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 Avanti\u00C2\u00A0 Polar\u00C2\u00A0 Lipids\u00C2\u00A0 (Alabaster\u00C2\u00A0 AL,\u00C2\u00A0 USA):\u00C2\u00A0 1,2\u00E2\u0080\u0090 distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphocholine\u00C2\u00A0 (DSPC),\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090phosphoethanolamine\u00E2\u0080\u0090 N\u00E2\u0080\u0090[methoxy(polyethylene\u00C2\u00A0 glycol)\u00E2\u0080\u00902000]\u00C2\u00A0 (DSPE\u00E2\u0080\u0090PEG),\u00C2\u00A0 and\u00C2\u00A0 1,2\u00E2\u0080\u0090distearoyl\u00E2\u0080\u0090sn\u00E2\u0080\u0090glycero\u00E2\u0080\u00903\u00E2\u0080\u0090 phosphoethanolamine\u00E2\u0080\u0090N\u00E2\u0080\u0090[biotinyl(polyethylene\u00C2\u00A0 glycol)\u00E2\u0080\u00902000]\u00C2\u00A0 (DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin).\u00C2\u00A0 \u00C2\u00A0 [3H]cholesteryl\u00C2\u00A0 hexadecyl\u00C2\u00A0ether\u00C2\u00A0([3H]CHE)\u00C2\u00A0and\u00C2\u00A0Pico\u00E2\u0080\u0090Fluor\u00C2\u00A015\u00C2\u00A0scintillation\u00C2\u00A0fluid\u00C2\u00A0were\u00C2\u00A0from\u00C2\u00A0Perkin\u00E2\u0080\u0090Elmer\u00C2\u00A0Life\u00C2\u00A0Sciences\u00C2\u00A0 (Woodbridge\u00C2\u00A0 ON,\u00C2\u00A0 Canada).\u00C2\u00A0 \u00C2\u00A0 NeutrAvidin\u00C2\u00A0 (Neut)\u00C2\u00A0 and\u00C2\u00A0 immobilized\u00C2\u00A0 iminodiacetic\u00C2\u00A0 acid\u00C2\u00A0 gel\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0from\u00C2\u00A0Pierce/Thermo\u00C2\u00A0Fisher\u00C2\u00A0Scientific\u00C2\u00A0(Rockford\u00C2\u00A0IL,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0Rituximab\u00C2\u00A0(Rtx)\u00C2\u00A0and\u00C2\u00A0trastuzumab\u00C2\u00A0 (Trz)\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 BC\u00C2\u00A0 Cancer\u00C2\u00A0 Agency\u00C2\u00A0 pharmacy\u00C2\u00A0 (Vancouver\u00C2\u00A0 BC,\u00C2\u00A0 Canada).\u00C2\u00A0 \u00C2\u00A0 N\u00E2\u0080\u0090 Hydroxysuccinimide\u00C2\u00A0ester\u00E2\u0080\u0090dPEG4\u00E2\u0080\u0090biotin\u00C2\u00A0 (NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin)\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0 from\u00C2\u00A0TimTec\u00C2\u00A0 (Newark\u00C2\u00A0DE,\u00C2\u00A0 USA).\u00C2\u00A0 \u00C2\u00A0Disposable\u00C2\u00A0PD\u00E2\u0080\u009010\u00C2\u00A0columns\u00C2\u00A0 (which\u00C2\u00A0contain\u00C2\u00A0Sephadex\u00C2\u00A0G\u00E2\u0080\u009025\u00C2\u00A0medium)\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0GE\u00C2\u00A0 Healthcare\u00C2\u00A0 Life\u00C2\u00A0 Sciences\u00C2\u00A0 (Piscataway\u00C2\u00A0 NJ,\u00C2\u00A0 USA).\u00C2\u00A0 \u00C2\u00A0 4\u00E2\u0080\u0090Hydroxyazobenzene\u00E2\u0080\u00902\u00E2\u0080\u0090carboxylic\u00C2\u00A0 acid\u00C2\u00A0 (HABA)/avidin\u00C2\u00A0 reagent\u00C2\u00A0 was\u00C2\u00A0 purchased\u00C2\u00A0 from\u00C2\u00A0 Sigma\u00E2\u0080\u0090Aldrich\u00C2\u00A0 (St.\u00C2\u00A0 Louis\u00C2\u00A0 MO,\u00C2\u00A0 USA)\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 lyophilized\u00C2\u00A0 powder.\u00C2\u00A0 \u00C2\u00A0ATTO\u00E2\u0080\u0090TAG\u00C2\u00A03\u00E2\u0080\u0090(4\u00E2\u0080\u0090carboxybenzoyl)quinoline\u00E2\u0080\u00902\u00E2\u0080\u0090carboxaldehyde\u00C2\u00A0(CBQCA)\u00C2\u00A0was\u00C2\u00A0from\u00C2\u00A0 Invitrogen\u00C2\u00A0 (Burlington\u00C2\u00A0ON,\u00C2\u00A0Canada).\u00C2\u00A0 \u00C2\u00A0Unless\u00C2\u00A0noted\u00C2\u00A0otherwise,\u00C2\u00A0all\u00C2\u00A0other\u00C2\u00A0 reagents\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0Sigma\u00E2\u0080\u0090 Aldrich\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0quality\u00C2\u00A0available.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A053\u00C2\u00A0\u00C2\u00A0 3.3.2\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00ADmicelles\u00C2\u00A0 Separate\u00C2\u00A0micellar\u00C2\u00A0 suspensions\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0and\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0by\u00C2\u00A0gently\u00C2\u00A0 dissolving\u00C2\u00A0the\u00C2\u00A0lipids\u00C2\u00A0in\u00C2\u00A0HBS\u00C2\u00A0(25\u00C2\u00A0mM\u00C2\u00A0HEPES,\u00C2\u00A0150\u00C2\u00A0mM\u00C2\u00A0NaCl,\u00C2\u00A0pH\u00C2\u00A07.4).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0two\u00C2\u00A0suspensions\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0 mixed\u00C2\u00A0 in\u00C2\u00A0different\u00C2\u00A0mole\u00C2\u00A0 ratios\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0 to\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0corresponding\u00C2\u00A0 to\u00C2\u00A0different\u00C2\u00A0desired\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP\u00C2\u00A0formulations.\u00C2\u00A0 \u00C2\u00A0Separately,\u00C2\u00A0with\u00C2\u00A0vortexing,\u00C2\u00A0the\u00C2\u00A0micellar\u00C2\u00A0suspension\u00C2\u00A0was\u00C2\u00A0slowly\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0 threefold\u00C2\u00A0mole\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00A0over\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin.\u00C2\u00A0 \u00C2\u00A0Suspensions\u00C2\u00A0were\u00C2\u00A0vortexed\u00C2\u00A0 for\u00C2\u00A030\u00C2\u00A0s\u00C2\u00A0 longer,\u00C2\u00A0 then\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A0room\u00C2\u00A0temperature\u00C2\u00A0with\u00C2\u00A0stirring.\u00C2\u00A0 3.3.3\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00ADLNP\u00C2\u00A0 Using\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 procedure\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 2.3.2,\u00C2\u00A0 SUVs\u00C2\u00A0 composed\u00C2\u00A0 of\u00C2\u00A0 DSPC\u00C2\u00A0 and\u00C2\u00A0 cholesterol\u00C2\u00A0 (Chol)\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A02:1\u00C2\u00A0and\u00C2\u00A0 labeled\u00C2\u00A0with\u00C2\u00A0[3H]CHE\u00C2\u00A0 (0.009\u00C2\u00A0\u00C2\u00B5Ci/\u00C2\u00B5mol\u00C2\u00A0 liposomal\u00C2\u00A0 lipid)\u00C2\u00A0 were\u00C2\u00A0prepared\u00C2\u00A0by\u00C2\u00A0extrusion.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0all\u00C2\u00A0 cases\u00C2\u00A0when\u00C2\u00A0producing\u00C2\u00A0Neut\u00E2\u0080\u0090LNPs,\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0added\u00C2\u00A0 moles\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0+\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0(in\u00C2\u00A0Neut\u00E2\u0080\u0090micelles)\u00C2\u00A0was\u00C2\u00A04\u00C2\u00A0%\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0moles\u00C2\u00A0of\u00C2\u00A0DSPC\u00C2\u00A0(in\u00C2\u00A0 SUV).\u00C2\u00A0\u00C2\u00A0SUV\u00C2\u00A0and\u00C2\u00A0Neut\u00E2\u0080\u0090micelles\u00C2\u00A0were\u00C2\u00A0heated\u00C2\u00A0separately\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A065\u00C2\u00A0oC\u00C2\u00A0water\u00C2\u00A0bath\u00C2\u00A0for\u00C2\u00A010\u00C2\u00A0min\u00C2\u00A0with\u00C2\u00A0stirring,\u00C2\u00A0 then\u00C2\u00A0were\u00C2\u00A0combined\u00C2\u00A0with\u00C2\u00A0vortexing\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A060\u00C2\u00A0min\u00C2\u00A0with\u00C2\u00A0stirring.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0being\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0 return\u00C2\u00A0to\u00C2\u00A0room\u00C2\u00A0temperature\u00C2\u00A0(with\u00C2\u00A0stirring),\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0preparations\u00C2\u00A0were\u00C2\u00A0purified\u00C2\u00A0on\u00C2\u00A0Sepharose\u00C2\u00A0CL\u00E2\u0080\u0090 4B\u00C2\u00A0 columns\u00C2\u00A0 pre\u00E2\u0080\u0090equilibrated\u00C2\u00A0 with\u00C2\u00A0 HBS.\u00C2\u00A0 \u00C2\u00A0 Lipid\u00C2\u00A0 concentrations\u00C2\u00A0 were\u00C2\u00A0 determined\u00C2\u00A0 with\u00C2\u00A0 liquid\u00C2\u00A0 scintillation\u00C2\u00A0counting,\u00C2\u00A0and\u00C2\u00A0Neut\u00C2\u00A0concentrations\u00C2\u00A0were\u00C2\u00A0determined\u00C2\u00A0using\u00C2\u00A0CBQCA\u00C2\u00A0(see\u00C2\u00A0below).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0Neut\u00C2\u00A0molecules\u00C2\u00A0 per\u00C2\u00A0 SUV\u00C2\u00A0was\u00C2\u00A0 calculated\u00C2\u00A0 by\u00C2\u00A0 dividing\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0Neut\u00C2\u00A0 molecules\u00C2\u00A0per\u00C2\u00A0liter\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0SUV\u00C2\u00A0per\u00C2\u00A0liter.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0latter\u00C2\u00A0quantity\u00C2\u00A0was\u00C2\u00A0calculated\u00C2\u00A0assuming\u00C2\u00A0that\u00C2\u00A0 each\u00C2\u00A0DSPC\u00C2\u00A0molecule\u00C2\u00A0occupies\u00C2\u00A00.815\u00C2\u00A0nm2\u00C2\u00A0of\u00C2\u00A0surface\u00C2\u00A0area\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0bilayer\u00C2\u00A0of\u00C2\u00A0DSPC/Chol\u00C2\u00A0(mole\u00C2\u00A0ratio\u00C2\u00A02:1)\u00C2\u00A0 SUV144\u00C2\u00A0and\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0bilayer\u00C2\u00A0 thickness\u00C2\u00A0 in\u00C2\u00A0 these\u00C2\u00A0SUV\u00C2\u00A0 is\u00C2\u00A04.9\u00C2\u00A0nm152.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0a\u00C2\u00A0100\u00E2\u0080\u0090nm\u00C2\u00A0diameter\u00C2\u00A0SUV,\u00C2\u00A0 this\u00C2\u00A0 corresponds\u00C2\u00A0to\u00C2\u00A06.98\u00C2\u00A0\u00C3\u0097\u00C2\u00A0104\u00C2\u00A0DSPC\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0SUV.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0SUV\u00C2\u00A0per\u00C2\u00A0liter\u00C2\u00A0was\u00C2\u00A0calculated\u00C2\u00A0by\u00C2\u00A0 dividing\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0DSPC\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0liter\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0DSPC\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0SUV.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A054\u00C2\u00A0\u00C2\u00A0 3.3.4\u00C2\u00A0 CBQCA\u00C2\u00A0 assay\u00C2\u00A0 for\u00C2\u00A0measuring\u00C2\u00A0protein\u00C2\u00A0 concentration\u00C2\u00A0 in\u00C2\u00A0 solution\u00C2\u00A0 or\u00C2\u00A0Ab\u00C2\u00ADLNP\u00C2\u00A0 suspension\u00C2\u00A0 \u00C2\u00A0 Solutions\u00C2\u00A0of\u00C2\u00A0borate\u00C2\u00A0 (100\u00C2\u00A0mM\u00C2\u00A0Na2B4O7\u00E2\u0080\u00A210H2O,\u00C2\u00A0pH\u00C2\u00A09.3)\u00C2\u00A0and\u00C2\u00A0KCN\u00C2\u00A0(20\u00C2\u00A0mM)\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0 in\u00C2\u00A0 ultrapure\u00C2\u00A0water.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0KCN/borate\u00C2\u00A0solution\u00C2\u00A0was\u00C2\u00A0then\u00C2\u00A0prepared\u00C2\u00A0by\u00C2\u00A0mixing\u00C2\u00A025\u00C2\u00A0volumes\u00C2\u00A0of\u00C2\u00A0borate\u00C2\u00A0and\u00C2\u00A01\u00C2\u00A0 volume\u00C2\u00A0of\u00C2\u00A0KCN.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plate\u00C2\u00A0 layout\u00C2\u00A0was\u00C2\u00A0prepared\u00C2\u00A0 for\u00C2\u00A0each\u00C2\u00A0assay,\u00C2\u00A0which\u00C2\u00A0 included\u00C2\u00A0separate\u00C2\u00A0Rtx\u00C2\u00A0 and\u00C2\u00A0Neut\u00C2\u00A0standards\u00C2\u00A0over\u00C2\u00A0appropriate\u00C2\u00A0concentration\u00C2\u00A0ranges,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0triplicates\u00C2\u00A0of\u00C2\u00A0unknowns.\u00C2\u00A0 \u00C2\u00A0To\u00C2\u00A0 each\u00C2\u00A0sample\u00E2\u0080\u0090containing\u00C2\u00A0well,\u00C2\u00A0130\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0KCN/borate\u00C2\u00A0were\u00C2\u00A0added,\u00C2\u00A0 followed\u00C2\u00A0by\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0standard\u00C2\u00A0or\u00C2\u00A0 unknown\u00C2\u00A0sample.\u00C2\u00A0\u00C2\u00A0Finally,\u00C2\u00A0a\u00C2\u00A040\u00C2\u00A0mM\u00C2\u00A0stock\u00C2\u00A0solution\u00C2\u00A0of\u00C2\u00A0ATTO\u00E2\u0080\u0090TAG\u00C2\u00A0CBQCA\u00C2\u00A0in\u00C2\u00A0dimethyl\u00C2\u00A0sulfoxide\u00C2\u00A0(DMSO)\u00C2\u00A0 was\u00C2\u00A0diluted\u00C2\u00A0tenfold\u00C2\u00A0in\u00C2\u00A0borate,\u00C2\u00A0and\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0resulting\u00C2\u00A0solution\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0wells.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0plate\u00C2\u00A0 was\u00C2\u00A0 covered\u00C2\u00A0 with\u00C2\u00A0 aluminum\u00C2\u00A0 foil\u00C2\u00A0 and\u00C2\u00A0 incubated\u00C2\u00A0 for\u00C2\u00A0 1\u00C2\u00A0 h\u00C2\u00A0 at\u00C2\u00A0 room\u00C2\u00A0 temperature\u00C2\u00A0 with\u00C2\u00A0 shaking.\u00C2\u00A0\u00C2\u00A0 Fluorescence\u00C2\u00A0 emission\u00C2\u00A0 intensities\u00C2\u00A0were\u00C2\u00A0 then\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0 at\u00C2\u00A0550\u00C2\u00A0nm\u00C2\u00A0with\u00C2\u00A0 an\u00C2\u00A0 excitation\u00C2\u00A0 wavelength\u00C2\u00A0 of\u00C2\u00A0 465\u00C2\u00A0 nm\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 BMG\u00C2\u00A0 Labtechnologies\u00C2\u00A0 Fluorstar\u00C2\u00A0 plate\u00C2\u00A0 reader.\u00C2\u00A0 \u00C2\u00A0 Unknown\u00C2\u00A0 concentrations\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Neut\u00C2\u00A0were\u00C2\u00A0calculated\u00C2\u00A0from\u00C2\u00A0their\u00C2\u00A0respective\u00C2\u00A0standard\u00C2\u00A0curves.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 3.3.5\u00C2\u00A0 Ammonium\u00C2\u00A0 ferrothiocyanate\u00C2\u00A0 assay\u00C2\u00A0 for\u00C2\u00A0 measuring\u00C2\u00A0 polyethylene\u00C2\u00A0 glycol\u00C2\u00A0 content\u00C2\u00A0in\u00C2\u00A0LNPs\u00C2\u00A0\u00C2\u00A0 Ammonium\u00C2\u00A0ferrothiocyanate\u00C2\u00A0(AF)\u00C2\u00A0was\u00C2\u00A0prepared\u00C2\u00A0by\u00C2\u00A0dissolving\u00C2\u00A030.4\u00C2\u00A0g\u00C2\u00A0of\u00C2\u00A0NH4SCN\u00C2\u00A0and\u00C2\u00A027.0\u00C2\u00A0g\u00C2\u00A0of\u00C2\u00A0 FeCl3\u00E2\u0080\u00A26H2O\u00C2\u00A0in\u00C2\u00A0ultrapure\u00C2\u00A0water\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0final\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A01.0\u00C2\u00A0L.\u00C2\u00A0\u00C2\u00A0Standard\u00C2\u00A0solutions\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0(0\u00C2\u00A0to\u00C2\u00A0500\u00C2\u00A0 \u00C2\u00B5M)\u00C2\u00A0and\u00C2\u00A0DSPC\u00C2\u00A0(0\u00C2\u00A0to\u00C2\u00A04.5\u00C2\u00A0mM)\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0from\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0micelles\u00C2\u00A0and\u00C2\u00A0DSPC/Chol\u00C2\u00A0(2:1)\u00C2\u00A0SUV.\u00C2\u00A0 \u00C2\u00A0In\u00C2\u00A0 1.5\u00C2\u00A0mL\u00C2\u00A0 Eppendorf\u00C2\u00A0 tubes,\u00C2\u00A0 500\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 of\u00C2\u00A0 CHCl3\u00C2\u00A0were\u00C2\u00A0 added,\u00C2\u00A0 followed\u00C2\u00A0 by\u00C2\u00A0 500\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 of\u00C2\u00A0 AF\u00C2\u00A0 and\u00C2\u00A0 50\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 of\u00C2\u00A0 standard\u00C2\u00A0or\u00C2\u00A0unknown\u00C2\u00A0solution\u00C2\u00A0(assayed\u00C2\u00A0in\u00C2\u00A0triplicate).\u00C2\u00A0\u00C2\u00A0Tubes\u00C2\u00A0were\u00C2\u00A0closed\u00C2\u00A0and\u00C2\u00A0mixed\u00C2\u00A0vigorously\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 plate\u00C2\u00A0shaker\u00C2\u00A0for\u00C2\u00A030\u00C2\u00A0min,\u00C2\u00A0then\u00C2\u00A0tubes\u00C2\u00A0were\u00C2\u00A0spun\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0microcentrifuge\u00C2\u00A0at\u00C2\u00A013000\u00C2\u00A0rpm\u00C2\u00A0for\u00C2\u00A02\u00C2\u00A0min,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0 lower\u00C2\u00A0CHCl3\u00C2\u00A0layers\u00C2\u00A0(495\u00C2\u00A0\u00C2\u00B5L)\u00C2\u00A0were\u00C2\u00A0pipetted\u00C2\u00A0into\u00C2\u00A0separate\u00C2\u00A0Eppendorf\u00C2\u00A0tubes.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0absorbances\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0 CHCl3\u00C2\u00A0solutions\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0at\u00C2\u00A0530\u00C2\u00A0nm.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0expected\u00C2\u00A0absorbances\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0unknown\u00C2\u00A0samples\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0 were\u00C2\u00A0 calculated\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 known\u00C2\u00A0 DSPC\u00C2\u00A0 concentrations\u00C2\u00A0 from\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A055\u00C2\u00A0\u00C2\u00A0 scintillation\u00C2\u00A0 counting,\u00C2\u00A0 and\u00C2\u00A0 excess\u00C2\u00A0 absorbances\u00C2\u00A0 at\u00C2\u00A0 530\u00C2\u00A0 nm\u00C2\u00A0 in\u00C2\u00A0 each\u00C2\u00A0 case\u00C2\u00A0 were\u00C2\u00A0 attributed\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0of\u00C2\u00A0PEG.\u00C2\u00A0\u00C2\u00A0PEG\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0unknowns\u00C2\u00A0were\u00C2\u00A0calculated\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0excess\u00C2\u00A0absorbance\u00C2\u00A0 values.\u00C2\u00A0\u00C2\u00A0 3.3.6\u00C2\u00A0 Ab\u00C2\u00A0 biotinylation\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 nickel\u00C2\u00A0 immobilized\u00C2\u00A0metal\u00C2\u00A0 affinity\u00C2\u00A0 chromatography\u00C2\u00A0 column\u00C2\u00A0 Immobilized\u00C2\u00A0iminodiacetic\u00C2\u00A0acid\u00C2\u00A0gel\u00C2\u00A0was\u00C2\u00A0transferred\u00C2\u00A0into\u00C2\u00A0a\u00C2\u00A01.5\u00C2\u00A0mL\u00C2\u00A0polypropylene\u00C2\u00A0column\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0 final\u00C2\u00A0 gel\u00C2\u00A0 bed\u00C2\u00A0 volume\u00C2\u00A0 of\u00C2\u00A0 500\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 then\u00C2\u00A0 rinsed\u00C2\u00A0with\u00C2\u00A0 5.0\u00C2\u00A0mL\u00C2\u00A0 of\u00C2\u00A0 ultrapure\u00C2\u00A0water.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 gel\u00C2\u00A0was\u00C2\u00A0 then\u00C2\u00A0 chelated\u00C2\u00A0with\u00C2\u00A0nickel\u00C2\u00A0by\u00C2\u00A0adding\u00C2\u00A05.0\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A050\u00C2\u00A0mM\u00C2\u00A0ammonium\u00C2\u00A0nickel(II)\u00C2\u00A0sulfate\u00C2\u00A0and\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0 additional\u00C2\u00A05.0\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0water.\u00C2\u00A0\u00C2\u00A0If\u00C2\u00A0not\u00C2\u00A0used\u00C2\u00A0immediately,\u00C2\u00A0the\u00C2\u00A0resultant\u00C2\u00A0nickel\u00C2\u00A0immobilized\u00C2\u00A0metal\u00C2\u00A0affinity\u00C2\u00A0 chromatography\u00C2\u00A0(NIMAC)\u00C2\u00A0columns\u00C2\u00A0were\u00C2\u00A0stored\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0oC\u00C2\u00A0in\u00C2\u00A00.02\u00C2\u00A0%\u00C2\u00A0sodium\u00C2\u00A0azide.\u00C2\u00A0\u00C2\u00A0Otherwise,\u00C2\u00A0columns\u00C2\u00A0 were\u00C2\u00A0equilibrated\u00C2\u00A0with\u00C2\u00A05.0\u00C2\u00A0mL\u00C2\u00A0binding\u00C2\u00A0buffer\u00C2\u00A0 (BB;\u00C2\u00A025\u00C2\u00A0mM\u00C2\u00A0HEPES,\u00C2\u00A01\u00C2\u00A0M\u00C2\u00A0NaCl,\u00C2\u00A0pH\u00C2\u00A07.4).\u00C2\u00A0 \u00C2\u00A0Separately,\u00C2\u00A0 500\u00EF\u0080\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A010\u00C2\u00A0mg/mL\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A01250\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A02x\u00C2\u00A0BB\u00C2\u00A0and\u00C2\u00A0750\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0ultrapure\u00C2\u00A0water.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0future\u00C2\u00A0 analysis,\u00C2\u00A0100\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0 this\u00C2\u00A0solution\u00C2\u00A0were\u00C2\u00A0set\u00C2\u00A0aside,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 remaining\u00C2\u00A02400\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0were\u00C2\u00A0 loaded\u00C2\u00A0onto\u00C2\u00A0 the\u00C2\u00A0 column\u00C2\u00A0by\u00C2\u00A0eluting\u00C2\u00A0the\u00C2\u00A0solution\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0gel\u00C2\u00A0bed\u00C2\u00A0twice.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0column\u00C2\u00A0was\u00C2\u00A0then\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A04\u00C2\u00A0x\u00C2\u00A0 500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0BB.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0biotinylation,\u00C2\u00A0 the\u00C2\u00A0desired\u00C2\u00A0mole\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0 measured\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0stock\u00C2\u00A0DMSO\u00C2\u00A0solution\u00C2\u00A0and\u00C2\u00A0was\u00C2\u00A0diluted\u00C2\u00A0to\u00C2\u00A01.0\u00C2\u00A0mL\u00C2\u00A0in\u00C2\u00A0cold\u00C2\u00A0BB.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0 NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0was\u00C2\u00A0equilibrated\u00C2\u00A0across\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0by\u00C2\u00A0adding\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0solution\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0column,\u00C2\u00A0 allowing\u00C2\u00A0the\u00C2\u00A0eluent\u00C2\u00A0to\u00C2\u00A0drain\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0solution,\u00C2\u00A0vortexing\u00C2\u00A0briefly,\u00C2\u00A0then\u00C2\u00A0another\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0 aliquot\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0column.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0five\u00C2\u00A0such\u00C2\u00A0aliquots,\u00C2\u00A0the\u00C2\u00A0entire\u00C2\u00A01.0\u00C2\u00A0mL\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0and\u00C2\u00A0allowed\u00C2\u00A0 to\u00C2\u00A0drain\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0by\u00C2\u00A0gravity\u00C2\u00A0for\u00C2\u00A045\u00C2\u00A0min,\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0being\u00C2\u00A0refilled\u00C2\u00A0as\u00C2\u00A0necessary\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0 drained\u00C2\u00A0solution.\u00C2\u00A0\u00C2\u00A0Next,\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0was\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A05\u00C2\u00A0x\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0BB\u00C2\u00A0to\u00C2\u00A0remove\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin.\u00C2\u00A0\u00C2\u00A0Rtx\u00C2\u00A0 was\u00C2\u00A0either\u00C2\u00A0eluted\u00C2\u00A0with\u00C2\u00A0imidazole\u00C2\u00A0or\u00C2\u00A0ethylene\u00C2\u00A0diamine\u00C2\u00A0tetraacetic\u00C2\u00A0acid\u00C2\u00A0(EDTA).\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0imidazole\u00C2\u00A0elution,\u00C2\u00A0 the\u00C2\u00A0 column\u00C2\u00A0was\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 3\u00C2\u00A0 x\u00C2\u00A0 500\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 elution\u00C2\u00A0 buffer\u00C2\u00A0 (EB;\u00C2\u00A0 25\u00C2\u00A0mM\u00C2\u00A0HEPES,\u00C2\u00A0 1\u00C2\u00A0M\u00C2\u00A0NaCl,\u00C2\u00A0 200\u00C2\u00A0mM\u00C2\u00A0 imidazole,\u00C2\u00A0pH\u00C2\u00A07.4).\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0final\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0aliquot\u00C2\u00A0of\u00C2\u00A0EB\u00C2\u00A0was\u00C2\u00A0passed\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0three\u00C2\u00A0times,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A056\u00C2\u00A0\u00C2\u00A0 four\u00C2\u00A0resulting\u00C2\u00A0fractions\u00C2\u00A0were\u00C2\u00A0pooled.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0elution\u00C2\u00A0with\u00C2\u00A0EDTA,\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0was\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A04\u00C2\u00A0x\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0 stripping\u00C2\u00A0buffer\u00C2\u00A0 (SB;\u00C2\u00A025\u00C2\u00A0mM\u00C2\u00A0HEPES,\u00C2\u00A01\u00C2\u00A0M\u00C2\u00A0NaCl,\u00C2\u00A050\u00C2\u00A0mM\u00C2\u00A0 EDTA,\u00C2\u00A0pH\u00C2\u00A07.4)\u00C2\u00A0 and\u00C2\u00A0 all\u00C2\u00A0 four\u00C2\u00A0 fractions\u00C2\u00A0were\u00C2\u00A0 pooled.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0CBQCA\u00C2\u00A0assay\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0 groups\u00C2\u00A0 per\u00C2\u00A0 Rtx\u00C2\u00A0molecule\u00C2\u00A0was\u00C2\u00A0 calculated\u00C2\u00A0 using\u00C2\u00A0 HABA/avidin\u00C2\u00A0 (see\u00C2\u00A0 below)\u00C2\u00A0 and\u00C2\u00A0 CBQCA\u00C2\u00A0 assay\u00C2\u00A0 data.\u00C2\u00A0\u00C2\u00A0 Columns\u00C2\u00A0were\u00C2\u00A0stripped\u00C2\u00A0of\u00C2\u00A0Ni2+\u00C2\u00A0by\u00C2\u00A0treating\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0total\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A05.0\u00C2\u00A0mL\u00C2\u00A0SB,\u00C2\u00A0then\u00C2\u00A0re\u00E2\u0080\u0090chelated\u00C2\u00A0with\u00C2\u00A0 nickel\u00C2\u00A0and\u00C2\u00A0stored\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0above.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 3.3.7\u00C2\u00A0 Ab\u00C2\u00A0biotinylation\u00C2\u00A0in\u00C2\u00A0solution\u00C2\u00A0 As\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0procedure\u00C2\u00A0above,\u00C2\u00A0500\u00EF\u0080\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A010\u00C2\u00A0mg/mL\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A01250\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A02x\u00C2\u00A0BB\u00C2\u00A0 and\u00C2\u00A0 750\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 of\u00C2\u00A0 ultrapure\u00C2\u00A0 water.\u00C2\u00A0 \u00C2\u00A0 Depending\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 mole\u00C2\u00A0 excess\u00C2\u00A0 of\u00C2\u00A0 NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 to\u00C2\u00A0 Ab,\u00C2\u00A0 the\u00C2\u00A0 appropriate\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0stock\u00C2\u00A0solution\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0in\u00C2\u00A0DMSO\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0and\u00C2\u00A0diluted\u00C2\u00A0to\u00C2\u00A01.0\u00C2\u00A0 mL\u00C2\u00A0 in\u00C2\u00A0 cold\u00C2\u00A0 BB.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 solutions\u00C2\u00A0 were\u00C2\u00A0 combined\u00C2\u00A0 and\u00C2\u00A0 incubated\u00C2\u00A0 at\u00C2\u00A0 room\u00C2\u00A0 temperature\u00C2\u00A0for\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0with\u00C2\u00A0stirring.\u00C2\u00A0\u00C2\u00A0Biotinylated\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0purified\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0PD\u00E2\u0080\u009010\u00C2\u00A0column\u00C2\u00A0pre\u00E2\u0080\u0090equilibrated\u00C2\u00A0 with\u00C2\u00A0 BB.\u00C2\u00A0 \u00C2\u00A0 Fractions\u00C2\u00A0 (500\u00C2\u00A0 \u00C2\u00B5L)\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090containing\u00C2\u00A0 fractions\u00C2\u00A0 (as\u00C2\u00A0 determined\u00C2\u00A0 by\u00C2\u00A0 measuring\u00C2\u00A0absorbances\u00C2\u00A0at\u00C2\u00A0280\u00C2\u00A0nm)\u00C2\u00A0were\u00C2\u00A0pooled.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Rtx\u00C2\u00A0molecule\u00C2\u00A0was\u00C2\u00A0 calculated\u00C2\u00A0as\u00C2\u00A0per\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0method.\u00C2\u00A0 3.3.8\u00C2\u00A0 HABA/avidin\u00C2\u00A0assay\u00C2\u00A0for\u00C2\u00A0measuring\u00C2\u00A0the\u00C2\u00A0extent\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0biotinylation\u00C2\u00A0 HABA/avidin\u00C2\u00A0reagent\u00C2\u00A0was\u00C2\u00A0reconstituted\u00C2\u00A0with\u00C2\u00A0ultrapure\u00C2\u00A0water\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0instructions\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0manufacturer.\u00C2\u00A0 \u00C2\u00A0 Biotin\u00C2\u00A0 standards\u00C2\u00A0 ranging\u00C2\u00A0 from\u00C2\u00A0 0\u00C2\u00A0 to\u00C2\u00A0 150\u00C2\u00A0 \u00C2\u00B5M\u00C2\u00A0were\u00C2\u00A0 prepared\u00C2\u00A0 in\u00C2\u00A0 BB,\u00C2\u00A0 EB,\u00C2\u00A0 or\u00C2\u00A0 SB\u00C2\u00A0 depending\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0biotinylation\u00C2\u00A0and\u00C2\u00A0elution\u00C2\u00A0 (see\u00C2\u00A0above).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0a\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plate,\u00C2\u00A0140\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0 HABA/avidin\u00C2\u00A0solution\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0wells\u00C2\u00A0as\u00C2\u00A0required,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0absorbances\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0wells\u00C2\u00A0were\u00C2\u00A0read\u00C2\u00A0at\u00C2\u00A0 500\u00C2\u00A0nm\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0Thermo\u00C2\u00A0Scientific\u00C2\u00A0absorbance\u00C2\u00A0microplate\u00C2\u00A0reader.\u00C2\u00A0 \u00C2\u00A0Next,\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0each\u00C2\u00A0 standard\u00C2\u00A0or\u00C2\u00A0unknown\u00C2\u00A0sample\u00C2\u00A0(assayed\u00C2\u00A0in\u00C2\u00A0triplicate)\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0wells.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0plate\u00C2\u00A0was\u00C2\u00A0shaken\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 plate\u00C2\u00A0 shaker\u00C2\u00A0 for\u00C2\u00A0 3\u00C2\u00A0min\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 absorbances\u00C2\u00A0were\u00C2\u00A0 read\u00C2\u00A0 again\u00C2\u00A0 at\u00C2\u00A0 500\u00C2\u00A0 nm\u00C2\u00A0 in\u00C2\u00A0 triplicate.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A057\u00C2\u00A0\u00C2\u00A0 average\u00C2\u00A0absorbance\u00C2\u00A0of\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0after\u00C2\u00A0sample\u00C2\u00A0addition\u00C2\u00A0was\u00C2\u00A0subtracted\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0absorbance\u00C2\u00A0before\u00C2\u00A0 to\u00C2\u00A0obtain\u00C2\u00A0\u00EF\u0081\u0084A,\u00C2\u00A0which\u00C2\u00A0was\u00C2\u00A0plotted\u00C2\u00A0against\u00C2\u00A0biotin\u00C2\u00A0 concentration\u00C2\u00A0 to\u00C2\u00A0generate\u00C2\u00A0a\u00C2\u00A0 standard\u00C2\u00A0 curve.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 number\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Rtx\u00C2\u00A0molecule\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0by\u00C2\u00A0dividing\u00C2\u00A0the\u00C2\u00A0biotin\u00C2\u00A0concentration\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0 Rtx\u00C2\u00A0concentration\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0CBQCA\u00C2\u00A0assay.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 3.3.9\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0Coupling\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADbiotin\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00ADLNP\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0desired\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00A0(in\u00C2\u00A0Neut\u00E2\u0080\u0090LNP),\u00C2\u00A0the\u00C2\u00A0necessary\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0dropwise\u00C2\u00A0with\u00C2\u00A0vortexing\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0solution\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0prepared\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0 procedure\u00C2\u00A0above.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0coupling\u00C2\u00A0reaction\u00C2\u00A0was\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0proceed\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0oC\u00C2\u00A0with\u00C2\u00A0stirring\u00C2\u00A0for\u00C2\u00A01\u00C2\u00A0to\u00C2\u00A072\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0resulting\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0were\u00C2\u00A0purified\u00C2\u00A0over\u00C2\u00A0Sepharose\u00C2\u00A0CL\u00E2\u0080\u00904B\u00C2\u00A0columns\u00C2\u00A0pre\u00E2\u0080\u0090equilibrated\u00C2\u00A0with\u00C2\u00A0HBS.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0 mean\u00C2\u00A0 vesicle\u00C2\u00A0 diameter\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0 between\u00C2\u00A0 125\u00C2\u00A0 and\u00C2\u00A0 140\u00C2\u00A0 nm\u00C2\u00A0 as\u00C2\u00A0 determined\u00C2\u00A0 by\u00C2\u00A0QLS.\u00C2\u00A0 \u00C2\u00A0 Lipid\u00C2\u00A0 concentrations\u00C2\u00A0 in\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 were\u00C2\u00A0 determined\u00C2\u00A0 with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP\u00C2\u00A0 by\u00C2\u00A0 liquid\u00C2\u00A0 scintillation\u00C2\u00A0 counting,\u00C2\u00A0and\u00C2\u00A0protein\u00C2\u00A0concentrations\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0using\u00C2\u00A0CBQCA.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00A0 in\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0calculated\u00C2\u00A0from\u00C2\u00A0lipid\u00C2\u00A0concentrations\u00C2\u00A0by\u00C2\u00A0assuming\u00C2\u00A0no\u00C2\u00A0change\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0Neut\u00E2\u0080\u0090to\u00E2\u0080\u0090DSPC\u00C2\u00A0ratio\u00C2\u00A0 as\u00C2\u00A0a\u00C2\u00A0 result\u00C2\u00A0of\u00C2\u00A0 coupling.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0expected\u00C2\u00A0 contribution\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0CBQCA\u00C2\u00A0 signal\u00C2\u00A0by\u00C2\u00A0Neut\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0 based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0Neut\u00C2\u00A0standard\u00C2\u00A0curve,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0excess\u00C2\u00A0signal\u00C2\u00A0was\u00C2\u00A0attributed\u00C2\u00A0to\u00C2\u00A0Rtx,\u00C2\u00A0whose\u00C2\u00A0concentration\u00C2\u00A0 was\u00C2\u00A0calculated\u00C2\u00A0from\u00C2\u00A0this\u00C2\u00A0excess\u00C2\u00A0signal.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0determined\u00C2\u00A0 using\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0assumptions\u00C2\u00A0as\u00C2\u00A0for\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0above\u00C2\u00A0(Section\u00C2\u00A03.3.3).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 3.4\u00C2\u00A0 Results\u00C2\u00A0 3.4.1\u00C2\u00A0 Overview\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 improved\u00C2\u00A0methodology\u00C2\u00A0 for\u00C2\u00A0creating\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0 valence\u00C2\u00A0 Given\u00C2\u00A0 the\u00C2\u00A0 considerations\u00C2\u00A0 outlined\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 3.2,\u00C2\u00A0 we\u00C2\u00A0 have\u00C2\u00A0 developed\u00C2\u00A0 and\u00C2\u00A0 optimized\u00C2\u00A0 an\u00C2\u00A0 improved\u00C2\u00A0methodology\u00C2\u00A0 for\u00C2\u00A0 producing\u00C2\u00A0 smaller\u00C2\u00A0 amounts\u00C2\u00A0 of\u00C2\u00A0 several\u00C2\u00A0 different\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 valences\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A058\u00C2\u00A0\u00C2\u00A0 manner\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0time\u00E2\u0080\u0090efficient,\u00C2\u00A0reproducible,\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0high\u00C2\u00A0yields\u00C2\u00A0of\u00C2\u00A0coupled\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0Table\u00C2\u00A03.1\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0new\u00C2\u00A0methodology\u00C2\u00A0provides\u00C2\u00A0 yields\u00C2\u00A0of\u00C2\u00A0 coupled\u00C2\u00A0Ab\u00C2\u00A0 as\u00C2\u00A0high\u00C2\u00A0 as\u00C2\u00A0 80%\u00C2\u00A0with\u00C2\u00A0 far\u00C2\u00A0 less\u00C2\u00A0Ab\u00C2\u00A0 modification\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0previous\u00C2\u00A0post\u00E2\u0080\u0090insertion\u00C2\u00A0method,\u00C2\u00A0which\u00C2\u00A0produces\u00C2\u00A0overall\u00C2\u00A0yields\u00C2\u00A0of\u00C2\u00A059%.\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A03.1\u00C2\u00A0\u00C2\u00A0Comparison\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0yields\u00C2\u00A0between\u00C2\u00A0current\u00C2\u00A0and\u00C2\u00A0previous\u00C2\u00A0methods\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0productiona.\u00C2\u00A0 Improved\u00C2\u00A0biotin\u00E2\u0080\u0090Neut\u00C2\u00A0 methodology\u00C2\u00A0(Figure\u00C2\u00A03.1)\u00C2\u00A0 Recovered\u00C2\u00A0Ab\u00C2\u00A0/\u00C2\u00A0%\u00C2\u00A0 Cumulative\u00C2\u00A0yield\u00C2\u00A0/\u00C2\u00A0%\u00C2\u00A0 Notes\u00C2\u00A0 Ab\u00C2\u00A0biotinylation\u00C2\u00A0(NIMAC)\u00C2\u00A0 96\u00C2\u00A0\u00C2\u00B1\u00C2\u00A06\u00C2\u00A0 96\u00C2\u00A0\u00C2\u00B1\u00C2\u00A06\u00C2\u00A0 1\u00E2\u0080\u00902\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Ab\u00C2\u00A0 Coupling\u00C2\u00A0to\u00C2\u00A0Neut\u00E2\u0080\u0090LNPb\u00C2\u00A0 84\u00C2\u00A0\u00C2\u00B1\u00C2\u00A06\u00C2\u00A0 80\u00C2\u00A0\u00C2\u00B1\u00C2\u00A07\u00C2\u00A0 Ab\u00C2\u00A0loss\u00C2\u00A0from\u00C2\u00A0purification\u00C2\u00A0 only\u00C2\u00A0 Previous\u00C2\u00A0thiol\u00E2\u0080\u0090Mal\u00C2\u00A0 post\u00E2\u0080\u0090insertion\u00C2\u00A0method\u00C2\u00A0 Recovered\u00C2\u00A0Ab\u00C2\u00A0/\u00C2\u00A0%\u00C2\u00A0 Cumulative\u00C2\u00A0yield\u00C2\u00A0/\u00C2\u00A0%\u00C2\u00A0 Notes\u00C2\u00A0 Ab\u00C2\u00A0thiolationc\u00C2\u00A0 80\u00C2\u00A0\u00C2\u00B1\u00C2\u00A04\u00C2\u00A0 80\u00C2\u00A0\u00C2\u00B1\u00C2\u00A04\u00C2\u00A0 3\u00E2\u0080\u00907\u00C2\u00A0thiol\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Ab\u00C2\u00A0 Coupling\u00C2\u00A0to\u00C2\u00A0micelles\u00C2\u00A0 and\u00C2\u00A0post\u00E2\u0080\u0090insertion\u00C2\u00A0of\u00C2\u00A0Abd\u00C2\u00A0 74\u00C2\u00A0\u00C2\u00B1\u00C2\u00A06\u00C2\u00A0 59\u00C2\u00A0\u00C2\u00B1\u00C2\u00A06\u00C2\u00A0 Ab\u00C2\u00A0loss\u00C2\u00A0from\u00C2\u00A0incomplete\u00C2\u00A0 coupling,\u00C2\u00A0post\u00E2\u0080\u0090insertion,\u00C2\u00A0 and\u00C2\u00A0purification\u00C2\u00A0 aAll\u00C2\u00A0values\u00C2\u00A0are\u00C2\u00A0averaged\u00C2\u00A0over\u00C2\u00A0three\u00C2\u00A0experiments\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0SD.\u00C2\u00A0 bMole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00A0=\u00C2\u00A00.5;\u00C2\u00A0coupling\u00C2\u00A0time\u00C2\u00A0=\u00C2\u00A072\u00C2\u00A0h.\u00C2\u00A0 cUsing\u00C2\u00A0Traut\u00E2\u0080\u0099s\u00C2\u00A0reagent\u00C2\u00A0 followed\u00C2\u00A0by\u00C2\u00A0purification\u00C2\u00A0over\u00C2\u00A0a\u00C2\u00A0PD\u00E2\u0080\u009010\u00C2\u00A0column;\u00C2\u00A0 information\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0manufacturer\u00C2\u00A0states\u00C2\u00A0that\u00C2\u00A070\u00E2\u0080\u0090 95%\u00C2\u00A0recovery\u00C2\u00A0is\u00C2\u00A0typical.\u00C2\u00A0 dAb\u00E2\u0080\u0090to\u00E2\u0080\u0090DSPC\u00C2\u00A0 ratio\u00C2\u00A0 after\u00C2\u00A0 purification\u00C2\u00A0 was\u00C2\u00A0 ~88%\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 input\u00C2\u00A0 amount,\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 Ishida\u00C2\u00A0 et\u00C2\u00A0 al.142,\u00C2\u00A0 representing\u00C2\u00A0 incomplete\u00C2\u00A0 coupling\u00C2\u00A0and\u00C2\u00A0post\u00E2\u0080\u0090insertion.\u00C2\u00A0 \u00C2\u00A0Ab\u00C2\u00A0 (coupled\u00C2\u00A0 to\u00C2\u00A0 liposomes)\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0 subsequently\u00C2\u00A0 lost\u00C2\u00A0during\u00C2\u00A0purification\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 Sepharose\u00C2\u00A0CL\u00E2\u0080\u00904B\u00C2\u00A0column.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 3.1\u00C2\u00A0 summarizes\u00C2\u00A0 the\u00C2\u00A0 five\u00C2\u00A0 major\u00C2\u00A0 steps\u00C2\u00A0 of\u00C2\u00A0 methodology,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 ensuing\u00C2\u00A0 sections\u00C2\u00A0 describe\u00C2\u00A0 them\u00C2\u00A0 in\u00C2\u00A0 greater\u00C2\u00A0 detail.\u00C2\u00A0 \u00C2\u00A0 First,\u00C2\u00A0 [1]\u00C2\u00A0 DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0 and\u00C2\u00A0 DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 are\u00C2\u00A0 dissolved\u00C2\u00A0 in\u00C2\u00A0 HBS,\u00C2\u00A0 producing\u00C2\u00A0a\u00C2\u00A0suspension\u00C2\u00A0of\u00C2\u00A0micelles.\u00C2\u00A0\u00C2\u00A0[2]\u00C2\u00A0Biotin\u00C2\u00A0present\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0micelles\u00C2\u00A0is\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0 Neut,\u00C2\u00A0a\u00C2\u00A0deglycosylated\u00C2\u00A0analog\u00C2\u00A0of\u00C2\u00A0streptavidin\u00C2\u00A0with\u00C2\u00A0 low\u00C2\u00A0nonspecific\u00C2\u00A0binding.\u00C2\u00A0 \u00C2\u00A0 [3]\u00C2\u00A0Micellar\u00C2\u00A0 lipids\u00C2\u00A0and\u00C2\u00A0 Neut\u00E2\u0080\u0090lipid\u00C2\u00A0 complexes\u00C2\u00A0 become\u00C2\u00A0 post\u00E2\u0080\u0090inserted\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 outer\u00C2\u00A0 leaflet\u00C2\u00A0 of\u00C2\u00A0 SUVs\u00C2\u00A0 to\u00C2\u00A0 produce\u00C2\u00A0Neut\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0 SUVs\u00C2\u00A0are\u00C2\u00A0composed\u00C2\u00A0of\u00C2\u00A0DSPC/Chol\u00C2\u00A0(mole\u00C2\u00A0ratio\u00C2\u00A02:1;\u00C2\u00A0Chol\u00C2\u00A0not\u00C2\u00A0shown),\u00C2\u00A0and\u00C2\u00A0Neut\u00C2\u00A0is\u00C2\u00A0post\u00E2\u0080\u0090inserted\u00C2\u00A0rather\u00C2\u00A0 than\u00C2\u00A0Ab,\u00C2\u00A0which\u00C2\u00A0eliminates\u00C2\u00A0Ab\u00C2\u00A0wastage\u00C2\u00A0during\u00C2\u00A0post\u00E2\u0080\u0090insertion.\u00C2\u00A0\u00C2\u00A0[4]\u00C2\u00A0Separately,\u00C2\u00A0Ab\u00C2\u00A0is\u00C2\u00A0biotinylated\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 NIMAC\u00C2\u00A0support\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0Ab\u00E2\u0080\u0090biotin,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0well\u00E2\u0080\u0090controlled\u00C2\u00A0and\u00C2\u00A0reproducible\u00C2\u00A0Ab\u00C2\u00A0modification\u00C2\u00A0 with\u00C2\u00A0 very\u00C2\u00A0 high\u00C2\u00A0 Ab\u00C2\u00A0 recovery.\u00C2\u00A0 \u00C2\u00A0 [5]\u00C2\u00A0 Ab\u00E2\u0080\u0090biotin\u00C2\u00A0 and\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP\u00C2\u00A0 are\u00C2\u00A0 combined,\u00C2\u00A0 producing\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0 of\u00C2\u00A0 defined\u00C2\u00A0valence.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A059\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.1\u00C2\u00A0\u00C2\u00A0Overview\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0improved\u00C2\u00A0methodology\u00C2\u00A0for\u00C2\u00A0preparing\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs.\u00C2\u00A0 Please\u00C2\u00A0see\u00C2\u00A0text\u00C2\u00A0for\u00C2\u00A0details.\u00C2\u00A0 \u00C2\u00A0 3.4.2\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00ADLNP\u00C2\u00A0and\u00C2\u00A0measurement\u00C2\u00A0of\u00C2\u00A0protein\u00C2\u00A0content\u00C2\u00A0 Initially,\u00C2\u00A0a\u00C2\u00A0mixture\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0and\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0was\u00C2\u00A0post\u00E2\u0080\u0090inserted\u00C2\u00A0 into\u00C2\u00A0SUVs\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0 biotin\u00E2\u0080\u0090LNPs\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0numbers\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0LNP.\u00C2\u00A0 \u00C2\u00A0Separately,\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0biotinylated\u00C2\u00A0and\u00C2\u00A0 SUV Neut\u00E2\u0080\u0090LNP Ab\u00E2\u0080\u0090LNP Ab\u00E2\u0080\u0090biotinAb Biotin\u00E2\u0080\u0090Micelles Neut\u00E2\u0080\u0090Micelles DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin DSPE\u00E2\u0080\u0090PEG [1] [3] [2] [4] [5] HBS excess\u00C2\u00A0 Neut NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0/ NIMAC \u00E2\u0080\u00A2\u00C2\u00A060\u00C2\u00A0\u00C2\u00A0 saturated\u00C2\u00A0with\u00C2\u00A0Neut\u00C2\u00A0to\u00C2\u00A0make\u00C2\u00A0Ab\u00E2\u0080\u0090Neut,\u00C2\u00A0which\u00C2\u00A0was\u00C2\u00A0then\u00C2\u00A0coupled\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0various\u00C2\u00A0biotin\u00E2\u0080\u0090LNPs\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valence.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0approach\u00C2\u00A0was\u00C2\u00A0problematic,\u00C2\u00A0however,\u00C2\u00A0since\u00C2\u00A0each\u00C2\u00A0Neut\u00C2\u00A0molecule\u00C2\u00A0 contains\u00C2\u00A0 four\u00C2\u00A0biotin\u00E2\u0080\u0090binding\u00C2\u00A0 sites,\u00C2\u00A0and\u00C2\u00A0adding\u00C2\u00A0Neut\u00C2\u00A0 to\u00C2\u00A0Ab\u00E2\u0080\u0090biotin\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0mixture\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090Neut\u00C2\u00A0 species\u00C2\u00A0 containing\u00C2\u00A0between\u00C2\u00A00\u00C2\u00A0and\u00C2\u00A04\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0Neut.\u00C2\u00A0 \u00C2\u00A0Yields\u00C2\u00A0were\u00C2\u00A0 reduced\u00C2\u00A0because\u00C2\u00A0 (Ab)4\u00E2\u0080\u0090 Neut\u00C2\u00A0 could\u00C2\u00A0not\u00C2\u00A0bind\u00C2\u00A0 to\u00C2\u00A0biotin\u00E2\u0080\u0090LNP,\u00C2\u00A0 and\u00C2\u00A0 separation\u00C2\u00A0of\u00C2\u00A0 (Ab)4\u00E2\u0080\u0090Neut\u00C2\u00A0 and\u00C2\u00A0 free\u00C2\u00A0Neut\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0desired\u00C2\u00A0 species\u00C2\u00A0was\u00C2\u00A0 inefficient.\u00C2\u00A0 \u00C2\u00A0Moreover,\u00C2\u00A0some\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0contained\u00C2\u00A0more\u00C2\u00A0than\u00C2\u00A0one\u00C2\u00A0biotin\u00C2\u00A0group\u00C2\u00A0and\u00C2\u00A0 thus\u00C2\u00A0 bound\u00C2\u00A0more\u00C2\u00A0 than\u00C2\u00A0 one\u00C2\u00A0Neut\u00C2\u00A0molecule,\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 complex\u00C2\u00A0mixture\u00C2\u00A0 of\u00C2\u00A0 adducts\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown).\u00C2\u00A0 It\u00C2\u00A0was\u00C2\u00A0 therefore\u00C2\u00A0more\u00C2\u00A0efficient\u00C2\u00A0 to\u00C2\u00A0couple\u00C2\u00A0Neut\u00C2\u00A0 to\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin.\u00C2\u00A0 \u00C2\u00A0Before\u00C2\u00A0post\u00E2\u0080\u0090insertion,\u00C2\u00A0 biotin\u00E2\u0080\u0090micelles\u00C2\u00A0were\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0threefold\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0Neut,\u00C2\u00A0forming\u00C2\u00A0Neut\u00E2\u0080\u0090micelles\u00C2\u00A0(reaction\u00C2\u00A0[2]\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A03.1).\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0post\u00E2\u0080\u0090insertion,\u00C2\u00A0a\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A04\u00C2\u00A0mol\u00C2\u00A0%\u00C2\u00A0PEGylated\u00C2\u00A0lipid\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0DSPC;\u00C2\u00A0 this\u00C2\u00A0percentage\u00C2\u00A0was\u00C2\u00A0selected\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0basis\u00C2\u00A0that\u00C2\u00A0higher\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0(above\u00C2\u00A0~7\u00C2\u00A0%)\u00C2\u00A0are\u00C2\u00A0known\u00C2\u00A0 to\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0unwanted\u00C2\u00A0effects\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0dissociation\u00C2\u00A0back\u00C2\u00A0to\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0micelles153.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00A0 molecules\u00C2\u00A0added\u00C2\u00A0per\u00C2\u00A0SUV\u00C2\u00A0was\u00C2\u00A0controlled\u00C2\u00A0by\u00C2\u00A0preparing\u00C2\u00A0Neut\u00E2\u0080\u0090micelles\u00C2\u00A0containing\u00C2\u00A0different\u00C2\u00A0 fractions\u00C2\u00A0 of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090Neut\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0(reaction\u00C2\u00A0[3]\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A03.1)\u00C2\u00A0and\u00C2\u00A0several\u00C2\u00A0different\u00C2\u00A0post\u00E2\u0080\u0090 insertion\u00C2\u00A0reactions\u00C2\u00A0were\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0simultaneously.\u00C2\u00A0\u00C2\u00A0Using\u00C2\u00A0this\u00C2\u00A0strategy,\u00C2\u00A0Figure\u00C2\u00A03.2A\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0 number\u00C2\u00A0of\u00C2\u00A0added\u00C2\u00A0Neut\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0SUV\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0controlled\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0precise\u00C2\u00A0manner.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 Neut\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0depended\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 initial\u00C2\u00A0mole\u00C2\u00A0percent\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0used;\u00C2\u00A011\u00C2\u00A0 Neut\u00C2\u00A0per\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0using\u00C2\u00A00.5\u00C2\u00A0mole\u00C2\u00A0%\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin,\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00A0per\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP\u00C2\u00A0increased\u00C2\u00A0to\u00C2\u00A0130\u00C2\u00A0when\u00C2\u00A020\u00C2\u00A0mole\u00C2\u00A0%\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0all\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0 shown\u00C2\u00A0conditions).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A061\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.2\u00C2\u00A0\u00C2\u00A0Control\u00C2\u00A0over\u00C2\u00A0Neut\u00C2\u00A0and\u00C2\u00A0PEG\u00C2\u00A0content\u00C2\u00A0during\u00C2\u00A0the\u00C2\u00A0preparation\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNPs.\u00C2\u00A0 (A)\u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Neut\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0(product\u00C2\u00A0of\u00C2\u00A0step\u00C2\u00A0[3]\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A03.1)\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0controlled\u00C2\u00A0by\u00C2\u00A0varying\u00C2\u00A0the\u00C2\u00A0mole\u00C2\u00A0 percent\u00C2\u00A0 of\u00C2\u00A0 DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 present\u00C2\u00A0 in\u00C2\u00A0 biotin\u00E2\u0080\u0090micelles\u00C2\u00A0 (step\u00C2\u00A0 [1]).\u00C2\u00A0 \u00C2\u00A0 (B)\u00C2\u00A0 The\u00C2\u00A0 Neut\u00C2\u00A0 content\u00C2\u00A0 of\u00C2\u00A0 Neut\u00E2\u0080\u0090micelles\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 significantly\u00C2\u00A0 influence\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0 amount\u00C2\u00A0 of\u00C2\u00A0 post\u00E2\u0080\u0090inserted\u00C2\u00A0 PEGylated\u00C2\u00A0 lipid\u00C2\u00A0 in\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP;\u00C2\u00A0 in\u00C2\u00A0 all\u00C2\u00A0 cases,\u00C2\u00A0 approximately\u00C2\u00A0 4\u00C2\u00A0%\u00C2\u00A0 PEGylated\u00C2\u00A0lipid\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0DSPC.\u00C2\u00A0\u00C2\u00A0Values\u00C2\u00A0for\u00C2\u00A0mole\u00C2\u00A0%\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0in\u00C2\u00A0(A)\u00C2\u00A0and\u00C2\u00A0(B)\u00C2\u00A0are\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0 total\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0moles\u00C2\u00A0of\u00C2\u00A0PEGylated\u00C2\u00A0lipid\u00C2\u00A0(DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0+\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin).\u00C2\u00A0 \u00C2\u00A0 Protein\u00C2\u00A0content\u00C2\u00A0in\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0other\u00C2\u00A0cases\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0CBQCA\u00C2\u00A0assay\u00C2\u00A0(see\u00C2\u00A0 Section\u00C2\u00A03.3.5).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0direct\u00C2\u00A0advantage\u00C2\u00A0of\u00C2\u00A0employing\u00C2\u00A0this\u00C2\u00A0fluorescence\u00E2\u0080\u0090based\u00C2\u00A0assay\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0no\u00C2\u00A0 interference\u00C2\u00A0 from\u00C2\u00A0 liposomes\u00C2\u00A0 or\u00C2\u00A0micelles.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 verify\u00C2\u00A0 the\u00C2\u00A0 lack\u00C2\u00A0 of\u00C2\u00A0 interference,\u00C2\u00A0mixtures\u00C2\u00A0 of\u00C2\u00A0 known\u00C2\u00A0 amounts\u00C2\u00A0of\u00C2\u00A0DSPC/Chol\u00C2\u00A0SUV,\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG,\u00C2\u00A0and\u00C2\u00A0Ab\u00C2\u00A0were\u00C2\u00A0 created\u00C2\u00A0 in\u00C2\u00A0various\u00C2\u00A0 combinations,\u00C2\u00A0and\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0Ab\u00C2\u00A0concentrations\u00C2\u00A0obtained\u00C2\u00A0were\u00C2\u00A0equivalent\u00C2\u00A0 regardless\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 lipids\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 is\u00C2\u00A0 in\u00C2\u00A0contrast\u00C2\u00A0 to\u00C2\u00A0other\u00C2\u00A0protein\u00C2\u00A0assays\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0bicinchoninic\u00C2\u00A0acid\u00C2\u00A0 (BCA)\u00C2\u00A0assay\u00C2\u00A0 (see\u00C2\u00A0Section\u00C2\u00A02.3.2),\u00C2\u00A0an\u00C2\u00A0absorbance\u00E2\u0080\u0090based\u00C2\u00A0assay\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 commonly\u00C2\u00A0employed\u00C2\u00A0 to\u00C2\u00A0measure\u00C2\u00A0protein\u00C2\u00A0 content\u00C2\u00A0 in\u00C2\u00A0 liposomes154,\u00C2\u00A0 155,\u00C2\u00A0 although\u00C2\u00A0 variable\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 interference\u00C2\u00A0 from\u00C2\u00A0 lipids\u00C2\u00A0 are\u00C2\u00A0 commonly\u00C2\u00A0 observed,\u00C2\u00A0even\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0detergents156,\u00C2\u00A0157.\u00C2\u00A0 3.4.3\u00C2\u00A0 Direct\u00C2\u00A0measurement\u00C2\u00A0of\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0post\u00C2\u00ADinserted\u00C2\u00A0polyethylene\u00C2\u00A0glycol\u00C2\u00A0in\u00C2\u00A0Neut\u00C2\u00AD LNP\u00C2\u00A0 To\u00C2\u00A0enhance\u00C2\u00A0the\u00C2\u00A0reproducibility\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0procedure\u00C2\u00A0by\u00C2\u00A0thoroughly\u00C2\u00A0characterizing\u00C2\u00A0 intermediate\u00C2\u00A0 products,\u00C2\u00A0 the\u00C2\u00A0PEG\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0quantified\u00C2\u00A0 following\u00C2\u00A0post\u00E2\u0080\u0090insertion.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0 this,\u00C2\u00A0we\u00C2\u00A0 developed\u00C2\u00A0an\u00C2\u00A0assay\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0measurement\u00C2\u00A0of\u00C2\u00A0PEG\u00C2\u00A0content\u00C2\u00A0 in\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0or\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 0 50 100 150 0.5 5 10 20 N N eu t pe r\u00C2\u00A0N eu t\u00E2\u0080\u0090L N P\u00C2\u00A0 Mole\u00C2\u00A0%\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 A 2 3 4 5 0.5 5 10 20 M ol e\u00C2\u00A0% \u00C2\u00A0PE G w it h\u00C2\u00A0r es pe ct \u00C2\u00A0to \u00C2\u00A0DS PC \u00C2\u00A0 Mole\u00C2\u00A0%\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin B \u00E2\u0080\u00A2\u00C2\u00A062\u00C2\u00A0\u00C2\u00A0 method\u00C2\u00A0originally\u00C2\u00A0described\u00C2\u00A0for\u00C2\u00A0quantifying\u00C2\u00A0PEG\u00C2\u00A0 in\u00C2\u00A0PEGylated\u00C2\u00A0proteins151\u00C2\u00A0(see\u00C2\u00A0Section\u00C2\u00A03.3.6).\u00C2\u00A0 \u00C2\u00A0Table\u00C2\u00A0 3.2\u00C2\u00A0shows\u00C2\u00A0the\u00C2\u00A0PEG\u00C2\u00A0content\u00C2\u00A0during\u00C2\u00A0a\u00C2\u00A0sample\u00C2\u00A0purification\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNP,\u00C2\u00A0and\u00C2\u00A0as\u00C2\u00A0expected,\u00C2\u00A0the\u00C2\u00A0fractions\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 largest\u00C2\u00A0 mean\u00C2\u00A0 diameter\u00C2\u00A0 (those\u00C2\u00A0 containing\u00C2\u00A0 the\u00C2\u00A0 most\u00C2\u00A0 PEG)\u00C2\u00A0 were\u00C2\u00A0 eluted\u00C2\u00A0 first\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 Sepharose\u00C2\u00A0CL\u00E2\u0080\u00904B\u00C2\u00A0column.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0pooled\u00C2\u00A0fraction\u00C2\u00A0contained\u00C2\u00A0close\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0PEGylated\u00C2\u00A0lipid\u00C2\u00A0that\u00C2\u00A0 was\u00C2\u00A0input\u00C2\u00A0(4\u00C2\u00A0%\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0DSPC).\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A03.2B,\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0samples\u00C2\u00A0indicated\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0PEG\u00C2\u00A0 content\u00C2\u00A0was\u00C2\u00A0 consistently\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 range\u00C2\u00A0 (3.5\u00E2\u0080\u00934.1\u00C2\u00A0mole\u00C2\u00A0%\u00C2\u00A0PEG\u00C2\u00A0with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0DSPC),\u00C2\u00A0 regardless\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0mole\u00C2\u00A0%\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0initially\u00C2\u00A0used\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0shown\u00C2\u00A0conditions\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A03.2B,\u00C2\u00A0except\u00C2\u00A0between\u00C2\u00A00.5\u00C2\u00A0and\u00C2\u00A05\u00C2\u00A0mole\u00C2\u00A0%\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin,\u00C2\u00A0where\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.05).\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0assay\u00C2\u00A0was\u00C2\u00A0 also\u00C2\u00A0verified\u00C2\u00A0by\u00C2\u00A0mixing\u00C2\u00A0different\u00C2\u00A0known\u00C2\u00A0quantities\u00C2\u00A0of\u00C2\u00A0DSPC/Chol\u00C2\u00A0SUV\u00C2\u00A0and\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00C2\u00A0micelles,\u00C2\u00A0and\u00C2\u00A0 excellent\u00C2\u00A0 agreement\u00C2\u00A0 (within\u00C2\u00A0 5\u00C2\u00A0%)\u00C2\u00A0was\u00C2\u00A0 found\u00C2\u00A0 between\u00C2\u00A0 the\u00C2\u00A0measured\u00C2\u00A0 and\u00C2\u00A0 known\u00C2\u00A0 values\u00C2\u00A0 for\u00C2\u00A0 PEG\u00C2\u00A0 concentrations\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0DSPC\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0 Table\u00C2\u00A03.2\u00C2\u00A0\u00C2\u00A0PEG\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A00.5\u00C2\u00A0mL\u00C2\u00A0fractions\u00C2\u00A0during\u00C2\u00A0purification\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090SUVa.\u00C2\u00A0 Column\u00C2\u00A0 fraction\u00C2\u00A0 [DSPC]\u00C2\u00A0 /\u00C2\u00A0mM\u00C2\u00A0 Mole\u00C2\u00A0%\u00C2\u00A0PEG wrt\u00C2\u00A0DSPCb\u00C2\u00A0 Column\u00C2\u00A0 fraction\u00C2\u00A0 [DSPC]\u00C2\u00A0 /\u00C2\u00A0mM\u00C2\u00A0 Mole\u00C2\u00A0%\u00C2\u00A0PEG wrt\u00C2\u00A0DSPCb\u00C2\u00A0 1\u00C2\u00A0 3.5\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.3\u00C2\u00A0 4.4\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 5\u00C2\u00A0 5.0\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 3.7\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 2\u00C2\u00A0 9.0\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.7\u00C2\u00A0 4.4\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 6\u00C2\u00A0 3.2\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.3\u00C2\u00A0 3.7\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 3\u00C2\u00A0 9.8\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.7\u00C2\u00A0 3.9\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 pooled\u00C2\u00A0 6.7\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 3.8\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 4\u00C2\u00A0 6.2\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.5\u00C2\u00A0 3.8\u00C2\u00A0\u00C2\u00B1\u00C2\u00A00.4\u00C2\u00A0 aAll\u00C2\u00A0values\u00C2\u00A0are\u00C2\u00A0\u00C2\u00B1 SD.\u00C2\u00A0 bwrt:\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to.\u00C2\u00A0 \u00C2\u00A0 3.4.4\u00C2\u00A0 Ab\u00C2\u00A0 biotinylation\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 nickel\u00C2\u00A0 immobilized\u00C2\u00A0metal\u00C2\u00A0 affinity\u00C2\u00A0 chromatography\u00C2\u00A0 support\u00C2\u00A0 A\u00C2\u00A0major\u00C2\u00A0drawback\u00C2\u00A0of\u00C2\u00A0thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0chemistry\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0need\u00C2\u00A0to\u00C2\u00A0extensively\u00C2\u00A0thiolate\u00C2\u00A0the\u00C2\u00A0Ab,\u00C2\u00A0as\u00C2\u00A0 outlined\u00C2\u00A0above.\u00C2\u00A0 \u00C2\u00A0To\u00C2\u00A0minimally\u00C2\u00A0biotinylate\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0well\u00E2\u0080\u0090controlled\u00C2\u00A0manner\u00C2\u00A0before\u00C2\u00A0coupling\u00C2\u00A0 it\u00C2\u00A0to\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP,\u00C2\u00A0 the\u00C2\u00A0water\u00E2\u0080\u0090soluble\u00C2\u00A0 compound\u00C2\u00A0 NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0was\u00C2\u00A0 employed.\u00C2\u00A0 \u00C2\u00A0When\u00C2\u00A0 the\u00C2\u00A0 biotinylation\u00C2\u00A0 reaction\u00C2\u00A0was\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0in\u00C2\u00A0solution,\u00C2\u00A0variable\u00C2\u00A0results\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A063\u00C2\u00A0\u00C2\u00A0 groups\u00C2\u00A0 per\u00C2\u00A0 Ab\u00C2\u00A0 molecule,\u00C2\u00A0 so\u00C2\u00A0 to\u00C2\u00A0 enhance\u00C2\u00A0 yields\u00C2\u00A0 and\u00C2\u00A0 reproducibility,\u00C2\u00A0 an\u00C2\u00A0 alternative\u00C2\u00A0 strategy\u00C2\u00A0 was\u00C2\u00A0 employed\u00C2\u00A0which\u00C2\u00A0made\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0NIMAC\u00C2\u00A0support149\u00C2\u00A0(reaction\u00C2\u00A0[4]\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A03.1).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0schematic\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0biotinylation\u00C2\u00A0process\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0column\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A03.3A\u00C2\u00A0&\u00C2\u00A0 B.\u00C2\u00A0\u00C2\u00A0Loading\u00C2\u00A0takes\u00C2\u00A0place\u00C2\u00A0by\u00C2\u00A0eluting\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0solution\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0support,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0contains\u00C2\u00A0a\u00C2\u00A0 highly\u00C2\u00A0 conserved\u00C2\u00A0 cluster\u00C2\u00A0 of\u00C2\u00A0 histidine\u00C2\u00A0 residues\u00C2\u00A0 that\u00C2\u00A0 binds\u00C2\u00A0 specifically\u00C2\u00A0 to\u00C2\u00A0Ni2+\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 column158,\u00C2\u00A0159.\u00C2\u00A0\u00C2\u00A0 Biotinylation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 immobilized\u00C2\u00A0Ab\u00C2\u00A0then\u00C2\u00A0occurs\u00C2\u00A0by\u00C2\u00A0eluting\u00C2\u00A0an\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0solution\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0 column,\u00C2\u00A0 and\u00C2\u00A0 purification\u00C2\u00A0 consists\u00C2\u00A0 of\u00C2\u00A0 simply\u00C2\u00A0 washing\u00C2\u00A0 the\u00C2\u00A0 column.\u00C2\u00A0 \u00C2\u00A0 Ab\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 eluted\u00C2\u00A0 either\u00C2\u00A0 by\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0solution\u00C2\u00A0containing\u00C2\u00A0imidazole\u00C2\u00A0(the\u00C2\u00A0side\u00C2\u00A0chain\u00C2\u00A0of\u00C2\u00A0histidine),\u00C2\u00A0which\u00C2\u00A0competes\u00C2\u00A0with\u00C2\u00A0Ab\u00C2\u00A0 binding\u00C2\u00A0to\u00C2\u00A0Ni2+,\u00C2\u00A0or\u00C2\u00A0through\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0EDTA,\u00C2\u00A0which\u00C2\u00A0removes\u00C2\u00A0Ni2+\u00C2\u00A0along\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0bound\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0No\u00C2\u00A0 differences\u00C2\u00A0were\u00C2\u00A0found\u00C2\u00A0between\u00C2\u00A0imidazole\u00C2\u00A0and\u00C2\u00A0EDTA\u00C2\u00A0elution\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0yield\u00C2\u00A0of\u00C2\u00A0recovered\u00C2\u00A0Ab\u00E2\u0080\u0090biotin\u00C2\u00A0 or\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0bind\u00C2\u00A0to\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0Imidazole\u00C2\u00A0elution\u00C2\u00A0was\u00C2\u00A0 therefore\u00C2\u00A0employed\u00C2\u00A0to\u00C2\u00A0avoid\u00C2\u00A0contamination\u00C2\u00A0with\u00C2\u00A0Ni2+,\u00C2\u00A0whose\u00C2\u00A0toxicity\u00C2\u00A0is\u00C2\u00A0well\u00C2\u00A0documented160.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0was\u00C2\u00A0 also\u00C2\u00A0 found\u00C2\u00A0 that\u00C2\u00A0 imidazole\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 interfere\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 CBQCA\u00C2\u00A0 or\u00C2\u00A0 HABA/avidin\u00C2\u00A0 assays\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown).\u00C2\u00A0 \u00C2\u00A0Overall,\u00C2\u00A0 imidazole\u00C2\u00A0elution\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0very\u00C2\u00A0high\u00C2\u00A0 recovery\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0 (96\u00C2\u00A0%;\u00C2\u00A0Table\u00C2\u00A03.1)\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 entire\u00C2\u00A0 biotinylation\u00C2\u00A0 scheme\u00C2\u00A0 takes\u00C2\u00A0 place\u00C2\u00A0 under\u00C2\u00A0 very\u00C2\u00A0mild\u00C2\u00A0 conditions\u00C2\u00A0 (room\u00C2\u00A0 temperature,\u00C2\u00A0 pH\u00C2\u00A0 7.4),\u00C2\u00A0 important\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0conservation\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0structure\u00C2\u00A0and\u00C2\u00A0function.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A03.3C\u00C2\u00A0compares\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0added\u00C2\u00A0per\u00C2\u00A0Rtx\u00C2\u00A0molecule\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0 of\u00C2\u00A0different\u00C2\u00A0excesses\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin,\u00C2\u00A0using\u00C2\u00A0either\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0support\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0or\u00C2\u00A0by\u00C2\u00A0carrying\u00C2\u00A0 out\u00C2\u00A0 the\u00C2\u00A0 reaction\u00C2\u00A0 in\u00C2\u00A0 solution\u00C2\u00A0 (white\u00C2\u00A0 bars).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 general,\u00C2\u00A0 the\u00C2\u00A0 degree\u00C2\u00A0 of\u00C2\u00A0Rtx\u00C2\u00A0 biotinylation\u00C2\u00A0 in\u00C2\u00A0 solution\u00C2\u00A0 increased\u00C2\u00A0 substantially\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 excess\u00C2\u00A0 of\u00C2\u00A0 NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin,\u00C2\u00A0whereas\u00C2\u00A0 biotinylation\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 NIMAC\u00C2\u00A0 column\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0more\u00C2\u00A0mild\u00C2\u00A0increases.\u00C2\u00A0\u00C2\u00A0When\u00C2\u00A0biotinylation\u00C2\u00A0was\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0in\u00C2\u00A0solution,\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0 of\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Rtx\u00C2\u00A0increased\u00C2\u00A0from\u00C2\u00A00.23\u00C2\u00A0to\u00C2\u00A016\u00C2\u00A0(~70\u00E2\u0080\u0090fold)\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A05\u00E2\u0080\u0090fold\u00C2\u00A0molar\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090 biotin\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A050\u00E2\u0080\u0090fold\u00C2\u00A0excess\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0all\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0conditions\u00C2\u00A0 in\u00C2\u00A0solution).\u00C2\u00A0 \u00C2\u00A0When\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0 column\u00C2\u00A0was\u00C2\u00A0employed\u00C2\u00A0instead\u00C2\u00A0over\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0range\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0concentrations,\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0 of\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Rtx\u00C2\u00A0molecule\u00C2\u00A0increased\u00C2\u00A0from\u00C2\u00A00.90\u00C2\u00A0to\u00C2\u00A03.8\u00C2\u00A0(~4\u00E2\u0080\u0090fold;\u00C2\u00A0p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A05\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A010\u00E2\u0080\u0090 \u00E2\u0080\u00A2\u00C2\u00A064\u00C2\u00A0\u00C2\u00A0 fold\u00C2\u00A0excesses,\u00C2\u00A0 and\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A010\u00E2\u0080\u0090fold\u00C2\u00A0 and\u00C2\u00A0higher\u00C2\u00A0excesses).\u00C2\u00A0 \u00C2\u00A0Greater\u00C2\u00A0 control\u00C2\u00A0over\u00C2\u00A0 the\u00C2\u00A0 biotinylation\u00C2\u00A0 process\u00C2\u00A0was\u00C2\u00A0 therefore\u00C2\u00A0 achieved\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 NIMAC\u00C2\u00A0 column\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 reaction\u00C2\u00A0 in\u00C2\u00A0 solution,\u00C2\u00A0 since\u00C2\u00A0 small\u00C2\u00A0 unavoidable\u00C2\u00A0 variations\u00C2\u00A0 in\u00C2\u00A0 reagent\u00C2\u00A0 concentrations\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 produce\u00C2\u00A0 large\u00C2\u00A0 fluctuations\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0degree\u00C2\u00A0of\u00C2\u00A0biotinylation\u00C2\u00A0using\u00C2\u00A0NIMAC,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0more\u00C2\u00A0reproducible\u00C2\u00A0procedure.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 3.3\u00C2\u00A0 \u00C2\u00A0 Ab\u00C2\u00A0 biotinylation\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0NIMAC\u00C2\u00A0 column\u00C2\u00A0 allows\u00C2\u00A0 for\u00C2\u00A0 easy\u00C2\u00A0 purification,\u00C2\u00A0 high\u00C2\u00A0 recovery,\u00C2\u00A0 and\u00C2\u00A0 precise\u00C2\u00A0 degree\u00C2\u00A0 of\u00C2\u00A0 biotinylation.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 (A)\u00C2\u00A0Schematic\u00C2\u00A0representation\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0concentration\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0eluent\u00C2\u00A0and\u00C2\u00A0(B)\u00C2\u00A0mass\u00C2\u00A0of\u00C2\u00A0immobilized\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0NIMAC\u00C2\u00A0column\u00C2\u00A0 as\u00C2\u00A0a\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0total\u00C2\u00A0volume\u00C2\u00A0eluted\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0column\u00C2\u00A0during\u00C2\u00A0biotinylation.\u00C2\u00A0\u00C2\u00A0During\u00C2\u00A0loading,\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0solution\u00C2\u00A0binds\u00C2\u00A0to\u00C2\u00A0 immobilized\u00C2\u00A0Ni2+\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0column.\u00C2\u00A0\u00C2\u00A0Reaction\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0takes\u00C2\u00A0place\u00C2\u00A0by\u00C2\u00A0allowing\u00C2\u00A0a\u00C2\u00A0solution\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0flow\u00C2\u00A0through\u00C2\u00A0 the\u00C2\u00A0column,\u00C2\u00A0then\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0washed\u00C2\u00A0away.\u00C2\u00A0\u00C2\u00A0Finally,\u00C2\u00A0Rtx\u00C2\u00A0elution\u00C2\u00A0is\u00C2\u00A0performed\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0solution\u00C2\u00A0containing\u00C2\u00A0imidazole,\u00C2\u00A0which\u00C2\u00A0releases\u00C2\u00A0 biotinylated\u00C2\u00A0Rtx\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0column.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0(C)\u00C2\u00A0The\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0added\u00C2\u00A0per\u00C2\u00A0Rtx\u00C2\u00A0molecule\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0for\u00C2\u00A0different\u00C2\u00A0excesses\u00C2\u00A0 of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin.\u00C2\u00A0\u00C2\u00A0Black\u00C2\u00A0bars:\u00C2\u00A0NIMAC\u00C2\u00A0column;\u00C2\u00A0white\u00C2\u00A0bars:\u00C2\u00A0biotinylation\u00C2\u00A0in\u00C2\u00A0solution.\u00C2\u00A0 \u00C2\u00A0 0 2 4 6 8 10 12m as s of im m ob iliz ed ri tu x / m g 0 1 2 3 4 5 6 0 2 4 6 8 10 12 [ ] 0 20 40 60 80 100 Volume\u00C2\u00A0eluted\u00C2\u00A0/\u00C2\u00A0mL [b io ti n] el ue nt \u00C2\u00A0/\u00C2\u00A0\u00CE\u00BC M m R tx on \u00C2\u00A0co lu m n\u00C2\u00A0\u00C2\u00A0/ \u00C2\u00A0mg Load React Elute A B 0 4 8 12 16 20 5 10 25 50 N bi ot in pe r\u00C2\u00A0R tx \u00E2\u0080\u0090bi ot in \u00C2\u00A0m ol ec ul e Mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0 0 1 2 3 4 5 10 C \u00E2\u0080\u00A2\u00C2\u00A065\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0minimize\u00C2\u00A0the\u00C2\u00A0overall\u00C2\u00A0modification\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0to\u00C2\u00A0obtain\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0homogeneous\u00C2\u00A0 Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0preparation,\u00C2\u00A0biotinylation\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0NIMAC\u00C2\u00A0 support\u00C2\u00A0was\u00C2\u00A0henceforth\u00C2\u00A0performed\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A010\u00E2\u0080\u0090 fold\u00C2\u00A0molar\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0resulted\u00C2\u00A0 in\u00C2\u00A0high\u00C2\u00A0yields\u00C2\u00A0of\u00C2\u00A0samples\u00C2\u00A0of\u00C2\u00A0 Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0containing,\u00C2\u00A0on\u00C2\u00A0average,\u00C2\u00A0between\u00C2\u00A01\u00C2\u00A0and\u00C2\u00A02\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Rtx\u00C2\u00A0molecule\u00C2\u00A0(Figure\u00C2\u00A03.3C).\u00C2\u00A0 3.4.5\u00C2\u00A0 Different\u00C2\u00A0Ab\u00C2\u00ADLNP\u00C2\u00A0 valences\u00C2\u00A0 are\u00C2\u00A0 achieved\u00C2\u00A0 by\u00C2\u00A0 taking\u00C2\u00A0 advantage\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 slow\u00C2\u00A0 association\u00C2\u00A0dynamics\u00C2\u00A0between\u00C2\u00A0Ab\u00C2\u00ADbiotin\u00C2\u00A0and\u00C2\u00A0Neut\u00C2\u00ADLNP\u00C2\u00A0 The\u00C2\u00A0final\u00C2\u00A0step\u00C2\u00A0in\u00C2\u00A0creating\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0involved\u00C2\u00A0coupling\u00C2\u00A0Ab\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0carrying\u00C2\u00A0out\u00C2\u00A0 this\u00C2\u00A0coupling\u00C2\u00A0reaction,\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0uncovered\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0association\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0and\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0actually\u00C2\u00A0a\u00C2\u00A0 relatively\u00C2\u00A0slow\u00C2\u00A0process;\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0continued\u00C2\u00A0to\u00C2\u00A0 increase\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0 start\u00C2\u00A0of\u00C2\u00A0coupling.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0was\u00C2\u00A0surprising\u00C2\u00A0since\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0between\u00C2\u00A0biotin\u00C2\u00A0and\u00C2\u00A0Neut\u00C2\u00A0 is\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 strongest\u00C2\u00A0noncovalent\u00C2\u00A0 interactions\u00C2\u00A0known\u00C2\u00A0(KD\u00C2\u00A0=\u00C2\u00A010\u00E2\u0080\u009015\u00C2\u00A0M)161,\u00C2\u00A0and\u00C2\u00A0mixing\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0and\u00C2\u00A0free\u00C2\u00A0Neut\u00C2\u00A0 in\u00C2\u00A0 solution\u00C2\u00A0often\u00C2\u00A0causes\u00C2\u00A0the\u00C2\u00A0immediate\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0precipitate,\u00C2\u00A0indicating\u00C2\u00A0very\u00C2\u00A0rapid\u00C2\u00A0association.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 significant\u00C2\u00A0 reduction\u00C2\u00A0 in\u00C2\u00A0 coupling\u00C2\u00A0 dynamics\u00C2\u00A0 has\u00C2\u00A0 not\u00C2\u00A0 been\u00C2\u00A0 previously\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 parallel\u00C2\u00A0application,\u00C2\u00A0and\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0key\u00C2\u00A0feature\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0described\u00C2\u00A0here.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0provides\u00C2\u00A0a\u00C2\u00A0practical\u00C2\u00A0 way\u00C2\u00A0 of\u00C2\u00A0 obtaining\u00C2\u00A0 different\u00C2\u00A0 valences\u00C2\u00A0 of\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs:\u00C2\u00A0 fractions\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 coupling\u00C2\u00A0 reaction\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 stopped\u00C2\u00A0at\u00C2\u00A0 consecutively\u00C2\u00A0 longer\u00C2\u00A0 time\u00C2\u00A0points,\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 successively\u00C2\u00A0higher\u00C2\u00A0valences.\u00C2\u00A0 \u00C2\u00A0Figure\u00C2\u00A03.4\u00C2\u00A0 shows\u00C2\u00A0three\u00C2\u00A0different\u00C2\u00A0sets\u00C2\u00A0of\u00C2\u00A0reaction\u00C2\u00A0conditions\u00C2\u00A0(mole\u00C2\u00A0ratios\u00C2\u00A0of\u00C2\u00A0added\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut),\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0 cases,\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0moles\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0associated\u00C2\u00A0to\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0increased\u00C2\u00A0from\u00C2\u00A0an\u00C2\u00A0incubation\u00C2\u00A0time\u00C2\u00A0 of\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0to\u00C2\u00A072\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0added\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00A0(in\u00C2\u00A0Neut\u00E2\u0080\u0090LNP)\u00C2\u00A0was\u00C2\u00A00.5,\u00C2\u00A0 the\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0coupled\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00A0(in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP)\u00C2\u00A0was\u00C2\u00A00.25\u00C2\u00A0after\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A00.47\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05);\u00C2\u00A0 when\u00C2\u00A0the\u00C2\u00A0added\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0was\u00C2\u00A0increased\u00C2\u00A0to\u00C2\u00A02.0,\u00C2\u00A0the\u00C2\u00A0coupled\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0 Neut\u00C2\u00A0was\u00C2\u00A00.28\u00C2\u00A0after\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A01.4\u00C2\u00A0after\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.005).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0slow\u00C2\u00A0kinetic\u00C2\u00A0of\u00C2\u00A0association\u00C2\u00A0 therefore\u00C2\u00A0 provide\u00C2\u00A0great\u00C2\u00A0versatility\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0since\u00C2\u00A0many\u00C2\u00A0different\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0valences\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0prepared\u00C2\u00A0 from\u00C2\u00A0one\u00C2\u00A0or\u00C2\u00A0two\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0preparations\u00C2\u00A0and\u00C2\u00A0one\u00C2\u00A0sample\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090biotin.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A066\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A03.4\u00C2\u00A0\u00C2\u00A0Control\u00C2\u00A0over\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0when\u00C2\u00A0coupling\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00E2\u0080\u0090LNP.\u00C2\u00A0 The\u00C2\u00A0final\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0controlled\u00C2\u00A0by\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0added\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00A0(in\u00C2\u00A0Neut\u00E2\u0080\u0090 LNP)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0length\u00C2\u00A0of\u00C2\u00A0time\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0coupling\u00C2\u00A0reaction\u00C2\u00A0is\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0proceed.\u00C2\u00A0\u00C2\u00A0Note\u00C2\u00A0that\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0value\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0horizontal\u00C2\u00A0 axis,\u00C2\u00A0a\u00C2\u00A0100\u00C2\u00A0%\u00C2\u00A0coupling\u00C2\u00A0efficiency\u00C2\u00A0 is\u00C2\u00A0represented\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0value\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0vertical\u00C2\u00A0axis.\u00C2\u00A0 \u00C2\u00A0Reaction\u00C2\u00A0 times:\u00C2\u00A0black\u00C2\u00A0bars:\u00C2\u00A01\u00C2\u00A0h;\u00C2\u00A0 white\u00C2\u00A0bars:\u00C2\u00A024\u00C2\u00A0h;\u00C2\u00A0hatched\u00C2\u00A0bars:\u00C2\u00A048\u00C2\u00A0h;\u00C2\u00A0dotted\u00C2\u00A0bars:\u00C2\u00A072\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 3.4\u00C2\u00A0 indicates\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0 general,\u00C2\u00A0 for\u00C2\u00A0 equivalent\u00C2\u00A0 reaction\u00C2\u00A0 times\u00C2\u00A0 of\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 or\u00C2\u00A0 longer,\u00C2\u00A0 the\u00C2\u00A0 amount\u00C2\u00A0of\u00C2\u00A0associated\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0increases\u00C2\u00A0when\u00C2\u00A0higher\u00C2\u00A0excesses\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0(with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0Neut)\u00C2\u00A0 are\u00C2\u00A0added.\u00C2\u00A0 \u00C2\u00A0Using\u00C2\u00A0 these\u00C2\u00A0higher\u00C2\u00A0mole\u00C2\u00A0excesses,\u00C2\u00A0however,\u00C2\u00A0had\u00C2\u00A0 the\u00C2\u00A0disadvantage\u00C2\u00A0of\u00C2\u00A0 lower\u00C2\u00A0yields;\u00C2\u00A0 in\u00C2\u00A0 general,\u00C2\u00A0 the\u00C2\u00A0 coupling\u00C2\u00A0 efficiency\u00C2\u00A0 increased\u00C2\u00A0 as\u00C2\u00A0 less\u00C2\u00A0 Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0 was\u00C2\u00A0 added\u00C2\u00A0 with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0 Neut.\u00C2\u00A0\u00C2\u00A0 Importantly,\u00C2\u00A0coupling\u00C2\u00A0efficiencies\u00C2\u00A0close\u00C2\u00A0to\u00C2\u00A0100%\u00C2\u00A0(represented\u00C2\u00A0by\u00C2\u00A0equal\u00C2\u00A0values\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0horizontal\u00C2\u00A0and\u00C2\u00A0 vertical\u00C2\u00A0axes)\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut\u00C2\u00A0of\u00C2\u00A00.5\u00C2\u00A0and\u00C2\u00A0by\u00C2\u00A0allowing\u00C2\u00A0the\u00C2\u00A0reaction\u00C2\u00A0 to\u00C2\u00A0proceed\u00C2\u00A0for\u00C2\u00A072\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0(A\u00C2\u00A0100%\u00C2\u00A0coupling\u00C2\u00A0efficiency\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0represent\u00C2\u00A0a\u00C2\u00A0100%\u00C2\u00A0yield\u00C2\u00A0since\u00C2\u00A0Ab,\u00C2\u00A0coupled\u00C2\u00A0 to\u00C2\u00A0Ab\u00E2\u0080\u0090LNP,\u00C2\u00A0will\u00C2\u00A0invariably\u00C2\u00A0be\u00C2\u00A0lost\u00C2\u00A0during\u00C2\u00A0ensuing\u00C2\u00A0purification.)\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A03.4\u00C2\u00A0also\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0although\u00C2\u00A0Neut\u00C2\u00A0has\u00C2\u00A0 four\u00C2\u00A0biotin\u00E2\u0080\u0090binding\u00C2\u00A0 sites\u00C2\u00A0 (three\u00C2\u00A0available\u00C2\u00A0 to\u00C2\u00A0 bind\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin),\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0bound\u00C2\u00A0a\u00C2\u00A0maximum\u00C2\u00A0of\u00C2\u00A01.4\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0Neut.\u00C2\u00A0\u00C2\u00A0Although\u00C2\u00A0this\u00C2\u00A0 stoichiometry\u00C2\u00A0likely\u00C2\u00A0resulted\u00C2\u00A0from\u00C2\u00A0PEG\u00C2\u00A0blocking\u00C2\u00A0the\u00C2\u00A0biotin\u00E2\u0080\u0090binding\u00C2\u00A0sites\u00C2\u00A0on\u00C2\u00A0Neut148,\u00C2\u00A0one\u00C2\u00A0concern\u00C2\u00A0was\u00C2\u00A0 Co up le d\u00C2\u00A0m ol e\u00C2\u00A0r at io \u00C2\u00A0of \u00C2\u00A0Rt x\u00C2\u00A0t o\u00C2\u00A0N eu t (in \u00C2\u00A0Rt x\u00E2\u0080\u0090L N P) Added\u00C2\u00A0mole\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0to\u00C2\u00A0Neut (in\u00C2\u00A0Neut\u00E2\u0080\u0090LNP) 2.0 1.5 1.0 0.5 0 0.5\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A01.0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A02.0 \u00E2\u0080\u00A2\u00C2\u00A067\u00C2\u00A0\u00C2\u00A0 that\u00C2\u00A0aggregation\u00C2\u00A0might\u00C2\u00A0occur\u00C2\u00A0over\u00C2\u00A0time\u00C2\u00A0from\u00C2\u00A0unbound\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0on\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0and\u00C2\u00A0free\u00C2\u00A0biotin\u00E2\u0080\u0090 binding\u00C2\u00A0sites\u00C2\u00A0on\u00C2\u00A0Neut.\u00C2\u00A0 \u00C2\u00A0Thus\u00C2\u00A0 in\u00C2\u00A0an\u00C2\u00A0effort\u00C2\u00A0 to\u00C2\u00A0reduce\u00C2\u00A0 the\u00C2\u00A0available\u00C2\u00A0biotin\u00E2\u0080\u0090biding\u00C2\u00A0sites,\u00C2\u00A0some\u00C2\u00A0samples\u00C2\u00A0 were\u00C2\u00A0prepared\u00C2\u00A0where\u00C2\u00A0 excess\u00C2\u00A0 free\u00C2\u00A0biotin\u00C2\u00A0was\u00C2\u00A0 added\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0end\u00C2\u00A0of\u00C2\u00A0 coupling\u00C2\u00A0 and\u00C2\u00A0 incubated\u00C2\u00A0 for\u00C2\u00A01h\u00C2\u00A0 before\u00C2\u00A0purification.\u00C2\u00A0\u00C2\u00A0Over\u00C2\u00A0the\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0one\u00C2\u00A0month,\u00C2\u00A0the\u00C2\u00A0sizes\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0prepared\u00C2\u00A0with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0 addition\u00C2\u00A0of\u00C2\u00A0biotin\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0at\u00C2\u00A0regular\u00C2\u00A0 intervals\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0 found\u00C2\u00A0 to\u00C2\u00A0remain\u00C2\u00A0constant\u00C2\u00A0at\u00C2\u00A0~130\u00C2\u00A0 nm\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0preparations\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0found\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0similar\u00C2\u00A0and\u00C2\u00A0we\u00C2\u00A0therefore\u00C2\u00A0concluded\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0were\u00C2\u00A0stable\u00C2\u00A0without\u00C2\u00A0the\u00C2\u00A0 addition\u00C2\u00A0of\u00C2\u00A0excess\u00C2\u00A0biotin.\u00C2\u00A0 \u00C2\u00A0 3.5\u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0 3.5.1\u00C2\u00A0 Discussion\u00C2\u00A0 The\u00C2\u00A0 work\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 chapter\u00C2\u00A0 has\u00C2\u00A0 uncovered\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 kinetics\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 association\u00C2\u00A0 between\u00C2\u00A0biotinylated\u00C2\u00A0protein\u00C2\u00A0and\u00C2\u00A0PEGylated\u00C2\u00A0 liposomes\u00C2\u00A0containing\u00C2\u00A0an\u00C2\u00A0avidin\u00C2\u00A0analog\u00C2\u00A0are\u00C2\u00A0surprisingly\u00C2\u00A0 slow.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 slow\u00C2\u00A0association\u00C2\u00A0kinetics\u00C2\u00A0 comprise\u00C2\u00A0a\u00C2\u00A0 central\u00C2\u00A0 feature\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 improved\u00C2\u00A0methodology\u00C2\u00A0 for\u00C2\u00A0 preparing\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0 described\u00C2\u00A0 here.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 avidin\u00E2\u0080\u0090biotin\u00C2\u00A0 interactions\u00C2\u00A0 to\u00C2\u00A0 couple\u00C2\u00A0 proteins\u00C2\u00A0 to\u00C2\u00A0 liposomes\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0a\u00C2\u00A0new\u00C2\u00A0 concept,\u00C2\u00A0but\u00C2\u00A0one\u00C2\u00A0 reason\u00C2\u00A0why\u00C2\u00A0 the\u00C2\u00A0 slow\u00C2\u00A0kinetics\u00C2\u00A0have\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0previously\u00C2\u00A0 described\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 that,\u00C2\u00A0 by\u00C2\u00A0 and\u00C2\u00A0 large,\u00C2\u00A0 this\u00C2\u00A0 type\u00C2\u00A0 of\u00C2\u00A0 coupling\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 performed\u00C2\u00A0 using\u00C2\u00A0 liposomes\u00C2\u00A0 lacking\u00C2\u00A0PEG147,\u00C2\u00A0162,\u00C2\u00A0163.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0aqueous\u00C2\u00A0 solution,\u00C2\u00A0however,\u00C2\u00A0a\u00C2\u00A0 similar\u00C2\u00A0phenomenon\u00C2\u00A0 involving\u00C2\u00A0biotinylated\u00C2\u00A0 PEG\u00C2\u00A0 and\u00C2\u00A0 avidin\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 attributed\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 steric\u00C2\u00A0effect\u00C2\u00A0where\u00C2\u00A0 the\u00C2\u00A0PEG\u00C2\u00A0 chains\u00C2\u00A0effectively\u00C2\u00A0block\u00C2\u00A0 the\u00C2\u00A0 biotin\u00E2\u0080\u0090binding\u00C2\u00A0sites\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0avidin\u00C2\u00A0molecules148.\u00C2\u00A0 \u00C2\u00A0One\u00C2\u00A0unanswered\u00C2\u00A0question\u00C2\u00A0 is\u00C2\u00A0how\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0 PEG\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 liposome\u00C2\u00A0affects\u00C2\u00A0 the\u00C2\u00A0kinetics\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0 coupling\u00C2\u00A0 reactions,\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0 those\u00C2\u00A0 that\u00C2\u00A0 employ\u00C2\u00A0thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0chemistry.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A068\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 effect\u00C2\u00A0of\u00C2\u00A0PEG\u00C2\u00A0blocking\u00C2\u00A0 lends\u00C2\u00A0 significant\u00C2\u00A0 efficiency\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0methodology\u00C2\u00A0because\u00C2\u00A0many\u00C2\u00A0 different\u00C2\u00A0valences\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0produced\u00C2\u00A0from\u00C2\u00A0single\u00C2\u00A0batches\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0Ab\u00E2\u0080\u0090biotin;\u00C2\u00A0the\u00C2\u00A0two\u00C2\u00A0reagents\u00C2\u00A0 need\u00C2\u00A0only\u00C2\u00A0be\u00C2\u00A0mixed\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0proportions\u00C2\u00A0and\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A0different\u00C2\u00A0times\u00C2\u00A0between\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A072\u00C2\u00A0h\u00C2\u00A0 to\u00C2\u00A0produce\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0(Figure\u00C2\u00A03.4).\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0in\u00C2\u00A0contrast\u00C2\u00A0to\u00C2\u00A0having\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0Neut\u00E2\u0080\u0090 LNP\u00C2\u00A0for\u00C2\u00A0every\u00C2\u00A0desired\u00C2\u00A0valence,\u00C2\u00A0which\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0cumbersome\u00C2\u00A0and\u00C2\u00A0time\u00E2\u0080\u0090consuming.\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0noted\u00C2\u00A0that\u00C2\u00A0one\u00C2\u00A0case\u00C2\u00A0where\u00C2\u00A0this\u00C2\u00A0more\u00C2\u00A0time\u00E2\u0080\u0090consuming\u00C2\u00A0strategy\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0desirable\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0 situation\u00C2\u00A0of\u00C2\u00A0severely\u00C2\u00A0 limited\u00C2\u00A0Ab\u00C2\u00A0availability;\u00C2\u00A0this\u00C2\u00A0 is\u00C2\u00A0because\u00C2\u00A0the\u00C2\u00A0reaction\u00C2\u00A0conditions\u00C2\u00A0 leading\u00C2\u00A0to\u00C2\u00A0100%\u00C2\u00A0 coupling\u00C2\u00A0efficiencies\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0employed\u00C2\u00A0 for\u00C2\u00A0every\u00C2\u00A0valence\u00C2\u00A0 (mole\u00C2\u00A0 ratio\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090biotin\u00C2\u00A0 to\u00C2\u00A0Neut\u00C2\u00A0=\u00C2\u00A00.5;\u00C2\u00A0 Figure\u00C2\u00A0 3.4).\u00C2\u00A0 \u00C2\u00A0 Overall,\u00C2\u00A0 the\u00C2\u00A0 methodology\u00C2\u00A0 is\u00C2\u00A0 therefore\u00C2\u00A0 versatile\u00C2\u00A0 and\u00C2\u00A0 easily\u00C2\u00A0 adaptable\u00C2\u00A0 to\u00C2\u00A0 different\u00C2\u00A0 circumstances\u00C2\u00A0of\u00C2\u00A0time\u00C2\u00A0and\u00C2\u00A0Ab\u00C2\u00A0availability.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Two\u00C2\u00A0other\u00C2\u00A0 features\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0methodology\u00C2\u00A0 also\u00C2\u00A0 served\u00C2\u00A0 to\u00C2\u00A0 improve\u00C2\u00A0 the\u00C2\u00A0 yields\u00C2\u00A0of\u00C2\u00A0 coupled\u00C2\u00A0Ab\u00C2\u00A0 (Table\u00C2\u00A03.1)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0reproducibility\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0procedure:\u00C2\u00A0elimination\u00C2\u00A0of\u00C2\u00A0thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0chemistry,\u00C2\u00A0and\u00C2\u00A0 use\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0NIMAC\u00C2\u00A0support\u00C2\u00A0 to\u00C2\u00A0biotinylate\u00C2\u00A0 the\u00C2\u00A0Ab.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0NIMAC\u00C2\u00A0column\u00C2\u00A0enabled\u00C2\u00A0a\u00C2\u00A0very\u00C2\u00A0high\u00C2\u00A0yield\u00C2\u00A0of\u00C2\u00A0 recovered\u00C2\u00A0biotinylated\u00C2\u00A0Ab\u00C2\u00A0(96\u00C2\u00A0%)\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0more\u00C2\u00A0homogeneous\u00C2\u00A0product\u00C2\u00A0with\u00C2\u00A0only\u00C2\u00A01\u00C2\u00A0to\u00C2\u00A02\u00C2\u00A0modified\u00C2\u00A0 amine\u00C2\u00A0groups\u00C2\u00A0per\u00C2\u00A0Ab\u00C2\u00A0 (using\u00C2\u00A0a\u00C2\u00A010\u00E2\u0080\u0090fold\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090PEG\u00E2\u0080\u0090biotin;\u00C2\u00A0Figure\u00C2\u00A03.3)\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A03\u00E2\u0080\u00907\u00C2\u00A0 thiol\u00C2\u00A0 groups\u00C2\u00A0per\u00C2\u00A0Ab\u00C2\u00A0using\u00C2\u00A0thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0coupling.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0NIMAC\u00C2\u00A0method\u00C2\u00A0also\u00C2\u00A0has\u00C2\u00A0the\u00C2\u00A0advantage\u00C2\u00A0of\u00C2\u00A0very\u00C2\u00A0 mild\u00C2\u00A0reaction\u00C2\u00A0and\u00C2\u00A0elution\u00C2\u00A0conditions;\u00C2\u00A0unlike\u00C2\u00A0the\u00C2\u00A0thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0method,\u00C2\u00A0all\u00C2\u00A0steps\u00C2\u00A0take\u00C2\u00A0place\u00C2\u00A0at\u00C2\u00A0pH\u00C2\u00A0 7.4\u00C2\u00A0at\u00C2\u00A0room\u00C2\u00A0temperature,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0aid\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0conservation\u00C2\u00A0of\u00C2\u00A0native\u00C2\u00A0Ab\u00C2\u00A0structure.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Table\u00C2\u00A03.1,\u00C2\u00A0thiol\u00E2\u0080\u0090maleimide\u00C2\u00A0coupling\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0post\u00E2\u0080\u0090insertion\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0may\u00C2\u00A0exhibit\u00C2\u00A0 a\u00C2\u00A0 high\u00C2\u00A0 coupling\u00C2\u00A0 efficiency,\u00C2\u00A0 but\u00C2\u00A0 the\u00C2\u00A0 start\u00E2\u0080\u0090to\u00E2\u0080\u0090finish\u00C2\u00A0 yield\u00C2\u00A0 of\u00C2\u00A0 Ab\u00C2\u00A0 is\u00C2\u00A0 much\u00C2\u00A0 lower.\u00C2\u00A0 \u00C2\u00A0 Replacing\u00C2\u00A0 thiol\u00E2\u0080\u0090 maleimide\u00C2\u00A0chemistry\u00C2\u00A0also\u00C2\u00A0improved\u00C2\u00A0reproducibility\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0inherent\u00C2\u00A0problem\u00C2\u00A0that\u00C2\u00A0only\u00C2\u00A0one\u00C2\u00A0DSPE\u00E2\u0080\u0090 PEG\u00E2\u0080\u0090maleimide\u00C2\u00A0molecule\u00C2\u00A0 is\u00C2\u00A0 required\u00C2\u00A0 per\u00C2\u00A0Ab,\u00C2\u00A0 but\u00C2\u00A0 higher\u00E2\u0080\u0090order\u00C2\u00A0 adducts\u00C2\u00A0 inevitably\u00C2\u00A0 result\u00C2\u00A0 since\u00C2\u00A0 the\u00C2\u00A0 reaction\u00C2\u00A0must\u00C2\u00A0be\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0under\u00C2\u00A0an\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0DSPE\u00E2\u0080\u0090PEG\u00E2\u0080\u0090maleimide142.\u00C2\u00A0 \u00C2\u00A0Exposed\u00C2\u00A0thiol\u00C2\u00A0groups\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0Ab\u00C2\u00A0are\u00C2\u00A0also\u00C2\u00A0known\u00C2\u00A0 to\u00C2\u00A0undergo\u00C2\u00A0oxidation\u00C2\u00A0 into\u00C2\u00A0disulfide\u00C2\u00A0bonds164,\u00C2\u00A0which\u00C2\u00A0along\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0higher\u00E2\u0080\u0090 order\u00C2\u00A0 adducts,\u00C2\u00A0 not\u00C2\u00A0 only\u00C2\u00A0 affect\u00C2\u00A0 Ab\u00C2\u00A0 structure\u00C2\u00A0 and\u00C2\u00A0 function\u00C2\u00A0 but\u00C2\u00A0 also\u00C2\u00A0 constitute\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A069\u00C2\u00A0\u00C2\u00A0 interliposomal\u00C2\u00A0crosslinking.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0 further\u00C2\u00A0problem\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0maleimide\u00C2\u00A0groups\u00C2\u00A0are\u00C2\u00A0particularly\u00C2\u00A0prone\u00C2\u00A0 to\u00C2\u00A0 hydrolysis\u00C2\u00A0at\u00C2\u00A0elevated\u00C2\u00A0temperatures\u00C2\u00A0(such\u00C2\u00A0as\u00C2\u00A065\u00C2\u00A0oC,\u00C2\u00A0used\u00C2\u00A0for\u00C2\u00A0extrusion)\u00C2\u00A0and\u00C2\u00A0at\u00C2\u00A0pH\u00C2\u00A0levels\u00C2\u00A0above\u00C2\u00A07\u00C2\u00A0(ref.\u00C2\u00A0 165).\u00C2\u00A0 \u00C2\u00A0 With\u00C2\u00A0 the\u00C2\u00A0 improved\u00C2\u00A0 methodology,\u00C2\u00A0 the\u00C2\u00A0 issues\u00C2\u00A0 of\u00C2\u00A0 nonhomogeneous\u00C2\u00A0 thiolation\u00C2\u00A0 and\u00C2\u00A0 adduct\u00C2\u00A0 stoichiometry,\u00C2\u00A0 disulfide\u00C2\u00A0 bond\u00C2\u00A0 formation,\u00C2\u00A0 and\u00C2\u00A0 maleimide\u00C2\u00A0 hydrolysis\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 eliminated\u00C2\u00A0 and\u00C2\u00A0 reproducibility\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0improved.\u00C2\u00A0 The\u00C2\u00A0 improved\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0preparation\u00C2\u00A0methodology\u00C2\u00A0may\u00C2\u00A0serve\u00C2\u00A0to\u00C2\u00A0 identify\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0that\u00C2\u00A0 are\u00C2\u00A0more\u00C2\u00A0efficacious\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0configuration\u00C2\u00A0and\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0the\u00C2\u00A0optimal\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0such\u00C2\u00A0Abs;\u00C2\u00A0it\u00C2\u00A0 has\u00C2\u00A0been\u00C2\u00A0estimated\u00C2\u00A0that\u00C2\u00A0valences\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0~400\u00C2\u00A0(200\u00C2\u00A0IgG)\u00C2\u00A0can\u00C2\u00A0theoretically\u00C2\u00A0be\u00C2\u00A0achieved\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0100\u00C2\u00A0nm\u00E2\u0080\u0090 diameter\u00C2\u00A0 liposome141\u00C2\u00A0 although\u00C2\u00A0 the\u00C2\u00A0 highest\u00C2\u00A0 valence\u00C2\u00A0 achieved\u00C2\u00A0 in\u00C2\u00A0 sufficient\u00C2\u00A0 yield\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 current\u00C2\u00A0 improved\u00C2\u00A0methodology\u00C2\u00A0was\u00C2\u00A0250.\u00C2\u00A0\u00C2\u00A0Optimal\u00C2\u00A0valence\u00C2\u00A0information\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0translated\u00C2\u00A0into\u00C2\u00A0non\u00E2\u0080\u0090liposomal\u00C2\u00A0 multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0where\u00C2\u00A0it\u00C2\u00A0might\u00C2\u00A0otherwise\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0difficult\u00C2\u00A0or\u00C2\u00A0time\u00E2\u0080\u0090consuming\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0 many\u00C2\u00A0different\u00C2\u00A0valences.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0alternative\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0may\u00C2\u00A0include\u00C2\u00A0Ab\u00E2\u0080\u0090coupled\u00C2\u00A0gold\u00C2\u00A0 nanoshells96,\u00C2\u00A0 Ab\u00E2\u0080\u0090dendrimer\u00C2\u00A0 conjugates95,\u00C2\u00A0 polymeric\u00C2\u00A0 nanoparticles166,\u00C2\u00A0 or\u00C2\u00A0 different\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 multifunctional\u00C2\u00A0 nanoparticles167.\u00C2\u00A0 \u00C2\u00A0 Using\u00C2\u00A0 liposomes\u00C2\u00A0 to\u00C2\u00A0 create\u00C2\u00A0 multivalent\u00C2\u00A0 Abs\u00C2\u00A0 also\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 necessarily\u00C2\u00A0 exclude\u00C2\u00A0 the\u00C2\u00A0 possibility\u00C2\u00A0 of\u00C2\u00A0 their\u00C2\u00A0 application\u00C2\u00A0 to\u00C2\u00A0 immunoliposomes\u00C2\u00A0 that\u00C2\u00A0 contain\u00C2\u00A0 encapsulated\u00C2\u00A0drug.\u00C2\u00A0 \u00C2\u00A0 Indeed,\u00C2\u00A0 it\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0noted\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0methodology\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0very\u00C2\u00A0useful\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0of\u00C2\u00A0 targeted\u00C2\u00A0 liposomal\u00C2\u00A0 formulations\u00C2\u00A0where\u00C2\u00A0 the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0varying\u00C2\u00A0 the\u00C2\u00A0protein\u00E2\u0080\u0090to\u00E2\u0080\u0090lipid\u00C2\u00A0 ratio\u00C2\u00A0are\u00C2\u00A0being\u00C2\u00A0studied.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 3.5.2\u00C2\u00A0 Conclusions\u00C2\u00A0 The\u00C2\u00A0 methodology\u00C2\u00A0 described\u00C2\u00A0 above\u00C2\u00A0 provides\u00C2\u00A0 a\u00C2\u00A0 means\u00C2\u00A0 of\u00C2\u00A0 studying\u00C2\u00A0 therapeutic\u00C2\u00A0 Ab\u00C2\u00A0 multivalency\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0Abs\u00C2\u00A0under\u00C2\u00A0development\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0efficacious\u00C2\u00A0as\u00C2\u00A0 multivalent\u00C2\u00A0 rather\u00C2\u00A0 than\u00C2\u00A0 bivalent\u00C2\u00A0 molecules.\u00C2\u00A0 \u00C2\u00A0 Compared\u00C2\u00A0 to\u00C2\u00A0 previous\u00C2\u00A0 methods,\u00C2\u00A0 the\u00C2\u00A0 improved\u00C2\u00A0 methodology\u00C2\u00A0 described\u00C2\u00A0 here\u00C2\u00A0 is\u00C2\u00A0more\u00C2\u00A0 time\u00E2\u0080\u0090efficient\u00C2\u00A0 and\u00C2\u00A0 reproducible,\u00C2\u00A0 and\u00C2\u00A0 also\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 higher\u00C2\u00A0 yields\u00C2\u00A0of\u00C2\u00A0coupled\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0With\u00C2\u00A0this\u00C2\u00A0optimized\u00C2\u00A0methodology,\u00C2\u00A0studies\u00C2\u00A0which\u00C2\u00A0examine\u00C2\u00A0how\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A070\u00C2\u00A0\u00C2\u00A0 therapeutic\u00C2\u00A0Ab\u00C2\u00A0affects\u00C2\u00A0its\u00C2\u00A0efficacy\u00C2\u00A0can\u00C2\u00A0now\u00C2\u00A0be\u00C2\u00A0carried\u00C2\u00A0out.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090 LNP\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A0 employed\u00C2\u00A0 to\u00C2\u00A0 identify\u00C2\u00A0 the\u00C2\u00A0optimal\u00C2\u00A0 valence\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Ab\u00C2\u00A0 and\u00C2\u00A0 to\u00C2\u00A0probe\u00C2\u00A0 the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 multivalent\u00C2\u00A0 drug.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 resulting\u00C2\u00A0 information\u00C2\u00A0 could\u00C2\u00A0 potentially\u00C2\u00A0 be\u00C2\u00A0 applied\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 development\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0liposomal\u00C2\u00A0and\u00C2\u00A0non\u00E2\u0080\u0090liposomal\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A071\u00C2\u00A0\u00C2\u00A0 4\u00C2\u00A0 Unique\u00C2\u00A0biological\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0rituximab\u00C2\u00ADlipid\u00C2\u00A0nanoparticles\u00C2\u00A0 4.1\u00C2\u00A0 Synopsis\u00C2\u00A0 This\u00C2\u00A0chapter\u00C2\u00A0examines\u00C2\u00A0the\u00C2\u00A0properties\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0prepared\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A0 improved\u00C2\u00A0methodology\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A03.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0properties\u00C2\u00A0 include\u00C2\u00A0binding\u00C2\u00A0characteristics\u00C2\u00A0to\u00C2\u00A0 CD20+\u00C2\u00A0lymphoma\u00C2\u00A0cells,\u00C2\u00A0distribution\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface,\u00C2\u00A0modulation\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0expression,\u00C2\u00A0 cytotoxicity\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0constructs,\u00C2\u00A0and\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0to\u00C2\u00A0directly\u00C2\u00A0induce\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0 4.2\u00C2\u00A0 Background\u00C2\u00A0 4.2.1\u00C2\u00A0 Properties\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0anti\u00C2\u00ADCD20\u00C2\u00A0Abs\u00C2\u00A0 Various\u00C2\u00A0designs\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0Abs\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0described,\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 general,\u00C2\u00A0 these\u00C2\u00A0 constructs\u00C2\u00A0exhibit\u00C2\u00A0superior\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0target\u00C2\u00A0cells\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0elevated\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0some\u00C2\u00A0cases\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0\u00C2\u00A0Evidence\u00C2\u00A0of\u00C2\u00A0superior\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0CD20+\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0provided\u00C2\u00A0by\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 constructs\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090gold\u00C2\u00A0 nanoshells96\u00C2\u00A0 and\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 multivalent\u00C2\u00A0 branched\u00C2\u00A0copolymer\u00E2\u0080\u0090Fab\u00C2\u00A0conjugates15.\u00C2\u00A0\u00C2\u00A0Enhanced\u00C2\u00A0binding\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0increased\u00C2\u00A0avidity\u00C2\u00A0of\u00C2\u00A0 multivalent\u00C2\u00A0ligands\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0usually\u00C2\u00A0observed,\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A01.6.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0spite\u00C2\u00A0of\u00C2\u00A0observations\u00C2\u00A0of\u00C2\u00A0 improved\u00C2\u00A0binding,\u00C2\u00A0 studies\u00C2\u00A0are\u00C2\u00A0 scarce\u00C2\u00A0 regarding\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0 interaction\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0target\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0 is\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cell,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0 known\u00C2\u00A0whether\u00C2\u00A0all\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0construct\u00C2\u00A0are\u00C2\u00A0bound\u00C2\u00A0 to\u00C2\u00A0CD20,\u00C2\u00A0or\u00C2\u00A0 if\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0 fraction\u00C2\u00A0 that\u00C2\u00A0 remains\u00C2\u00A0unbound.\u00C2\u00A0 Several\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 described\u00C2\u00A0 therapeutic\u00C2\u00A0 improvements\u00C2\u00A0 in\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 constructs\u00C2\u00A0 compared\u00C2\u00A0to\u00C2\u00A0bivalent\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0generally\u00C2\u00A0understood\u00C2\u00A0that\u00C2\u00A0this\u00C2\u00A0results\u00C2\u00A0from\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0 CD20\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0\u00C2\u00A0Ghetie\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0created\u00C2\u00A0tetravalent\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0F(ab\u00E2\u0080\u00B2)2\u00C2\u00A0homodimers,\u00C2\u00A0which\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A072\u00C2\u00A0\u00C2\u00A0 inhibited\u00C2\u00A0 the\u00C2\u00A0 growth\u00C2\u00A0of\u00C2\u00A0 several\u00C2\u00A0different\u00C2\u00A0B\u00E2\u0080\u0090lymphoma\u00C2\u00A0 cell\u00C2\u00A0 lines\u00C2\u00A0 in\u00C2\u00A0 vitro\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0 apoptosis,\u00C2\u00A0while\u00C2\u00A0monomeric\u00C2\u00A0Rtx\u00C2\u00A0had\u00C2\u00A0no\u00C2\u00A0such\u00C2\u00A0effect.\u00C2\u00A0\u00C2\u00A0Apoptosis\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0Fc\u00C2\u00A0 receptors\u00C2\u00A0nor\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0density\u00C2\u00A0of\u00C2\u00A0cell\u00E2\u0080\u0090surface\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0homodimers\u00C2\u00A0sensitized\u00C2\u00A0 drug\u00E2\u0080\u0090resistant\u00C2\u00A0 CD20+\u00C2\u00A0 B\u00E2\u0080\u0090lymphoma\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 chemotherapy88.\u00C2\u00A0 \u00C2\u00A0 Zhang\u00C2\u00A0 and\u00C2\u00A0 colleagues\u00C2\u00A0 prepared\u00C2\u00A0 Rtx\u00E2\u0080\u0090 dextran\u00C2\u00A0polymers\u00C2\u00A0(valence\u00C2\u00A0~10)\u00C2\u00A0which\u00C2\u00A0induced\u00C2\u00A0significantly\u00C2\u00A0elevated\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0several\u00C2\u00A0CD20+\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0in\u00C2\u00A0CD20\u00E2\u0080\u0093\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0lines.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0polymer\u00C2\u00A0also\u00C2\u00A0showed\u00C2\u00A0an\u00C2\u00A0extended\u00C2\u00A0circulation\u00C2\u00A0half\u00E2\u0080\u0090 life\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 producing\u00C2\u00A0 a\u00C2\u00A0 marked\u00C2\u00A0 regression\u00C2\u00A0 in\u00C2\u00A0 CD20+\u00C2\u00A0 lymphoma\u00C2\u00A0 xenografts,\u00C2\u00A0while\u00C2\u00A0Rtx\u00C2\u00A0dimer\u00C2\u00A0and\u00C2\u00A0monomer\u00C2\u00A0showed\u00C2\u00A0 little\u00C2\u00A0effect\u00C2\u00A0 in\u00C2\u00A0vivo75.\u00C2\u00A0 \u00C2\u00A0Trivalent\u00C2\u00A0and\u00C2\u00A0 tetravalent\u00C2\u00A0 Rtx\u00C2\u00A0 constructs\u00C2\u00A0 obtained\u00C2\u00A0 through\u00C2\u00A0 protein\u00C2\u00A0 engineering\u00C2\u00A0 were\u00C2\u00A0 prepared\u00C2\u00A0 by\u00C2\u00A0 Miller\u00C2\u00A0 et\u00C2\u00A0 al.;\u00C2\u00A0 many\u00C2\u00A0 configurations\u00C2\u00A0of\u00C2\u00A0constructs\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0with\u00C2\u00A0tandem\u00C2\u00A0Fab\u00C2\u00A0repeats\u00C2\u00A0(with\u00C2\u00A0or\u00C2\u00A0without\u00C2\u00A0Fc\u00C2\u00A0regions)\u00C2\u00A0in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0mimic\u00C2\u00A0 crosslinked\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0Bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 induced\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0while\u00C2\u00A0many\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 multivalent\u00C2\u00A0 constructs\u00C2\u00A0 induced\u00C2\u00A0 elevated\u00C2\u00A0 apoptosis\u00C2\u00A0without\u00C2\u00A0 crosslinking89.\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 final\u00C2\u00A0 example\u00C2\u00A0 from\u00C2\u00A0 Rossi\u00C2\u00A0 and\u00C2\u00A0 colleagues\u00C2\u00A0 involves\u00C2\u00A0 the\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 technique\u00C2\u00A0 known\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 Dock\u00E2\u0080\u0090and\u00E2\u0080\u0090Lock\u00C2\u00A0method\u00C2\u00A0 for\u00C2\u00A0 creating\u00C2\u00A0 hexavalent\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 constructs\u00C2\u00A0 that\u00C2\u00A0 comprise\u00C2\u00A0 six\u00C2\u00A0 Fab\u00C2\u00A0 fragments\u00C2\u00A0 and\u00C2\u00A0 one\u00C2\u00A0 Fc\u00C2\u00A0 fragment.\u00C2\u00A0\u00C2\u00A0 These\u00C2\u00A0constructs\u00C2\u00A0inhibited\u00C2\u00A0proliferation\u00C2\u00A0in\u00C2\u00A0CD20+\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0without\u00C2\u00A0a\u00C2\u00A0crosslinking\u00C2\u00A0Ab,\u00C2\u00A0and\u00C2\u00A0they\u00C2\u00A0 induced\u00C2\u00A0ADCC\u00C2\u00A0although\u00C2\u00A0CDC\u00C2\u00A0was\u00C2\u00A0completely\u00C2\u00A0abolished90.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 These\u00C2\u00A0 studies\u00C2\u00A0 demonstrate\u00C2\u00A0 clear\u00C2\u00A0 benefits\u00C2\u00A0 to\u00C2\u00A0 employing\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 constructs\u00C2\u00A0 as\u00C2\u00A0 opposed\u00C2\u00A0 to\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 Importantly,\u00C2\u00A0 nearly\u00C2\u00A0 all\u00C2\u00A0 reports\u00C2\u00A0 on\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 have\u00C2\u00A0 employed\u00C2\u00A0 constructs\u00C2\u00A0 with\u00C2\u00A0 valences\u00C2\u00A0 that\u00C2\u00A0 generally\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 exceed\u00C2\u00A0 10.\u00C2\u00A0 \u00C2\u00A0 Higher\u00C2\u00A0 valences\u00C2\u00A0 are\u00C2\u00A0 occasionally\u00C2\u00A0 employed\u00C2\u00A0when\u00C2\u00A0different\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0nanoparticle\u00C2\u00A0formulations\u00C2\u00A0are\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0Ab,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0valences\u00C2\u00A0 of\u00C2\u00A0such\u00C2\u00A0constructs\u00C2\u00A0are\u00C2\u00A0undefined\u00C2\u00A0and/or\u00C2\u00A0quite\u00C2\u00A0heterogeneous.\u00C2\u00A0 \u00C2\u00A0Multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0 hand,\u00C2\u00A0represent\u00C2\u00A0constructs\u00C2\u00A0of\u00C2\u00A0defined\u00C2\u00A0valence,\u00C2\u00A0and\u00C2\u00A0valences\u00C2\u00A0as\u00C2\u00A0high\u00C2\u00A0as\u00C2\u00A0250\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0obtained\u00C2\u00A0 in\u00C2\u00A0 sufficient\u00C2\u00A0quantities\u00C2\u00A0for\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0experiments.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A073\u00C2\u00A0\u00C2\u00A0 4.2.2\u00C2\u00A0 Hypotheses\u00C2\u00A0 concerning\u00C2\u00A0 the\u00C2\u00A0 observed\u00C2\u00A0 increases\u00C2\u00A0 in\u00C2\u00A0 apoptosis\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0 Rtx\u00C2\u00ADLNP\u00C2\u00A0\u00C2\u00A0 Previous\u00C2\u00A0work\u00C2\u00A0has\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0valence\u00C2\u00A0~90\u00C2\u00A0induce\u00C2\u00A0significantly\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 target\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 (Fig.\u00C2\u00A0 2.2C\u00C2\u00A0&\u00C2\u00A0 D)7,\u00C2\u00A016.\u00C2\u00A0 There\u00C2\u00A0 are\u00C2\u00A0 two\u00C2\u00A0 possible\u00C2\u00A0scenarios\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0this\u00C2\u00A0observed\u00C2\u00A0enhancement\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action:\u00C2\u00A0(1)\u00C2\u00A0 the\u00C2\u00A0chemical\u00C2\u00A0modification\u00C2\u00A0imparted\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0and\u00C2\u00A0coupling\u00C2\u00A0to\u00C2\u00A0liposomes\u00C2\u00A0induces\u00C2\u00A0an\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 apoptotic\u00C2\u00A0potency\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0itself,\u00C2\u00A0or\u00C2\u00A0(2)\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Ab,\u00C2\u00A0through\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0its\u00C2\u00A0target\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane,\u00C2\u00A0 is\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 enhanced\u00C2\u00A0 apoptosis\u00C2\u00A0 through\u00C2\u00A0 an\u00C2\u00A0 undefined\u00C2\u00A0 mechanism.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0equivalent\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0scenario\u00C2\u00A0 (1)\u00C2\u00A0would\u00C2\u00A0result\u00C2\u00A0 in\u00C2\u00A0equal\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valence\u00C2\u00A0because\u00C2\u00A0the\u00C2\u00A0effect\u00C2\u00A0is\u00C2\u00A0due\u00C2\u00A0specifically\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Ab;\u00C2\u00A0this\u00C2\u00A0would\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 valence\u00E2\u0080\u0090independent\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 scenario\u00C2\u00A0 (2)\u00C2\u00A0would\u00C2\u00A0 show\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 apoptosis\u00C2\u00A0 levels\u00C2\u00A0 because\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 is\u00C2\u00A0 correlated\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 degree\u00C2\u00A0 of\u00C2\u00A0 crosslinking\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0is\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0impart;\u00C2\u00A0higher\u00C2\u00A0valences\u00C2\u00A0can\u00C2\u00A0hypercrosslink\u00C2\u00A0more\u00C2\u00A0appreciably\u00C2\u00A0than\u00C2\u00A0 lower\u00C2\u00A0valences.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 These\u00C2\u00A0 scenarios\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 tested\u00C2\u00A0 by\u00C2\u00A0 creating\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 valence\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 methodology\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 3\u00C2\u00A0 and\u00C2\u00A0 adding\u00C2\u00A0 them\u00C2\u00A0 to\u00C2\u00A0 CD20+\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 whether\u00C2\u00A0the\u00C2\u00A0resulting\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0are\u00C2\u00A0valence\u00E2\u0080\u0090independent\u00C2\u00A0or\u00C2\u00A0\u00E2\u0080\u0090dependent.\u00C2\u00A0 \u00C2\u00A0With\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0 to\u00C2\u00A0reproducibly\u00C2\u00A0prepare\u00C2\u00A0specific\u00C2\u00A0valences\u00C2\u00A0over\u00C2\u00A0a\u00C2\u00A0wide\u00C2\u00A0range\u00C2\u00A0(2\u00C2\u00A0to\u00C2\u00A0250),\u00C2\u00A0we\u00C2\u00A0are\u00C2\u00A0able\u00C2\u00A0to\u00C2\u00A0carry\u00C2\u00A0out\u00C2\u00A0one\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 first\u00C2\u00A0 studies\u00C2\u00A0 that\u00C2\u00A0 systematically\u00C2\u00A0 examines\u00C2\u00A0 the\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 increasing\u00C2\u00A0 degrees\u00C2\u00A0 of\u00C2\u00A0 target\u00C2\u00A0 hypercrosslinking\u00C2\u00A0brought\u00C2\u00A0about\u00C2\u00A0by\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Abs.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A074\u00C2\u00A0\u00C2\u00A0 4.3\u00C2\u00A0\u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0 4.3.1\u00C2\u00A0 Materials\u00C2\u00A0 Rituximab\u00C2\u00A0(Rtx)\u00C2\u00A0and\u00C2\u00A0trastuzumab\u00C2\u00A0(Trz)\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0BC\u00C2\u00A0Cancer\u00C2\u00A0Agency\u00C2\u00A0pharmacy\u00C2\u00A0 (Vancouver\u00C2\u00A0 BC,\u00C2\u00A0 Canada).\u00C2\u00A0 \u00C2\u00A0 Hanks\u00E2\u0080\u0099\u00C2\u00A0 balanced\u00C2\u00A0 salt\u00C2\u00A0 solution\u00C2\u00A0 (modified,\u00C2\u00A0 without\u00C2\u00A0 phenol\u00C2\u00A0 red)\u00C2\u00A0 was\u00C2\u00A0 purchased\u00C2\u00A0 from\u00C2\u00A0 Stemcell\u00C2\u00A0 Technologies\u00C2\u00A0 (Vancouver\u00C2\u00A0 BC,\u00C2\u00A0 Canada).\u00C2\u00A0 \u00C2\u00A0 AlamarBlue,\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00C2\u00A0 binding\u00C2\u00A0 buffer,\u00C2\u00A0 recombinant\u00C2\u00A0 human\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00C2\u00A0 labelled\u00C2\u00A0with\u00C2\u00A0 fluorescein\u00C2\u00A0 isothiocyanate\u00C2\u00A0 (Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC),\u00C2\u00A0 and\u00C2\u00A0an\u00C2\u00A0Alexa\u00C2\u00A0Fluor\u00C2\u00A0647\u00C2\u00A0 (A647)\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0 labeling\u00C2\u00A0kit\u00C2\u00A0were\u00C2\u00A0 from\u00C2\u00A0 Invitrogen\u00C2\u00A0 (Burlington\u00C2\u00A0 ON,\u00C2\u00A0 Canada).\u00C2\u00A0 \u00C2\u00A0 FITC\u00E2\u0080\u0090labeled\u00C2\u00A0 anti\u00E2\u0080\u0090Rtx\u00C2\u00A0 Ab\u00C2\u00A0 (FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx),\u00C2\u00A0 A647\u00E2\u0080\u0090labeled\u00C2\u00A0 monoclonal\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 Ab\u00C2\u00A0 (A647\u00E2\u0080\u0090mCD20),\u00C2\u00A0 and\u00C2\u00A0 A647\u00E2\u0080\u0090labeled\u00C2\u00A0mouse\u00C2\u00A0 IgG2a\u00C2\u00A0 negative\u00C2\u00A0 control\u00C2\u00A0 Ab\u00C2\u00A0 (A647\u00E2\u0080\u0090neg)\u00C2\u00A0 were\u00C2\u00A0 from\u00C2\u00A0 AbD\u00C2\u00A0 Serotec\u00C2\u00A0 (Kidlington,\u00C2\u00A0UK).\u00C2\u00A0 \u00C2\u00A0Glass\u00C2\u00A0 cover\u00C2\u00A0 slips\u00C2\u00A0 (8\u00C2\u00A0mm)\u00C2\u00A0 and\u00C2\u00A0 paraformaldehyde\u00C2\u00A0were\u00C2\u00A0 purchased\u00C2\u00A0 from\u00C2\u00A0 Electron\u00C2\u00A0 Microscopy\u00C2\u00A0 Sciences.\u00C2\u00A0 \u00C2\u00A0 Phycoerythrin\u00E2\u0080\u0090conjugated\u00C2\u00A0 anti\u00E2\u0080\u0090human\u00E2\u0080\u0090IgG\u00C2\u00A0 Ab\u00C2\u00A0 (PE\u00E2\u0080\u0090anti\u00E2\u0080\u0090IgG)\u00C2\u00A0 and\u00C2\u00A0 polyclonal\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 Ab\u00C2\u00A0 were\u00C2\u00A0 purchased\u00C2\u00A0 from\u00C2\u00A0 Santa\u00C2\u00A0 Cruz\u00C2\u00A0 Biotechnology\u00C2\u00A0 (Santa\u00C2\u00A0 Cruz\u00C2\u00A0 CA,\u00C2\u00A0 USA).\u00C2\u00A0\u00C2\u00A0 FluorSave\u00C2\u00A0 reagent\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0Calbiochem\u00C2\u00A0 (Billerica\u00C2\u00A0MA,\u00C2\u00A0USA).\u00C2\u00A0Unless\u00C2\u00A0noted\u00C2\u00A0otherwise,\u00C2\u00A0all\u00C2\u00A0 other\u00C2\u00A0reagents\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Sigma\u00E2\u0080\u0090Aldrich\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0quality\u00C2\u00A0available.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 4.3.2\u00C2\u00A0 Cell\u00C2\u00A0lines\u00C2\u00A0 Ramos\u00C2\u00A0cells\u00C2\u00A0(a\u00C2\u00A0Burkitt\u00E2\u0080\u0099s\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0line)\u00C2\u00A0were\u00C2\u00A0a\u00C2\u00A0kind\u00C2\u00A0gift\u00C2\u00A0from\u00C2\u00A0Dr.\u00C2\u00A0Robert\u00C2\u00A0Kay\u00C2\u00A0(Terry\u00C2\u00A0Fox\u00C2\u00A0 Laboratory,\u00C2\u00A0Vancouver\u00C2\u00A0BC,\u00C2\u00A0Canada),\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0mantle\u00C2\u00A0cell\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0generously\u00C2\u00A0provided\u00C2\u00A0 by\u00C2\u00A0 Dr.\u00C2\u00A0 Zeev\u00C2\u00A0 Estrov\u00C2\u00A0 (University\u00C2\u00A0 of\u00C2\u00A0 Texas)\u00C2\u00A0 and\u00C2\u00A0 previously\u00C2\u00A0 characterized127.\u00C2\u00A0 \u00C2\u00A0 Both\u00C2\u00A0 cell\u00C2\u00A0 lines\u00C2\u00A0 were\u00C2\u00A0 maintained\u00C2\u00A0 in\u00C2\u00A0 RPMI\u00C2\u00A0 1640\u00C2\u00A0 medium\u00C2\u00A0 from\u00C2\u00A0 Stemcell\u00C2\u00A0 Technologies\u00C2\u00A0 (Vancouver\u00C2\u00A0 BC,\u00C2\u00A0 Canada)\u00C2\u00A0 supplemented\u00C2\u00A0with\u00C2\u00A010\u00C2\u00A0%\u00C2\u00A0(v/v)\u00C2\u00A0fetal\u00C2\u00A0bovine\u00C2\u00A0serum\u00C2\u00A0and\u00C2\u00A02\u00C2\u00A0mM\u00C2\u00A0L\u00E2\u0080\u0090glutamine\u00C2\u00A0from\u00C2\u00A0Invitrogen,\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0 penicillin\u00E2\u0080\u0090streptomysin\u00C2\u00A0from\u00C2\u00A0Stemcell\u00C2\u00A0Technologies.\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0maintained\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0\u00C2\u00B0C\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0humidified\u00C2\u00A0 atmosphere\u00C2\u00A0containing\u00C2\u00A05%\u00C2\u00A0CO2.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A075\u00C2\u00A0\u00C2\u00A0 4.3.3\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNPs\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0and\u00C2\u00A0summarized\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 3.1.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Three\u00C2\u00A0 separate\u00C2\u00A0 batches\u00C2\u00A0 of\u00C2\u00A0 four\u00C2\u00A0 valences\u00C2\u00A0 each\u00C2\u00A0 were\u00C2\u00A0 prepared\u00C2\u00A0 over\u00C2\u00A0 the\u00C2\u00A0 course\u00C2\u00A0 of\u00C2\u00A0 approximately\u00C2\u00A0six\u00C2\u00A0weeks\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0resulting\u00C2\u00A0valences\u00C2\u00A0ranged\u00C2\u00A0 from\u00C2\u00A013\u00C2\u00A0to\u00C2\u00A0165.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0valences\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0 concentrations\u00C2\u00A0in\u00C2\u00A0each\u00C2\u00A0preparation\u00C2\u00A0were\u00C2\u00A0quantified\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0previous\u00C2\u00A0chapter.\u00C2\u00A0 4.3.4\u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0free\u00C2\u00A0CD20\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 or\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0examine\u00C2\u00A0the\u00C2\u00A0relationship\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0CD20+\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0free\u00C2\u00A0from\u00C2\u00A0Rtx,\u00C2\u00A01.25\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0250\u00C2\u00A0 \u00CE\u00BCL\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 ranging\u00C2\u00A0 from\u00C2\u00A00\u00E2\u0080\u009330\u00C2\u00A0 \u00CE\u00BCg.\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0a\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0 incubation\u00C2\u00A0 at\u00C2\u00A0 37\u00C2\u00A0 oC,\u00C2\u00A0 the\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 transferred\u00C2\u00A0 to\u00C2\u00A0 Eppendorf\u00C2\u00A0 tubes,\u00C2\u00A0 washed\u00C2\u00A0 with\u00C2\u00A0 cold\u00C2\u00A0 PBSB\u00C2\u00A0 (phosphate\u00E2\u0080\u0090buffered\u00C2\u00A0saline\u00C2\u00A0(PBS)\u00C2\u00A0+\u00C2\u00A00.1%\u00C2\u00A0bovine\u00C2\u00A0serum\u00C2\u00A0albumin,\u00C2\u00A0pH\u00C2\u00A07.4),\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A020\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0PBSB,\u00C2\u00A0 and\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A01\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0each\u00C2\u00A0of\u00C2\u00A0FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090mCD20.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0latter\u00C2\u00A0Ab\u00C2\u00A0is\u00C2\u00A0blocked\u00C2\u00A0from\u00C2\u00A0binding\u00C2\u00A0 to\u00C2\u00A0CD20\u00C2\u00A0if\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0already\u00C2\u00A0bound,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0former\u00C2\u00A0one\u00C2\u00A0measures\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0staining\u00C2\u00A0for\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0on\u00C2\u00A0 ice,\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 washed\u00C2\u00A0 with\u00C2\u00A0 PBSB,\u00C2\u00A0 then\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0 300\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 PBSB,\u00C2\u00A0 filtered\u00C2\u00A0 through\u00C2\u00A0 35\u00C2\u00A0 \u00C2\u00B5m\u00C2\u00A0 strainers,\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0FACSCalibur\u00C2\u00A0flow\u00C2\u00A0cytometer\u00C2\u00A0(Becton\u00C2\u00A0Dickinson,\u00C2\u00A0San\u00C2\u00A0Jose,\u00C2\u00A0CA)\u00C2\u00A0collecting\u00C2\u00A0 10,000\u00C2\u00A0events\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0sample.\u00C2\u00A0 \u00C2\u00A0Fluorescence\u00C2\u00A0 intensities\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0analysis\u00C2\u00A0using\u00C2\u00A0FlowJo\u00C2\u00A0 software,\u00C2\u00A0and\u00C2\u00A0 triplicates\u00C2\u00A0were\u00C2\u00A0averaged\u00C2\u00A0 to\u00C2\u00A0obtain\u00C2\u00A0mean\u00C2\u00A0 fluorescence\u00C2\u00A0 intensities\u00C2\u00A0 (MFIs).\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0each\u00C2\u00A0 dose,\u00C2\u00A0the\u00C2\u00A0MFI\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0appropriate\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0was\u00C2\u00A0subtracted\u00C2\u00A0from\u00C2\u00A0each\u00C2\u00A0measured\u00C2\u00A0MFI\u00C2\u00A0value\u00C2\u00A0 before\u00C2\u00A0being\u00C2\u00A0scaled\u00C2\u00A0relative\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0maximum\u00C2\u00A0value\u00C2\u00A0 for\u00C2\u00A0each\u00C2\u00A0stain.\u00C2\u00A0 \u00C2\u00A0Data\u00C2\u00A0values\u00C2\u00A0are\u00C2\u00A0reported\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0 cases\u00C2\u00A0as\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0standard\u00C2\u00A0deviation\u00C2\u00A0(SD).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A076\u00C2\u00A0\u00C2\u00A0 4.3.5\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0Confocal\u00C2\u00A0laser\u00C2\u00ADscanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0of\u00C2\u00A0treated\u00C2\u00A0cells\u00C2\u00A0 Ramos\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0divided\u00C2\u00A0 in\u00C2\u00A048\u00E2\u0080\u0090well\u00C2\u00A0plates\u00C2\u00A0with\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium\u00C2\u00A0 per\u00C2\u00A0well.\u00C2\u00A0\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0and\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0diluted\u00C2\u00A0to\u00C2\u00A0obtain\u00C2\u00A0equal\u00C2\u00A0Rtx\u00C2\u00A0concentrations\u00C2\u00A0across\u00C2\u00A0all\u00C2\u00A0samples,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 required\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0or\u00C2\u00A0 free\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0calculated.\u00C2\u00A0 \u00C2\u00A0Generally,\u00C2\u00A0 the\u00C2\u00A0 treatment\u00C2\u00A0volume\u00C2\u00A0 was\u00C2\u00A0between\u00C2\u00A0100\u00C2\u00A0and\u00C2\u00A0200\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0for\u00C2\u00A0a\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0treatment\u00C2\u00A0times\u00C2\u00A0of\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A048\u00C2\u00A0h,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 spun\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0microcentrifuge\u00C2\u00A0at\u00C2\u00A07000\u00C2\u00A0 rpm\u00C2\u00A0 for\u00C2\u00A015\u00C2\u00A0 s,\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0 serum\u00E2\u0080\u0090free\u00C2\u00A0medium\u00C2\u00A0 (also\u00C2\u00A0 free\u00C2\u00A0of\u00C2\u00A0L\u00E2\u0080\u0090 glutamine\u00C2\u00A0and\u00C2\u00A0penicillin\u00E2\u0080\u0090streptomysin),\u00C2\u00A0then\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0cold\u00C2\u00A0serum\u00E2\u0080\u0090free\u00C2\u00A0medium.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0 a\u00C2\u00A0cold\u00C2\u00A0room,\u00C2\u00A0a\u00C2\u00A050\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0suspension\u00C2\u00A0(5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0104\u00C2\u00A0cells)\u00C2\u00A0was\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0Eppendorf\u00C2\u00A0tube\u00C2\u00A0 and\u00C2\u00A01\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0PE\u00E2\u0080\u0090anti\u00E2\u0080\u0090IgG\u00C2\u00A0was\u00C2\u00A0added.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0suspension\u00C2\u00A0was\u00C2\u00A0mixed\u00C2\u00A0and\u00C2\u00A0transferred\u00C2\u00A0onto\u00C2\u00A0an\u00C2\u00A08\u00C2\u00A0mm\u00C2\u00A0poly\u00E2\u0080\u0090L\u00E2\u0080\u0090 lysine\u00C2\u00A0 glass\u00C2\u00A0 cover\u00C2\u00A0 slip\u00C2\u00A0onto\u00C2\u00A0which\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 allowed\u00C2\u00A0 to\u00C2\u00A0 adhere\u00C2\u00A0 for\u00C2\u00A0 1\u00C2\u00A0h\u00C2\u00A0on\u00C2\u00A0 ice.\u00C2\u00A0 \u00C2\u00A0 Liquid\u00C2\u00A0was\u00C2\u00A0 then\u00C2\u00A0 removed\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0cover\u00C2\u00A0slip,\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0gently\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0then\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0fixed\u00C2\u00A0by\u00C2\u00A0applying\u00C2\u00A0a\u00C2\u00A075\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0drop\u00C2\u00A0of\u00C2\u00A04%\u00C2\u00A0paraformaldehyde\u00C2\u00A0for\u00C2\u00A020\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A0room\u00C2\u00A0temperature.\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0fixing,\u00C2\u00A0the\u00C2\u00A0cover\u00C2\u00A0slip\u00C2\u00A0was\u00C2\u00A0 washed\u00C2\u00A0with\u00C2\u00A0 HBS\u00C2\u00A0 before\u00C2\u00A0mounting\u00C2\u00A0 onto\u00C2\u00A0 a\u00C2\u00A0 glass\u00C2\u00A0 slide\u00C2\u00A0 using\u00C2\u00A0 10\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 of\u00C2\u00A0 FluorSave.\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0were\u00C2\u00A0 allowed\u00C2\u00A0 to\u00C2\u00A0dry\u00C2\u00A0overnight\u00C2\u00A0 in\u00C2\u00A0closed\u00C2\u00A0Petri\u00C2\u00A0dishes\u00C2\u00A0protected\u00C2\u00A0 from\u00C2\u00A0 light,\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0 imaged\u00C2\u00A0within\u00C2\u00A0 two\u00C2\u00A0 days\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 Bio\u00E2\u0080\u0090Rad\u00C2\u00A0 2100MP\u00C2\u00A0 confocal\u00C2\u00A0 and\u00C2\u00A0 multiphoton\u00C2\u00A0 microscope\u00C2\u00A0 controlled\u00C2\u00A0 by\u00C2\u00A0 Zeiss/Biorad\u00C2\u00A0 Lasersharp\u00C2\u00A0 2000\u00C2\u00A0 software.\u00C2\u00A0 \u00C2\u00A0 Negligible\u00C2\u00A0 staining\u00C2\u00A0 was\u00C2\u00A0 observed\u00C2\u00A0 in\u00C2\u00A0 fluorescence\u00C2\u00A0 images\u00C2\u00A0 of\u00C2\u00A0 control\u00C2\u00A0 samples\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0not\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 4.3.6\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0Measurement\u00C2\u00A0of\u00C2\u00A0total\u00C2\u00A0CD20\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0treated\u00C2\u00A0cells\u00C2\u00A0 A\u00C2\u00A0polyclonal\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0labeled\u00C2\u00A0with\u00C2\u00A0A647\u00C2\u00A0using\u00C2\u00A0an\u00C2\u00A0antibody\u00C2\u00A0labeling\u00C2\u00A0kit\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0instructions.\u00C2\u00A0\u00C2\u00A0Unlike\u00C2\u00A0the\u00C2\u00A0monoclonal\u00C2\u00A0A647\u00E2\u0080\u0090mCD20\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A04.3.4,\u00C2\u00A0the\u00C2\u00A0 resulting\u00C2\u00A0A647\u00E2\u0080\u0090labeled\u00C2\u00A0polyclonal\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0Ab\u00C2\u00A0(A647\u00E2\u0080\u0090pCD20)\u00C2\u00A0exhibited\u00C2\u00A0equivalent\u00C2\u00A0staining\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0and\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0Ramos\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0divided\u00C2\u00A0in\u00C2\u00A048\u00E2\u0080\u0090well\u00C2\u00A0 plates\u00C2\u00A0with\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0medium\u00C2\u00A0per\u00C2\u00A0well,\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0an\u00C2\u00A0 incubation\u00C2\u00A0of\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A048\u00C2\u00A0h,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0PI\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090pCD20,\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A077\u00C2\u00A0\u00C2\u00A0 analyzed\u00C2\u00A0 using\u00C2\u00A0 flow\u00C2\u00A0 cytometry,\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 4.3.4.\u00C2\u00A0 \u00C2\u00A0 Before\u00C2\u00A0 analysis,\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 resuspended\u00C2\u00A0in\u00C2\u00A0PBSB\u00C2\u00A0containing\u00C2\u00A00.5\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0propidium\u00C2\u00A0iodide\u00C2\u00A0(PI),\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090pCD20\u00C2\u00A0MFI\u00C2\u00A0values\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0 cases\u00C2\u00A0were\u00C2\u00A0calculated\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0data\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0(PI\u00E2\u0080\u0093)\u00C2\u00A0populations.\u00C2\u00A0 \u00C2\u00A0Here\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0subsequent\u00C2\u00A0 cases,\u00C2\u00A0two\u00E2\u0080\u0090tailed\u00C2\u00A0unpaired\u00C2\u00A0Student\u00E2\u0080\u0099s\u00C2\u00A0t\u00E2\u0080\u0090tests\u00C2\u00A0were\u00C2\u00A0applied\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0significance\u00C2\u00A0of\u00C2\u00A00.05.\u00C2\u00A0 4.3.7\u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0mitochondrial\u00C2\u00A0activity\u00C2\u00A0using\u00C2\u00A0AlamarBlue\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0 evaluate\u00C2\u00A0 the\u00C2\u00A0 cytotoxicity\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs,\u00C2\u00A0 AlamarBlue\u00C2\u00A0was\u00C2\u00A0 employed\u00C2\u00A0 to\u00C2\u00A0measure\u00C2\u00A0 mitochondrial\u00C2\u00A0activity\u00C2\u00A0in\u00C2\u00A0samples\u00C2\u00A0of\u00C2\u00A0treated\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0a\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plate,\u00C2\u00A05\u00C2\u00A0\u00EF\u0082\u00B4\u00C2\u00A0103\u00C2\u00A0Ramos\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0 100\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0wells,\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A050\u00C2\u00A0\u00C2\u00B5L/well\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0ranging\u00C2\u00A0 from\u00C2\u00A00.2\u00C2\u00A0to\u00C2\u00A02000\u00C2\u00A0ng/well.\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0a\u00C2\u00A096\u00C2\u00A0h\u00C2\u00A0 incubation,\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0of\u00C2\u00A0AlamarBlue\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0per\u00C2\u00A0well,\u00C2\u00A0and\u00C2\u00A0 plates\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 for\u00C2\u00A0 a\u00C2\u00A0 further\u00C2\u00A0 6\u00C2\u00A0 h\u00C2\u00A0 before\u00C2\u00A0 fluorescence\u00C2\u00A0measurements\u00C2\u00A0were\u00C2\u00A0 obtained\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 Fluorstar\u00C2\u00A0 plate\u00C2\u00A0 reader\u00C2\u00A0 with\u00C2\u00A0 excitation\u00C2\u00A0 and\u00C2\u00A0 emission\u00C2\u00A0 wavelengths\u00C2\u00A0 of\u00C2\u00A0 544\u00C2\u00A0 nm\u00C2\u00A0 and\u00C2\u00A0 590\u00C2\u00A0 nm,\u00C2\u00A0 respectively.\u00C2\u00A0\u00C2\u00A0Larger\u00C2\u00A0emission\u00C2\u00A0intensities\u00C2\u00A0corresponded\u00C2\u00A0to\u00C2\u00A0higher\u00C2\u00A0mitochondrial\u00C2\u00A0activities\u00C2\u00A0and\u00C2\u00A0greater\u00C2\u00A0 fractions\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0cells.\u00C2\u00A0 4.3.8\u00C2\u00A0 Annexin\u00C2\u00ADV/PI\u00C2\u00A0apoptosis\u00C2\u00A0assay\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0 This\u00C2\u00A0assay\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A02.3.6,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0modifications\u00C2\u00A0described\u00C2\u00A0here\u00C2\u00A0apply\u00C2\u00A0to\u00C2\u00A0 this\u00C2\u00A0and\u00C2\u00A0all\u00C2\u00A0subsequent\u00C2\u00A0chapters.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A010\u00C2\u00A0 \u00C2\u00B5g\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0 time\u00E2\u0080\u0090course\u00C2\u00A0experiments,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0 oC\u00C2\u00A0 for\u00C2\u00A04\u00C2\u00A0 to\u00C2\u00A072\u00C2\u00A0h,\u00C2\u00A0and\u00C2\u00A0all\u00C2\u00A0 other\u00C2\u00A0experiments\u00C2\u00A0involved\u00C2\u00A0a\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0incubation\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC.\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0equivalent\u00C2\u00A0mass\u00C2\u00A0of\u00C2\u00A0Trz\u00C2\u00A0 were\u00C2\u00A0 used\u00C2\u00A0 as\u00C2\u00A0 negative\u00C2\u00A0 controls,\u00C2\u00A0 and\u00C2\u00A0 for\u00C2\u00A0 positive\u00C2\u00A0 controls,\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 4.0\u00C2\u00A0 \u00C2\u00B5M\u00C2\u00A0 camptothecin\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC\u00C2\u00A0 for\u00C2\u00A04\u00C2\u00A0h.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0analysis,\u00C2\u00A0samples\u00C2\u00A0were\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A01.5\u00C2\u00A0mL\u00C2\u00A0Eppendorf\u00C2\u00A0tubes\u00C2\u00A0 and\u00C2\u00A0spun\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0microcentrifuge\u00C2\u00A0at\u00C2\u00A07000\u00C2\u00A0rpm\u00C2\u00A0for\u00C2\u00A015\u00C2\u00A0s.\u00C2\u00A0 \u00C2\u00A0Pellets\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0cold\u00C2\u00A0Hanks\u00E2\u0080\u0099\u00C2\u00A0 balanced\u00C2\u00A0salt\u00C2\u00A0solution.\u00C2\u00A0\u00C2\u00A0Different\u00C2\u00A0positive\u00C2\u00A0control\u00C2\u00A0samples\u00C2\u00A0were\u00C2\u00A0unstained,\u00C2\u00A0single\u00E2\u0080\u0090stained,\u00C2\u00A0or\u00C2\u00A0double\u00E2\u0080\u0090 stained\u00C2\u00A0with\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0and/or\u00C2\u00A0propidium\u00C2\u00A0 iodide\u00C2\u00A0 (PI);\u00C2\u00A0all\u00C2\u00A0other\u00C2\u00A0 samples\u00C2\u00A0were\u00C2\u00A0double\u00E2\u0080\u0090stained.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A078\u00C2\u00A0\u00C2\u00A0 Double\u00C2\u00A0staining\u00C2\u00A0consisted\u00C2\u00A0of\u00C2\u00A0resuspending\u00C2\u00A0the\u00C2\u00A0pellets\u00C2\u00A0in\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0(5\u00C2\u00A0\u00C2\u00B5L)\u00C2\u00A0and\u00C2\u00A0binding\u00C2\u00A0buffer\u00C2\u00A0(40\u00C2\u00A0 \u00C2\u00B5L),\u00C2\u00A0incubating\u00C2\u00A0at\u00C2\u00A0room\u00C2\u00A0temperature\u00C2\u00A0for\u00C2\u00A020\u00C2\u00A0min,\u00C2\u00A0then\u00C2\u00A0adding\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0cold\u00C2\u00A00.5\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0solution\u00C2\u00A0of\u00C2\u00A0 PI\u00C2\u00A0in\u00C2\u00A0Hanks\u00E2\u0080\u0099.\u00C2\u00A0 \u00C2\u00A0Samples\u00C2\u00A0were\u00C2\u00A0strained\u00C2\u00A0through\u00C2\u00A050\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0strainers,\u00C2\u00A0kept\u00C2\u00A0on\u00C2\u00A0ice,\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0within\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0 on\u00C2\u00A0a\u00C2\u00A0Becton\u00C2\u00A0Dickinson\u00C2\u00A0 FACSCalibur\u00C2\u00A0 flow\u00C2\u00A0 cytometer.\u00C2\u00A0 \u00C2\u00A0Compensation\u00C2\u00A0 values\u00C2\u00A0were\u00C2\u00A0 set\u00C2\u00A0with\u00C2\u00A0positive\u00C2\u00A0 controls\u00C2\u00A0 and\u00C2\u00A0 at\u00C2\u00A0 least\u00C2\u00A0 10,000\u00C2\u00A0 events\u00C2\u00A0were\u00C2\u00A0 recorded\u00C2\u00A0 for\u00C2\u00A0 each\u00C2\u00A0 sample.\u00C2\u00A0 \u00C2\u00A0 Early\u00C2\u00A0 apoptotic\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 defined\u00C2\u00A0 as\u00C2\u00A0 PI\u00C2\u00A0 negative\u00C2\u00A0 and\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0 positive,\u00C2\u00A0 necrotic\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 positive\u00C2\u00A0 for\u00C2\u00A0 both\u00C2\u00A0 fluorophores,\u00C2\u00A0and\u00C2\u00A0viable\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0negative\u00C2\u00A0for\u00C2\u00A0both.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 4.4\u00C2\u00A0\u00C2\u00A0 Results\u00C2\u00A0 4.4.1\u00C2\u00A0 Rtx\u00C2\u00ADLNPs\u00C2\u00A0 exhibit\u00C2\u00A0 unique\u00C2\u00A0 binding\u00C2\u00A0 properties\u00C2\u00A0 to\u00C2\u00A0 CD20+\u00C2\u00A0 target\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0 assess\u00C2\u00A0differences\u00C2\u00A0 in\u00C2\u00A0binding\u00C2\u00A0 properties\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 bivalent\u00C2\u00A0Rtx,\u00C2\u00A0 two\u00C2\u00A0different\u00C2\u00A0CD20+\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0were\u00C2\u00A0employed:\u00C2\u00A0Ramos,\u00C2\u00A0a\u00C2\u00A0Burkitt\u00E2\u0080\u0099s\u00C2\u00A0 lymphoma\u00C2\u00A0cell\u00C2\u00A0 line,\u00C2\u00A0 and\u00C2\u00A0Z138,\u00C2\u00A0a\u00C2\u00A0mantle\u00C2\u00A0cell\u00C2\u00A0 lymphoma\u00C2\u00A0cell\u00C2\u00A0 line.\u00C2\u00A0 \u00C2\u00A0Two\u00C2\u00A0values\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry:\u00C2\u00A0the\u00C2\u00A0 amount\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0that\u00C2\u00A0became\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cells,\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx\u00C2\u00A0Ab,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0free\u00C2\u00A0from\u00C2\u00A0bound\u00C2\u00A0Rtx,\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0A647\u00E2\u0080\u0090mCD20\u00C2\u00A0Ab\u00C2\u00A0(see\u00C2\u00A0Section\u00C2\u00A04.3.4).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0 Rtx\u00C2\u00A0 to\u00C2\u00A0CD20\u00C2\u00A0completely\u00C2\u00A0 inhibits\u00C2\u00A0 the\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090mCD20,\u00C2\u00A0 thereby\u00C2\u00A0providing\u00C2\u00A0a\u00C2\u00A0practical\u00C2\u00A0way\u00C2\u00A0of\u00C2\u00A0 measuring\u00C2\u00A0unbound\u00C2\u00A0CD20.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0 titration\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0 first\u00C2\u00A0performed\u00C2\u00A0across\u00C2\u00A0a\u00C2\u00A00\u00E2\u0080\u009330\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose\u00C2\u00A0 range\u00C2\u00A0 given\u00C2\u00A0 to\u00C2\u00A01.25\u00C2\u00A0 \u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.1A,\u00C2\u00A0 the\u00C2\u00A0 amount\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0 (white\u00C2\u00A0bars)\u00C2\u00A0began\u00C2\u00A0to\u00C2\u00A0decrease\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A00.03\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0and\u00C2\u00A0above\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A00.01\u00C2\u00A0and\u00C2\u00A00.03\u00C2\u00A0 \u00C2\u00B5g\u00C2\u00A0doses),\u00C2\u00A0and\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0above\u00C2\u00A00.3\u00C2\u00A0\u00C2\u00B5g,\u00C2\u00A0essentially\u00C2\u00A0all\u00C2\u00A0cell\u00E2\u0080\u0090surface\u00C2\u00A0CD20\u00C2\u00A0molecules\u00C2\u00A0were\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0 Rtx\u00C2\u00A0as\u00C2\u00A0reflected\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0relative\u00C2\u00A0MFI\u00C2\u00A0values\u00C2\u00A0of\u00C2\u00A0zero\u00C2\u00A0(within\u00C2\u00A0error),\u00C2\u00A0which\u00C2\u00A0represent\u00C2\u00A0MFI\u00C2\u00A0values\u00C2\u00A0roughly\u00C2\u00A0 equal\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0of\u00C2\u00A0negative\u00C2\u00A0isotype\u00C2\u00A0controls.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0(black\u00C2\u00A0bars),\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A079\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.1\u00C2\u00A0\u00C2\u00A0Levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0show\u00C2\u00A0an\u00C2\u00A0inverse\u00C2\u00A0correlation\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0 but\u00C2\u00A0not\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 In\u00C2\u00A0all\u00C2\u00A0cases,\u00C2\u00A01.25\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0250\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0then\u00C2\u00A0analyzed\u00C2\u00A0for\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0 Rtx\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0and\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0(white\u00C2\u00A0bars).\u00C2\u00A0\u00C2\u00A0(A)\u00C2\u00A0The\u00C2\u00A0indicated\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0given,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0show\u00C2\u00A0 an\u00C2\u00A0inverse\u00C2\u00A0correlation\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20.\u00C2\u00A0\u00C2\u00A0(B)\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP(158),\u00C2\u00A0and\u00C2\u00A0although\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0relationship\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A0there\u00C2\u00A0was\u00C2\u00A0still\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0 proportion\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0dose.\u00C2\u00A0 \u00C2\u00A0(C)\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valence\u00C2\u00A0were\u00C2\u00A0administered\u00C2\u00A0to\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A03\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose.\u00C2\u00A0\u00C2\u00A0Lower\u00C2\u00A0valences\u00C2\u00A0showed\u00C2\u00A0similar\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0bivalent\u00C2\u00A0Rtx,\u00C2\u00A0but\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0was\u00C2\u00A0increased\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0 same\u00C2\u00A0dose,\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0also\u00C2\u00A0increased.\u00C2\u00A0\u00C2\u00A0Higher\u00C2\u00A0valences\u00C2\u00A0also\u00C2\u00A0showed\u00C2\u00A0more\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0 Black\u00C2\u00A0bars:\u00C2\u00A0FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx\u00C2\u00A0staining;\u00C2\u00A0white\u00C2\u00A0bars:\u00C2\u00A0A647\u00E2\u0080\u0090mCD20\u00C2\u00A0staining.\u00C2\u00A0\u00C2\u00A0MFI:\u00C2\u00A0mean\u00C2\u00A0fluorescence\u00C2\u00A0intensity.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u009020 0 20 40 60 80 100 120 0 0.01 0.03 0.1 0.3 1 3 10 30 Re la ti ve \u00C2\u00A0M FI Dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0/\u00C2\u00A0\u00CE\u00BCg A \u00E2\u0080\u009020 0 20 40 60 80 100 120 0 1 3 10 30 Re la ti ve \u00C2\u00A0M FI Dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0(in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP)\u00C2\u00A0/\u00C2\u00A0\u00CE\u00BCg B \u00E2\u0080\u009020 0 20 40 60 80 100 120 0 2 19 32 75 113 158 Re la ti ve \u00C2\u00A0M FI Valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP C \u00E2\u0080\u00A2\u00C2\u00A080\u00C2\u00A0\u00C2\u00A0 measurable\u00C2\u00A0amounts\u00C2\u00A0appeared\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A00.03\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0and\u00C2\u00A0above,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 at\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A03\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0and\u00C2\u00A0above\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.005\u00C2\u00A0between\u00C2\u00A01\u00C2\u00A0and\u00C2\u00A03\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0doses;\u00C2\u00A0p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A03,\u00C2\u00A010,\u00C2\u00A0and\u00C2\u00A030\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0 doses).\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0doses\u00C2\u00A0between\u00C2\u00A00.1\u00E2\u0080\u00930.3\u00C2\u00A0\u00C2\u00B5g,\u00C2\u00A0there\u00C2\u00A0were\u00C2\u00A0measurable\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0free\u00C2\u00A0 CD20,\u00C2\u00A0 indicated\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0only\u00C2\u00A0partially\u00C2\u00A0saturated\u00C2\u00A0at\u00C2\u00A0these\u00C2\u00A0doses,\u00C2\u00A0and\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0 point\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0half\u00E2\u0080\u0090saturated\u00C2\u00A0(equal\u00C2\u00A0values\u00C2\u00A0of\u00C2\u00A0white\u00C2\u00A0and\u00C2\u00A0black\u00C2\u00A0bars)\u00C2\u00A0lied\u00C2\u00A0between\u00C2\u00A0these\u00C2\u00A0 two\u00C2\u00A0 doses.\u00C2\u00A0 \u00C2\u00A0 Apart\u00C2\u00A0 from\u00C2\u00A0 Rtx\u00C2\u00A0 saturation\u00C2\u00A0 data\u00C2\u00A0 in\u00C2\u00A0 Ramos\u00C2\u00A0 cells,\u00C2\u00A0 this\u00C2\u00A0 figure\u00C2\u00A0 illustrates\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 Abs\u00C2\u00A0 employed\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 assay\u00C2\u00A0 function\u00C2\u00A0 as\u00C2\u00A0 expected,\u00C2\u00A0 given\u00C2\u00A0 the\u00C2\u00A0 excellent\u00C2\u00A0 inverse\u00C2\u00A0 correlation\u00C2\u00A0 observed\u00C2\u00A0 between\u00C2\u00A0 the\u00C2\u00A0data\u00C2\u00A0reported\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 two\u00C2\u00A0Abs.\u00C2\u00A0 \u00C2\u00A0Similar\u00C2\u00A0data\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A0Z138\u00C2\u00A0cell\u00C2\u00A0 line\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0similar\u00C2\u00A0titration\u00C2\u00A0was\u00C2\u00A0next\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0using\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0of\u00C2\u00A0valence\u00C2\u00A0158\u00C2\u00A0(Rtx\u00E2\u0080\u0090LNP(158)),\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0 data\u00C2\u00A0 that\u00C2\u00A0was\u00C2\u00A0 obtained\u00C2\u00A0 is\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.1B.\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0 shown\u00C2\u00A0 for\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx,\u00C2\u00A0 the\u00C2\u00A0 measured\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0detected\u00C2\u00A0using\u00C2\u00A0FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx\u00C2\u00A0increased\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0 increased,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0dose\u00C2\u00A0at\u00C2\u00A0which\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0saturated\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0found\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 higher\u00C2\u00A0than\u00C2\u00A0that\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0the\u00C2\u00A0unscaled\u00C2\u00A0MFI\u00C2\u00A0measured\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP(158)\u00C2\u00A0 (10\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose)\u00C2\u00A0was\u00C2\u00A0984\u00C2\u00A0after\u00C2\u00A0subtracting\u00C2\u00A0negative\u00C2\u00A0 isotype\u00C2\u00A0control;\u00C2\u00A0 this\u00C2\u00A0value\u00C2\u00A0 is\u00C2\u00A010.0\u00C2\u00A0 times\u00C2\u00A0 higher\u00C2\u00A0 than\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 value\u00C2\u00A0 for\u00C2\u00A0bound\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0dose\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A04.1A\u00C2\u00A0 (98.7).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0bind\u00C2\u00A0 significantly\u00C2\u00A0more\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 bivalent\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0spite\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0higher\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx,\u00C2\u00A0Figure\u00C2\u00A04.1B\u00C2\u00A0shows\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0that\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0have\u00C2\u00A0Rtx\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0it\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A03\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0and\u00C2\u00A0above\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A03,\u00C2\u00A010,\u00C2\u00A0and\u00C2\u00A0 30\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0doses),\u00C2\u00A0which\u00C2\u00A0includes\u00C2\u00A0saturating\u00C2\u00A0doses.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A030\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(158),\u00C2\u00A041%\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0is\u00C2\u00A0free\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0Therefore,\u00C2\u00A0at\u00C2\u00A0saturating\u00C2\u00A0doses,\u00C2\u00A0although\u00C2\u00A0significantly\u00C2\u00A0 higher\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0are\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0bivalent\u00C2\u00A0Rtx,\u00C2\u00A0only\u00C2\u00A0a\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0physically\u00C2\u00A0binds\u00C2\u00A0to\u00C2\u00A0CD20,\u00C2\u00A0since\u00C2\u00A0under\u00C2\u00A0one\u00C2\u00A0half\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0CD20\u00C2\u00A0molecules\u00C2\u00A0 on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0are\u00C2\u00A0free\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0Note\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0scaling\u00C2\u00A0of\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A081\u00C2\u00A0\u00C2\u00A0 black\u00C2\u00A0bars\u00C2\u00A0is\u00C2\u00A0different\u00C2\u00A0between\u00C2\u00A0Figures\u00C2\u00A04.1A\u00E2\u0080\u0093C,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0scaling\u00C2\u00A0of\u00C2\u00A0white\u00C2\u00A0bars\u00C2\u00A0is\u00C2\u00A0roughly\u00C2\u00A0the\u00C2\u00A0same;\u00C2\u00A0the\u00C2\u00A0 white\u00C2\u00A0bars\u00C2\u00A0are\u00C2\u00A0scaled\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0black\u00C2\u00A0bars\u00C2\u00A0are\u00C2\u00A0scaled\u00C2\u00A0according\u00C2\u00A0 to\u00C2\u00A0the\u00C2\u00A0maximum\u00C2\u00A0measured\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx.\u00C2\u00A0 Finally,\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 equivalent\u00C2\u00A0 doses\u00C2\u00A0 (3\u00C2\u00A0 \u00C2\u00B5g)\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 of\u00C2\u00A0 different\u00C2\u00A0 valence,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0properties\u00C2\u00A0were\u00C2\u00A0examined\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0way.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A03\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose\u00C2\u00A0was\u00C2\u00A0selected\u00C2\u00A0 because\u00C2\u00A0as\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.1B,\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0saturated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0at\u00C2\u00A0this\u00C2\u00A0dose,\u00C2\u00A0but\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 were\u00C2\u00A0 just\u00C2\u00A0 saturated,\u00C2\u00A0 as\u00C2\u00A0 indicated\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.1A.\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 4.1C\u00C2\u00A0 summarizes\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0valences.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0first\u00C2\u00A0feature\u00C2\u00A0worth\u00C2\u00A0noting\u00C2\u00A0is\u00C2\u00A0 that\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0the\u00C2\u00A0lower\u00C2\u00A0valences\u00C2\u00A0(19\u00C2\u00A0and\u00C2\u00A032)\u00C2\u00A0show\u00C2\u00A0equal\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A0 0.05)\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0roughly\u00C2\u00A0equivalent\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0measured\u00C2\u00A0for\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0(albeit\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0these\u00C2\u00A0 valences\u00C2\u00A0and\u00C2\u00A0valence\u00C2\u00A02).\u00C2\u00A0\u00C2\u00A0Valences\u00C2\u00A02,\u00C2\u00A019,\u00C2\u00A0and\u00C2\u00A032,\u00C2\u00A0however,\u00C2\u00A0show\u00C2\u00A0increasing\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0 as\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0 increases\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0valences).\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0titration\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A0 4.1B,\u00C2\u00A0this\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0not\u00C2\u00A0all\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0time,\u00C2\u00A0 but\u00C2\u00A0that\u00C2\u00A0at\u00C2\u00A0any\u00C2\u00A0given\u00C2\u00A0time\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0associated\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0LNP\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0CD20.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 At\u00C2\u00A0 this\u00C2\u00A0 same\u00C2\u00A0 dose,\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 increased\u00C2\u00A0 above\u00C2\u00A0 32,\u00C2\u00A0more\u00C2\u00A0 and\u00C2\u00A0more\u00C2\u00A0 bound\u00C2\u00A0 Rtx\u00C2\u00A0was\u00C2\u00A0 measured;\u00C2\u00A0for\u00C2\u00A0example,\u00C2\u00A0the\u00C2\u00A0unscaled\u00C2\u00A0MFI\u00C2\u00A0for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(32)\u00C2\u00A0was\u00C2\u00A0133,\u00C2\u00A0while\u00C2\u00A0that\u00C2\u00A0for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(113)\u00C2\u00A0was\u00C2\u00A0 343,\u00C2\u00A0representing\u00C2\u00A0a\u00C2\u00A02.6\u00E2\u0080\u0090fold\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0Rtx\u00C2\u00A0signal\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.0001)\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0given\u00C2\u00A0the\u00C2\u00A0 same\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0Because\u00C2\u00A0the\u00C2\u00A0dose\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0employed\u00C2\u00A0 is\u00C2\u00A0below\u00C2\u00A0saturating\u00C2\u00A0 levels,\u00C2\u00A0this\u00C2\u00A0suggests\u00C2\u00A0a\u00C2\u00A0 greater\u00C2\u00A0avidity\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0higher\u00C2\u00A0 valence.\u00C2\u00A0 \u00C2\u00A0 Finally,\u00C2\u00A0even\u00C2\u00A0 though\u00C2\u00A0 the\u00C2\u00A0measured\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0 bound\u00C2\u00A0Rtx\u00C2\u00A0increased\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0valence,\u00C2\u00A0so\u00C2\u00A0did\u00C2\u00A0the\u00C2\u00A0measured\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20,\u00C2\u00A0which\u00C2\u00A0was\u00C2\u00A0as\u00C2\u00A0 high\u00C2\u00A0 as\u00C2\u00A0 58%\u00C2\u00A0 unbound\u00C2\u00A0 CD20\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 highest\u00C2\u00A0 valence\u00C2\u00A0 of\u00C2\u00A0 158.\u00C2\u00A0 \u00C2\u00A0 Valences\u00C2\u00A0 of\u00C2\u00A0 113\u00C2\u00A0 and\u00C2\u00A0 158\u00C2\u00A0 showed\u00C2\u00A0 equivalent\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 bound\u00C2\u00A0Rtx\u00C2\u00A0 and\u00C2\u00A0 unbound\u00C2\u00A0 CD20\u00C2\u00A0 (p\u00C2\u00A0 >\u00C2\u00A0 0.05\u00C2\u00A0 for\u00C2\u00A0 both\u00C2\u00A0 parameters).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 further\u00C2\u00A0 suggests\u00C2\u00A0that\u00C2\u00A0as\u00C2\u00A0opposed\u00C2\u00A0to\u00C2\u00A0bivalent\u00C2\u00A0Rtx,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0spread\u00C2\u00A0evenly\u00C2\u00A0across\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface,\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0 of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 causes\u00C2\u00A0 some\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD20\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0present\u00C2\u00A0 in\u00C2\u00A0very\u00C2\u00A0high\u00C2\u00A0 local\u00C2\u00A0 concentrations,\u00C2\u00A0 while\u00C2\u00A0 some\u00C2\u00A0 CD20\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 bound\u00C2\u00A0 to\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 other\u00C2\u00A0words,\u00C2\u00A0 this\u00C2\u00A0 supports\u00C2\u00A0 the\u00C2\u00A0 existence\u00C2\u00A0 of\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A082\u00C2\u00A0\u00C2\u00A0 clusters\u00C2\u00A0that\u00C2\u00A0form\u00C2\u00A0between\u00C2\u00A0a\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0 membrane.\u00C2\u00A0 \u00C2\u00A0 4.4.2\u00C2\u00A0 Rtx\u00C2\u00ADenriched\u00C2\u00A0domains\u00C2\u00A0are\u00C2\u00A0 found\u00C2\u00A0on\u00C2\u00A0cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0on\u00C2\u00A0 those\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0 To\u00C2\u00A0examine\u00C2\u00A0the\u00C2\u00A0organization\u00C2\u00A0of\u00C2\u00A0Rtx/CD20\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0the\u00C2\u00A0distribution\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0was\u00C2\u00A0 investigated\u00C2\u00A0using\u00C2\u00A0 confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0 were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0for\u00C2\u00A0different\u00C2\u00A0times\u00C2\u00A0then\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0anti\u00E2\u0080\u0090Rtx\u00C2\u00A0Ab\u00C2\u00A0to\u00C2\u00A0label\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0surface.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0images\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0obtained,\u00C2\u00A0as\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.2,\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00C2\u00A0became\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0as\u00C2\u00A0soon\u00C2\u00A0as\u00C2\u00A015\u00C2\u00A0min\u00C2\u00A0after\u00C2\u00A0treatment,\u00C2\u00A0and\u00C2\u00A0remained\u00C2\u00A0associated\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0 later.\u00C2\u00A0 \u00C2\u00A0Rtx\u00C2\u00A0 remained\u00C2\u00A0 associated\u00C2\u00A0up\u00C2\u00A0 to\u00C2\u00A0 48\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0 and\u00C2\u00A0 no\u00C2\u00A0 staining\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 (not\u00C2\u00A0 shown).\u00C2\u00A0 \u00C2\u00A0 Cells\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 free\u00C2\u00A0 Rtx\u00C2\u00A0 showed\u00C2\u00A0 a\u00C2\u00A0 relatively\u00C2\u00A0 uniform\u00C2\u00A0 distribution\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0over\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cell,\u00C2\u00A0but\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0exhibited\u00C2\u00A0Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0 domains\u00C2\u00A0with\u00C2\u00A0significantly\u00C2\u00A0elevated\u00C2\u00A0fluorescence\u00C2\u00A0intensities\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0(arrows\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.2).\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0size\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0domains\u00C2\u00A0was\u00C2\u00A0<\u00C2\u00A0500\u00C2\u00A0nm\u00C2\u00A0and\u00C2\u00A0they\u00C2\u00A0were\u00C2\u00A0visible\u00C2\u00A0at\u00C2\u00A0all\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0between\u00C2\u00A015\u00C2\u00A0min\u00C2\u00A0and\u00C2\u00A0 24\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0although\u00C2\u00A0 they\u00C2\u00A0were\u00C2\u00A0most\u00C2\u00A0prevalent\u00C2\u00A0after\u00C2\u00A045\u00C2\u00A0min.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0 this\u00C2\u00A0 time\u00C2\u00A0 point,\u00C2\u00A0there\u00C2\u00A0were\u00C2\u00A0between\u00C2\u00A00\u00C2\u00A0and\u00C2\u00A0~15\u00C2\u00A0domains\u00C2\u00A0per\u00C2\u00A0cell,\u00C2\u00A0as\u00C2\u00A0found\u00C2\u00A0by\u00C2\u00A0counting\u00C2\u00A0the\u00C2\u00A0domains\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0z\u00E2\u0080\u0090stacked\u00C2\u00A0images\u00C2\u00A0(on\u00C2\u00A0average,\u00C2\u00A010\u00C2\u00A0slices\u00C2\u00A0per\u00C2\u00A0cell).\u00C2\u00A0\u00C2\u00A0No\u00C2\u00A0domains\u00C2\u00A0were\u00C2\u00A0present\u00C2\u00A0on\u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0at\u00C2\u00A0 any\u00C2\u00A0 time,\u00C2\u00A0 but\u00C2\u00A0 a\u00C2\u00A0 qualitative\u00C2\u00A0 rise\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 domains\u00C2\u00A0 per\u00C2\u00A0 cell\u00C2\u00A0was\u00C2\u00A0 observed\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 increased:\u00C2\u00A0 on\u00C2\u00A0 average,\u00C2\u00A0 valence\u00C2\u00A0 53\u00C2\u00A0 showed\u00C2\u00A0 ~5\u00C2\u00A0 domains/cell\u00C2\u00A0 and\u00C2\u00A0 valence\u00C2\u00A0 121\u00C2\u00A0 showed\u00C2\u00A0 ~15\u00C2\u00A0 domains/cell.\u00C2\u00A0 \u00C2\u00A0Overall,\u00C2\u00A0these\u00C2\u00A0 images\u00C2\u00A0support\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0data\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.1\u00C2\u00A0which\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0for\u00C2\u00A0 equivalent\u00C2\u00A0doses,\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0higher\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0bound\u00C2\u00A0more\u00C2\u00A0Rtx\u00C2\u00A0but\u00C2\u00A0had\u00C2\u00A0more\u00C2\u00A0 unbound\u00C2\u00A0 CD20.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 result\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0 domains\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0greater\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0domains\u00C2\u00A0being\u00C2\u00A0formed\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0increases.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A083\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.2\u00C2\u00A0\u00C2\u00A0Representative\u00C2\u00A0confocal\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0images\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0distribution\u00C2\u00A0on\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 The\u00C2\u00A0distribution\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0 is\u00C2\u00A0shown\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0 indicated\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0 early\u00C2\u00A0as\u00C2\u00A015\u00C2\u00A0min\u00C2\u00A0after\u00C2\u00A0treatment,\u00C2\u00A0and\u00C2\u00A0more\u00C2\u00A0pronouncedly\u00C2\u00A0at\u00C2\u00A045\u00C2\u00A0min,\u00C2\u00A0Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0domains\u00C2\u00A0appeared\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(arrows)\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0those\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Bar:\u00C2\u00A05\u00C2\u00A0\u00C2\u00B5m.\u00C2\u00A0 \u00C2\u00A0 4.4.3\u00C2\u00A0 Expression\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0change\u00C2\u00A0substantially\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00AD LNP\u00C2\u00A0 The\u00C2\u00A0 previous\u00C2\u00A0 two\u00C2\u00A0 sections\u00C2\u00A0 showed\u00C2\u00A0 that\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 dose\u00C2\u00A0 of\u00C2\u00A0 Rtx,\u00C2\u00A0 CD20+\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 bound\u00C2\u00A0more\u00C2\u00A0Rtx\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 increased.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 increased\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0occurred\u00C2\u00A0 although\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0was\u00C2\u00A0free\u00C2\u00A0from\u00C2\u00A0any\u00C2\u00A0bound\u00C2\u00A0Rtx,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0of\u00C2\u00A0Rtx/CD20\u00E2\u0080\u0090enriched\u00C2\u00A0 clusters\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0 \u00C2\u00A0One\u00C2\u00A0 important\u00C2\u00A0possibility\u00C2\u00A0 that\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0this\u00C2\u00A0effect\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0may\u00C2\u00A0cause\u00C2\u00A0an\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0 cell\u00C2\u00A0surface\u00C2\u00A0after\u00C2\u00A0binding,\u00C2\u00A0which\u00C2\u00A0would\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0help\u00C2\u00A0explain\u00C2\u00A0the\u00C2\u00A0 increased\u00C2\u00A0binding\u00C2\u00A0capacity\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 Valence: 2 (Rtx) 53 121 15\u00C2\u00A0min\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A045\u00C2\u00A0min\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A024\u00C2\u00A0h \u00E2\u0080\u00A2\u00C2\u00A084\u00C2\u00A0\u00C2\u00A0 but\u00C2\u00A0 also\u00C2\u00A0 the\u00C2\u00A0 large\u00C2\u00A0 fraction\u00C2\u00A0 of\u00C2\u00A0 unbound\u00C2\u00A0 CD20.\u00C2\u00A0 \u00C2\u00A0 Note,\u00C2\u00A0 however,\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.1\u00C2\u00A0was\u00C2\u00A0 obtained\u00C2\u00A0at\u00C2\u00A0a\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A0of\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 total\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 was\u00C2\u00A0 therefore\u00C2\u00A0 measured\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0at\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A048\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0was\u00C2\u00A0done\u00C2\u00A0using\u00C2\u00A0an\u00C2\u00A0 A647\u00E2\u0080\u0090labeled\u00C2\u00A0 polyclonal\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 Ab\u00C2\u00A0 (A647\u00E2\u0080\u0090pCD20;\u00C2\u00A0 see\u00C2\u00A0 Section\u00C2\u00A0 4.3.6).\u00C2\u00A0 \u00C2\u00A0 Unlike\u00C2\u00A0 the\u00C2\u00A0monoclonal\u00C2\u00A0 A647\u00E2\u0080\u0090mCD20\u00C2\u00A0used\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A04.4.1,\u00C2\u00A0no\u00C2\u00A0measurable\u00C2\u00A0 interference\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0A647\u00E2\u0080\u0090pCD20\u00C2\u00A0 signal\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0time\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 is\u00C2\u00A0summarized\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A0 4.3.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0all\u00C2\u00A0time\u00C2\u00A0points,\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0(light\u00C2\u00A0gray\u00C2\u00A0 bars)\u00C2\u00A0were\u00C2\u00A0 roughly\u00C2\u00A0equivalent\u00C2\u00A0 to\u00C2\u00A0 those\u00C2\u00A0 in\u00C2\u00A0cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0as\u00C2\u00A0 indicated\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0MFI\u00C2\u00A0 values\u00C2\u00A0of\u00C2\u00A0~1.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0time\u00C2\u00A0point,\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0after\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(52)\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(170)\u00C2\u00A0treatment\u00C2\u00A0 was\u00C2\u00A0maximal;\u00C2\u00A0measured\u00C2\u00A0MFI\u00C2\u00A0values\u00C2\u00A0were\u00C2\u00A01.7\u00C2\u00A0and\u00C2\u00A01.9\u00C2\u00A0 times\u00C2\u00A0 those\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0HBS\u00E2\u0080\u0090treated\u00C2\u00A0 cells,\u00C2\u00A0 respectively.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0 in\u00C2\u00A0PI\u00E2\u0080\u0093\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0sustained\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A048\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment.\u00C2\u00A0\u00C2\u00A0 Between\u00C2\u00A0all\u00C2\u00A0treatments\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0and\u00C2\u00A0including\u00C2\u00A08\u00C2\u00A0h,\u00C2\u00A0there\u00C2\u00A0was\u00C2\u00A0no\u00C2\u00A0significant\u00C2\u00A0difference\u00C2\u00A0between\u00C2\u00A0any\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0treatments\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0all\u00C2\u00A0possible\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0treatments).\u00C2\u00A0\u00C2\u00A0With\u00C2\u00A0the\u00C2\u00A0exception\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0 time\u00C2\u00A0point,\u00C2\u00A0 there\u00C2\u00A0was\u00C2\u00A0no\u00C2\u00A0 significant\u00C2\u00A0difference\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0measured\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0after\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP(52)\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(170)\u00C2\u00A0 treatment\u00C2\u00A0 (p\u00C2\u00A0 <\u00C2\u00A0 0.05\u00C2\u00A0 only\u00C2\u00A0 at\u00C2\u00A0 16\u00C2\u00A0 h).\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0 16\u00C2\u00A0 h\u00C2\u00A0 and\u00C2\u00A0 longer,\u00C2\u00A0 however,\u00C2\u00A0 the\u00C2\u00A0 differences\u00C2\u00A0between\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0either\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0treatment\u00C2\u00A0were\u00C2\u00A0significant\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(52)\u00C2\u00A0and\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.01\u00C2\u00A0between\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(170)\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0cases).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0is\u00C2\u00A0unlikely,\u00C2\u00A0however,\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0after\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0treatment\u00C2\u00A0(~1.5\u00C2\u00A0 times\u00C2\u00A0those\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx)\u00C2\u00A0explain\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.1\u00C2\u00A0showing\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A010.0\u00E2\u0080\u0090 fold\u00C2\u00A0higher\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0bivalent\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0modest\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0 CD20\u00C2\u00A0expression\u00C2\u00A0cannot\u00C2\u00A0account\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0high\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0 (up\u00C2\u00A0 to\u00C2\u00A058%)\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0 measured\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0therefore\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0support\u00C2\u00A0the\u00C2\u00A0hypothesis\u00C2\u00A0that\u00C2\u00A0 the\u00C2\u00A0results\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0Figures\u00C2\u00A04.1\u00C2\u00A0&\u00C2\u00A04.2\u00C2\u00A0were\u00C2\u00A0an\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0upregulation.\u00C2\u00A0 \u00C2\u00A0They\u00C2\u00A0suggest\u00C2\u00A0 instead\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A085\u00C2\u00A0\u00C2\u00A0 that\u00C2\u00A0 a\u00C2\u00A0 reorganization\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 occurred\u00C2\u00A0 following\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 treatment,\u00C2\u00A0 forming\u00C2\u00A0CD20/Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0microdomains\u00C2\u00A0that\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0 constructs.\u00C2\u00A0 \u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.3\u00C2\u00A0\u00C2\u00A0Time\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 Using\u00C2\u00A0 a\u00C2\u00A0 polyclonal\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 Ab\u00C2\u00A0 (A647\u00E2\u0080\u0090pCD20),\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0 CD20\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 of\u00C2\u00A0 PI\u00E2\u0080\u0093\u00C2\u00A0 cells\u00C2\u00A0was\u00C2\u00A0 measured\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0times\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0MFI:\u00C2\u00A0mean\u00C2\u00A0fluorescence\u00C2\u00A0intensity.\u00C2\u00A0 \u00C2\u00A0 4.4.4\u00C2\u00A0 Cytotoxicity\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNPs\u00C2\u00A0in\u00C2\u00A0two\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0 To\u00C2\u00A0assess\u00C2\u00A0 the\u00C2\u00A0cytotoxicity\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0a\u00C2\u00A0range\u00C2\u00A0of\u00C2\u00A0valences\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0 to\u00C2\u00A0Ramos\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0 lymphoma\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0 Five\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0 selected\u00C2\u00A0 (0.2\u00E2\u0080\u00932000\u00C2\u00A0ng;\u00C2\u00A0 see\u00C2\u00A0 Section\u00C2\u00A0 4.3.7)\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 binding\u00C2\u00A0data\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A04.1\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0on\u00C2\u00A0doses\u00C2\u00A0 employed\u00C2\u00A0 in\u00C2\u00A0previous\u00C2\u00A0 studies74,\u00C2\u00A0168,\u00C2\u00A0169.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0for\u00C2\u00A096\u00C2\u00A0h\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0various\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0preparations\u00C2\u00A0or\u00C2\u00A0with\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0(valence\u00C2\u00A02)\u00C2\u00A0at\u00C2\u00A0each\u00C2\u00A0dose.\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A0 4.4\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 cell\u00C2\u00A0 lines\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 highest\u00C2\u00A0 dose\u00C2\u00A0 (2.0\u00C2\u00A0 \u00C2\u00B5g),\u00C2\u00A0 all\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 valences\u00C2\u00A0were\u00C2\u00A0 significantly\u00C2\u00A0more\u00C2\u00A0cytotoxic\u00C2\u00A0than\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.0005\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0valences).\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0this\u00C2\u00A0dose,\u00C2\u00A039%\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0viable\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0cells,\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0\u00E2\u0089\u00A4\u00C2\u00A01%\u00C2\u00A0viable\u00C2\u00A0cells\u00C2\u00A0 for\u00C2\u00A0all\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dose,\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0showed\u00C2\u00A086%\u00C2\u00A0viability\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0control\u00C2\u00A0after\u00C2\u00A0Rtx\u00C2\u00A0 treatment,\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A025\u00E2\u0080\u009342%\u00C2\u00A0viability\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0 1 2 3 1 4 8 16 24 36 48 M FI \u00C2\u00A0(A 64 7\u00E2\u0080\u0090p CD 20 )\u00C2\u00A0re la ti ve \u00C2\u00A0to \u00C2\u00A0HB S Time\u00C2\u00A0/\u00C2\u00A0h 2 52 170 Valence: \u00E2\u0080\u00A2\u00C2\u00A086\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.4\u00C2\u00A0\u00C2\u00A0Particularly\u00C2\u00A0at\u00C2\u00A0higher\u00C2\u00A0doses,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0are\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0cytotoxic\u00C2\u00A0than\u00C2\u00A0equivalent\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plate,\u00C2\u00A05\u00C2\u00A0\u00EF\u0082\u00B4\u00C2\u00A0103\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0100\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A050\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium\u00C2\u00A0containing\u00C2\u00A0the\u00C2\u00A0dose\u00C2\u00A0 of\u00C2\u00A0Rtx\u00C2\u00A0 indicated\u00C2\u00A0 above.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 control\u00C2\u00A0 consisted\u00C2\u00A0 of\u00C2\u00A0medium\u00C2\u00A0only.\u00C2\u00A0 \u00C2\u00A0Plates\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 for\u00C2\u00A0 96\u00C2\u00A0h\u00C2\u00A0 and\u00C2\u00A0 cell\u00C2\u00A0 viability\u00C2\u00A0was\u00C2\u00A0 measured\u00C2\u00A0using\u00C2\u00A0AlamarBlue.\u00C2\u00A0 \u00C2\u00A0 Overall,\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0more\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cytotoxic\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0and\u00C2\u00A0for\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0 2.0\u00C2\u00A0ng\u00C2\u00A0and\u00C2\u00A0above,\u00C2\u00A0all\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0valences\u00C2\u00A0were\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0cytotoxic\u00C2\u00A0than\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.025\u00C2\u00A0for\u00C2\u00A0 all\u00C2\u00A0valences).\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A0200\u00C2\u00A0ng\u00C2\u00A0dose,\u00C2\u00A0there\u00C2\u00A0were\u00C2\u00A049%\u00C2\u00A0viable\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0Rtx\u00C2\u00A0treatment,\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0\u00E2\u0089\u00A4\u00C2\u00A05%\u00C2\u00A0 for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.0005\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0valences).\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0Z138\u00C2\u00A0cells,\u00C2\u00A0significant\u00C2\u00A0differences\u00C2\u00A0between\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00C2\u00A0treatment\u00C2\u00A0began\u00C2\u00A0to\u00C2\u00A0appear\u00C2\u00A0at\u00C2\u00A020\u00C2\u00A0ng\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0121:\u00C2\u00A089%\u00C2\u00A0for\u00C2\u00A0Rtx,\u00C2\u00A074%\u00C2\u00A0for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(121)\u00C2\u00A0(p\u00C2\u00A0 <\u00C2\u00A00.005).\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A0200\u00C2\u00A0ng\u00C2\u00A0dose,\u00C2\u00A0all\u00C2\u00A0valences\u00C2\u00A0showed\u00C2\u00A0significant\u00C2\u00A0difference\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A0 0.05).\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 data\u00C2\u00A0 demonstrate\u00C2\u00A0 that\u00C2\u00A0 regardless\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 equal\u00C2\u00A0 doses\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 were\u00C2\u00A0 administered,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0are\u00C2\u00A0significantly\u00C2\u00A0more\u00C2\u00A0cytotoxic\u00C2\u00A0than\u00C2\u00A0Rtx.\u00C2\u00A0 4.4.5\u00C2\u00A0 Time\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 well\u00E2\u0080\u0090documented\u00C2\u00A0 that\u00C2\u00A0 Rtx\u00C2\u00A0 induces\u00C2\u00A0 direct\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 when\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 crosslinked\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0reagent\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0an\u00C2\u00A0anti\u00E2\u0080\u0090human\u00E2\u0080\u0090IgG\u00C2\u00A0secondary\u00C2\u00A0antibody7,\u00C2\u00A074\u00E2\u0080\u009076.\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0 determine\u00C2\u00A0 if\u00C2\u00A0 the\u00C2\u00A0 cytotoxicity\u00C2\u00A0 observed\u00C2\u00A0 above\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 directly\u00C2\u00A0 inducing\u00C2\u00A0 0.0 0.4 0.8 1.2 0.1 10 1000 M FI \u00C2\u00A0(re la ti ve \u00C2\u00A0to \u00C2\u00A0co nt ro l) Dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0/\u00C2\u00A0ng A Ramos 0.0 0.4 0.8 1.2 0.1 10 1000 M FI \u00C2\u00A0(re la ti ve \u00C2\u00A0to \u00C2\u00A0co nt ro l) Dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0/\u00C2\u00A0ng B 2 19 53 72 121 Z138 Valence: \u00E2\u0080\u00A2\u00C2\u00A087\u00C2\u00A0\u00C2\u00A0 apoptosis\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 own\u00C2\u00A0 without\u00C2\u00A0 hypercrosslinking,\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 two\u00C2\u00A0 cell\u00C2\u00A0 lines\u00C2\u00A0was\u00C2\u00A0 examined.\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 4.5A\u00C2\u00A0 illustrates\u00C2\u00A0 the\u00C2\u00A0 flow\u00C2\u00A0 cytometric\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC/PI\u00C2\u00A0assay\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0employed.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0figure\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0after\u00C2\u00A0treating\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0 similar\u00C2\u00A0 to\u00C2\u00A0 those\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0 confirming\u00C2\u00A0 that\u00C2\u00A0Rtx,\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0of\u00C2\u00A0crosslinking,\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0elicit\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0vitro.\u00C2\u00A0 \u00C2\u00A0Figure\u00C2\u00A04.5A\u00C2\u00A0also\u00C2\u00A0demonstrates\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 precursors\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0and\u00C2\u00A0Neut\u00E2\u0080\u0090LNP)\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0on\u00C2\u00A0their\u00C2\u00A0own,\u00C2\u00A0and\u00C2\u00A0neither\u00C2\u00A0 did\u00C2\u00A0a\u00C2\u00A0mixture\u00C2\u00A0of\u00C2\u00A0unbiotinylated\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Neut\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0 treating\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0onset\u00C2\u00A0of\u00C2\u00A0significant\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0(bottom\u00C2\u00A0rightmost\u00C2\u00A0plot\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.5A).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 indicates\u00C2\u00A0that\u00C2\u00A0as\u00C2\u00A0opposed\u00C2\u00A0to\u00C2\u00A0bivalent\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 induces\u00C2\u00A0apoptosis\u00C2\u00A0on\u00C2\u00A0 its\u00C2\u00A0own\u00C2\u00A0 in\u00C2\u00A0target\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 depends\u00C2\u00A0 on\u00C2\u00A0 many\u00C2\u00A0 Rtx\u00C2\u00A0 molecules\u00C2\u00A0 being\u00C2\u00A0 associated\u00C2\u00A0 together\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 multivalent\u00C2\u00A0Rtx\u00C2\u00A0construct.\u00C2\u00A0 The\u00C2\u00A0time\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0measured\u00C2\u00A0by\u00C2\u00A0analyzing\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0 with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0two\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0at\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A048\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0 Figure\u00C2\u00A04.5B,\u00C2\u00A0the\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0(Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC+\u00C2\u00A0/\u00C2\u00A0PI\u00E2\u0080\u0093)\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0 with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(63)\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0increased\u00C2\u00A0sharply\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment,\u00C2\u00A0and\u00C2\u00A0after\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0subsequently\u00C2\u00A0decreased\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A016\u00C2\u00A0and\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0for\u00C2\u00A0 both\u00C2\u00A0 treatments).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0decrease\u00C2\u00A0after\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0was\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0 increased\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0necrotic\u00C2\u00A0 cells\u00C2\u00A0(Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC+\u00C2\u00A0/\u00C2\u00A0PI+;\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0(valence\u00C2\u00A00)\u00C2\u00A0and\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 show\u00C2\u00A0peak\u00C2\u00A0levels\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment;\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0for\u00C2\u00A0both\u00C2\u00A0HBS\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0at\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0were\u00C2\u00A0 equivalent\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0at\u00C2\u00A00\u00C2\u00A0h\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0cases),\u00C2\u00A0but\u00C2\u00A0slight\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0apoptosis\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0 with\u00C2\u00A0HBS\u00C2\u00A0or\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0at\u00C2\u00A0time\u00C2\u00A0points\u00C2\u00A0of\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A0longer\u00C2\u00A0(from\u00C2\u00A02.9%\u00C2\u00A0at\u00C2\u00A00\u00C2\u00A0h\u00C2\u00A0to\u00C2\u00A08.2%\u00C2\u00A0at\u00C2\u00A048\u00C2\u00A0h).\u00C2\u00A0\u00C2\u00A0 However,\u00C2\u00A0at\u00C2\u00A0all\u00C2\u00A0 time\u00C2\u00A0points,\u00C2\u00A0HBS\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0 treatment\u00C2\u00A0resulted\u00C2\u00A0 in\u00C2\u00A0equivalent\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 (p\u00C2\u00A0>\u00C2\u00A0 0.05).\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0separate\u00C2\u00A0experiment\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0also\u00C2\u00A0exhibited\u00C2\u00A0maximum\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0at\u00C2\u00A0 a\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0Based\u00C2\u00A0on\u00C2\u00A0these\u00C2\u00A0data,\u00C2\u00A0the\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A0was\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A088\u00C2\u00A0\u00C2\u00A0 considered\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0the\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A0when\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0considered\u00C2\u00A0maximal\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.5\u00C2\u00A0\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0directly\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0individual\u00C2\u00A0precursors\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0do\u00C2\u00A0not.\u00C2\u00A0 (A)\u00C2\u00A0To\u00C2\u00A0measure\u00C2\u00A0apoptosis\u00C2\u00A0levels,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0and\u00C2\u00A0PI\u00C2\u00A0and\u00C2\u00A0analyzed\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry.\u00C2\u00A0\u00C2\u00A0Viable\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0negative\u00C2\u00A0 for\u00C2\u00A0both\u00C2\u00A0dyes\u00C2\u00A0 (lower\u00C2\u00A0 left\u00C2\u00A0quadrant),\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC+\u00C2\u00A0and\u00C2\u00A0PI\u00E2\u0080\u0093\u00C2\u00A0 (lower\u00C2\u00A0 right\u00C2\u00A0 quadrant),\u00C2\u00A0and\u00C2\u00A0necrotic\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0positive\u00C2\u00A0for\u00C2\u00A0both\u00C2\u00A0dyes\u00C2\u00A0(upper\u00C2\u00A0right\u00C2\u00A0quadrant).\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0this\u00C2\u00A0example,\u00C2\u00A05\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A020\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0Rtx\u00C2\u00A0dose\u00C2\u00A0(or\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0Neut\u00E2\u0080\u0090LNP\u00C2\u00A0equivalent\u00C2\u00A0to\u00C2\u00A0that\u00C2\u00A0given\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0treatment)\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00C2\u00A0treatment\u00C2\u00A0exhibited\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0and\u00C2\u00A0necrotic\u00C2\u00A0cells\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0all\u00C2\u00A0other\u00C2\u00A0 treatments.\u00C2\u00A0 \u00C2\u00A0 (B)\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0Rtx,\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 (10\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0Rtx\u00C2\u00A0dose\u00C2\u00A0given\u00C2\u00A0 to\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0 medium)\u00C2\u00A0and\u00C2\u00A0percentages\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0times\u00C2\u00A0post\u00E2\u0080\u0090treatment.\u00C2\u00A0 \u00C2\u00A0Maximum\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 A 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 104 103 102 101 100 104 103 102 101 100 90.1%90.6%91.0% 61.5%90.5%89.3% 8.4% 2.0% 2.4% 11.6%F lu or es ce nc e\u00C2\u00A0i nt en si ty \u00C2\u00A0\u00C2\u00A0(P I) Fluorescence\u00C2\u00A0intensity\u00C2\u00A0 \u00C2\u00A0(Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC) 7.4% Neut\u00E2\u0080\u0090LNP HBS Rtx Rtx\u00E2\u0080\u0090biotin Rtx\u00C2\u00A0+\u00C2\u00A0 Neut\u00E2\u0080\u0090LNP Rtx\u00E2\u0080\u0090LNP (val.\u00C2\u00A0121) 5.3% 6.9% 26.7%6.5% 2.5%3.9%1.4% 0 25 50 75 0 8 16 24 36 48 % \u00C2\u00A0of \u00C2\u00A0ea rl y\u00C2\u00A0a po pt ot ic \u00C2\u00A0ce lls Time\u00C2\u00A0/\u00C2\u00A0h 0 2 63 158 Valence: RamosB \u00E2\u0080\u00A2\u00C2\u00A089\u00C2\u00A0\u00C2\u00A0 apoptosis\u00C2\u00A0occurred\u00C2\u00A0approximately\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 regardless\u00C2\u00A0of\u00C2\u00A0valence,\u00C2\u00A0while\u00C2\u00A0 free\u00C2\u00A0Rtx\u00C2\u00A0 showed\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0at\u00C2\u00A0all\u00C2\u00A0time\u00C2\u00A0points.\u00C2\u00A0 4.4.6\u00C2\u00A0 Levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 At\u00C2\u00A0the\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A0where\u00C2\u00A0maximum\u00C2\u00A0apoptosis\u00C2\u00A0occurred\u00C2\u00A0(24\u00C2\u00A0h),\u00C2\u00A0the\u00C2\u00A0apoptotic\u00C2\u00A0potency\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 various\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0valences\u00C2\u00A0was\u00C2\u00A0measured.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0 a\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0either\u00C2\u00A0in\u00C2\u00A0its\u00C2\u00A0bivalent\u00C2\u00A0form\u00C2\u00A0or\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0valences.\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0(valence\u00C2\u00A00).\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0an\u00C2\u00A0 incubation\u00C2\u00A0of\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0fractions\u00C2\u00A0 were\u00C2\u00A0measured\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0previous\u00C2\u00A0section,\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.6.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0figure\u00C2\u00A0clearly\u00C2\u00A0illustrates\u00C2\u00A0 that\u00C2\u00A0 higher\u00C2\u00A0 valences\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 have\u00C2\u00A0 a\u00C2\u00A0 much\u00C2\u00A0 stronger\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 directly\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0cells,\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 is\u00C2\u00A0given\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0cases.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 figure\u00C2\u00A0also\u00C2\u00A0shows\u00C2\u00A0an\u00C2\u00A0 inverse\u00C2\u00A0correlation\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0apoptotic\u00C2\u00A0populations\u00C2\u00A0(black\u00C2\u00A0and\u00C2\u00A0white\u00C2\u00A0symbols)\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0 cell\u00C2\u00A0 lines,\u00C2\u00A0 indicating\u00C2\u00A0 a\u00C2\u00A0 shift\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 viable\u00C2\u00A0 population\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 early\u00C2\u00A0 apoptotic\u00C2\u00A0 population\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 exposure\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A04.6\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0is\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090treated\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0even\u00C2\u00A0 though\u00C2\u00A0all\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0given\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0(A)\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0(B)\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0free\u00C2\u00A0Rtx\u00C2\u00A0(valence\u00C2\u00A02)\u00C2\u00A0or\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0 of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0Valence\u00C2\u00A00\u00C2\u00A0refers\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0HBS.\u00C2\u00A0\u00C2\u00A0Percentages\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0and\u00C2\u00A0viable\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0 Figure\u00C2\u00A04.5.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0 the\u00C2\u00A0higher\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 induced\u00C2\u00A0elevated\u00C2\u00A0apoptosis\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 lower\u00C2\u00A0 valences,\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 by\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 is\u00C2\u00A0 valence\u00E2\u0080\u0090dependent.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 Ramos\u00C2\u00A0 cells,\u00C2\u00A0maximum\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0occurred\u00C2\u00A0at\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0approximately\u00C2\u00A0100\u00C2\u00A0and\u00C2\u00A0higher.\u00C2\u00A0 0 25 50 75 100 0 50 100 150 200 % \u00C2\u00A0of \u00C2\u00A0to ta l\u00C2\u00A0ce ll\u00C2\u00A0p op ul at io n Valence Ramos 0 25 50 75 100 0 100 200 % \u00C2\u00A0of \u00C2\u00A0to ta l\u00C2\u00A0ce ll\u00C2\u00A0p op ul at io n Valence Z138 viable early\u00C2\u00A0 apoptotic \u00E2\u0080\u00A2\u00C2\u00A090\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0Ramos\u00C2\u00A0cell\u00C2\u00A0line\u00C2\u00A0(Figure\u00C2\u00A04.6A)\u00C2\u00A0was\u00C2\u00A0more\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNPs\u00C2\u00A0 than\u00C2\u00A0 the\u00C2\u00A0Z138\u00C2\u00A0 line\u00C2\u00A0 (Figure\u00C2\u00A04.6B).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 (valence\u00C2\u00A02)\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 4.5%\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0and\u00C2\u00A0 this\u00C2\u00A0value\u00C2\u00A0 increased\u00C2\u00A0 to\u00C2\u00A021%\u00C2\u00A0 for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(19)\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.001),\u00C2\u00A0 while\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0Z138\u00C2\u00A0 line,\u00C2\u00A0 low\u00C2\u00A0equivalent\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A075\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A0 0.05\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0valences\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0and\u00C2\u00A0including\u00C2\u00A075),\u00C2\u00A0and\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0until\u00C2\u00A0a\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0113\u00C2\u00A0when\u00C2\u00A0higher\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0 (18%,\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A06.5%\u00C2\u00A0 for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(75);\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.05).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0Ramos\u00C2\u00A0 cells,\u00C2\u00A0 maximum\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0(65%)\u00C2\u00A0occurred\u00C2\u00A0at\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0and\u00C2\u00A0above\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0valences),\u00C2\u00A0but\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0unclear\u00C2\u00A0whether\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0plateau\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0Z138\u00C2\u00A0cells,\u00C2\u00A0although\u00C2\u00A0p\u00C2\u00A0 >\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0113,\u00C2\u00A0152,\u00C2\u00A0and\u00C2\u00A0165.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0valences\u00C2\u00A0 show\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0of\u00C2\u00A0 22%,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0significantly\u00C2\u00A0lower\u00C2\u00A0than\u00C2\u00A0those\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0further\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0Z138\u00C2\u00A0 cell\u00C2\u00A0line\u00C2\u00A0is\u00C2\u00A0less\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Ramos\u00C2\u00A0line.\u00C2\u00A0 Overall,\u00C2\u00A0 these\u00C2\u00A0data\u00C2\u00A0 confirm\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0apoptotic\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 is\u00C2\u00A0 significantly\u00C2\u00A0elevated\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 are\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 in\u00C2\u00A0 two\u00C2\u00A0 different\u00C2\u00A0 lymphoma\u00C2\u00A0 cell\u00C2\u00A0 lines.\u00C2\u00A0 \u00C2\u00A0 Increasing\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 is\u00C2\u00A0 therefore\u00C2\u00A0 a\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0 improving\u00C2\u00A0 the\u00C2\u00A0 therapeutic\u00C2\u00A0 activity\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0by\u00C2\u00A0enhancing\u00C2\u00A0 its\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0Ramos\u00C2\u00A0 cell\u00C2\u00A0 line\u00C2\u00A0also\u00C2\u00A0 show\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0optimal\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0100,\u00C2\u00A0since\u00C2\u00A0no\u00C2\u00A0further\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0apoptosis\u00C2\u00A0occurred\u00C2\u00A0at\u00C2\u00A0 higher\u00C2\u00A0valences.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 implies\u00C2\u00A0that,\u00C2\u00A0 in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action,\u00C2\u00A0there\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0no\u00C2\u00A0 additional\u00C2\u00A0therapeutic\u00C2\u00A0benefit\u00C2\u00A0of\u00C2\u00A0using\u00C2\u00A0valences\u00C2\u00A0higher\u00C2\u00A0than\u00C2\u00A0100.\u00C2\u00A0\u00C2\u00A0 4.5\u00C2\u00A0\u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0 4.5.1\u00C2\u00A0 Discussion\u00C2\u00A0 This\u00C2\u00A0 chapter\u00C2\u00A0 has\u00C2\u00A0 demonstrated\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 methodology\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 3\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 employed\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0constructs\u00C2\u00A0that\u00C2\u00A0possess\u00C2\u00A0unique\u00C2\u00A0biological\u00C2\u00A0properties\u00C2\u00A0(Figures\u00C2\u00A0 4.1\u00E2\u0080\u00934.3)\u00C2\u00A0and\u00C2\u00A0significantly\u00C2\u00A0 improved\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0(Figures\u00C2\u00A04.4\u00E2\u0080\u00934.6).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0chapter\u00C2\u00A0also\u00C2\u00A0proves\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A091\u00C2\u00A0\u00C2\u00A0 the\u00C2\u00A0concept\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0employed\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0Abs\u00C2\u00A0that\u00C2\u00A0show\u00C2\u00A0enhanced\u00C2\u00A0responses\u00C2\u00A0 when\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0multivalent,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0also\u00C2\u00A0to\u00C2\u00A0identify\u00C2\u00A0the\u00C2\u00A0optimal\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0such\u00C2\u00A0multivalent\u00C2\u00A0 constructs\u00C2\u00A0(valence\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0for\u00C2\u00A0multivalent\u00C2\u00A0Rtx;\u00C2\u00A0Figure\u00C2\u00A04.6A).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0constructs\u00C2\u00A0warrant\u00C2\u00A0further\u00C2\u00A0study\u00C2\u00A0 due\u00C2\u00A0to\u00C2\u00A0their\u00C2\u00A0potential\u00C2\u00A0in\u00C2\u00A0improving\u00C2\u00A0therapeutic\u00C2\u00A0antibody\u00E2\u0080\u0090based\u00C2\u00A0cancer\u00C2\u00A0treatments.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0interaction\u00C2\u00A0between\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0and\u00C2\u00A0CD20+\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0uncovered\u00C2\u00A0here\u00C2\u00A0 provides\u00C2\u00A0interesting\u00C2\u00A0clues\u00C2\u00A0regarding\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0Figure\u00C2\u00A04.1A\u00C2\u00A0shows\u00C2\u00A0 that\u00C2\u00A0 increased\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0 correlated\u00C2\u00A0with\u00C2\u00A0decreased\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0unbound\u00C2\u00A0CD20,\u00C2\u00A0 until\u00C2\u00A0 all\u00C2\u00A0 CD20\u00C2\u00A0 became\u00C2\u00A0 saturated\u00C2\u00A0 with\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 Saturating\u00C2\u00A0 cells\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.1B,\u00C2\u00A0 however,\u00C2\u00A0 showed\u00C2\u00A0 that\u00C2\u00A0 10\u00E2\u0080\u0090fold\u00C2\u00A0more\u00C2\u00A0 Rtx\u00C2\u00A0was\u00C2\u00A0 bound\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 cells\u00C2\u00A0 although\u00C2\u00A0 41%\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0was\u00C2\u00A0 unoccupied,\u00C2\u00A0 indicating\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0proportion\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0CD20.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0spherical\u00C2\u00A0structure\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0interacting\u00C2\u00A0with\u00C2\u00A0CD20\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0\u00C2\u00A0 When\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cell,\u00C2\u00A0the\u00C2\u00A0Rtx\u00C2\u00A0molecules\u00C2\u00A0closest\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0accessible\u00C2\u00A0 to\u00C2\u00A0CD20\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0opposite\u00C2\u00A0side\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0Rtx\u00C2\u00A0molecules\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0opposite\u00C2\u00A0 side\u00C2\u00A0may\u00C2\u00A0therefore\u00C2\u00A0not\u00C2\u00A0bind\u00C2\u00A0directly\u00C2\u00A0to\u00C2\u00A0CD20\u00C2\u00A0although\u00C2\u00A0they\u00C2\u00A0remain\u00C2\u00A0associated\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0construct.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Moreover,\u00C2\u00A0Figure\u00C2\u00A04.1C\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0higher\u00C2\u00A0 valences\u00C2\u00A0results\u00C2\u00A0 in\u00C2\u00A0elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0unbound\u00C2\u00A0CD20.\u00C2\u00A0 \u00C2\u00A0Although\u00C2\u00A0this\u00C2\u00A0may\u00C2\u00A0seem\u00C2\u00A0 counterintuitive,\u00C2\u00A0 it\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 explained\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 for\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs,\u00C2\u00A0 equal\u00C2\u00A0 doses\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 represent\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0given\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0Because\u00C2\u00A0the\u00C2\u00A0higher\u00C2\u00A0valences\u00C2\u00A0have\u00C2\u00A0more\u00C2\u00A0 Rtx\u00C2\u00A0molecules\u00C2\u00A0per\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0administered\u00C2\u00A0deceases\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0increases,\u00C2\u00A0 when\u00C2\u00A0the\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0held\u00C2\u00A0constant.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 data\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.1C,\u00C2\u00A0 when\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.6,\u00C2\u00A0 show\u00C2\u00A0 that\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 saturates\u00C2\u00A0all\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0and\u00C2\u00A0induces\u00C2\u00A0low\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis,\u00C2\u00A0while\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 engages\u00C2\u00A0only\u00C2\u00A0a\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0available\u00C2\u00A0CD20\u00C2\u00A0and\u00C2\u00A0induces\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0is\u00C2\u00A0therefore\u00C2\u00A0 not\u00C2\u00A0 simply\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 interactions\u00C2\u00A0 that\u00C2\u00A0 provides\u00C2\u00A0 the\u00C2\u00A0 stimulus\u00C2\u00A0 for\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A092\u00C2\u00A0\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 data\u00C2\u00A0 imply\u00C2\u00A0 that\u00C2\u00A0 instead,\u00C2\u00A0 high\u00C2\u00A0 local\u00C2\u00A0 concentrations\u00C2\u00A0 of\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0microdomains\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.2,\u00C2\u00A0elicit\u00C2\u00A0the\u00C2\u00A0apoptotic\u00C2\u00A0signal.\u00C2\u00A0\u00C2\u00A0Higher\u00C2\u00A0local\u00C2\u00A0 concentrations\u00C2\u00A0 of\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 are\u00C2\u00A0 provided\u00C2\u00A0 by\u00C2\u00A0more\u00C2\u00A0 extensive\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 of\u00C2\u00A0 CD20,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 facilitated\u00C2\u00A0by\u00C2\u00A0higher\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 The\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0113\u00C2\u00A0and\u00C2\u00A0158\u00C2\u00A0exhibited\u00C2\u00A0equivalent\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0unbound\u00C2\u00A0 CD20\u00C2\u00A0(Figure\u00C2\u00A04.1C),\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.6A\u00C2\u00A0&\u00C2\u00A0B,\u00C2\u00A0they\u00C2\u00A0were\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0equivalent\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0parameters).\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0maximum\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 observed\u00C2\u00A0above\u00C2\u00A0the\u00C2\u00A0optimal\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.6\u00C2\u00A0are\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0equivalent\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0 Rtx\u00C2\u00A0 and\u00C2\u00A0 unengaged\u00C2\u00A0 CD20;\u00C2\u00A0 in\u00C2\u00A0 other\u00C2\u00A0words,\u00C2\u00A0 it\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0maximum\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0 achievable\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0correlated\u00C2\u00A0with\u00C2\u00A0maximum\u00C2\u00A0apoptosis\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 We\u00C2\u00A0 showed\u00C2\u00A0 specifically\u00C2\u00A0 that\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 are\u00C2\u00A0 considerably\u00C2\u00A0more\u00C2\u00A0 cytotoxic\u00C2\u00A0 than\u00C2\u00A0 bivalent\u00C2\u00A0Rtx\u00C2\u00A0 in\u00C2\u00A0 two\u00C2\u00A0 lymphoma\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0 (Figure\u00C2\u00A04.4)\u00C2\u00A0and\u00C2\u00A0 that\u00C2\u00A0 they\u00C2\u00A0 induce\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0 these\u00C2\u00A0cells\u00C2\u00A0 while\u00C2\u00A0 none\u00C2\u00A0 of\u00C2\u00A0 their\u00C2\u00A0 individual\u00C2\u00A0 constituents\u00C2\u00A0 do\u00C2\u00A0 (Figure\u00C2\u00A0 4.5A).\u00C2\u00A0 \u00C2\u00A0Moreover,\u00C2\u00A0 at\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 post\u00E2\u0080\u0090treatment,\u00C2\u00A0 when\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0were\u00C2\u00A0maximal\u00C2\u00A0(Figure\u00C2\u00A04.5B),\u00C2\u00A0the\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0 was\u00C2\u00A0directly\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0all\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0given\u00C2\u00A0equivalent\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0 Rtx\u00C2\u00A0 (Figure\u00C2\u00A04.6).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0clearly\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0by\u00C2\u00A0 increasing\u00C2\u00A0 the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0 its\u00C2\u00A0efficacy\u00C2\u00A0 is\u00C2\u00A0 improved\u00C2\u00A0 through\u00C2\u00A0 an\u00C2\u00A0 augmentation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 has\u00C2\u00A0 not\u00C2\u00A0 been\u00C2\u00A0 previously\u00C2\u00A0 demonstrated\u00C2\u00A0 for\u00C2\u00A0 Rtx,\u00C2\u00A0 likely\u00C2\u00A0 because\u00C2\u00A0 until\u00C2\u00A0 now\u00C2\u00A0 there\u00C2\u00A0 has\u00C2\u00A0 not\u00C2\u00A0 been\u00C2\u00A0 a\u00C2\u00A0 precise\u00C2\u00A0methodology\u00C2\u00A0 for\u00C2\u00A0 creating\u00C2\u00A0defined\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0as\u00C2\u00A0high\u00C2\u00A0as\u00C2\u00A0those\u00C2\u00A0employed\u00C2\u00A0here.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0observation\u00C2\u00A0of\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0supports\u00C2\u00A0hypothesis\u00C2\u00A0(2)\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A0 4.2.2,\u00C2\u00A0where\u00C2\u00A0different\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0resulting\u00C2\u00A0from\u00C2\u00A0multivalency\u00C2\u00A0are\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0can\u00C2\u00A0therefore\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0study\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0action\u00C2\u00A0since\u00C2\u00A0one\u00C2\u00A0would\u00C2\u00A0also\u00C2\u00A0expect\u00C2\u00A0concomitant\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0signaling\u00C2\u00A0events\u00C2\u00A0that\u00C2\u00A0precede\u00C2\u00A0 apoptosis.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0further\u00C2\u00A0explored\u00C2\u00A0in\u00C2\u00A0Chapters\u00C2\u00A05\u00C2\u00A0and\u00C2\u00A06.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A093\u00C2\u00A0\u00C2\u00A0 4.5.2\u00C2\u00A0 Conclusions\u00C2\u00A0 The\u00C2\u00A0methodology\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0successfully\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0create\u00C2\u00A0many\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0that\u00C2\u00A0differ\u00C2\u00A0in\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 valence.\u00C2\u00A0 \u00C2\u00A0Multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 prepared\u00C2\u00A0with\u00C2\u00A0 this\u00C2\u00A0methodology\u00C2\u00A0 exhibit\u00C2\u00A0 unique\u00C2\u00A0 binding\u00C2\u00A0 properties\u00C2\u00A0 to\u00C2\u00A0 CD20+\u00C2\u00A0 lymphoma\u00C2\u00A0 cells,\u00C2\u00A0 where\u00C2\u00A0 more\u00C2\u00A0 Rtx\u00C2\u00A0 is\u00C2\u00A0 associated\u00C2\u00A0 to\u00C2\u00A0 cells\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 is\u00C2\u00A0 increased\u00C2\u00A0 although\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 significant\u00C2\u00A0 fraction\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0 that\u00C2\u00A0 remains\u00C2\u00A0 unassociated\u00C2\u00A0 with\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0 of\u00C2\u00A0 Rtx/CD20\u00E2\u0080\u0090enriched\u00C2\u00A0 domains\u00C2\u00A0 observed\u00C2\u00A0on\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0Bivalent\u00C2\u00A0Rtx\u00C2\u00A0saturates\u00C2\u00A0all\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0and\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0low\u00C2\u00A0 cytotoxicity\u00C2\u00A0and\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis,\u00C2\u00A0while\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0 saturate\u00C2\u00A0all\u00C2\u00A0CD20\u00C2\u00A0and\u00C2\u00A0 result\u00C2\u00A0in\u00C2\u00A0higher\u00C2\u00A0cytotoxicity\u00C2\u00A0and\u00C2\u00A0high\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0indicates\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0Rtx/CD20\u00C2\u00A0 interactions\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0determine\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx;\u00C2\u00A0the\u00C2\u00A0clustering\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0does\u00C2\u00A0 instead,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0 facilitated\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0results\u00C2\u00A0suggest\u00C2\u00A0 that\u00C2\u00A0it\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0beneficial\u00C2\u00A0to\u00C2\u00A0apply\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0to\u00C2\u00A0other\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0Trz,\u00C2\u00A0 which\u00C2\u00A0has\u00C2\u00A0also\u00C2\u00A0shown\u00C2\u00A0benefits\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0multivalency7,\u00C2\u00A089,\u00C2\u00A0109,\u00C2\u00A0110.\u00C2\u00A0 Moreover,\u00C2\u00A0 the\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 constructs\u00C2\u00A0 of\u00C2\u00A0 higher\u00C2\u00A0 valence\u00C2\u00A0 are\u00C2\u00A0 capable\u00C2\u00A0 of\u00C2\u00A0 greater\u00C2\u00A0 hypercrosslinking,\u00C2\u00A0and\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0to\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0all\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 given\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 dose\u00C2\u00A0 of\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 relationship\u00C2\u00A0 opens\u00C2\u00A0 the\u00C2\u00A0 door\u00C2\u00A0 to\u00C2\u00A0 studies\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 poorly\u00C2\u00A0 defined\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 because\u00C2\u00A0 signaling\u00C2\u00A0 events\u00C2\u00A0 that\u00C2\u00A0 precede\u00C2\u00A0and\u00C2\u00A0cause\u00C2\u00A0apoptosis\u00C2\u00A0would\u00C2\u00A0also\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0valence\u00E2\u0080\u0090dependent.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0methodology\u00C2\u00A0 therefore\u00C2\u00A0provides\u00C2\u00A0a\u00C2\u00A0framework\u00C2\u00A0for\u00C2\u00A0probing\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0molecular\u00C2\u00A0point\u00C2\u00A0 of\u00C2\u00A0view.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A094\u00C2\u00A0\u00C2\u00A0 5\u00C2\u00A0 A\u00C2\u00A0novel\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0rituximab\u00C2\u00A0 5.1\u00C2\u00A0 Synopsis\u00C2\u00A0 The\u00C2\u00A0previous\u00C2\u00A0 chapter\u00C2\u00A0 showed\u00C2\u00A0 that\u00C2\u00A0even\u00C2\u00A0 though\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A0given\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0at\u00C2\u00A0 equivalent\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0higher\u00C2\u00A0 level\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0are\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0constructs\u00C2\u00A0of\u00C2\u00A0higher\u00C2\u00A0valence.\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 chapter\u00C2\u00A0 describes\u00C2\u00A0 studies\u00C2\u00A0 undertaken\u00C2\u00A0 to\u00C2\u00A0 understand\u00C2\u00A0 the\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 5.2\u00C2\u00A0 Background\u00C2\u00A0 5.2.1\u00C2\u00A0 Extrinsic\u00C2\u00A0and\u00C2\u00A0intrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathways\u00C2\u00A0 Apoptosis\u00C2\u00A0occurs\u00C2\u00A0primarily\u00C2\u00A0through\u00C2\u00A0two\u00C2\u00A0distinct\u00C2\u00A0pathways,\u00C2\u00A0the\u00C2\u00A0intrinsic\u00C2\u00A0pathway\u00C2\u00A0(also\u00C2\u00A0known\u00C2\u00A0 as\u00C2\u00A0the\u00C2\u00A0mitochondrial\u00C2\u00A0or\u00C2\u00A0Bcl\u00E2\u0080\u00902\u00C2\u00A0family\u00C2\u00A0regulated\u00C2\u00A0pathway)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0extrinsic\u00C2\u00A0pathway82,\u00C2\u00A086,\u00C2\u00A087.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0two\u00C2\u00A0 pathways\u00C2\u00A0 involve\u00C2\u00A0 distinct\u00C2\u00A0 initiator\u00C2\u00A0 caspases\u00C2\u00A0 that\u00C2\u00A0 set\u00C2\u00A0 each\u00C2\u00A0 pathway\u00C2\u00A0 in\u00C2\u00A0 motion,\u00C2\u00A0 while\u00C2\u00A0 they\u00C2\u00A0 both\u00C2\u00A0 converge\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0effector\u00C2\u00A0caspases\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0caspase\u00E2\u0080\u00903,\u00C2\u00A0\u00E2\u0080\u00906,\u00C2\u00A0and\u00C2\u00A0\u00E2\u0080\u00907.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0intrinsic\u00C2\u00A0pathway\u00C2\u00A0 depends\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 activation\u00C2\u00A0of\u00C2\u00A0 caspase\u00E2\u0080\u00909,\u00C2\u00A0which\u00C2\u00A0occurs\u00C2\u00A0 after\u00C2\u00A0perturbation\u00C2\u00A0of\u00C2\u00A0mitochondria\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 cellular\u00C2\u00A0stress,\u00C2\u00A0growth\u00C2\u00A0factor\u00C2\u00A0withdrawal,\u00C2\u00A0or\u00C2\u00A0cytotoxic\u00C2\u00A0stimuli.\u00C2\u00A0 \u00C2\u00A0Death\u00C2\u00A0stimuli\u00C2\u00A0activate\u00C2\u00A0pro\u00E2\u0080\u0090apoptotic\u00C2\u00A0 Bcl\u00E2\u0080\u00902\u00C2\u00A0 family\u00C2\u00A0members,\u00C2\u00A0 leading\u00C2\u00A0 to\u00C2\u00A0disruption\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0outer\u00C2\u00A0mitochondrial\u00C2\u00A0membrane\u00C2\u00A0and\u00C2\u00A0 release\u00C2\u00A0of\u00C2\u00A0 cytochrome\u00C2\u00A0c\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0apoptosis\u00E2\u0080\u0090inducing\u00C2\u00A0proteins\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0cytosol.\u00C2\u00A0\u00C2\u00A0Cytochrome\u00C2\u00A0c,\u00C2\u00A0along\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0 adaptor\u00C2\u00A0protein\u00C2\u00A0APAF1,\u00C2\u00A0forms\u00C2\u00A0a\u00C2\u00A0complex\u00C2\u00A0termed\u00C2\u00A0the\u00C2\u00A0apoptosome,\u00C2\u00A0which\u00C2\u00A0activates\u00C2\u00A0caspase\u00E2\u0080\u00909,\u00C2\u00A0which\u00C2\u00A0 in\u00C2\u00A0 turn\u00C2\u00A0 leads\u00C2\u00A0 to\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 effector\u00C2\u00A0 caspases.\u00C2\u00A0 \u00C2\u00A0 Additional\u00C2\u00A0 caspase\u00E2\u0080\u0090independent\u00C2\u00A0 cell\u00C2\u00A0 death\u00C2\u00A0 processes\u00C2\u00A0regulated\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0Bcl\u00E2\u0080\u00902\u00C2\u00A0family\u00C2\u00A0have\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0described86.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0cancer\u00C2\u00A0treatment,\u00C2\u00A0apoptosis\u00C2\u00A0 is\u00C2\u00A0usually\u00C2\u00A0triggered\u00C2\u00A0 in\u00C2\u00A0oncogenic\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0chemotherapy\u00C2\u00A0and\u00C2\u00A0 irradiation\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0 intrinsic\u00C2\u00A0pathway,\u00C2\u00A0since\u00C2\u00A0these\u00C2\u00A0agents\u00C2\u00A0cause\u00C2\u00A0cellular\u00C2\u00A0damage.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0pathway\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A095\u00C2\u00A0\u00C2\u00A0 usually\u00C2\u00A0 involves\u00C2\u00A0 requires\u00C2\u00A0p53\u00C2\u00A0 function,\u00C2\u00A0but\u00C2\u00A0mutations\u00C2\u00A0or\u00C2\u00A0 inhibiting\u00C2\u00A0proteins\u00C2\u00A0 impair\u00C2\u00A0p53\u00C2\u00A0 function\u00C2\u00A0 in\u00C2\u00A0 many\u00C2\u00A0human\u00C2\u00A0cancers.\u00C2\u00A0 In\u00C2\u00A0 this\u00C2\u00A0way,\u00C2\u00A0 tumour\u00C2\u00A0cells\u00C2\u00A0evade\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0continue\u00C2\u00A0 to\u00C2\u00A0proliferate\u00C2\u00A0even\u00C2\u00A0 though\u00C2\u00A0 the\u00C2\u00A0 treatment\u00C2\u00A0 caused\u00C2\u00A0 genetic\u00C2\u00A0 instabilities82.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 demonstrated\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0 specifically;\u00C2\u00A0for\u00C2\u00A0example,\u00C2\u00A0the\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0intrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathway\u00C2\u00A0downstream\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00909\u00C2\u00A0 activation\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0cause\u00C2\u00A0resistance\u00C2\u00A0to\u00C2\u00A0chemotherapy\u00C2\u00A0in\u00C2\u00A0diffuse\u00C2\u00A0large\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0lymphomas170.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0the\u00C2\u00A0extrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathway,\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0critical\u00C2\u00A0initiator\u00C2\u00A0caspase.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0cleavage\u00C2\u00A0of\u00C2\u00A0 procaspase\u00E2\u0080\u00908\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0death\u00E2\u0080\u0090inducing\u00C2\u00A0signaling\u00C2\u00A0complex\u00C2\u00A0(DISC),\u00C2\u00A0a\u00C2\u00A0plasma\u00C2\u00A0membrane\u00E2\u0080\u0090associated\u00C2\u00A0 complex\u00C2\u00A0 of\u00C2\u00A0 specific\u00C2\u00A0 proteins\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 initiating\u00C2\u00A0 the\u00C2\u00A0 extrinsic\u00C2\u00A0 pathway.\u00C2\u00A0 \u00C2\u00A0 Activated\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0is\u00C2\u00A0released\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0cytosol\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0DISC\u00C2\u00A0and\u00C2\u00A0goes\u00C2\u00A0on\u00C2\u00A0to\u00C2\u00A0activate\u00C2\u00A0the\u00C2\u00A0effector\u00C2\u00A0caspases87,\u00C2\u00A0 171.\u00C2\u00A0 \u00C2\u00A0Crosstalk\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0 two\u00C2\u00A0 apoptosis\u00C2\u00A0pathways\u00C2\u00A0 is\u00C2\u00A0 also\u00C2\u00A0well\u00E2\u0080\u0090documented,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00E2\u0080\u0090 mediated\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0proapoptotic\u00C2\u00A0Bcl\u00E2\u0080\u00902\u00C2\u00A0family\u00C2\u00A0members,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0cytochrome\u00C2\u00A0c\u00C2\u00A0release.\u00C2\u00A0\u00C2\u00A0Such\u00C2\u00A0 amplification\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0extrinsic\u00C2\u00A0pathway\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0 intrinsic\u00C2\u00A0pathway\u00C2\u00A0 is\u00C2\u00A0required\u00C2\u00A0 in\u00C2\u00A0some\u00C2\u00A0cell\u00C2\u00A0types\u00C2\u00A0 but\u00C2\u00A0not\u00C2\u00A0 in\u00C2\u00A0others82.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0highlights\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 intrinsic\u00C2\u00A0and\u00C2\u00A0extrinsic\u00C2\u00A0pathways\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0distinct,\u00C2\u00A0but\u00C2\u00A0 interdependent.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 5.2.2\u00C2\u00A0 The\u00C2\u00A0 death\u00C2\u00ADinducing\u00C2\u00A0 signaling\u00C2\u00A0 complex\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0superfamily\u00C2\u00A0 Besides\u00C2\u00A0procaspase\u00E2\u0080\u00908,\u00C2\u00A0 there\u00C2\u00A0are\u00C2\u00A0 two\u00C2\u00A0other\u00C2\u00A0essential\u00C2\u00A0 components\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DISC:\u00C2\u00A0an\u00C2\u00A0adaptor\u00C2\u00A0 protein\u00C2\u00A0 (either\u00C2\u00A0 Fas\u00E2\u0080\u0090associated\u00C2\u00A0 death\u00C2\u00A0 domain\u00C2\u00A0 (FADD)\u00C2\u00A0 or\u00C2\u00A0 TNFR\u00E2\u0080\u0090associated\u00C2\u00A0 death\u00C2\u00A0 domain\u00C2\u00A0 (TRADD)),\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A0 death\u00C2\u00A0 receptor171,\u00C2\u00A0172.\u00C2\u00A0 \u00C2\u00A0Death\u00C2\u00A0 receptors\u00C2\u00A0 constitute\u00C2\u00A0 specific\u00C2\u00A0members\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 (TNFR)\u00C2\u00A0 superfamily.\u00C2\u00A0 \u00C2\u00A0 Discovery\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 TNFR\u00C2\u00A0 superfamily\u00C2\u00A0 was\u00C2\u00A0made\u00C2\u00A0 possible\u00C2\u00A0 by\u00C2\u00A0 observations\u00C2\u00A0 in\u00C2\u00A01868\u00C2\u00A0where\u00C2\u00A0 tumors\u00C2\u00A0 in\u00C2\u00A0 some\u00C2\u00A0patients\u00C2\u00A0 showed\u00C2\u00A0 regression\u00C2\u00A0 following\u00C2\u00A0acute\u00C2\u00A0bacterial\u00C2\u00A0 infection,\u00C2\u00A0but\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0until\u00C2\u00A01975\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0term\u00C2\u00A0\u00E2\u0080\u009Ctumor\u00E2\u0080\u0090necrosis\u00C2\u00A0factor\u00E2\u0080\u009D\u00C2\u00A0(TNF)\u00C2\u00A0was\u00C2\u00A0coined82,\u00C2\u00A0173.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0TNFR\u00C2\u00A0superfamily\u00C2\u00A0are\u00C2\u00A0critically\u00C2\u00A0involved\u00C2\u00A0in\u00C2\u00A0maintenance\u00C2\u00A0of\u00C2\u00A0homeostasis\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system.\u00C2\u00A0\u00C2\u00A0They\u00C2\u00A0are\u00C2\u00A0type\u00E2\u0080\u0090I\u00C2\u00A0transmembrane\u00C2\u00A0proteins\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0C\u00E2\u0080\u0090terminal\u00C2\u00A0intracellular\u00C2\u00A0tail,\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A096\u00C2\u00A0\u00C2\u00A0 a\u00C2\u00A0membrane\u00E2\u0080\u0090spanning\u00C2\u00A0region,\u00C2\u00A0and\u00C2\u00A0an\u00C2\u00A0extracellular\u00C2\u00A0ligand\u00E2\u0080\u0090binding\u00C2\u00A0N\u00E2\u0080\u0090terminal\u00C2\u00A0domain.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0hallmark\u00C2\u00A0 of\u00C2\u00A0TNFR\u00C2\u00A0superfamily\u00C2\u00A0members\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0cysteine\u00E2\u0080\u0090rich\u00C2\u00A0extracellular\u00C2\u00A0domains,\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 which\u00C2\u00A0can\u00C2\u00A0vary\u00C2\u00A0from\u00C2\u00A0one\u00C2\u00A0to\u00C2\u00A0six,\u00C2\u00A0and\u00C2\u00A0which\u00C2\u00A0define\u00C2\u00A0ligand\u00C2\u00A0specificity.\u00C2\u00A0\u00C2\u00A0Over\u00C2\u00A040\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0TNFR\u00C2\u00A0 superfamily\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0identified\u00C2\u00A0to\u00C2\u00A0date172,\u00C2\u00A0174.\u00C2\u00A0 5.2.3\u00C2\u00A0 Death\u00C2\u00A0receptors\u00C2\u00A0 Death\u00C2\u00A0receptors\u00C2\u00A0are\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0TNFR\u00C2\u00A0superfamily\u00C2\u00A0characterized\u00C2\u00A0by\u00C2\u00A0an\u00C2\u00A0~80\u00C2\u00A0amino\u00E2\u0080\u0090acid\u00C2\u00A0 cytoplasmic\u00C2\u00A0 \u00E2\u0080\u009Cdeath\u00C2\u00A0 domain\u00E2\u0080\u009D\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 essential\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0 When\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 receives\u00C2\u00A0a\u00C2\u00A0proapoptotic\u00C2\u00A0signal,\u00C2\u00A0the\u00C2\u00A0death\u00C2\u00A0domain\u00C2\u00A0is\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0recruiting\u00C2\u00A0FADD\u00C2\u00A0or\u00C2\u00A0TRADD,\u00C2\u00A0which\u00C2\u00A0 in\u00C2\u00A0turn\u00C2\u00A0recruits\u00C2\u00A0procaspase\u00E2\u0080\u00908,\u00C2\u00A0forming\u00C2\u00A0the\u00C2\u00A0DISC\u00C2\u00A0which\u00C2\u00A0leads\u00C2\u00A0to\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0activation175.\u00C2\u00A0\u00C2\u00A0There\u00C2\u00A0are\u00C2\u00A0six\u00C2\u00A0 death\u00C2\u00A0 receptors\u00C2\u00A0 that\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 identified\u00C2\u00A0 to\u00C2\u00A0date;\u00C2\u00A0 the\u00C2\u00A0 four\u00C2\u00A0most\u00C2\u00A0extensively\u00C2\u00A0 studied\u00C2\u00A0are\u00C2\u00A0CD120a\u00C2\u00A0 (also\u00C2\u00A0 known\u00C2\u00A0 as\u00C2\u00A0 TNFR1\u00C2\u00A0 or\u00C2\u00A0 TNFRSF1A),\u00C2\u00A0 CD95\u00C2\u00A0 (Fas\u00C2\u00A0 receptor,\u00C2\u00A0 APO\u00E2\u0080\u00901,\u00C2\u00A0 or\u00C2\u00A0 TNFRSF6),\u00C2\u00A0DR4\u00C2\u00A0 (TRAILR1\u00C2\u00A0 or\u00C2\u00A0 TNFRSF10A),\u00C2\u00A0 and\u00C2\u00A0 DR5\u00C2\u00A0 (TRAILR2,\u00C2\u00A0 APO\u00E2\u0080\u00902,\u00C2\u00A0 or\u00C2\u00A0 TNFRSF10B)174,\u00C2\u00A0 176.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 ligands\u00C2\u00A0 to\u00C2\u00A0 these\u00C2\u00A0 receptors,\u00C2\u00A0 respectively,\u00C2\u00A0are\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1,\u00C2\u00A0Fas\u00C2\u00A0 ligand\u00C2\u00A0(FasL),\u00C2\u00A0and\u00C2\u00A0Apo2L/TRAIL\u00C2\u00A0(for\u00C2\u00A0both\u00C2\u00A0DR4\u00C2\u00A0and\u00C2\u00A0DR5).\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0 ligands\u00C2\u00A0 comprise\u00C2\u00A0 a\u00C2\u00A0 group\u00C2\u00A0 of\u00C2\u00A0 complementary\u00C2\u00A0 cytokines\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0mainly\u00C2\u00A0 type\u00E2\u0080\u0090II\u00C2\u00A0 transmembrane\u00C2\u00A0 proteins,\u00C2\u00A0 although\u00C2\u00A0some\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0released\u00C2\u00A0as\u00C2\u00A0soluble\u00C2\u00A0cytokines\u00C2\u00A0upon\u00C2\u00A0proteolytic\u00C2\u00A0cleavage174,\u00C2\u00A0176.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Almost\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0TNF\u00C2\u00A0ligands\u00C2\u00A0are\u00C2\u00A0expressed\u00C2\u00A0only\u00C2\u00A0by\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system,\u00C2\u00A0including\u00C2\u00A0B\u00C2\u00A0 cells,\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0NK\u00C2\u00A0cells,\u00C2\u00A0monocytes\u00C2\u00A0and\u00C2\u00A0dendritic\u00C2\u00A0cells,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0TNFRs\u00C2\u00A0are\u00C2\u00A0expressed\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0wide\u00C2\u00A0variety\u00C2\u00A0 of\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0no\u00C2\u00A0cell\u00C2\u00A0type\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0body\u00C2\u00A0has\u00C2\u00A0yet\u00C2\u00A0been\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0express\u00C2\u00A0CD120a177.\u00C2\u00A0\u00C2\u00A0 There\u00C2\u00A0 is\u00C2\u00A0also\u00C2\u00A0another\u00C2\u00A0subset\u00C2\u00A0of\u00C2\u00A0TNFR\u00C2\u00A0superfamily\u00C2\u00A0members\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0decoy\u00C2\u00A0 receptors,\u00C2\u00A0which\u00C2\u00A0 lack\u00C2\u00A0 the\u00C2\u00A0death\u00C2\u00A0domain\u00C2\u00A0and\u00C2\u00A0 therefore\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0nonsignaling\u00C2\u00A0role;\u00C2\u00A0 they\u00C2\u00A0compete\u00C2\u00A0with\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0 for\u00C2\u00A0 ligand\u00C2\u00A0binding,\u00C2\u00A0thereby\u00C2\u00A0inhibiting\u00C2\u00A0death\u00C2\u00A0receptor\u00C2\u00A0function172.\u00C2\u00A0\u00C2\u00A0Finally,\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0two\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0 that\u00C2\u00A0have\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0mentioned,\u00C2\u00A0which\u00C2\u00A0in\u00C2\u00A0general\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0as\u00C2\u00A0effective\u00C2\u00A0at\u00C2\u00A0inducing\u00C2\u00A0apoptosis,\u00C2\u00A0are\u00C2\u00A0DR3\u00C2\u00A0 (TNFRSF12)\u00C2\u00A0and\u00C2\u00A0DR6\u00C2\u00A0(TNFRSF21)174,\u00C2\u00A0178.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A097\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0common\u00C2\u00A0 requirement\u00C2\u00A0 to\u00C2\u00A0 form\u00C2\u00A0 the\u00C2\u00A0DISC\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0oligomerization\u00C2\u00A0of\u00C2\u00A0death\u00C2\u00A0 receptors\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 facilitated\u00C2\u00A0 with\u00C2\u00A0 ligand\u00C2\u00A0 binding\u00C2\u00A0 and\u00C2\u00A0 which\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 receptor\u00C2\u00A0 trimerization172,\u00C2\u00A0 174.\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0 ligand\u00C2\u00A0 binding\u00C2\u00A0 was\u00C2\u00A0 originally\u00C2\u00A0 thought\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 DISC\u00C2\u00A0 formation,\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 substantial\u00C2\u00A0 evidence\u00C2\u00A0 showing\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0 of\u00C2\u00A0 preassembled\u00C2\u00A0 death\u00C2\u00A0 receptor\u00C2\u00A0 complexes\u00C2\u00A0 (such\u00C2\u00A0 as\u00C2\u00A0 ones\u00C2\u00A0 containing\u00C2\u00A0 CD95\u00C2\u00A0 and\u00C2\u00A0 CD120a)\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 ligand179.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0occur\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 interaction\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0membrane\u00E2\u0080\u0090distal\u00C2\u00A0 extracellular\u00C2\u00A0 cysteine\u00E2\u0080\u0090rich\u00C2\u00A0domain\u00C2\u00A0termed\u00C2\u00A0the\u00C2\u00A0preligand\u00C2\u00A0assembly\u00C2\u00A0domain180.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0ligand\u00E2\u0080\u0090independent\u00C2\u00A0activation\u00C2\u00A0 of\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0apoptosis\u00C2\u00A0is\u00C2\u00A0well\u00E2\u0080\u0090documented\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0cases181\u00E2\u0080\u0090186.\u00C2\u00A0 5.2.4\u00C2\u00A0 Deciphering\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 target\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 2.2,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 were\u00C2\u00A0 also\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.6.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 aims\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 experiments\u00C2\u00A0outlined\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0chapter\u00C2\u00A0were\u00C2\u00A0to\u00C2\u00A0 first\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0the\u00C2\u00A0observed\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0 caspase\u00E2\u0080\u0090dependent,\u00C2\u00A0and\u00C2\u00A0 if\u00C2\u00A0 so,\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0 the\u00C2\u00A0pattern\u00C2\u00A0of\u00C2\u00A0 caspase\u00C2\u00A0activation\u00C2\u00A0 to\u00C2\u00A0discern\u00C2\u00A0whether\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0 inducing\u00C2\u00A0apoptosis\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0intrinsic\u00C2\u00A0or\u00C2\u00A0extrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathway.\u00C2\u00A0 \u00C2\u00A0Based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 known\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0described,\u00C2\u00A0further\u00C2\u00A0experiments\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0the\u00C2\u00A0 specific\u00C2\u00A0 proteins\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0 Because\u00C2\u00A0 the\u00C2\u00A0 level\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 was\u00C2\u00A0 valence\u00E2\u0080\u0090dependent,\u00C2\u00A0 signaling\u00C2\u00A0 events\u00C2\u00A0 directly\u00C2\u00A0 preceding\u00C2\u00A0 apoptosis\u00C2\u00A0 (such\u00C2\u00A0 as\u00C2\u00A0 concentrations\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0proteins\u00C2\u00A0that\u00C2\u00A0 induce\u00C2\u00A0apoptosis)\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0valence\u00E2\u0080\u0090dependent.\u00C2\u00A0 \u00C2\u00A0Finally,\u00C2\u00A0 the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 liposomal\u00C2\u00A0component\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0 examined.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A098\u00C2\u00A0\u00C2\u00A0 5.3\u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0 5.3.1\u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 Vybrant\u00C2\u00A0 FAM\u00C2\u00A0 Caspase\u00E2\u0080\u00908\u00C2\u00A0 Assay\u00C2\u00A0 Kit,\u00C2\u00A0 Alexa\u00C2\u00A0 Fluor\u00C2\u00A0 488\u00E2\u0080\u0090labeled\u00C2\u00A0 cholera\u00C2\u00A0 toxin\u00C2\u00A0 subunit\u00C2\u00A0 B\u00C2\u00A0 (A488\u00E2\u0080\u0090CTX),\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00C2\u00A0 binding\u00C2\u00A0 buffer,\u00C2\u00A0 recombinant\u00C2\u00A0 human\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00C2\u00A0 labelled\u00C2\u00A0 with\u00C2\u00A0 fluorescein\u00C2\u00A0 isothiocyanate\u00C2\u00A0 (Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC),\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0Alexa\u00C2\u00A0Fluor\u00C2\u00A0647\u00C2\u00A0 (A647)\u00C2\u00A0monoclonal\u00C2\u00A0Ab\u00C2\u00A0 labeling\u00C2\u00A0 kit\u00C2\u00A0were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 Invitrogen\u00C2\u00A0 (Burlington\u00C2\u00A0ON,\u00C2\u00A0Canada).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0SensoLyte\u00C2\u00A0AFC\u00C2\u00A0 (7\u00E2\u0080\u0090amino\u00E2\u0080\u00904\u00E2\u0080\u0090trifluoromethyl\u00C2\u00A0 coumarin)\u00C2\u00A0Caspase\u00C2\u00A0Profiling\u00C2\u00A0Kit\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0 from\u00C2\u00A0AnaSpec\u00C2\u00A0 (Fremont\u00C2\u00A0CA,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 caspse\u00E2\u0080\u00908\u00C2\u00A0 inhibitor\u00C2\u00A0Z\u00E2\u0080\u0090IETD\u00E2\u0080\u0090FMK\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0R&D\u00C2\u00A0Systems\u00C2\u00A0(Minneapolis\u00C2\u00A0MN,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0 peptide\u00C2\u00A0Z\u00E2\u0080\u0090FA\u00E2\u0080\u0090FMK\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0Santa\u00C2\u00A0Cruz\u00C2\u00A0Biotechnology\u00C2\u00A0 (Santa\u00C2\u00A0Cruz\u00C2\u00A0CA,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0death\u00C2\u00A0 domain\u00C2\u00A0receptor\u00C2\u00A0antibody\u00C2\u00A0detection\u00C2\u00A0set\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0ProSci\u00C2\u00A0(Poway\u00C2\u00A0CA,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0A647\u00E2\u0080\u0090labeled\u00C2\u00A0 Abs\u00C2\u00A0against\u00C2\u00A0CD120a\u00C2\u00A0(A647\u00E2\u0080\u0090CD120a)\u00C2\u00A0and\u00C2\u00A0CD95\u00C2\u00A0(A647\u00E2\u0080\u0090CD95)\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0an\u00C2\u00A0A488\u00E2\u0080\u0090labeled\u00C2\u00A0anti\u00E2\u0080\u0090DR4\u00C2\u00A0Ab\u00C2\u00A0 (A488\u00E2\u0080\u0090DR4),\u00C2\u00A0an\u00C2\u00A0A488\u00E2\u0080\u0090labeled\u00C2\u00A0mouse\u00C2\u00A0 IgG1\u00C2\u00A0negative\u00C2\u00A0control,\u00C2\u00A0an\u00C2\u00A0A647\u00E2\u0080\u0090labeled\u00C2\u00A0mouse\u00C2\u00A0 IgG2a\u00C2\u00A0negative\u00C2\u00A0 control,\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0FITC\u00E2\u0080\u0090labeled\u00C2\u00A0anti\u00E2\u0080\u0090Rtx\u00C2\u00A0Ab\u00C2\u00A0(FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx)\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0AbD\u00C2\u00A0Serotec\u00C2\u00A0(Kidlington,\u00C2\u00A0 UK).\u00C2\u00A0 \u00C2\u00A0 Streptavidin\u00E2\u0080\u0090coated\u00C2\u00A0polystyrene\u00C2\u00A0microspheres\u00C2\u00A0with\u00C2\u00A0 a\u00C2\u00A0diameter\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0nm\u00C2\u00A0 (streptavidin\u00E2\u0080\u0090MS)\u00C2\u00A0 were\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0Bangs\u00C2\u00A0Laboratories\u00C2\u00A0(Fishers\u00C2\u00A0IN,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0DyLight\u00C2\u00A0649\u00E2\u0080\u0090conjugated\u00C2\u00A0goat\u00C2\u00A0anti\u00E2\u0080\u0090rabbit\u00E2\u0080\u0090 IgG\u00C2\u00A0 F(ab\u00E2\u0080\u0099)2\u00C2\u00A0 fragment\u00C2\u00A0 (D649\u00E2\u0080\u0090anti\u00E2\u0080\u0090rabbit\u00E2\u0080\u0090IgG)\u00C2\u00A0was\u00C2\u00A0 purchased\u00C2\u00A0 from\u00C2\u00A0 Jackson\u00C2\u00A0 ImmunoResearch\u00C2\u00A0 (West\u00C2\u00A0 Grove\u00C2\u00A0PA,\u00C2\u00A0USA).\u00C2\u00A0\u00C2\u00A0Disposable\u00C2\u00A0PD\u00E2\u0080\u009010\u00C2\u00A0columns\u00C2\u00A0(which\u00C2\u00A0contain\u00C2\u00A0Sephadex\u00C2\u00A0G\u00E2\u0080\u009025\u00C2\u00A0medium)\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0GE\u00C2\u00A0Healthcare\u00C2\u00A0Life\u00C2\u00A0Sciences\u00C2\u00A0(Piscataway\u00C2\u00A0NJ,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0Unless\u00C2\u00A0noted\u00C2\u00A0otherwise,\u00C2\u00A0all\u00C2\u00A0other\u00C2\u00A0reagents\u00C2\u00A0 were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Sigma\u00E2\u0080\u0090Aldrich\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0quality\u00C2\u00A0available.\u00C2\u00A0 \u00C2\u00A0 Ramos\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0were\u00C2\u00A0acquired\u00C2\u00A0and\u00C2\u00A0maintained\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A04.3.2.\u00C2\u00A0 5.3.2\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNPs\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0methodology\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0and\u00C2\u00A0summarized\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A03.1\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0 and\u00C2\u00A0characterize\u00C2\u00A0two\u00C2\u00A0separate\u00C2\u00A0batches\u00C2\u00A0(four\u00C2\u00A0valences\u00C2\u00A0each)\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0with\u00C2\u00A0valences\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0204.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A099\u00C2\u00A0\u00C2\u00A0 5.3.3\u00C2\u00A0 Assay\u00C2\u00A0for\u00C2\u00A0quantifying\u00C2\u00A0caspase\u00C2\u00AD8\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0treated\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 Vybrant\u00C2\u00A0 FAM\u00C2\u00A0 Caspase\u00E2\u0080\u00908\u00C2\u00A0 Assay\u00C2\u00A0 Kit\u00C2\u00A0 was\u00C2\u00A0 employed\u00C2\u00A0 to\u00C2\u00A0 quantify\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 in\u00C2\u00A0 treated\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0kit\u00C2\u00A0contains\u00C2\u00A0the\u00C2\u00A0cell\u00E2\u0080\u0090permeable\u00C2\u00A0and\u00C2\u00A0noncytotoxic\u00C2\u00A0peptide\u00C2\u00A0reagent\u00C2\u00A0FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK,\u00C2\u00A0 which\u00C2\u00A0contains\u00C2\u00A0an\u00C2\u00A0affinity\u00C2\u00A0peptide\u00C2\u00A0sequence\u00C2\u00A0specific\u00C2\u00A0 to\u00C2\u00A0activated\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0 (leucine\u00E2\u0080\u0090glutamic\u00C2\u00A0acid\u00E2\u0080\u0090 threonine\u00E2\u0080\u0090aspartic\u00C2\u00A0acid\u00C2\u00A0(LETD)).\u00C2\u00A0\u00C2\u00A0Upon\u00C2\u00A0association\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0peptide\u00C2\u00A0sequence,\u00C2\u00A0the\u00C2\u00A0probe\u00C2\u00A0covalently\u00C2\u00A0 binds\u00C2\u00A0 to\u00C2\u00A0 cleaved\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 fluoromethyl\u00C2\u00A0 ketone\u00C2\u00A0 (FMK)\u00C2\u00A0 moiety,\u00C2\u00A0 while\u00C2\u00A0 the\u00C2\u00A0 FAM\u00C2\u00A0 (carboxyfluorescein)\u00C2\u00A0reporter\u00C2\u00A0has\u00C2\u00A0excitation\u00C2\u00A0and\u00C2\u00A0emission\u00C2\u00A0wavelengths\u00C2\u00A0of\u00C2\u00A0approximately\u00C2\u00A0488\u00C2\u00A0nm\u00C2\u00A0and\u00C2\u00A0 530\u00C2\u00A0 nm,\u00C2\u00A0 respectively.\u00C2\u00A0 \u00C2\u00A0 Bound\u00C2\u00A0 FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK\u00C2\u00A0 remains\u00C2\u00A0 within\u00C2\u00A0 the\u00C2\u00A0 cell,\u00C2\u00A0 while\u00C2\u00A0 unbound\u00C2\u00A0 reagent\u00C2\u00A0 diffuses\u00C2\u00A0out\u00C2\u00A0and\u00C2\u00A0 is\u00C2\u00A0washed\u00C2\u00A0away.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A048\u00E2\u0080\u0090well\u00C2\u00A0plate,\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0250\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0 medium\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0and\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0Rtx,\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0(10\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0dose).\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0the\u00C2\u00A0time\u00E2\u0080\u0090course\u00C2\u00A0study,\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0 was\u00C2\u00A0 employed\u00C2\u00A0 and\u00C2\u00A0 incubation\u00C2\u00A0 was\u00C2\u00A0 allowed\u00C2\u00A0 to\u00C2\u00A0 proceed\u00C2\u00A0 for\u00C2\u00A0 different\u00C2\u00A0 times\u00C2\u00A0 at\u00C2\u00A0 6\u00C2\u00A0 h\u00C2\u00A0 intervals\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A048\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0the\u00C2\u00A0valence\u00E2\u0080\u0090dependence\u00C2\u00A0study,\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00C2\u00A0were\u00C2\u00A0employed\u00C2\u00A0and\u00C2\u00A0analysis\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0the\u00C2\u00A0treatment\u00C2\u00A0time,\u00C2\u00A08.3\u00C2\u00A0 \u00CE\u00BCL\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A030x\u00C2\u00A0FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK\u00C2\u00A0solution\u00C2\u00A0(prepared\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0kit\u00C2\u00A0instructions)\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0each\u00C2\u00A0well\u00C2\u00A0 and\u00C2\u00A0the\u00C2\u00A0plate\u00C2\u00A0was\u00C2\u00A0gently\u00C2\u00A0shaken\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0plate\u00C2\u00A0shaker\u00C2\u00A0for\u00C2\u00A03\u00C2\u00A0min.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0plate\u00C2\u00A0was\u00C2\u00A0then\u00C2\u00A0incubated\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC\u00C2\u00A0 and\u00C2\u00A05\u00C2\u00A0%\u00C2\u00A0CO2\u00C2\u00A0 for\u00C2\u00A060\u00C2\u00A0min,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0plate\u00C2\u00A0was\u00C2\u00A0mixed\u00C2\u00A0every\u00C2\u00A020\u00C2\u00A0min\u00C2\u00A0during\u00C2\u00A0the\u00C2\u00A0 incubation.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0 resuspended\u00C2\u00A0and\u00C2\u00A0transferred\u00C2\u00A0to\u00C2\u00A0Eppendorf\u00C2\u00A0tubes,\u00C2\u00A0spun\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0microcentrifuge\u00C2\u00A0at\u00C2\u00A07000\u00C2\u00A0rpm\u00C2\u00A0 for\u00C2\u00A015\u00C2\u00A0s,\u00C2\u00A0 washed\u00C2\u00A0 twice\u00C2\u00A0with\u00C2\u00A0wash\u00C2\u00A0 buffer\u00C2\u00A0 (500\u00C2\u00A0 \u00CE\u00BCL\u00C2\u00A0 and\u00C2\u00A0 300\u00C2\u00A0 \u00CE\u00BCL),\u00C2\u00A0 then\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0 350\u00C2\u00A0 \u00CE\u00BCL\u00C2\u00A0wash\u00C2\u00A0 buffer\u00C2\u00A0 containing\u00C2\u00A00.2\u00C2\u00A0\u00CE\u00BCg/mL\u00C2\u00A0propidium\u00C2\u00A0iodide\u00C2\u00A0(PI)\u00C2\u00A0before\u00C2\u00A0analysis\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0FACSCalibur\u00C2\u00A0flow\u00C2\u00A0cytometer\u00C2\u00A0(Becton\u00C2\u00A0 Dickinson,\u00C2\u00A0 San\u00C2\u00A0 Jose,\u00C2\u00A0 CA).\u00C2\u00A0 \u00C2\u00A0 FAM\u00C2\u00A0 fluorescence\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 FL1\u00C2\u00A0 channel.\u00C2\u00A0 \u00C2\u00A0Using\u00C2\u00A0 a\u00C2\u00A0 sample\u00C2\u00A0 where\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0was\u00C2\u00A0irreversibly\u00C2\u00A0inhibited\u00C2\u00A0using\u00C2\u00A0Z\u00E2\u0080\u0090IETD\u00E2\u0080\u0090FMK\u00C2\u00A0(cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0200\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0for\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0 at\u00C2\u00A037\u00C2\u00A0oC),\u00C2\u00A0a\u00C2\u00A0fluorescence\u00C2\u00A0intensity\u00C2\u00A0threshold\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0FL1\u00C2\u00A0channel\u00C2\u00A0was\u00C2\u00A0defined\u00C2\u00A0above\u00C2\u00A0which\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 considered\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0positive.\u00C2\u00A0 \u00C2\u00A0Treatments\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0 in\u00C2\u00A0 triplicate,\u00C2\u00A0and\u00C2\u00A0data\u00C2\u00A0here\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0 subsequent\u00C2\u00A0figures\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0as\u00C2\u00A0mean\u00C2\u00A0\u00C2\u00B1\u00C2\u00A0standard\u00C2\u00A0deviation\u00C2\u00A0(SD)\u00C2\u00A0unless\u00C2\u00A0noted\u00C2\u00A0otherwise.\u00C2\u00A0\u00C2\u00A0Two\u00E2\u0080\u0090tailed\u00C2\u00A0 unpaired\u00C2\u00A0Student\u00E2\u0080\u0099s\u00C2\u00A0t\u00E2\u0080\u0090tests\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0cases\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0significance\u00C2\u00A0of\u00C2\u00A00.05.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0100\u00C2\u00A0\u00C2\u00A0 5.3.4\u00C2\u00A0 Profiling\u00C2\u00A0of\u00C2\u00A0caspases\u00C2\u00A0and\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0caspase\u00C2\u00AD8\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 SensoLyte\u00C2\u00A0 AFC\u00C2\u00A0 Caspase\u00C2\u00A0 Profiling\u00C2\u00A0 Kit\u00C2\u00A0 was\u00C2\u00A0 used\u00C2\u00A0 according\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions\u00C2\u00A0 to\u00C2\u00A0measure\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 cleaved\u00C2\u00A0 caspases\u00C2\u00A01,\u00C2\u00A02,\u00C2\u00A03\u00C2\u00A0and\u00C2\u00A07\u00C2\u00A0 (3/7),\u00C2\u00A06,\u00C2\u00A08,\u00C2\u00A0and\u00C2\u00A09\u00C2\u00A0 in\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 different\u00C2\u00A0 treatments.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0kit\u00C2\u00A0contains\u00C2\u00A0a\u00C2\u00A0 substrates\u00C2\u00A0 specific\u00C2\u00A0 to\u00C2\u00A0each\u00C2\u00A0caspase\u00C2\u00A0 that\u00C2\u00A0contains\u00C2\u00A0an\u00C2\u00A0AFC\u00C2\u00A0 that\u00C2\u00A0when\u00C2\u00A0cleaved\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0substrate\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0caspase,\u00C2\u00A0has\u00C2\u00A0emission\u00C2\u00A0and\u00C2\u00A0excitation\u00C2\u00A0wavelengths\u00C2\u00A0of\u00C2\u00A0 approximately\u00C2\u00A0380\u00C2\u00A0nm\u00C2\u00A0and\u00C2\u00A0500\u00C2\u00A0nm,\u00C2\u00A0respectively.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A02\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A02.0\u00C2\u00A0mL\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0or\u00C2\u00A0an\u00C2\u00A080\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0 Rtx\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(150)\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0experiments\u00C2\u00A0involving\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0using\u00C2\u00A0Z\u00E2\u0080\u0090IETD\u00E2\u0080\u0090FMK\u00C2\u00A0or\u00C2\u00A0 Z\u00E2\u0080\u0090FA\u00E2\u0080\u0090FMK\u00C2\u00A0(negative\u00C2\u00A0control),\u00C2\u00A0200\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A02.0\u00C2\u00A0mM\u00C2\u00A0solution\u00C2\u00A0in\u00C2\u00A0cell\u00C2\u00A0culture\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0 cells\u00C2\u00A0which\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0 incubated\u00C2\u00A0for\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC\u00C2\u00A0before\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(150)\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 above.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0 positive\u00C2\u00A0 control\u00C2\u00A0 consisted\u00C2\u00A0 of\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 4.0\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0 camptothecin\u00C2\u00A0 for\u00C2\u00A0 4\u00C2\u00A0 h.\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0 the\u00C2\u00A0 incubation,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0collected\u00C2\u00A0by\u00C2\u00A0centrifugation\u00C2\u00A0at\u00C2\u00A0500g\u00C2\u00A0for\u00C2\u00A05\u00C2\u00A0min,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0pellet\u00C2\u00A0was\u00C2\u00A0resuspended\u00C2\u00A0 in\u00C2\u00A01.3\u00C2\u00A0mL\u00C2\u00A0of\u00C2\u00A0lysis\u00C2\u00A0buffer\u00C2\u00A0(provided\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0kit).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0suspension\u00C2\u00A0was\u00C2\u00A0rotated\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0rotating\u00C2\u00A0apparatus\u00C2\u00A0 for\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0oC,\u00C2\u00A0then\u00C2\u00A0centrifuged\u00C2\u00A0at\u00C2\u00A02500g\u00C2\u00A0for\u00C2\u00A010\u00C2\u00A0min\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0oC.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0resulting\u00C2\u00A0 caspase\u00E2\u0080\u0090containing\u00C2\u00A0supernatant\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0wells\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A096\u00E2\u0080\u0090well\u00C2\u00A0plate\u00C2\u00A0containing\u00C2\u00A0 the\u00C2\u00A0various\u00C2\u00A0 substrates\u00C2\u00A0dissolved\u00C2\u00A0 in\u00C2\u00A0assay\u00C2\u00A0buffer\u00C2\u00A0and\u00C2\u00A0dithiothreitol\u00C2\u00A0 (DTT).\u00C2\u00A0 \u00C2\u00A0Samples\u00C2\u00A0were\u00C2\u00A0 in\u00C2\u00A0 triplicate\u00C2\u00A0 for\u00C2\u00A0every\u00C2\u00A0 caspase\u00C2\u00A0 for\u00C2\u00A0each\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0Plates\u00C2\u00A0were\u00C2\u00A0 shaken\u00C2\u00A0and\u00C2\u00A0 read\u00C2\u00A0every\u00C2\u00A030\u00C2\u00A0min\u00C2\u00A0 for\u00C2\u00A0up\u00C2\u00A0 to\u00C2\u00A03\u00C2\u00A0h.\u00C2\u00A0 \u00C2\u00A0Average\u00C2\u00A0 fluorescence\u00C2\u00A0 intensities\u00C2\u00A0 were\u00C2\u00A0 calculated\u00C2\u00A0 after\u00C2\u00A0 subtracting\u00C2\u00A0 averages\u00C2\u00A0 of\u00C2\u00A0 blanks,\u00C2\u00A0 and\u00C2\u00A0 were\u00C2\u00A0 scaled\u00C2\u00A0 relative\u00C2\u00A0to\u00C2\u00A0HBS\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0caspase.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 Fluorstar\u00C2\u00A0 fluorescence\u00C2\u00A0 plate\u00C2\u00A0 reader\u00C2\u00A0was\u00C2\u00A0 calibrated\u00C2\u00A0 using\u00C2\u00A0 free\u00C2\u00A0 AFC,\u00C2\u00A0 and\u00C2\u00A0 fluorescence\u00C2\u00A0 readings\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0linear\u00C2\u00A0range\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0emission\u00C2\u00A0intensity\u00C2\u00A0versus\u00C2\u00A0AFC\u00C2\u00A0concentration\u00C2\u00A0plot.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 5.3.5\u00C2\u00A0 Measurement\u00C2\u00A0of\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0using\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0quantify\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0in\u00C2\u00A0treated\u00C2\u00A0cells,\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0or\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0 medium\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 a\u00C2\u00A0 10\u00C2\u00A0 \u00CE\u00BCg\u00C2\u00A0 dose\u00C2\u00A0 of\u00C2\u00A0 Rtx,\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 or\u00C2\u00A0 an\u00C2\u00A0 equivalent\u00C2\u00A0 volume\u00C2\u00A0 of\u00C2\u00A0HBS,\u00C2\u00A0 and\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0101\u00C2\u00A0\u00C2\u00A0 analyzed\u00C2\u00A0 at\u00C2\u00A0 different\u00C2\u00A0 times\u00C2\u00A0 after\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0 analysis\u00C2\u00A0 using\u00C2\u00A0 flow\u00C2\u00A0 cytometry,\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 transferred\u00C2\u00A0to\u00C2\u00A01.5\u00C2\u00A0mL\u00C2\u00A0Eppendorf\u00C2\u00A0tubes\u00C2\u00A0and\u00C2\u00A0spun\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0microcentrifuge\u00C2\u00A0at\u00C2\u00A07000\u00C2\u00A0rpm\u00C2\u00A0for\u00C2\u00A015\u00C2\u00A0s.\u00C2\u00A0\u00C2\u00A0Pellets\u00C2\u00A0 were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0 cold\u00C2\u00A0PBSB\u00C2\u00A0 (phosphate\u00E2\u0080\u0090buffered\u00C2\u00A0 saline\u00C2\u00A0 (PBS)\u00C2\u00A0+\u00C2\u00A00.1%\u00C2\u00A0bovine\u00C2\u00A0 serum\u00C2\u00A0albumin,\u00C2\u00A0pH\u00C2\u00A0 7.4)\u00C2\u00A0and\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A020\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0PBSB.\u00C2\u00A0\u00C2\u00A0An\u00C2\u00A0appropriate\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0each\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0and\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 stained\u00C2\u00A0 for\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0on\u00C2\u00A0 ice.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0 titration\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0on\u00C2\u00A0each\u00C2\u00A0Ab\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0saturation\u00C2\u00A0 in\u00C2\u00A0 each\u00C2\u00A0cell\u00C2\u00A0line\u00C2\u00A0and\u00C2\u00A0appropriate\u00C2\u00A0volumes\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00A0required\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0added\u00C2\u00A0(generally\u00C2\u00A0between\u00C2\u00A00.5\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A03.0\u00C2\u00A0\u00C2\u00B5L).\u00C2\u00A0\u00C2\u00A0 Primary\u00C2\u00A0labeled\u00C2\u00A0Abs\u00C2\u00A0consisted\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090CD95,\u00C2\u00A0A488\u00E2\u0080\u0090DR4,\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090CD120a,\u00C2\u00A0and\u00C2\u00A0nonlabeled\u00C2\u00A0primary\u00C2\u00A0 Abs\u00C2\u00A0 included\u00C2\u00A0anti\u00E2\u0080\u0090DR3,\u00C2\u00A0DR4,\u00C2\u00A0DR5,\u00C2\u00A0and\u00C2\u00A0DR6\u00C2\u00A0Abs\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0death\u00C2\u00A0domain\u00C2\u00A0 receptor\u00C2\u00A0Ab\u00C2\u00A0detection\u00C2\u00A0set\u00C2\u00A0 (see\u00C2\u00A0Section\u00C2\u00A05.3.1).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 After\u00C2\u00A0 staining\u00C2\u00A0 for\u00C2\u00A0 1\u00C2\u00A0 h\u00C2\u00A0 on\u00C2\u00A0 ice,\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 washed\u00C2\u00A0 with\u00C2\u00A0 cold\u00C2\u00A0 PBSB.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0 staining\u00C2\u00A0 with\u00C2\u00A0 a\u00C2\u00A0 secondary\u00C2\u00A0Ab\u00C2\u00A0(if\u00C2\u00A0required),\u00C2\u00A0pellets\u00C2\u00A0were\u00C2\u00A0resuspended\u00C2\u00A0in\u00C2\u00A020\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0cold\u00C2\u00A0PBSB\u00C2\u00A0containing\u00C2\u00A00.5\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0D649\u00E2\u0080\u0090anti\u00E2\u0080\u0090 rabbit\u00E2\u0080\u0090IgG,\u00C2\u00A0incubated\u00C2\u00A0for\u00C2\u00A0an\u00C2\u00A0additional\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0on\u00C2\u00A0ice,\u00C2\u00A0then\u00C2\u00A0washed\u00C2\u00A0again\u00C2\u00A0with\u00C2\u00A0cold\u00C2\u00A0PBSB.\u00C2\u00A0\u00C2\u00A0Pellets\u00C2\u00A0were\u00C2\u00A0 then\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0300\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0PBSB\u00C2\u00A0containing\u00C2\u00A00.5\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0propidium\u00C2\u00A0 iodide\u00C2\u00A0 (PI),\u00C2\u00A0 filtered\u00C2\u00A0 through\u00C2\u00A035\u00C2\u00A0 \u00C2\u00B5m\u00C2\u00A0 strainers,\u00C2\u00A0 and\u00C2\u00A0 analyzed\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 FACSCalibur\u00C2\u00A0 flow\u00C2\u00A0 cytometer\u00C2\u00A0 (Becton\u00C2\u00A0 Dickinson,\u00C2\u00A0 San\u00C2\u00A0 Jose,\u00C2\u00A0 CA)\u00C2\u00A0 collecting\u00C2\u00A010,000\u00C2\u00A0events\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0sample.\u00C2\u00A0 \u00C2\u00A0Appropriate\u00C2\u00A0single\u00E2\u0080\u0090stained\u00C2\u00A0controls\u00C2\u00A0were\u00C2\u00A0run\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0cases\u00C2\u00A0 to\u00C2\u00A0set\u00C2\u00A0compensation.\u00C2\u00A0 \u00C2\u00A0Fluorescence\u00C2\u00A0 intensities\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0analysis\u00C2\u00A0with\u00C2\u00A0FlowJo\u00C2\u00A0software,\u00C2\u00A0 and\u00C2\u00A0 triplicates\u00C2\u00A0were\u00C2\u00A0averaged\u00C2\u00A0 to\u00C2\u00A0obtain\u00C2\u00A0mean\u00C2\u00A0 fluorescence\u00C2\u00A0 intensities\u00C2\u00A0 (MFIs).\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0each\u00C2\u00A0dose,\u00C2\u00A0 the\u00C2\u00A0 MFI\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0appropriate\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0was\u00C2\u00A0subtracted\u00C2\u00A0 from\u00C2\u00A0each\u00C2\u00A0measured\u00C2\u00A0MFI\u00C2\u00A0value\u00C2\u00A0before\u00C2\u00A0 being\u00C2\u00A0scaled\u00C2\u00A0to\u00C2\u00A0obtain\u00C2\u00A0relative\u00C2\u00A0MFIs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 5.3.6\u00C2\u00A0 Annexin\u00C2\u00ADV/PI\u00C2\u00A0apoptosis\u00C2\u00A0assay\u00C2\u00A0and\u00C2\u00A0measurement\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 levels\u00C2\u00A0within\u00C2\u00A0 apoptotic\u00C2\u00A0subpopulations\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0apoptosis\u00C2\u00A0assay\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A04.3.8\u00C2\u00A0using\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0 105\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 500\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0medium\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A0 10\u00C2\u00A0 \u00C2\u00B5g\u00C2\u00A0 Rtx\u00C2\u00A0 dose,\u00C2\u00A0 and\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 co\u00E2\u0080\u0090stained\u00C2\u00A0with\u00C2\u00A0A647\u00E2\u0080\u0090 CD120a\u00C2\u00A0 (2\u00C2\u00A0 \u00CE\u00BCL\u00C2\u00A0 /\u00C2\u00A0 sample)\u00C2\u00A0during\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0 staining.\u00C2\u00A0 \u00C2\u00A0Quadrants\u00C2\u00A0were\u00C2\u00A0 set\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0102\u00C2\u00A0\u00C2\u00A0 plots\u00C2\u00A0of\u00C2\u00A0PI\u00C2\u00A0 versus\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0 fluorescence\u00C2\u00A0 intensity,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0quadrants\u00C2\u00A0were\u00C2\u00A0 set\u00C2\u00A0as\u00C2\u00A0 separate\u00C2\u00A0 gates\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0measure\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0within\u00C2\u00A0the\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0(PI\u00E2\u0080\u0093/Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC+),\u00C2\u00A0 necrotic\u00C2\u00A0 (PI+/Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC+),\u00C2\u00A0 and\u00C2\u00A0 viable\u00C2\u00A0 (PI\u00E2\u0080\u0093/Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00E2\u0080\u0093)\u00C2\u00A0 populations.\u00C2\u00A0 \u00C2\u00A0 Appropriate\u00C2\u00A0 single\u00E2\u0080\u0090 stained\u00C2\u00A0and\u00C2\u00A0double\u00E2\u0080\u0090stained\u00C2\u00A0positive\u00C2\u00A0controls\u00C2\u00A0(cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A04.0\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0camptothecin\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC\u00C2\u00A0for\u00C2\u00A04\u00C2\u00A0 h)\u00C2\u00A0were\u00C2\u00A0run\u00C2\u00A0to\u00C2\u00A0set\u00C2\u00A0compensation,\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090labeled\u00C2\u00A0IgG2a\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0set\u00C2\u00A0the\u00C2\u00A0FL4\u00C2\u00A0 detector\u00C2\u00A0voltage\u00C2\u00A0(for\u00C2\u00A0A647\u00C2\u00A0emission\u00C2\u00A0detection)\u00C2\u00A0such\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0negative\u00C2\u00A0signal\u00C2\u00A0occurred\u00C2\u00A0within\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0 decade.\u00C2\u00A0 \u00C2\u00A0Analysis\u00C2\u00A0of\u00C2\u00A0percentages\u00C2\u00A0within\u00C2\u00A0each\u00C2\u00A0population\u00C2\u00A0 and\u00C2\u00A0mean\u00C2\u00A0 fluorescence\u00C2\u00A0 intensities\u00C2\u00A0were\u00C2\u00A0 performed\u00C2\u00A0using\u00C2\u00A0FlowJo\u00C2\u00A0software.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 5.3.7\u00C2\u00A0 Confocal\u00C2\u00A0laser\u00C2\u00ADscanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0procedure\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A04.3.5,\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 exception\u00C2\u00A0of\u00C2\u00A0staining\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0microscope\u00C2\u00A0used.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0staining\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0using\u00C2\u00A0FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK,\u00C2\u00A0the\u00C2\u00A0 same\u00C2\u00A0procedure\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A05.3.4\u00C2\u00A0was\u00C2\u00A0used;\u00C2\u00A0 for\u00C2\u00A0CD120a\u00C2\u00A0staining\u00C2\u00A0 (either\u00C2\u00A0on\u00C2\u00A0 its\u00C2\u00A0own\u00C2\u00A0or\u00C2\u00A0 after\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 staining),\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0 20\u00C2\u00A0 \u00C2\u00B5L\u00C2\u00A0 cold\u00C2\u00A0 PBSB\u00C2\u00A0 containing\u00C2\u00A0 an\u00C2\u00A0 appropriate\u00C2\u00A0 volume\u00C2\u00A0(determined\u00C2\u00A0by\u00C2\u00A0titration\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0Section\u00C2\u00A05.3.6)\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0and\u00C2\u00A0 incubated\u00C2\u00A0on\u00C2\u00A0 ice\u00C2\u00A0for\u00C2\u00A030\u00C2\u00A0min,\u00C2\u00A0 then\u00C2\u00A05\u00C2\u00A0\u00C3\u0097\u00C2\u00A0104\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0suspended\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0 total\u00C2\u00A0of\u00C2\u00A050\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0cold\u00C2\u00A0serum\u00E2\u0080\u0090free\u00C2\u00A0medium\u00C2\u00A0and\u00C2\u00A0allowed\u00C2\u00A0 to\u00C2\u00A0 adhere\u00C2\u00A0on\u00C2\u00A0each\u00C2\u00A08\u00C2\u00A0mm\u00C2\u00A0poly\u00E2\u0080\u0090L\u00E2\u0080\u0090lysine\u00C2\u00A0glass\u00C2\u00A0cover\u00C2\u00A0slip\u00C2\u00A0for\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0on\u00C2\u00A0ice\u00C2\u00A0before\u00C2\u00A0washing\u00C2\u00A0the\u00C2\u00A0cover\u00C2\u00A0slips\u00C2\u00A0with\u00C2\u00A0 HBS\u00C2\u00A0and\u00C2\u00A0fixing\u00C2\u00A0with\u00C2\u00A0paraformaldehyde\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A04.3.5.\u00C2\u00A0\u00C2\u00A0Sample\u00C2\u00A0preparation\u00C2\u00A0proceeded\u00C2\u00A0 as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A04.3.5.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0 were\u00C2\u00A0 imaged\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0 confocal\u00C2\u00A0 Eclipse\u00C2\u00A0 TE2000\u00E2\u0080\u0090E\u00C2\u00A0 microscope\u00C2\u00A0 (Nikon\u00C2\u00A0 Instruments,\u00C2\u00A0 Melville\u00C2\u00A0NY,\u00C2\u00A0USA)\u00C2\u00A0controlled\u00C2\u00A0with\u00C2\u00A0EZ\u00E2\u0080\u0090C1\u00C2\u00A0software.\u00C2\u00A0 \u00C2\u00A0Acquired\u00C2\u00A0 images\u00C2\u00A0were\u00C2\u00A0converted\u00C2\u00A0to\u00C2\u00A0TIFF\u00C2\u00A0format\u00C2\u00A0 for\u00C2\u00A0presentation.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0103\u00C2\u00A0\u00C2\u00A0 5.3.8\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADMS\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 prepare\u00C2\u00A0 Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0 Rtx\u00C2\u00A0 was\u00C2\u00A0 biotinylated\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0 nickel\u00C2\u00A0 immobilized\u00C2\u00A0 metal\u00C2\u00A0 affinity\u00C2\u00A0 chromatography\u00C2\u00A0 (NIMAC)\u00C2\u00A0method\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A03.3.6\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A010\u00E2\u0080\u0090fold\u00C2\u00A0molar\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0NHS\u00E2\u0080\u0090 PEG\u00E2\u0080\u0090biotin\u00C2\u00A0 with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0 Rtx,\u00C2\u00A0 yielding\u00C2\u00A0 between\u00C2\u00A0 1\u00C2\u00A0 and\u00C2\u00A0 2\u00C2\u00A0 biotin\u00C2\u00A0 groups\u00C2\u00A0 per\u00C2\u00A0 Rtx\u00C2\u00A0 molecule.\u00C2\u00A0\u00C2\u00A0 Streptavidin\u00E2\u0080\u0090MS\u00C2\u00A0were\u00C2\u00A0exchanged\u00C2\u00A0into\u00C2\u00A0HBS\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0PD\u00E2\u0080\u009010\u00C2\u00A0column\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0remove\u00C2\u00A0surfactants\u00C2\u00A0and\u00C2\u00A0 EDTA.\u00C2\u00A0\u00C2\u00A0Based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0certificate\u00C2\u00A0of\u00C2\u00A0analysis\u00C2\u00A0provided\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0manufacturer,\u00C2\u00A0a\u00C2\u00A0fivefold\u00C2\u00A0molar\u00C2\u00A0excess\u00C2\u00A0of\u00C2\u00A0 Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0streptavidin\u00C2\u00A0present\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0streptavidin\u00E2\u0080\u0090MS\u00C2\u00A0suspension\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0produce\u00C2\u00A0Rtx\u00E2\u0080\u0090MS.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0suspension\u00C2\u00A0was\u00C2\u00A0 incubated\u00C2\u00A0with\u00C2\u00A0stirring\u00C2\u00A0at\u00C2\u00A0room\u00C2\u00A0 temperature\u00C2\u00A0 for\u00C2\u00A030\u00C2\u00A0min,\u00C2\u00A0 then\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0oC\u00C2\u00A0for\u00C2\u00A012\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0Unbound\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0was\u00C2\u00A0purified\u00C2\u00A0from\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0using\u00C2\u00A0Sepharose\u00C2\u00A0CL\u00E2\u0080\u00904B\u00C2\u00A0columns\u00C2\u00A0 pre\u00E2\u0080\u0090equilibrated\u00C2\u00A0with\u00C2\u00A0HBS.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 5.4\u00C2\u00A0 Results\u00C2\u00A0 5.4.1\u00C2\u00A0 Caspase\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00ADinduced\u00C2\u00A0apoptosis\u00C2\u00A0 To\u00C2\u00A0 determine\u00C2\u00A0 whether\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 proceeds\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 caspase\u00E2\u0080\u0090dependent\u00C2\u00A0 mechanism,\u00C2\u00A0 the\u00C2\u00A0caspase\u00E2\u0080\u00908\u00E2\u0080\u0090specific\u00C2\u00A0peptide\u00C2\u00A0 reagent\u00C2\u00A0FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK\u00C2\u00A0was\u00C2\u00A0employed\u00C2\u00A0 (Section\u00C2\u00A05.3.3).\u00C2\u00A0\u00C2\u00A0 Time\u00E2\u0080\u0090course\u00C2\u00A0 experiments\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 the\u00C2\u00A0 activation\u00C2\u00A0 state\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 caspase\u00C2\u00A0 in\u00C2\u00A0 treated\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0points\u00C2\u00A0after\u00C2\u00A0treatment.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.1A,\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0 activated\u00C2\u00A0 in\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0or\u00C2\u00A0Rtx,\u00C2\u00A0but\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0activated\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(165).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0positive\u00C2\u00A0 cells\u00C2\u00A0 increased\u00C2\u00A0until\u00C2\u00A030\u00C2\u00A0h\u00C2\u00A0 after\u00C2\u00A0 treatment,\u00C2\u00A0 after\u00C2\u00A0 which\u00C2\u00A0point\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0began\u00C2\u00A0to\u00C2\u00A0drop.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A030\u00C2\u00A0h\u00C2\u00A0time\u00C2\u00A0point,\u00C2\u00A083\u00C2\u00A0%\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0found\u00C2\u00A0to\u00C2\u00A0 be\u00C2\u00A0caspase\u00E2\u0080\u00908\u00E2\u0080\u0090positive\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A019\u00C2\u00A0%\u00C2\u00A0and\u00C2\u00A018\u00C2\u00A0%\u00C2\u00A0for\u00C2\u00A0HBS\u00C2\u00A0and\u00C2\u00A0Rtx,\u00C2\u00A0respectively\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.005\u00C2\u00A0between\u00C2\u00A0 both\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0and\u00C2\u00A0HBS/Rtx).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0correlate\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0apoptosis\u00C2\u00A0data\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.5B,\u00C2\u00A0which\u00C2\u00A0 shows\u00C2\u00A0a\u00C2\u00A0similar\u00C2\u00A0time\u00C2\u00A0course\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0one\u00C2\u00A0observed\u00C2\u00A0here.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0the\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00E2\u0080\u0090\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0104\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 5.1\u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 is\u00C2\u00A0 dependent\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 activation\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908.\u00C2\u00A0 (A)\u00C2\u00A0Levels\u00C2\u00A0of\u00C2\u00A0cleaved\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0are\u00C2\u00A0shown\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0time\u00C2\u00A0 in\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0different\u00C2\u00A0treatments.\u00C2\u00A0\u00C2\u00A0(B)\u00C2\u00A0Expression\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0 each\u00C2\u00A0 activated\u00C2\u00A0 caspase\u00C2\u00A0 are\u00C2\u00A0 shown\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 various\u00C2\u00A0 treatments\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 measured\u00C2\u00A0 fluorescence\u00C2\u00A0 values\u00C2\u00A0 after\u00C2\u00A0 subtracting\u00C2\u00A0 blanks\u00C2\u00A0 and\u00C2\u00A0 normalizing\u00C2\u00A0 to\u00C2\u00A0 HBS\u00E2\u0080\u0090treated\u00C2\u00A0 samples.\u00C2\u00A0\u00C2\u00A0 By\u00C2\u00A0treating\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0peptide\u00C2\u00A0inhibitor\u00C2\u00A0specific\u00C2\u00A0for\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0 (Casp\u00E2\u0080\u00908\u00C2\u00A0 inh.)\u00C2\u00A0 for\u00C2\u00A0 4\u00C2\u00A0 h\u00C2\u00A0 and\u00C2\u00A0 then\u00C2\u00A0 treating\u00C2\u00A0 the\u00C2\u00A0 cells\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00E2\u0080\u0090 LNP(150),\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0nearly\u00C2\u00A0all\u00C2\u00A0activated\u00C2\u00A0caspases\u00C2\u00A0were\u00C2\u00A0reduced\u00C2\u00A0 to\u00C2\u00A0 levels\u00C2\u00A0 similar\u00C2\u00A0 to\u00C2\u00A0 blanks.\u00C2\u00A0 (C)\u00C2\u00A0 Levels\u00C2\u00A0 of\u00C2\u00A0 cleaved\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 measured\u00C2\u00A0after\u00C2\u00A0treating\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0 0 25 50 75 100 0 20 40 60 % \u00C2\u00A0Ca sp as e\u00E2\u0080\u00908 \u00C2\u00A0po si ti ve \u00C2\u00A0ce lls Time\u00C2\u00A0/\u00C2\u00A0h 0 2 165 Valence: A \u00E2\u0080\u00900.5 0 0.5 1 1.5 2 2.5 3 1 2 3/7 6 8 9 Re la ti ve \u00C2\u00A0ex pr es si on Caspase 0 2 150 150\u00C2\u00A0+Casp\u00E2\u0080\u00908\u00C2\u00A0inh. Valence: B 0 20 40 60 80 0 2 32 75 113 158 % \u00C2\u00A0ca sp as e\u00E2\u0080\u00908 \u00C2\u00A0po si ti ve \u00C2\u00A0ce lls Valence C \u00E2\u0080\u00A2\u00C2\u00A0105\u00C2\u00A0\u00C2\u00A0 positive\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A05.1A\u00C2\u00A0 is\u00C2\u00A0roughly\u00C2\u00A0equal\u00C2\u00A0 to\u00C2\u00A0the\u00C2\u00A0sum\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0percentages\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0and\u00C2\u00A0 necrotic\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.6A.\u00C2\u00A0 The\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0apoptosis\u00C2\u00A0occurred\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0extrinsic\u00C2\u00A0apoptosis\u00C2\u00A0 pathway,\u00C2\u00A0which\u00C2\u00A0 involves\u00C2\u00A0 formation\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0DISC.\u00C2\u00A0 \u00C2\u00A0Next,\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0 the\u00C2\u00A0pattern\u00C2\u00A0of\u00C2\u00A0 activation\u00C2\u00A0of\u00C2\u00A0 other\u00C2\u00A0 caspases,\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 activated\u00C2\u00A0 caspases\u00C2\u00A0 1,\u00C2\u00A0 2,\u00C2\u00A0 3,\u00C2\u00A0 6,\u00C2\u00A0 7,\u00C2\u00A0 8,\u00C2\u00A0 and\u00C2\u00A0 9\u00C2\u00A0were\u00C2\u00A0 profiled\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 after\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0are\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A05.1B\u00C2\u00A0and\u00C2\u00A0they\u00C2\u00A0 indicate\u00C2\u00A0that\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 similar\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 caspase\u00C2\u00A0 activation\u00C2\u00A0 as\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 HBS\u00C2\u00A0 (no\u00C2\u00A0 drug),\u00C2\u00A0 but\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(150)\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0an\u00C2\u00A0approximately\u00C2\u00A0two\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0detected\u00C2\u00A0cleaved\u00C2\u00A0 caspase\u00C2\u00A0levels\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0caspases\u00C2\u00A0tested\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.005\u00C2\u00A0for\u00C2\u00A0every\u00C2\u00A0caspase\u00C2\u00A0between\u00C2\u00A0valences\u00C2\u00A00\u00C2\u00A0or\u00C2\u00A02\u00C2\u00A0and\u00C2\u00A0150).\u00C2\u00A0\u00C2\u00A0 A\u00C2\u00A0positive\u00C2\u00A0control\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0by\u00C2\u00A0treating\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A04.0\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0camptothecin\u00C2\u00A0for\u00C2\u00A04\u00C2\u00A0h,\u00C2\u00A0and\u00C2\u00A0these\u00C2\u00A0 samples\u00C2\u00A0showed\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0caspases\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0HBS,\u00C2\u00A0particularly\u00C2\u00A0the\u00C2\u00A0reading\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 caspase\u00C2\u00A03/7\u00C2\u00A0substrate,\u00C2\u00A0which\u00C2\u00A0was\u00C2\u00A0at\u00C2\u00A0 least\u00C2\u00A010\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0than\u00C2\u00A0all\u00C2\u00A0other\u00C2\u00A0caspases\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0expected\u00C2\u00A0since\u00C2\u00A0camptothecin\u00C2\u00A0 inhibits\u00C2\u00A0 topoisomerase\u00C2\u00A0 I,\u00C2\u00A0causing\u00C2\u00A0DNA\u00C2\u00A0damage\u00C2\u00A0 that\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 apoptosis\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 intrinsic\u00C2\u00A0pathway,\u00C2\u00A0which\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0 involve\u00C2\u00A0caspase\u00E2\u0080\u00908\u00E2\u0080\u0090dependent\u00C2\u00A0 initiation82,\u00C2\u00A0 187.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0is\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0caspases,\u00C2\u00A0cells\u00C2\u00A0 were\u00C2\u00A0 treated\u00C2\u00A0 for\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0with\u00C2\u00A0 Z\u00E2\u0080\u0090IETD\u00E2\u0080\u0090FMK,\u00C2\u00A0 a\u00C2\u00A0 cell\u00E2\u0080\u0090permeable,\u00C2\u00A0 irreversible\u00C2\u00A0peptide\u00C2\u00A0 inhibitor\u00C2\u00A0 specific\u00C2\u00A0 to\u00C2\u00A0 caspase\u00E2\u0080\u00908,\u00C2\u00A0followed\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(150).\u00C2\u00A0\u00C2\u00A0Figure\u00C2\u00A05.1B\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0inhibition\u00C2\u00A0 of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0dramatic\u00C2\u00A0reduction\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0caspases\u00C2\u00A0down\u00C2\u00A0to\u00C2\u00A0levels\u00C2\u00A0that\u00C2\u00A0 were\u00C2\u00A0 roughly\u00C2\u00A0 one\u00E2\u0080\u0090tenth\u00C2\u00A0 of\u00C2\u00A0 those\u00C2\u00A0 in\u00C2\u00A0 HBS\u00E2\u0080\u0090treated\u00C2\u00A0 cells\u00C2\u00A0 (caspases\u00C2\u00A0 3/7\u00C2\u00A0 and\u00C2\u00A0 6)\u00C2\u00A0 or\u00C2\u00A0 similar\u00C2\u00A0 to\u00C2\u00A0 blanks\u00C2\u00A0 (caspases\u00C2\u00A01,\u00C2\u00A02,\u00C2\u00A0and\u00C2\u00A09).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0caspase\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0 level\u00C2\u00A0of\u00C2\u00A0activation\u00C2\u00A0was\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0 itself,\u00C2\u00A0which\u00C2\u00A0 was\u00C2\u00A00.33\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0HBS\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.005\u00C2\u00A0for\u00C2\u00A0every\u00C2\u00A0caspase\u00C2\u00A0between\u00C2\u00A0valences\u00C2\u00A00\u00C2\u00A0or\u00C2\u00A02\u00C2\u00A0and\u00C2\u00A0150(Casp\u00E2\u0080\u00908\u00C2\u00A0 inh.)).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Ramos\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0equivalent\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0Z\u00E2\u0080\u0090FA\u00E2\u0080\u0090FMK,\u00C2\u00A0a\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0 peptide\u00C2\u00A0that\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0inhibit\u00C2\u00A0caspases,\u00C2\u00A0showed\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0cleaved\u00C2\u00A0caspases\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090 \u00E2\u0080\u00A2\u00C2\u00A0106\u00C2\u00A0\u00C2\u00A0 LNP(150)\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0 similar\u00C2\u00A0 to\u00C2\u00A0 those\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(150)\u00C2\u00A0 alone\u00C2\u00A0 (data\u00C2\u00A0 not\u00C2\u00A0 shown).\u00C2\u00A0\u00C2\u00A0 Apoptosis\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0significantly\u00C2\u00A0 reduced\u00C2\u00A0by\u00C2\u00A0 inhibiting\u00C2\u00A0caspase\u00E2\u0080\u00908;\u00C2\u00A0 treatment\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0Z\u00E2\u0080\u0090 IETD\u00E2\u0080\u0090FMK\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A011%\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0early\u00C2\u00A0apoptosis,\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A061%\u00C2\u00A0for\u00C2\u00A0 cells\u00C2\u00A0treated\u00C2\u00A0only\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.001),\u00C2\u00A0as\u00C2\u00A0measured\u00C2\u00A0using\u00C2\u00A0 flow\u00C2\u00A0cytometry\u00C2\u00A0 (Section\u00C2\u00A05.3.6;\u00C2\u00A0 data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0therefore\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0 caspase\u00E2\u0080\u0090dependent,\u00C2\u00A0and\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0initiating\u00C2\u00A0caspase.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Since\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0as\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.6,\u00C2\u00A0one\u00C2\u00A0would\u00C2\u00A0 also\u00C2\u00A0expect\u00C2\u00A0that\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0would\u00C2\u00A0show\u00C2\u00A0a\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0relationship.\u00C2\u00A0\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0therefore\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0levels\u00C2\u00A0were\u00C2\u00A0measured.\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A0 5.1C\u00C2\u00A0 confirms\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 activated\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 are\u00C2\u00A0 valence\u00E2\u0080\u0090dependent,\u00C2\u00A0with\u00C2\u00A0 higher\u00C2\u00A0 valences\u00C2\u00A0producing\u00C2\u00A0elevated\u00C2\u00A0 levels\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0 lower\u00C2\u00A0valences.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0or\u00C2\u00A0 Rtx\u00C2\u00A0 showed\u00C2\u00A0 16\u00C2\u00A0 %\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00E2\u0080\u0090positive\u00C2\u00A0 cells,\u00C2\u00A0 while\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 65\u00C2\u00A0 %\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00E2\u0080\u0090positive\u00C2\u00A0cells\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.001).\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0data,\u00C2\u00A0when\u00C2\u00A0taken\u00C2\u00A0together,\u00C2\u00A0reveal\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 induces\u00C2\u00A0 apoptosis\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0via\u00C2\u00A0the\u00C2\u00A0caspase\u00E2\u0080\u00908\u00E2\u0080\u0090dependent\u00C2\u00A0extrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathway.\u00C2\u00A0 5.4.2\u00C2\u00A0 Dependence\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 superfamily\u00C2\u00A0 member\u00C2\u00A0 expression\u00C2\u00A0on\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 As\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A05.2,\u00C2\u00A0the\u00C2\u00A0extrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathway\u00C2\u00A0involves\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DISC,\u00C2\u00A0 which\u00C2\u00A0normally\u00C2\u00A0contains\u00C2\u00A0specific\u00C2\u00A0members\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0TNFR\u00C2\u00A0superfamily.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0which\u00C2\u00A0 receptor(s),\u00C2\u00A0 if\u00C2\u00A0 any,\u00C2\u00A0 initiate\u00C2\u00A0 the\u00C2\u00A0 extrinsic\u00C2\u00A0 apoptosis\u00C2\u00A0 pathway\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 the\u00C2\u00A0 expression\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 death\u00C2\u00A0 domain\u00E2\u0080\u0090containing\u00C2\u00A0 death\u00C2\u00A0 receptors\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0 at\u00C2\u00A0 different\u00C2\u00A0 times\u00C2\u00A0 following\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0Rtx,\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0summarized\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.2\u00C2\u00A0show\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0 for\u00C2\u00A0 three\u00C2\u00A0key\u00C2\u00A0death\u00C2\u00A0 receptors:\u00C2\u00A0CD95\u00C2\u00A0 (Fas\u00C2\u00A0 receptor),\u00C2\u00A0death\u00C2\u00A0 receptor\u00C2\u00A04\u00C2\u00A0 (DR4),\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0 (TNFR\u00E2\u0080\u0090I).\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A05.2A\u00C2\u00A0 summarizes\u00C2\u00A0 the\u00C2\u00A0 fluorescence\u00C2\u00A0 intensities\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0Ramos\u00C2\u00A0 cell\u00C2\u00A0population\u00C2\u00A0 after\u00C2\u00A0staining\u00C2\u00A0with\u00C2\u00A0 fluorescently\u00C2\u00A0 labeled\u00C2\u00A0primary\u00C2\u00A0Abs\u00C2\u00A0against\u00C2\u00A0 the\u00C2\u00A0shown\u00C2\u00A0antigens.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0cell\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0107\u00C2\u00A0\u00C2\u00A0 population,\u00C2\u00A0DR4\u00C2\u00A0showed\u00C2\u00A0very\u00C2\u00A0low\u00C2\u00A0staining\u00C2\u00A0over\u00C2\u00A0all\u00C2\u00A0treatments,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0labeling\u00C2\u00A0of\u00C2\u00A0CD95\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0low\u00C2\u00A0 and\u00C2\u00A0 constant\u00C2\u00A0 except\u00C2\u00A0 at\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(165),\u00C2\u00A0where\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0MFI\u00C2\u00A0was\u00C2\u00A0 5.7\u00C2\u00A0 compared\u00C2\u00A0to\u00C2\u00A00.96\u00C2\u00A0for\u00C2\u00A0HBS\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0time\u00C2\u00A0point\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0most\u00C2\u00A0significant\u00C2\u00A0change\u00C2\u00A0was\u00C2\u00A0with\u00C2\u00A0 CD120a,\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0relative\u00C2\u00A0MFI\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0was\u00C2\u00A09.2\u00C2\u00A0after\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A021\u00C2\u00A0 after\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.005\u00C2\u00A0 in\u00C2\u00A0both\u00C2\u00A0cases\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0HBS\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 time\u00C2\u00A0point).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 total\u00C2\u00A0 staining\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD95,\u00C2\u00A0DR4,\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0were\u00C2\u00A0similar\u00C2\u00A0in\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.2\u00C2\u00A0\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0is\u00C2\u00A0dramatically\u00C2\u00A0elevated\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Using\u00C2\u00A0 flow\u00C2\u00A0 cytometry,\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 expression\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 indicated\u00C2\u00A0 death\u00C2\u00A0 receptors\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0 cell\u00C2\u00A0 population\u00C2\u00A0 of\u00C2\u00A0 treated\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 (A)\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 of\u00C2\u00A0 Ramos\u00C2\u00A0 (B)\u00C2\u00A0 and\u00C2\u00A0 Z138\u00C2\u00A0 (C)\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0 Plasma\u00C2\u00A0 membrane\u00C2\u00A0expression\u00C2\u00A0was\u00C2\u00A0obtained\u00C2\u00A0by\u00C2\u00A0measuring\u00C2\u00A0fluorescence\u00C2\u00A0intensities\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0PI\u00E2\u0080\u0090\u00C2\u00A0population.\u00C2\u00A0\u00C2\u00A0MFI,\u00C2\u00A0mean\u00C2\u00A0fluorescence\u00C2\u00A0 intensity.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0 4 8 12 16 20 24 0 4 24 0 4 24 0 4 24Re la ti ve \u00C2\u00A0M FI \u00C2\u00A0(to ta l\u00C2\u00A0ce ll\u00C2\u00A0p op ul at io n) Time\u00C2\u00A0/\u00C2\u00A0h CD95 Ramos DR4 CD120a A \u00E2\u0080\u00901 0 1 2 3 4 5 6 7 8 0 4 24 0 4 24 0 4 24 Re la ti ve \u00C2\u00A0M FI \u00C2\u00A0(P I\u00E2\u0080\u0093 po pu la ti on ) Time\u00C2\u00A0/\u00C2\u00A0h 0 2 165 Valence: Ramos CD95 DR4 CD120a B 0 1 2 3 4 5 6 7 8 0 4 24 0 4 24 0 4 24 Re la ti ve \u00C2\u00A0M FI \u00C2\u00A0(P I\u00E2\u0080\u0093 po pu la ti on ) Time\u00C2\u00A0/\u00C2\u00A0h 0 2 152 Valence: Z138 CD95 DR4 CD120a C \u00E2\u0080\u00A2\u00C2\u00A0108\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A05.2B\u00C2\u00A0&\u00C2\u00A0C\u00C2\u00A0show\u00C2\u00A0the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0each\u00C2\u00A0death\u00C2\u00A0receptor\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0 same\u00C2\u00A0 treatments,\u00C2\u00A0obtained\u00C2\u00A0by\u00C2\u00A0gating\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0 that\u00C2\u00A0exclude\u00C2\u00A0PI.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 same\u00C2\u00A0overall\u00C2\u00A0 trends\u00C2\u00A0were\u00C2\u00A0 observed\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0PI\u00E2\u0080\u0093\u00C2\u00A0populations\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0total\u00C2\u00A0cell\u00C2\u00A0populations,\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0being\u00C2\u00A0the\u00C2\u00A0 highest\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0the\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0tested.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0general,\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 change\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0start\u00C2\u00A0of\u00C2\u00A0treatment\u00C2\u00A0(0\u00C2\u00A0h)\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0or\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Comparing\u00C2\u00A0Figure\u00C2\u00A05.2A\u00C2\u00A0to\u00C2\u00A0 Figure\u00C2\u00A05.2B,\u00C2\u00A0one\u00C2\u00A0notable\u00C2\u00A0observation\u00C2\u00A0was\u00C2\u00A0that\u00C2\u00A0most\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0detected\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.2A\u00C2\u00A0after\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0 was\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0(relative\u00C2\u00A0MFI\u00C2\u00A0of\u00C2\u00A09.2\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.2A\u00C2\u00A0and\u00C2\u00A06.4\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.2B,\u00C2\u00A0 representing\u00C2\u00A0 70%\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 signal),\u00C2\u00A0while\u00C2\u00A0 the\u00C2\u00A0majority\u00C2\u00A0 after\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0was\u00C2\u00A0 associated\u00C2\u00A0with\u00C2\u00A0 intracellular\u00C2\u00A0 staining\u00C2\u00A0(relative\u00C2\u00A0MFI\u00C2\u00A0of\u00C2\u00A021\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.2A\u00C2\u00A0and\u00C2\u00A04.8\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.2B;\u00C2\u00A023%\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0signal).\u00C2\u00A0\u00C2\u00A0Also,\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0 CD120a\u00C2\u00A0 in\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0 (relative\u00C2\u00A0MFI\u00C2\u00A0of\u00C2\u00A06.4)\u00C2\u00A0were\u00C2\u00A0higher\u00C2\u00A0 than\u00C2\u00A0those\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0(relative\u00C2\u00A0MFI\u00C2\u00A04.8)\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05),\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0showed\u00C2\u00A0highest\u00C2\u00A0levels\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0(0.78\u00C2\u00A0for\u00C2\u00A0DR4\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.005),\u00C2\u00A01.6\u00C2\u00A0for\u00C2\u00A0CD95\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.0005;\u00C2\u00A0both\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0HBS)).\u00C2\u00A0\u00C2\u00A0Observations\u00C2\u00A0in\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0(Figure\u00C2\u00A05.2C)\u00C2\u00A0paralleled\u00C2\u00A0those\u00C2\u00A0in\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0exception\u00C2\u00A0 that\u00C2\u00A0the\u00C2\u00A0average\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0at\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(152)\u00C2\u00A0 were\u00C2\u00A0higher\u00C2\u00A0than\u00C2\u00A0those\u00C2\u00A0after\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0(5.5\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A03.6),\u00C2\u00A0albeit\u00C2\u00A0the\u00C2\u00A0difference\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0significant\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A0 0.05).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Levels\u00C2\u00A0of\u00C2\u00A0DR3,\u00C2\u00A0DR5,\u00C2\u00A0 and\u00C2\u00A0DR6\u00C2\u00A0 in\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 also\u00C2\u00A0 tested\u00C2\u00A0using\u00C2\u00A0 fluorescently\u00C2\u00A0 labeled\u00C2\u00A0 secondary\u00C2\u00A0Ab\u00C2\u00A0detection\u00C2\u00A0after\u00C2\u00A0staining\u00C2\u00A0with\u00C2\u00A0primary\u00C2\u00A0Abs\u00C2\u00A0(not\u00C2\u00A0shown);\u00C2\u00A0at\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0 DR3\u00C2\u00A0and\u00C2\u00A0DR6\u00C2\u00A0showed\u00C2\u00A0similar\u00C2\u00A0expression\u00C2\u00A0as\u00C2\u00A0DR4\u00C2\u00A0(total\u00C2\u00A0and\u00C2\u00A0PI\u00E2\u0080\u0093\u00C2\u00A0populations),\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0DR5\u00C2\u00A0 lied\u00C2\u00A0 between\u00C2\u00A0 those\u00C2\u00A0 of\u00C2\u00A0DR3/DR4/DR6\u00C2\u00A0 and\u00C2\u00A0 CD95,\u00C2\u00A0 i.e.\u00C2\u00A0 \u00C2\u00A0DR3\u00C2\u00A0 \u00E2\u0089\u0088\u00C2\u00A0DR4\u00C2\u00A0 \u00E2\u0089\u0088\u00C2\u00A0DR6\u00C2\u00A0 <\u00C2\u00A0DR5\u00C2\u00A0 <\u00C2\u00A0 CD95\u00C2\u00A0 <\u00C2\u00A0 CD120a.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0data\u00C2\u00A0 indicate\u00C2\u00A0 a\u00C2\u00A0 global\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0death\u00C2\u00A0 receptors\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 following\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0Rtx,\u00C2\u00A0with\u00C2\u00A0CD120a\u00C2\u00A0undergoing\u00C2\u00A0by\u00C2\u00A0far\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0significant\u00C2\u00A0 upregulation.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0indicates\u00C2\u00A0that\u00C2\u00A0CD120a\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0key\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0109\u00C2\u00A0\u00C2\u00A0 5.4.3\u00C2\u00A0 CD120a\u00C2\u00A0 is\u00C2\u00A0 equally\u00C2\u00A0 upregulated\u00C2\u00A0 in\u00C2\u00A0 viable\u00C2\u00A0 and\u00C2\u00A0 early\u00C2\u00A0 apoptotic\u00C2\u00A0 cells\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 With\u00C2\u00A0CD120a\u00C2\u00A0being\u00C2\u00A0the\u00C2\u00A0member\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0TNFR\u00C2\u00A0superfamily\u00C2\u00A0that\u00C2\u00A0 is\u00C2\u00A0most\u00C2\u00A0upregulated\u00C2\u00A0 in\u00C2\u00A0cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0we\u00C2\u00A0further\u00C2\u00A0 investigated\u00C2\u00A0which\u00C2\u00A0subpopulations\u00C2\u00A0(viable,\u00C2\u00A0apoptotic,\u00C2\u00A0or\u00C2\u00A0necrotic)\u00C2\u00A0 expressed\u00C2\u00A0elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A05.3.6,\u00C2\u00A0analysis\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0using\u00C2\u00A0 flow\u00C2\u00A0cytometry\u00C2\u00A0on\u00C2\u00A0HBS\u00E2\u0080\u0090,\u00C2\u00A0Rtx\u00E2\u0080\u0090,\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0triple\u00E2\u0080\u0090stained\u00C2\u00A0with\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC,\u00C2\u00A0 PI,\u00C2\u00A0and\u00C2\u00A0Alexa\u00C2\u00A0Fluor\u00C2\u00A0647\u00C2\u00A0 labeled\u00C2\u00A0anti\u00E2\u0080\u0090CD120a\u00C2\u00A0Ab\u00C2\u00A0(A647\u00E2\u0080\u0090CD120a).\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 leftmost\u00C2\u00A0plots\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A05.3,\u00C2\u00A0cell\u00C2\u00A0populations\u00C2\u00A0were\u00C2\u00A0gated\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0whether\u00C2\u00A0they\u00C2\u00A0were\u00C2\u00A0viable\u00C2\u00A0(V),\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0 (A),\u00C2\u00A0 or\u00C2\u00A0 necrotic\u00C2\u00A0 (N),\u00C2\u00A0 and\u00C2\u00A0 within\u00C2\u00A0 each\u00C2\u00A0 subpopulation,\u00C2\u00A0 the\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 is\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 corresponding\u00C2\u00A0dot\u00C2\u00A0plots.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.3\u00C2\u00A0\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0are\u00C2\u00A0elevated\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0apoptotic\u00C2\u00A0fractions\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0(A)\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0(B)\u00C2\u00A0for\u00C2\u00A08\u00C2\u00A0h,\u00C2\u00A0then\u00C2\u00A0triple\u00E2\u0080\u0090stained\u00C2\u00A0with\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC,\u00C2\u00A0PI,\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090 CD120a.\u00C2\u00A0\u00C2\u00A0Gates\u00C2\u00A0were\u00C2\u00A0set\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0quadrant\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0plot\u00C2\u00A0of\u00C2\u00A0PI\u00C2\u00A0versus\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0fluorescence\u00C2\u00A0intensities\u00C2\u00A0(shown\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0left\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0figure)\u00C2\u00A0using\u00C2\u00A0HBS\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0(not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0expression\u00C2\u00A0within\u00C2\u00A0each\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0three\u00C2\u00A0 relevant\u00C2\u00A0quadrants\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0(V,\u00C2\u00A0viable;\u00C2\u00A0A,\u00C2\u00A0early\u00C2\u00A0apoptotic;\u00C2\u00A0N,\u00C2\u00A0necrotic).\u00C2\u00A0\u00C2\u00A0Mean\u00C2\u00A0fluorescence\u00C2\u00A0intensities\u00C2\u00A0and\u00C2\u00A0percentages\u00C2\u00A0of\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0each\u00C2\u00A0population\u00C2\u00A0 are\u00C2\u00A0 indicated\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0dot\u00C2\u00A0plots.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 fluorescence\u00C2\u00A0 intensities\u00C2\u00A0 in\u00C2\u00A0 viable\u00C2\u00A0 and\u00C2\u00A0apoptotic\u00C2\u00A0 cells\u00C2\u00A0 (PI\u00E2\u0080\u0093)\u00C2\u00A0 V\u00C2\u00A0 FSC FI \u00C2\u00A0(A 64 7\u00E2\u0080\u0090C D 12 0a ) FSCFSCFI\u00C2\u00A0(Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC)\u00C2\u00A0 FI \u00C2\u00A0(PI ) V A\u00C2\u00A0 N Rtx A N 2.2\u00C2\u00A0%91.1\u00C2\u00A0% 6.6\u00C2\u00A0% 28.6 30.3 1598 V A\u00C2\u00A0 N Rtx\u00E2\u0080\u0090LNP(165) FI\u00C2\u00A0(Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC)\u00C2\u00A0 FI \u00C2\u00A0(PI ) V\u00C2\u00A0 A N FI \u00C2\u00A0(A 64 7\u00E2\u0080\u0090C D 12 0a ) FSC FSCFSC 40.9\u00C2\u00A0%40.0\u00C2\u00A0% 18.8\u00C2\u00A0% 157 166 1354 A B \u00E2\u0080\u00A2\u00C2\u00A0110\u00C2\u00A0\u00C2\u00A0 represent\u00C2\u00A0 the\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00E2\u0080\u0090bound\u00C2\u00A0 CD120a,\u00C2\u00A0while\u00C2\u00A0 CD120a\u00C2\u00A0 in\u00C2\u00A0 necrotic\u00C2\u00A0 cells\u00C2\u00A0 is\u00C2\u00A0 significantly\u00C2\u00A0 elevated,\u00C2\u00A0 presumably\u00C2\u00A0 from\u00C2\u00A0 intracellular,\u00C2\u00A0 extracellular,\u00C2\u00A0 and\u00C2\u00A0 nonspecific\u00C2\u00A0 staining.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 data\u00C2\u00A0 correlate\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 confocal\u00C2\u00A0 laser\u00E2\u0080\u0090 scanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0images\u00C2\u00A0shown\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0right\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0figure,\u00C2\u00A0where\u00C2\u00A0elevated\u00C2\u00A0staining\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0(red)\u00C2\u00A0is\u00C2\u00A0 visible\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00E2\u0080\u0090treated\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0necrotic\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0visible\u00C2\u00A0in\u00C2\u00A0both\u00C2\u00A0samples.\u00C2\u00A0\u00C2\u00A0FI,\u00C2\u00A0fluorescence\u00C2\u00A0intensity.\u00C2\u00A0\u00C2\u00A0Scale\u00C2\u00A0bars,\u00C2\u00A020\u00C2\u00A0 \u00CE\u00BCm.\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0experiment\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0 low\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0but\u00C2\u00A0higher\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165).\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0both\u00C2\u00A0cases,\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0 and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0fractions\u00C2\u00A0showed\u00C2\u00A0relatively\u00C2\u00A0equal\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0(shown\u00C2\u00A0directly\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0dot\u00C2\u00A0plots).\u00C2\u00A0 \u00C2\u00A0Since\u00C2\u00A0these\u00C2\u00A0fractions\u00C2\u00A0both\u00C2\u00A0constituted\u00C2\u00A0cells\u00C2\u00A0which\u00C2\u00A0excluded\u00C2\u00A0PI,\u00C2\u00A0these\u00C2\u00A0CD120a\u00C2\u00A0 levels\u00C2\u00A0 represent\u00C2\u00A0plasma\u00C2\u00A0membrane\u00E2\u0080\u0090bound\u00C2\u00A0(extracellular)\u00C2\u00A0CD120a.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0mean\u00C2\u00A0fluorescence\u00C2\u00A0intensity\u00C2\u00A0(MFI)\u00C2\u00A0 of\u00C2\u00A0an\u00C2\u00A0A647\u00E2\u0080\u0090labeled\u00C2\u00A0negative\u00C2\u00A0 isotype\u00C2\u00A0control\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0conditions\u00C2\u00A0was\u00C2\u00A014.4\u00C2\u00A0(not\u00C2\u00A0shown),\u00C2\u00A0and\u00C2\u00A0 when\u00C2\u00A0this\u00C2\u00A0value\u00C2\u00A0is\u00C2\u00A0subtracted\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0MFIs\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.3,\u00C2\u00A0the\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0(Figure\u00C2\u00A05.3B)\u00C2\u00A0is\u00C2\u00A010\u00E2\u0080\u0090 fold\u00C2\u00A0 higher\u00C2\u00A0 than\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0 Rtx\u00E2\u0080\u0090treated\u00C2\u00A0 cells\u00C2\u00A0 (Figure\u00C2\u00A0 5.3A).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 data\u00C2\u00A0 for\u00C2\u00A0HBS\u00E2\u0080\u0090treated\u00C2\u00A0 cells\u00C2\u00A0was\u00C2\u00A0 very\u00C2\u00A0 similar\u00C2\u00A0to\u00C2\u00A0that\u00C2\u00A0 for\u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0 is\u00C2\u00A0therefore\u00C2\u00A0not\u00C2\u00A0shown.\u00C2\u00A0 \u00C2\u00A0Regardless\u00C2\u00A0of\u00C2\u00A0treatment,\u00C2\u00A0necrotic\u00C2\u00A0 cells\u00C2\u00A0 showed\u00C2\u00A0much\u00C2\u00A0 higher\u00C2\u00A0 CD120a\u00C2\u00A0 staining,\u00C2\u00A0 possibly\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 an\u00C2\u00A0 internal\u00C2\u00A0 pool\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 and/or\u00C2\u00A0 nonspecific\u00C2\u00A0intracellular\u00C2\u00A0staining.\u00C2\u00A0\u00C2\u00A0 Confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy\u00C2\u00A0 images\u00C2\u00A0 seen\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 right\u00C2\u00A0 of\u00C2\u00A0 Figure\u00C2\u00A0 5.3\u00C2\u00A0 confirm\u00C2\u00A0the\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0data\u00C2\u00A0since\u00C2\u00A0they\u00C2\u00A0show\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0few\u00C2\u00A0necrotic\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0visible\u00C2\u00A0in\u00C2\u00A0 both\u00C2\u00A0cases\u00C2\u00A0with\u00C2\u00A0high\u00C2\u00A0staining\u00C2\u00A0throughout\u00C2\u00A0the\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 involves\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 CD120a\u00C2\u00A0would\u00C2\u00A0subsequently\u00C2\u00A0form\u00C2\u00A0DISCs\u00C2\u00A0with\u00C2\u00A0caspase\u00E2\u0080\u00908,\u00C2\u00A0 leading\u00C2\u00A0to\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0 CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0can\u00C2\u00A0only\u00C2\u00A0be\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0apoptosis\u00C2\u00A0if\u00C2\u00A0it\u00C2\u00A0precedes\u00C2\u00A0apoptosis,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0observation\u00C2\u00A0that\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0are\u00C2\u00A0equal\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0fractions\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090 treated\u00C2\u00A0cells\u00C2\u00A0does\u00C2\u00A0initially\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0occurs\u00C2\u00A0before\u00C2\u00A0the\u00C2\u00A0onset\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0111\u00C2\u00A0\u00C2\u00A0 5.4.4\u00C2\u00A0 Time\u00C2\u00A0course\u00C2\u00A0and\u00C2\u00A0valence\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0 Having\u00C2\u00A0established\u00C2\u00A0 that\u00C2\u00A0CD120a\u00C2\u00A0shows\u00C2\u00A0higher\u00C2\u00A0expression\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00E2\u0080\u0090treated\u00C2\u00A0lymphoma\u00C2\u00A0cells,\u00C2\u00A0we\u00C2\u00A0next\u00C2\u00A0determined\u00C2\u00A0the\u00C2\u00A0time\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0time\u00C2\u00A0 points\u00C2\u00A0 between\u00C2\u00A0 0\u00C2\u00A0 h\u00C2\u00A0 and\u00C2\u00A0 48\u00C2\u00A0 h,\u00C2\u00A0 plasma\u00E2\u0080\u0090membrane\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 were\u00C2\u00A0measured\u00C2\u00A0 using\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0by\u00C2\u00A0measuring\u00C2\u00A0 the\u00C2\u00A0 intensity\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 staining\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0PI\u00E2\u0080\u0093\u00C2\u00A0 cell\u00C2\u00A0population.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0 shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.4A,\u00C2\u00A0these\u00C2\u00A0experiments\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0increased\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0approximately\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0then\u00C2\u00A0decreased\u00C2\u00A0to\u00C2\u00A0relatively\u00C2\u00A0stable\u00C2\u00A0levels\u00C2\u00A0by\u00C2\u00A024\u00C2\u00A0 h\u00C2\u00A0 after\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 contrasted\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 signal\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0 cell\u00C2\u00A0 population,\u00C2\u00A0 which\u00C2\u00A0continued\u00C2\u00A0to\u00C2\u00A0 increase\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A048\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0Figure\u00C2\u00A05.2A\u00C2\u00A0&\u00C2\u00A0B\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0 shown).\u00C2\u00A0 The\u00C2\u00A0maximum\u00C2\u00A0observed\u00C2\u00A0values\u00C2\u00A0were\u00C2\u00A0an\u00C2\u00A018\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0(relative\u00C2\u00A0to\u00C2\u00A0HBS)\u00C2\u00A08\u00C2\u00A0 h\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.001\u00C2\u00A0between\u00C2\u00A0 this\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0 time\u00C2\u00A0points\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 treatment),\u00C2\u00A0and\u00C2\u00A0an\u00C2\u00A08.1\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0expression\u00C2\u00A0at\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(63)\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0 the\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0time\u00C2\u00A0point,\u00C2\u00A0and\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0time\u00C2\u00A0points).\u00C2\u00A0 \u00C2\u00A0By\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0these\u00C2\u00A0relative\u00C2\u00A0 expression\u00C2\u00A0values\u00C2\u00A0decreased\u00C2\u00A0 to\u00C2\u00A05.2\u00C2\u00A0 (valence\u00C2\u00A0158)\u00C2\u00A0and\u00C2\u00A03.8\u00C2\u00A0 (valence\u00C2\u00A063)\u00C2\u00A0with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0HBS\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0time\u00C2\u00A0point.\u00C2\u00A0 \u00C2\u00A0Increases\u00C2\u00A0in\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0 with\u00C2\u00A0Rtx,\u00C2\u00A0which\u00C2\u00A0showed\u00C2\u00A0levels\u00C2\u00A0equal\u00C2\u00A0to\u00C2\u00A0HBS\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0(relative\u00C2\u00A0MFI\u00C2\u00A0=\u00C2\u00A01\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.4A)\u00C2\u00A0at\u00C2\u00A0all\u00C2\u00A0time\u00C2\u00A0 points\u00C2\u00A0 (p\u00C2\u00A0 >\u00C2\u00A0 0.05).\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0 comparison\u00C2\u00A0 of\u00C2\u00A0 Figure\u00C2\u00A0 5.4A\u00C2\u00A0 to\u00C2\u00A0 Figure\u00C2\u00A0 4.5B\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0maximum\u00C2\u00A0 apoptosis\u00C2\u00A0 occurred\u00C2\u00A0 approximately\u00C2\u00A0 16\u00C2\u00A0 h\u00C2\u00A0 after\u00C2\u00A0 peak\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 CD120a\u00C2\u00A0 levels\u00C2\u00A0 (peak\u00C2\u00A0 CD120a\u00C2\u00A0 at\u00C2\u00A0 8h,\u00C2\u00A0 apoptosis\u00C2\u00A0 at\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 post\u00E2\u0080\u0090treatment).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 time\u00C2\u00A0 course\u00C2\u00A0 is\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 notion\u00C2\u00A0 that\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 treatment\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0leading\u00C2\u00A0to\u00C2\u00A0subsequent\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0112\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 5.4\u00C2\u00A0 \u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 levels\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 are\u00C2\u00A0 highest\u00C2\u00A0 at\u00C2\u00A0 8\u00C2\u00A0 h\u00C2\u00A0 post\u00E2\u0080\u0090treatment\u00C2\u00A0 and\u00C2\u00A0 are\u00C2\u00A0 valence\u00E2\u0080\u0090 dependent.\u00C2\u00A0\u00C2\u00A0 (A)\u00C2\u00A0The\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00E2\u0080\u0090bound\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0non\u00E2\u0080\u0090necrotic\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0 with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0causes\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0\u00C2\u00A0Peak\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0approximately\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment.\u00C2\u00A0 \u00C2\u00A0(B\u00C2\u00A0&\u00C2\u00A0C)\u00C2\u00A0The\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0(B)\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0(C)\u00C2\u00A0 cells\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0higher\u00C2\u00A0valence\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0enhanced\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0valence\u00E2\u0080\u0090 dependent\u00C2\u00A0process\u00C2\u00A0mirrors\u00C2\u00A0the\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.6.\u00C2\u00A0\u00C2\u00A0MFI\u00C2\u00A0values\u00C2\u00A0 are\u00C2\u00A0shown\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0histograms.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0 5 10 15 20 0 4 8 16 24 36 48 M FI \u00C2\u00A0(A 64 7\u00E2\u0080\u0090C D 12 0a )\u00C2\u00A0re la ti ve \u00C2\u00A0to \u00C2\u00A0HB S Time\u00C2\u00A0/\u00C2\u00A0h 2 63 158 Valence: A Valence 2 19 32 100 113 158 165 MFI 75.0 80.1 141 214 213 620 636 A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 fluorescence\u00C2\u00A0intensity Co un ts Ramos A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 fluorescence\u00C2\u00A0intensity Valence 2 32 63 75 113 152 165 MFI 16.2 17.0 23.7 16.7 41.7 96.6 73.5 Co un ts Z138B C \u00E2\u0080\u00A2\u00C2\u00A0113\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 determine\u00C2\u00A0 if\u00C2\u00A0 CD120a\u00C2\u00A0 upregulation\u00C2\u00A0 corresponds\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.6,\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0were\u00C2\u00A0next\u00C2\u00A0measured\u00C2\u00A0after\u00C2\u00A0 treating\u00C2\u00A0Ramos\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0various\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0valences.\u00C2\u00A0 \u00C2\u00A0Figure\u00C2\u00A05.4B\u00C2\u00A0&\u00C2\u00A0C\u00C2\u00A0illustrate\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0 all\u00C2\u00A0 cases,\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 higher\u00C2\u00A0 valences\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 more\u00C2\u00A0 elevated\u00C2\u00A0 CD120a\u00C2\u00A0 expression,\u00C2\u00A0even\u00C2\u00A0though\u00C2\u00A0all\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0given\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Measured\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0also\u00C2\u00A0 correlated\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0percentages\u00C2\u00A0of\u00C2\u00A0apoptotic\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.6\u00C2\u00A0 insofar\u00C2\u00A0as\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0 that\u00C2\u00A0 induced\u00C2\u00A0relatively\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0also\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0equal\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 plasma\u00C2\u00A0membrane.\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0the\u00C2\u00A0Z138\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0line\u00C2\u00A0was\u00C2\u00A0less\u00C2\u00A0sensitive\u00C2\u00A0to\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0 by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0and\u00C2\u00A0 increases\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 cell\u00C2\u00A0 line\u00C2\u00A0were\u00C2\u00A0more\u00C2\u00A0modest\u00C2\u00A0 than\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 Ramos\u00C2\u00A0cell\u00C2\u00A0line.\u00C2\u00A0\u00C2\u00A0Together,\u00C2\u00A0these\u00C2\u00A0data\u00C2\u00A0confirm\u00C2\u00A0that\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0appears\u00C2\u00A0to\u00C2\u00A0 play\u00C2\u00A0a\u00C2\u00A0central\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0 5.4.5\u00C2\u00A0 Colocalization\u00C2\u00A0of\u00C2\u00A0caspase\u00C2\u00AD8\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0 Next,\u00C2\u00A0confocal\u00C2\u00A0laser\u00E2\u0080\u0090scanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0images\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0dual\u00E2\u0080\u0090labeled\u00C2\u00A0with\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0and\u00C2\u00A0FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK,\u00C2\u00A0 the\u00C2\u00A0 latter\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0 labels\u00C2\u00A0cleaved\u00C2\u00A0caspsase\u00E2\u0080\u00908\u00C2\u00A0(Section\u00C2\u00A05.3.3).\u00C2\u00A0 \u00C2\u00A0FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK\u00C2\u00A0 is\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0 permeable,\u00C2\u00A0 but\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 not\u00C2\u00A0 permeabilized\u00C2\u00A0 prior\u00C2\u00A0 to\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 staining\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 visualize\u00C2\u00A0 CD120a\u00C2\u00A0only\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0 \u00C2\u00A0Representative\u00C2\u00A0 images\u00C2\u00A0 are\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 5.5\u00C2\u00A0 for\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0with\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A032,\u00C2\u00A075,\u00C2\u00A0and\u00C2\u00A0113.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0114\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.5\u00C2\u00A0\u00C2\u00A0Two\u00C2\u00A0slices\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0representative\u00C2\u00A0non\u00E2\u0080\u0090necrotic\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0 of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Activated\u00C2\u00A0caspase\u00E2\u0080\u00908\u00E2\u0080\u0090rich\u00C2\u00A0regions\u00C2\u00A0(green;\u00C2\u00A0FAM\u00E2\u0080\u0090LETD\u00E2\u0080\u0090FMK\u00C2\u00A0emission)\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0seen\u00C2\u00A0inside\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0colocalized\u00C2\u00A0with\u00C2\u00A0and\u00C2\u00A0directly\u00C2\u00A0 adjacent\u00C2\u00A0to\u00C2\u00A0CD120a\u00E2\u0080\u0090enriched\u00C2\u00A0domains\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0(red;\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0emission),\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0 Valence 32 75 113 \u00E2\u0080\u00A2\u00C2\u00A0115\u00C2\u00A0\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 are\u00C2\u00A0 both\u00C2\u00A0 DISC\u00C2\u00A0 constituents.\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 increased,\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 became\u00C2\u00A0 higher,\u00C2\u00A0 and\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 activation\u00C2\u00A0became\u00C2\u00A0increasingly\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0CD120a.\u00C2\u00A0\u00C2\u00A0Scale\u00C2\u00A0bars,\u00C2\u00A05\u00C2\u00A0\u00CE\u00BCm.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 images\u00C2\u00A0visually\u00C2\u00A0confirm\u00C2\u00A0 that\u00C2\u00A0greater\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0occurred\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0of\u00C2\u00A0higher\u00C2\u00A0valence,\u00C2\u00A0since\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0(red)\u00C2\u00A0became\u00C2\u00A0more\u00C2\u00A0 intense\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0 increased.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 images\u00C2\u00A0 also\u00C2\u00A0 show\u00C2\u00A0 specific\u00C2\u00A0 colocalization\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 and\u00C2\u00A0 caspase\u00E2\u0080\u00908,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 staining\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 intracellular\u00C2\u00A0 region\u00C2\u00A0directly\u00C2\u00A0adjacent\u00C2\u00A0 to\u00C2\u00A0CD120a\u00E2\u0080\u0090enriched\u00C2\u00A0domains\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane;\u00C2\u00A0 this\u00C2\u00A0 is\u00C2\u00A0particularly\u00C2\u00A0 the\u00C2\u00A0case\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0higher\u00C2\u00A0valences.\u00C2\u00A0 \u00C2\u00A0Procaspase\u00E2\u0080\u00908\u00C2\u00A0 is\u00C2\u00A0cleaved\u00C2\u00A0 to\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0while\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 bound\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00E2\u0080\u0090associated\u00C2\u00A0DISC;\u00C2\u00A0 after\u00C2\u00A0 cleavage,\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 leaves\u00C2\u00A0the\u00C2\u00A0inner\u00C2\u00A0leaflet\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0and\u00C2\u00A0enters\u00C2\u00A0the\u00C2\u00A0cytoplasm87,\u00C2\u00A0171.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0illustrated\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0images,\u00C2\u00A0which\u00C2\u00A0show\u00C2\u00A0an\u00C2\u00A0increasing\u00C2\u00A0degree\u00C2\u00A0of\u00C2\u00A0colocalized\u00C2\u00A0and\u00C2\u00A0adjacent\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 increased\u00C2\u00A0 from\u00C2\u00A032\u00C2\u00A0 to\u00C2\u00A0113.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0 experiments\u00C2\u00A0 visually\u00C2\u00A0 imply\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 treatment\u00C2\u00A0 results\u00C2\u00A0in\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0DISC\u00C2\u00A0formation\u00C2\u00A0with\u00C2\u00A0procaspase\u00E2\u0080\u00908,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0ultimately\u00C2\u00A0cleaved\u00C2\u00A0to\u00C2\u00A0 caspase\u00E2\u0080\u00908,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0initiation\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 5.4.6\u00C2\u00A0 CD120a\u00C2\u00A0 upregulation\u00C2\u00A0 and\u00C2\u00A0 direct\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 result\u00C2\u00A0 from\u00C2\u00A0 Rtx\u00C2\u00A0 multivalency\u00C2\u00A0and\u00C2\u00A0not\u00C2\u00A0the\u00C2\u00A0liposomal\u00C2\u00A0component\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0 To\u00C2\u00A0 more\u00C2\u00A0 fully\u00C2\u00A0 understand\u00C2\u00A0 why\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 induces\u00C2\u00A0 CD120a\u00C2\u00A0 upregulation,\u00C2\u00A0 CD120a/procaspase\u00E2\u0080\u00908\u00C2\u00A0DISC\u00C2\u00A0formation,\u00C2\u00A0and\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0equal\u00C2\u00A0 doses\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0 important\u00C2\u00A0 to\u00C2\u00A0 examine\u00C2\u00A0 the\u00C2\u00A0 role\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 liposome\u00C2\u00A0 in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A01.2.2,\u00C2\u00A0 the\u00C2\u00A0 liposome\u00C2\u00A0 serves\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 scaffold\u00C2\u00A0 for\u00C2\u00A0bridging\u00C2\u00A0many\u00C2\u00A0Rtx\u00C2\u00A0molecules\u00C2\u00A0 together\u00C2\u00A0 into\u00C2\u00A0 a\u00C2\u00A0 multivalent\u00C2\u00A0configuration,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0distinct\u00C2\u00A0from\u00C2\u00A0traditional\u00C2\u00A0applications\u00C2\u00A0of\u00C2\u00A0 liposome\u00C2\u00A0technology.\u00C2\u00A0 \u00C2\u00A0If\u00C2\u00A0 the\u00C2\u00A0liposome\u00C2\u00A0serves\u00C2\u00A0only\u00C2\u00A0a\u00C2\u00A0structural\u00C2\u00A0role\u00C2\u00A0without\u00C2\u00A0contributing\u00C2\u00A0to\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0or\u00C2\u00A0induction\u00C2\u00A0 of\u00C2\u00A0apoptosis,\u00C2\u00A0then\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0effects\u00C2\u00A0should\u00C2\u00A0also\u00C2\u00A0be\u00C2\u00A0present\u00C2\u00A0when\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00C2\u00A0constructs\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0based\u00C2\u00A0on\u00C2\u00A0liposomes.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0116\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0an\u00C2\u00A0alternative\u00C2\u00A0 to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs,\u00C2\u00A0polystyrene\u00C2\u00A0microspheres\u00C2\u00A0 (MS)\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0diameter\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0nm\u00C2\u00A0 were\u00C2\u00A0employed\u00C2\u00A0 to\u00C2\u00A0 create\u00C2\u00A0multivalent\u00C2\u00A0Rtx;\u00C2\u00A0 these\u00C2\u00A0MS\u00C2\u00A0have\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0diameter\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0SUV\u00C2\u00A0used\u00C2\u00A0 to\u00C2\u00A0 prepare\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 mirror\u00C2\u00A0 the\u00C2\u00A0 preparation\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 employed\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 3,\u00C2\u00A0 Rtx\u00C2\u00A0 was\u00C2\u00A0 biotinylated\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0NIMAC\u00C2\u00A0column\u00C2\u00A0under\u00C2\u00A0identical\u00C2\u00A0conditions\u00C2\u00A0(Section\u00C2\u00A03.3.6),\u00C2\u00A0and\u00C2\u00A0streptavidin\u00E2\u0080\u0090coupled\u00C2\u00A0 MS\u00C2\u00A0 (streptavidin\u00E2\u0080\u0090MS)\u00C2\u00A0were\u00C2\u00A0 saturated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin,\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A05.3.8.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 resulting\u00C2\u00A0 Rtx\u00E2\u0080\u0090MS\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0dose\u00E2\u0080\u0090response\u00C2\u00A0curve\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.6A.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0figure\u00C2\u00A0 illustrates\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0induced\u00C2\u00A0significantly\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0 Rtx,\u00C2\u00A0which\u00C2\u00A0mirrors\u00C2\u00A0the\u00C2\u00A0behavior\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0a\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A04.1\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0Rtx\u00C2\u00A0(in\u00C2\u00A0Rtx\u00E2\u0080\u0090MS),\u00C2\u00A043%\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 undergoing\u00C2\u00A0 early\u00C2\u00A0 apoptosis\u00C2\u00A0 after\u00C2\u00A0 24\u00C2\u00A0 h,\u00C2\u00A0 while\u00C2\u00A0 for\u00C2\u00A0 doses\u00C2\u00A0 of\u00C2\u00A0 20.6\u00C2\u00A0 \u00C2\u00B5g\u00C2\u00A0 and\u00C2\u00A0 higher,\u00C2\u00A0 equal\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0for\u00C2\u00A0all\u00C2\u00A0doses\u00C2\u00A0(~60%);\u00C2\u00A0this\u00C2\u00A0is\u00C2\u00A0in\u00C2\u00A0contrast\u00C2\u00A0to\u00C2\u00A03\u00E2\u0080\u00934%\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 at\u00C2\u00A0 all\u00C2\u00A0 doses\u00C2\u00A0 tested\u00C2\u00A0 (not\u00C2\u00A0 shown).\u00C2\u00A0 \u00C2\u00A0Maximum\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 early\u00C2\u00A0 apoptosis\u00C2\u00A0 observed\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0were\u00C2\u00A0similar\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0measured\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0as\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A04.6A.\u00C2\u00A0\u00C2\u00A0Another\u00C2\u00A0parallel\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0two\u00C2\u00A0figures\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0inverse\u00C2\u00A0relationship\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0viable\u00C2\u00A0 and\u00C2\u00A0 apoptotic\u00C2\u00A0 cell\u00C2\u00A0 fractions,\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 in\u00C2\u00A0both\u00C2\u00A0 cases\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0dying\u00C2\u00A0 via\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0not\u00C2\u00A0 another\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 cell\u00C2\u00A0 death\u00C2\u00A0 that\u00C2\u00A0 would\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 viable\u00C2\u00A0 cells\u00C2\u00A0 directly\u00C2\u00A0 becoming\u00C2\u00A0 necrotic.\u00C2\u00A0\u00C2\u00A0 Treatment\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0or\u00C2\u00A0 streptavidin\u00E2\u0080\u0090MS\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0equivalent\u00C2\u00A0 to\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0(not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0117\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A05.6\u00C2\u00A0\u00C2\u00A0Apoptosis\u00C2\u00A0via\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0an\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0liposomal\u00C2\u00A0component\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0formulation\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP.\u00C2\u00A0\u00C2\u00A0 (A)\u00C2\u00A0Dose\u00E2\u0080\u0090response\u00C2\u00A0curve\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A01.25\u00C2\u00A0\u00EF\u0082\u00B4\u00C2\u00A0105\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0250\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0medium\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0for\u00C2\u00A024\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0(B)\u00C2\u00A0Similar\u00C2\u00A0to\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 different\u00C2\u00A0 doses\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090MS\u00C2\u00A0 exhibited\u00C2\u00A0 increased\u00C2\u00A0 apoptosis\u00C2\u00A0 alongside\u00C2\u00A0 elevated\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0CD120a\u00C2\u00A0 levels.\u00C2\u00A0 \u00C2\u00A0Both\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0apoptosis\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A012h\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0 (compared\u00C2\u00A0 to\u00C2\u00A0 24h\u00C2\u00A0 in\u00C2\u00A0 (A))\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 simultaneously\u00C2\u00A0 measure\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 and\u00C2\u00A0 early\u00C2\u00A0 apoptosis,\u00C2\u00A0 which\u00C2\u00A0 show\u00C2\u00A0 maximum\u00C2\u00A0values\u00C2\u00A0at\u00C2\u00A08h\u00C2\u00A0and\u00C2\u00A024h,\u00C2\u00A0respectively.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Cells\u00C2\u00A0were\u00C2\u00A0next\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0region\u00C2\u00A0where\u00C2\u00A0dose\u00E2\u0080\u0090dependent\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0apoptosis\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0(below\u00C2\u00A0a\u00C2\u00A025\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0Rtx\u00C2\u00A0dose).\u00C2\u00A0 \u00C2\u00A0Levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 and\u00C2\u00A0 apoptosis\u00C2\u00A0 were\u00C2\u00A0 measured\u00C2\u00A0 simultaneously\u00C2\u00A0 with\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0 12\u00C2\u00A0 h\u00C2\u00A0 after\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 resulting\u00C2\u00A0 data,\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 5.6B,\u00C2\u00A0 show\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 dose\u00E2\u0080\u0090dependent\u00C2\u00A0 increases\u00C2\u00A0 in\u00C2\u00A0 apoptosis\u00C2\u00A0 correspond\u00C2\u00A0 to\u00C2\u00A0elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0early\u00C2\u00A0 apoptosis\u00C2\u00A0increased\u00C2\u00A0from\u00C2\u00A06.9\u00C2\u00A0%\u00C2\u00A0to\u00C2\u00A016\u00C2\u00A0%\u00C2\u00A0(2.3\u00E2\u0080\u0090fold;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.005)\u00C2\u00A0from\u00C2\u00A00\u00C2\u00A0to\u00C2\u00A013.2\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0Rtx\u00C2\u00A0(in\u00C2\u00A0Rtx\u00E2\u0080\u0090MS)\u00C2\u00A0while\u00C2\u00A0 the\u00C2\u00A0MFI\u00C2\u00A0 from\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 increased\u00C2\u00A0 from\u00C2\u00A043.7\u00C2\u00A0 to\u00C2\u00A0402\u00C2\u00A0 (9.2\u00E2\u0080\u0090fold;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.0001)\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0dose\u00C2\u00A0 difference.\u00C2\u00A0 \u00C2\u00A0 Each\u00C2\u00A0 successively\u00C2\u00A0 higher\u00C2\u00A0 dose\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 figure\u00C2\u00A0 shows\u00C2\u00A0 an\u00C2\u00A0 elevated\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 next\u00C2\u00A0 lowest\u00C2\u00A0 dose\u00C2\u00A0 (p\u00C2\u00A0 <\u00C2\u00A0 0.05\u00C2\u00A0 between\u00C2\u00A0 all\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0MFIs).\u00C2\u00A0 \u00C2\u00A0However,\u00C2\u00A0 not\u00C2\u00A0 all\u00C2\u00A0 measured\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0show\u00C2\u00A0differences\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0statistically\u00C2\u00A0significant;\u00C2\u00A0p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0for\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0 0\u00E2\u0080\u00933.3\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0 and\u00C2\u00A06.6\u00E2\u0080\u009313.2\u00C2\u00A0\u00C2\u00B5g,\u00C2\u00A0but\u00C2\u00A0p\u00C2\u00A0 <\u00C2\u00A00.05\u00C2\u00A0 for\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0doses\u00C2\u00A0when\u00C2\u00A0one\u00C2\u00A0dose\u00C2\u00A0 is\u00C2\u00A0 from\u00C2\u00A0each\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0 0 25 50 75 100 0 25 50 75 100 % \u00C2\u00A0of \u00C2\u00A0to ta l\u00C2\u00A0ce ll\u00C2\u00A0p op ul at io n Dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0(in\u00C2\u00A0Rtx\u00E2\u0080\u0090MS)\u00C2\u00A0/\u00C2\u00A0\u00C2\u00B5g Viable E.\u00C2\u00A0apoptotic A 0 1 2 3 4 0 1.7 3.3 6.6 13.2 Re la ti ve \u00C2\u00A0va lu es Dose\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0(in\u00C2\u00A0Rtx\u00E2\u0080\u0090MS)\u00C2\u00A0/\u00C2\u00A0\u00C2\u00B5g CD120a\u00C2\u00A0exp. E.\u00C2\u00A0apoptotic B \u00E2\u0080\u00A2\u00C2\u00A0118\u00C2\u00A0\u00C2\u00A0 groups.\u00C2\u00A0\u00C2\u00A0Note\u00C2\u00A0that\u00C2\u00A0apoptosis\u00C2\u00A0values\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.6B\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0at\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.6A;\u00C2\u00A0this\u00C2\u00A0was\u00C2\u00A0necessary\u00C2\u00A0to\u00C2\u00A0simultaneously\u00C2\u00A0measure\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0 and\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0cells,\u00C2\u00A0which\u00C2\u00A0show\u00C2\u00A0maximum\u00C2\u00A0values\u00C2\u00A0at\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A024\u00C2\u00A0h,\u00C2\u00A0respectively.\u00C2\u00A0\u00C2\u00A0Overall,\u00C2\u00A0 these\u00C2\u00A0results\u00C2\u00A0demonstrate\u00C2\u00A0 that\u00C2\u00A0a\u00C2\u00A0 liposome\u00C2\u00A0specifically\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0required\u00C2\u00A0 to\u00C2\u00A0achieve\u00C2\u00A0beneficial\u00C2\u00A0effects\u00C2\u00A0 from\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0because\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0and\u00C2\u00A0maximum\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0were\u00C2\u00A0 reproduced\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0non\u00E2\u0080\u0090liposomal\u00C2\u00A0formulation.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 5.5\u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0 5.5.1\u00C2\u00A0 Discussion\u00C2\u00A0 The\u00C2\u00A0molecular\u00C2\u00A0basis\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0poorly\u00C2\u00A0defined,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0work\u00C2\u00A0 in\u00C2\u00A0this\u00C2\u00A0chapter\u00C2\u00A0provides\u00C2\u00A0some\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0evidence\u00C2\u00A0explaining\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0 observed\u00C2\u00A0when\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0hypercrosslinked\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0when\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0bivalent\u00C2\u00A0(Sections\u00C2\u00A01.4.3\u00C2\u00A0and\u00C2\u00A01.6.1).\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0uncovered\u00C2\u00A0here\u00C2\u00A0 consists\u00C2\u00A0of\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0 CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 followed\u00C2\u00A0 by\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 (consistent\u00C2\u00A0 with\u00C2\u00A0 CD120a/procaspase\u00E2\u0080\u00908\u00C2\u00A0DISC\u00C2\u00A0formation)\u00C2\u00A0and\u00C2\u00A0subsequent\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 is\u00C2\u00A0 supported\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 observation\u00C2\u00A0 that\u00C2\u00A0 of\u00C2\u00A0 all\u00C2\u00A0 the\u00C2\u00A0 known\u00C2\u00A0 death\u00C2\u00A0 receptors,\u00C2\u00A0CD120a\u00C2\u00A0 showed\u00C2\u00A0 the\u00C2\u00A0highest\u00C2\u00A0upregulation\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 (Figure\u00C2\u00A05.2).\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 was\u00C2\u00A0upregulated\u00C2\u00A0equally\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0 (Figure\u00C2\u00A0 5.3),\u00C2\u00A0 suggesting\u00C2\u00A0 that\u00C2\u00A0 upregulation\u00C2\u00A0 preceded\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 was\u00C2\u00A0 confirmed\u00C2\u00A0 with\u00C2\u00A0 time\u00E2\u0080\u0090course\u00C2\u00A0 experiments\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.4A\u00C2\u00A0which\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0peak\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0occurred\u00C2\u00A08\u00C2\u00A0h\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0while\u00C2\u00A0maximum\u00C2\u00A0 apoptosis\u00C2\u00A0was\u00C2\u00A0 observed\u00C2\u00A0 24\u00C2\u00A0 h\u00C2\u00A0 after\u00C2\u00A0 treatment,\u00C2\u00A0 as\u00C2\u00A0 indicated\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.5B.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0time\u00C2\u00A0course\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0measured\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0activated\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0over\u00C2\u00A0time\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A05.1A.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0119\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0Moreover,\u00C2\u00A0 the\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 (Figure\u00C2\u00A05.4B\u00C2\u00A0&\u00C2\u00A0C)\u00C2\u00A0corresponded\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0(Figure\u00C2\u00A05.1C)\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 4.6.\u00C2\u00A0 \u00C2\u00A0 Finally,\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 CD120a\u00E2\u0080\u0090 dependent\u00C2\u00A0mechanism\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0two\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0(Ramos\u00C2\u00A0and\u00C2\u00A0Z138),\u00C2\u00A0and\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 regardless\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 type\u00C2\u00A0 of\u00C2\u00A0multivalent\u00C2\u00A0 construct\u00C2\u00A0 used\u00C2\u00A0 (Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 or\u00C2\u00A0 Rtx\u00E2\u0080\u0090MS;\u00C2\u00A0 Figure\u00C2\u00A0 5.6).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Overall,\u00C2\u00A0this\u00C2\u00A0data\u00C2\u00A0supports\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx.\u00C2\u00A0 Given\u00C2\u00A0 that\u00C2\u00A0 CD120a\u00C2\u00A0 is\u00C2\u00A0 expressed\u00C2\u00A0 in\u00C2\u00A0 all\u00C2\u00A0 human\u00C2\u00A0 cell\u00C2\u00A0 types\u00C2\u00A0 (Section\u00C2\u00A0 5.2.3),\u00C2\u00A0 there\u00C2\u00A0 are\u00C2\u00A0 innumerable\u00C2\u00A0 reported\u00C2\u00A0 instances\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0pathogenesis\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0wide\u00C2\u00A0 variety\u00C2\u00A0of\u00C2\u00A0 diseases.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0its\u00C2\u00A0upregulation\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0cardiovascular\u00C2\u00A0diseases\u00C2\u00A0in\u00C2\u00A0diabetic\u00C2\u00A0 rats188,\u00C2\u00A0ocular\u00C2\u00A0allergic\u00C2\u00A0inflammation189,\u00C2\u00A0and\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0for\u00C2\u00A0chronic\u00C2\u00A0hepatitis\u00C2\u00A0C190.\u00C2\u00A0\u00C2\u00A0Various\u00C2\u00A0 viruses,\u00C2\u00A0bacteria,\u00C2\u00A0and\u00C2\u00A0parasites\u00C2\u00A0also\u00C2\u00A0act\u00C2\u00A0by\u00C2\u00A0modulating\u00C2\u00A0the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0CD120a,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0 caspase\u00E2\u0080\u0090dependent\u00C2\u00A0 apoptosis\u00C2\u00A0 or\u00C2\u00A0 NF\u00E2\u0080\u0090\u00CE\u00BAB\u00E2\u0080\u0090induced\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 inflammatory\u00C2\u00A0 or\u00C2\u00A0 antiapoptotic\u00C2\u00A0 genes191.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0oncology\u00C2\u00A0specifically,\u00C2\u00A0CD120a\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0upregulated\u00C2\u00A0by\u00C2\u00A0estrogen\u00C2\u00A0in\u00C2\u00A0human\u00C2\u00A0 breast\u00C2\u00A0adipose\u00C2\u00A0fibroblasts,\u00C2\u00A0serving\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0mechanism\u00C2\u00A0that\u00C2\u00A0supports\u00C2\u00A0breast\u00C2\u00A0tumor\u00C2\u00A0growth192.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0 upregulated\u00C2\u00A0on\u00C2\u00A0endothelial\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0by\u00C2\u00A0endothelial\u00C2\u00A0monocyte\u00C2\u00A0activating\u00C2\u00A0polypeptide\u00E2\u0080\u0090 II,\u00C2\u00A0a\u00C2\u00A0 tumor\u00E2\u0080\u0090derived\u00C2\u00A0 inflammatory\u00C2\u00A0cytokine193.\u00C2\u00A0 \u00C2\u00A0Finally,\u00C2\u00A0CD120a\u00C2\u00A0was\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0upregulated\u00C2\u00A0after\u00C2\u00A0 treating\u00C2\u00A0chronic\u00C2\u00A0lymphocytic\u00C2\u00A0leukemia\u00C2\u00A0(CLL)\u00C2\u00A0B\u00E2\u0080\u0090cells\u00C2\u00A0with\u00C2\u00A0fludarabine,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0apoptosis194.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0molecular\u00C2\u00A0basis\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0described\u00C2\u00A0as\u00C2\u00A0 a\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0sensitizing\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0its\u00C2\u00A0ligand,\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0(refs.\u00C2\u00A0172,\u00C2\u00A0189,\u00C2\u00A0193).\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0test\u00C2\u00A0whether\u00C2\u00A0cells\u00C2\u00A0showed\u00C2\u00A0 increased\u00C2\u00A0sensitivity\u00C2\u00A0to\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0following\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0soluble\u00C2\u00A0TNF\u00E2\u0080\u0090 \u00CE\u00B1\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0and\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0all\u00C2\u00A0cases,\u00C2\u00A0regardless\u00C2\u00A0of\u00C2\u00A0whether\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0added,\u00C2\u00A0the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A00.01\u00C2\u00A0ng/mL\u00C2\u00A0to\u00C2\u00A01\u00C2\u00A0\u00CE\u00BCg/mL\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0 apoptosis\u00C2\u00A0equivalent\u00C2\u00A0to\u00C2\u00A0treatments\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0120\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 strongly\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 through\u00C2\u00A0 CD120a\u00C2\u00A0 is\u00C2\u00A0 ligand\u00E2\u0080\u0090 independent,\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0other\u00C2\u00A0 reports\u00C2\u00A0of\u00C2\u00A0 ligand\u00E2\u0080\u0090independent\u00C2\u00A0death\u00C2\u00A0 receptor\u00C2\u00A0activation181\u00E2\u0080\u0090186.\u00C2\u00A0\u00C2\u00A0 Although\u00C2\u00A0 previous\u00C2\u00A0 reports\u00C2\u00A0 have\u00C2\u00A0 indicated\u00C2\u00A0 that\u00C2\u00A0 soluble\u00C2\u00A0 TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0 associates\u00C2\u00A0 with\u00C2\u00A0 CD120a\u00C2\u00A0 while\u00C2\u00A0 membrane\u00E2\u0080\u0090bound\u00C2\u00A0 TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0 associates\u00C2\u00A0 with\u00C2\u00A0 CD120b\u00C2\u00A0 (which\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 contain\u00C2\u00A0 a\u00C2\u00A0 death\u00C2\u00A0 domain)195,\u00C2\u00A0 ultimately\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 soluble\u00C2\u00A0 and\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00E2\u0080\u0090bound\u00C2\u00A0 TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0 in\u00C2\u00A0 treated\u00C2\u00A0 and\u00C2\u00A0 untreated\u00C2\u00A0 samples\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 should\u00C2\u00A0 be\u00C2\u00A0 measured\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 firmly\u00C2\u00A0 establish\u00C2\u00A0 ligand\u00C2\u00A0 independence\u00C2\u00A0 of\u00C2\u00A0 this\u00C2\u00A0 signaling\u00C2\u00A0process.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Together,\u00C2\u00A0this\u00C2\u00A0chapter\u00C2\u00A0provides\u00C2\u00A0a\u00C2\u00A0proof\u00E2\u0080\u0090of\u00E2\u0080\u0090concept\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0can\u00C2\u00A0 be\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0many\u00C2\u00A0valences\u00C2\u00A0for\u00C2\u00A0use\u00C2\u00A0 in\u00C2\u00A0studying\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090 LNPs.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0also\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0non\u00E2\u0080\u0090liposomal\u00C2\u00A0 formulations\u00C2\u00A0exhibit\u00C2\u00A0equivalent\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0 to\u00C2\u00A0Ab\u00E2\u0080\u0090 LNPs\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 indicates\u00C2\u00A0 that\u00C2\u00A0 information\u00C2\u00A0obtained\u00C2\u00A0using\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0Ab\u00C2\u00A0(for\u00C2\u00A0example,\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0at\u00C2\u00A0which\u00C2\u00A0optimal\u00C2\u00A0activity\u00C2\u00A0 is\u00C2\u00A0observed)\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0employed\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0 formulations\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0 that\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0 utilize\u00C2\u00A0 liposomes.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0useful\u00C2\u00A0 in\u00C2\u00A0cases\u00C2\u00A0where\u00C2\u00A0preparing\u00C2\u00A0many\u00C2\u00A0different\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0specific\u00C2\u00A0 non\u00E2\u0080\u0090liposomal\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0is\u00C2\u00A0difficult\u00C2\u00A0or\u00C2\u00A0not\u00C2\u00A0feasible.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0multivalent\u00C2\u00A0 therapeutic\u00C2\u00A0Abs\u00C2\u00A0 can\u00C2\u00A0 show\u00C2\u00A0enhanced\u00C2\u00A0efficacies\u00C2\u00A0 regardless\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 specific\u00C2\u00A0formulation\u00C2\u00A0(Figure\u00C2\u00A05.6)\u00C2\u00A0further\u00C2\u00A0supports\u00C2\u00A0the\u00C2\u00A0conclusions\u00C2\u00A0of\u00C2\u00A0Chapter\u00C2\u00A04\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0improved\u00C2\u00A0 therapeutic\u00C2\u00A0effects\u00C2\u00A0were\u00C2\u00A0driven\u00C2\u00A0by\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Ab.\u00C2\u00A0 \u00C2\u00A0It\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0 envisioned\u00C2\u00A0that\u00C2\u00A0another\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0would\u00C2\u00A0entail\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Ab\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0 reagent\u00C2\u00A0 that\u00C2\u00A0hypercrosslinks\u00C2\u00A0 the\u00C2\u00A0Ab.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A01.4.3,\u00C2\u00A0Rtx\u00C2\u00A0elicits\u00C2\u00A0higher\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 when\u00C2\u00A0 bivalent\u00C2\u00A0 crosslinking\u00C2\u00A0 is\u00C2\u00A0 achieved\u00C2\u00A0 using\u00C2\u00A0 a\u00C2\u00A0 2oAb.\u00C2\u00A0 \u00C2\u00A0 Another\u00C2\u00A0 conceivable\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0may\u00C2\u00A0occur\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0present\u00C2\u00A0on\u00C2\u00A0lymphoma\u00C2\u00A0 cells\u00C2\u00A0 interacts\u00C2\u00A0with\u00C2\u00A0 Fc\u00CE\u00B3R\u00C2\u00A0 on\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 constitutes\u00C2\u00A0 a\u00C2\u00A0multivalent\u00C2\u00A0 interaction\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0ADCC\u00C2\u00A0(Sections\u00C2\u00A01.4.2\u00C2\u00A0&\u00C2\u00A02.2.2),\u00C2\u00A0but\u00C2\u00A0it\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 to\u00C2\u00A0contribute\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0results\u00C2\u00A0from\u00C2\u00A0this\u00C2\u00A0chapter\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0121\u00C2\u00A0\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 if\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 can\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 Rtx\u00E2\u0080\u0090treated\u00C2\u00A0 cells\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0 mechanism,\u00C2\u00A0then\u00C2\u00A0they\u00C2\u00A0are\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0Rtx\u00C2\u00A0sufficiently\u00C2\u00A0to\u00C2\u00A0directly\u00C2\u00A0 induce\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0vivo.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0would\u00C2\u00A0 imply\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0applicable\u00C2\u00A0to\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 but\u00C2\u00A0 also\u00C2\u00A0 to\u00C2\u00A0 regular\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 therapy,\u00C2\u00A0 shedding\u00C2\u00A0 light\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 poorly\u00C2\u00A0 defined\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0drug.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 5.5.2\u00C2\u00A0 Conclusions\u00C2\u00A0 The\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0constructs,\u00C2\u00A0including\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0 involves\u00C2\u00A0 the\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 resulting\u00C2\u00A0 from\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 and\u00C2\u00A0 resultant\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 Inhibition\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0significantly\u00C2\u00A0decreased\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 other\u00C2\u00A0activated\u00C2\u00A0caspases\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0 of\u00C2\u00A0DISCs\u00C2\u00A0that\u00C2\u00A0include\u00C2\u00A0both\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0procaspase\u00E2\u0080\u00908,\u00C2\u00A0and\u00C2\u00A0confocal\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0images\u00C2\u00A0 showed\u00C2\u00A0 CD120a/caspase\u00E2\u0080\u00908\u00C2\u00A0 colocalization\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00C2\u00A0 present\u00C2\u00A0 in\u00C2\u00A0 intracellular\u00C2\u00A0 regions\u00C2\u00A0 adjacent\u00C2\u00A0 to\u00C2\u00A0 CD120a\u00E2\u0080\u0090enriched\u00C2\u00A0 domains\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface.\u00C2\u00A0 \u00C2\u00A0 Higher\u00C2\u00A0 valences\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 which\u00C2\u00A0 induced\u00C2\u00A0greater\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis,\u00C2\u00A0also\u00C2\u00A0caused\u00C2\u00A0more\u00C2\u00A0significant\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0caspase\u00E2\u0080\u00908\u00C2\u00A0 activation,\u00C2\u00A0supporting\u00C2\u00A0earlier\u00C2\u00A0conclusions\u00C2\u00A0that\u00C2\u00A0more\u00C2\u00A0extensive\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0drives\u00C2\u00A0this\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action.\u00C2\u00A0\u00C2\u00A0Overall,\u00C2\u00A0this\u00C2\u00A0work\u00C2\u00A0provides\u00C2\u00A0a\u00C2\u00A0proof\u00E2\u0080\u0090of\u00E2\u0080\u0090concept\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0in\u00C2\u00A0 Chapter\u00C2\u00A0 3\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 prepare\u00C2\u00A0many\u00C2\u00A0 valences\u00C2\u00A0 of\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0 for\u00C2\u00A0 use\u00C2\u00A0 in\u00C2\u00A0mechanistic\u00C2\u00A0 studies,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0results\u00C2\u00A0of\u00C2\u00A0such\u00C2\u00A0studies\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0applied\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0both\u00C2\u00A0 liposomal\u00C2\u00A0and\u00C2\u00A0 non\u00E2\u0080\u0090liposomal\u00C2\u00A0multivalent\u00C2\u00A0formulations.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0122\u00C2\u00A0\u00C2\u00A0 6\u00C2\u00A0 In\u00C2\u00A0vivo\u00C2\u00A0relevance\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0CD120a\u00C2\u00ADdependent\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0 rituximab\u00C2\u00A0 6.1\u00C2\u00A0 Synopsis\u00C2\u00A0 The\u00C2\u00A0previous\u00C2\u00A0chapter\u00C2\u00A0identified\u00C2\u00A0a\u00C2\u00A0novel\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0that\u00C2\u00A0 involves\u00C2\u00A0the\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0ensuing\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0By\u00C2\u00A0considering\u00C2\u00A0recent\u00C2\u00A0work\u00C2\u00A0concerning\u00C2\u00A0 the\u00C2\u00A0molecular\u00C2\u00A0biology\u00C2\u00A0of\u00C2\u00A0CD120a,\u00C2\u00A0this\u00C2\u00A0chapter\u00C2\u00A0examines\u00C2\u00A0whether\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0mechanism\u00C2\u00A0 is\u00C2\u00A0plausible\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 after\u00C2\u00A0 normal\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 therapy.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 results\u00C2\u00A0 suggest\u00C2\u00A0 a\u00C2\u00A0 possible\u00C2\u00A0 treatment\u00C2\u00A0 strategy\u00C2\u00A0 for\u00C2\u00A0 improving\u00C2\u00A0the\u00C2\u00A0sensitivity\u00C2\u00A0of\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0Rtx.\u00C2\u00A0 6.2\u00C2\u00A0 Background\u00C2\u00A0 6.2.1\u00C2\u00A0 The\u00C2\u00A0biology\u00C2\u00A0and\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 Unlike\u00C2\u00A0other\u00C2\u00A0death\u00C2\u00A0 receptors\u00C2\u00A0 (discussed\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A05.2.3),\u00C2\u00A0CD120a\u00C2\u00A0 is\u00C2\u00A0primarily\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 mediating\u00C2\u00A0 inflammation\u00C2\u00A0and\u00C2\u00A0not\u00C2\u00A0cell\u00C2\u00A0death174.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0often\u00C2\u00A0activated\u00C2\u00A0by\u00C2\u00A0 its\u00C2\u00A0 ligand,\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1,\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0 trivalent\u00C2\u00A0ligand\u00C2\u00A0found\u00C2\u00A0in\u00C2\u00A0two\u00C2\u00A0forms,\u00C2\u00A0a\u00C2\u00A0soluble\u00C2\u00A0form\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0plasma\u00C2\u00A0membrane\u00E2\u0080\u0090bound\u00C2\u00A0form.\u00C2\u00A0 \u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0is\u00C2\u00A0 also\u00C2\u00A0specific\u00C2\u00A0to\u00C2\u00A0another\u00C2\u00A0TNFR\u00C2\u00A0superfamily\u00C2\u00A0member,\u00C2\u00A0CD120b\u00C2\u00A0(also\u00C2\u00A0known\u00C2\u00A0as\u00C2\u00A0TNFR2),\u00C2\u00A0which\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0a\u00C2\u00A0 death\u00C2\u00A0receptor\u00C2\u00A0since\u00C2\u00A0it\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0possess\u00C2\u00A0an\u00C2\u00A0intracellular\u00C2\u00A0death\u00C2\u00A0domain172.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0suggested\u00C2\u00A0that\u00C2\u00A0 the\u00C2\u00A0soluble\u00C2\u00A0form\u00C2\u00A0primarily\u00C2\u00A0activates\u00C2\u00A0CD120a\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0membrane\u00E2\u0080\u0090bound\u00C2\u00A0form\u00C2\u00A0activates\u00C2\u00A0CD120b195,\u00C2\u00A0 but\u00C2\u00A0CD120a\u00C2\u00A0appears\u00C2\u00A0to\u00C2\u00A0dominate\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0signaling\u00C2\u00A0in\u00C2\u00A0most\u00C2\u00A0cell\u00C2\u00A0types174,\u00C2\u00A0175.\u00C2\u00A0 TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0 plays\u00C2\u00A0 a\u00C2\u00A0 key\u00C2\u00A0 role\u00C2\u00A0 in\u00C2\u00A0 inflammation\u00C2\u00A0 and\u00C2\u00A0 immunity,\u00C2\u00A0 as\u00C2\u00A0 well\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 proliferation\u00C2\u00A0 and\u00C2\u00A0 differentiation\u00C2\u00A0of\u00C2\u00A0many\u00C2\u00A0different\u00C2\u00A0cell\u00C2\u00A0types177.\u00C2\u00A0\u00C2\u00A0Like\u00C2\u00A0almost\u00C2\u00A0all\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0TNFR\u00C2\u00A0ligands,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0expressed\u00C2\u00A0by\u00C2\u00A0 cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 immune\u00C2\u00A0system,\u00C2\u00A0 including\u00C2\u00A0B\u00C2\u00A0cells,\u00C2\u00A0T\u00C2\u00A0cells,\u00C2\u00A0NK\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0macrophages.\u00C2\u00A0 \u00C2\u00A0CD120a,\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 other\u00C2\u00A0hand,\u00C2\u00A0is\u00C2\u00A0expressed\u00C2\u00A0by\u00C2\u00A0every\u00C2\u00A0cell\u00C2\u00A0type\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0body\u00C2\u00A0(Section\u00C2\u00A05.2.3),\u00C2\u00A0and\u00C2\u00A0although\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0TNFR\u00C2\u00A0 family\u00C2\u00A0members\u00C2\u00A0are\u00C2\u00A0expressed\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0wide\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0cells,\u00C2\u00A0 they\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0done\u00C2\u00A0 so\u00C2\u00A0as\u00C2\u00A0ubiquitously\u00C2\u00A0as\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0123\u00C2\u00A0\u00C2\u00A0 CD120a177.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 almost\u00C2\u00A0 exclusively\u00C2\u00A0 expressed\u00C2\u00A0 at\u00C2\u00A0 constitutively\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0 and\u00C2\u00A0 is\u00C2\u00A0 controlled\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 noninducible\u00C2\u00A0promoter174.\u00C2\u00A0\u00C2\u00A0 CD120a\u00C2\u00A0 and\u00C2\u00A0other\u00C2\u00A0TNFRs\u00C2\u00A0 contain\u00C2\u00A0 cysteine\u00E2\u0080\u0090rich\u00C2\u00A0 repeats\u00C2\u00A0 in\u00C2\u00A0 their\u00C2\u00A0 extracellular\u00C2\u00A0domains\u00C2\u00A0 that\u00C2\u00A0 mediate\u00C2\u00A0homophilic\u00C2\u00A0 interactions\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0 ligand.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0preligand\u00C2\u00A0assembly\u00C2\u00A0domain\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0 membrane\u00E2\u0080\u0090distal\u00C2\u00A0first\u00C2\u00A0cysteine\u00E2\u0080\u0090rich\u00C2\u00A0domain,\u00C2\u00A0and\u00C2\u00A0 it\u00C2\u00A0enables\u00C2\u00A0TNFRs\u00C2\u00A0to\u00C2\u00A0form\u00C2\u00A0homotrimers180.\u00C2\u00A0 \u00C2\u00A0Binding\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0trivalent\u00C2\u00A0ligand\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0preformed\u00C2\u00A0complexes\u00C2\u00A0induces\u00C2\u00A0intracellular\u00C2\u00A0signal\u00C2\u00A0transduction.\u00C2\u00A0\u00C2\u00A0Ligand\u00C2\u00A0 binding\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0always\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0receptor,\u00C2\u00A0however,\u00C2\u00A0since\u00C2\u00A0signal\u00C2\u00A0transduction\u00C2\u00A0of\u00C2\u00A0 CD120a\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0reported\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0ligand181\u00E2\u0080\u0090186.\u00C2\u00A0\u00C2\u00A0 Once\u00C2\u00A0activated,\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0 inducing\u00C2\u00A0both\u00C2\u00A0proapoptotic\u00C2\u00A0and\u00C2\u00A0antiapoptotic\u00C2\u00A0signals.\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 further\u00C2\u00A0 detail\u00C2\u00A0 below,\u00C2\u00A0 the\u00C2\u00A0 signal\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 transmitted\u00C2\u00A0 is\u00C2\u00A0 ultimately\u00C2\u00A0 dictated\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 compartmentalization\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane175.\u00C2\u00A0 \u00C2\u00A0 Within\u00C2\u00A0 minutes\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 engagement\u00C2\u00A0 in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1\u00C2\u00A0or\u00C2\u00A0 independent\u00C2\u00A0of\u00C2\u00A0 ligand,\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0two\u00C2\u00A0complexes\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0formed:\u00C2\u00A0 Complex\u00C2\u00A0 I,\u00C2\u00A0which\u00C2\u00A0 regulates\u00C2\u00A0 the\u00C2\u00A0expression\u00C2\u00A0of\u00C2\u00A0antiapoptotic\u00C2\u00A0proteins,\u00C2\u00A0or\u00C2\u00A0Complex\u00C2\u00A0 II,\u00C2\u00A0which\u00C2\u00A0 triggers\u00C2\u00A0 cell\u00C2\u00A0death196,\u00C2\u00A0197.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Activation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0induces\u00C2\u00A0the\u00C2\u00A0recruitment\u00C2\u00A0of\u00C2\u00A0specific\u00C2\u00A0proteins\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 (TRADD,\u00C2\u00A0RIP1,\u00C2\u00A0TRAF2,\u00C2\u00A0and\u00C2\u00A0c\u00E2\u0080\u0090IAP1/2),\u00C2\u00A0resulting\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0Complex\u00C2\u00A0 I.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0proteins\u00C2\u00A0then\u00C2\u00A0 undergo\u00C2\u00A0post\u00E2\u0080\u0090translational\u00C2\u00A0modifications\u00C2\u00A0that\u00C2\u00A0lead\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0translocation\u00C2\u00A0of\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0nucleus\u00C2\u00A0and\u00C2\u00A0 transcription\u00C2\u00A0of\u00C2\u00A0antiapoptotic\u00C2\u00A0 target\u00C2\u00A0genes\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0 cFLIP,\u00C2\u00A0 cIAP\u00E2\u0080\u00901,\u00C2\u00A0 cIAP\u00E2\u0080\u00902,\u00C2\u00A0TRAF1,\u00C2\u00A0and\u00C2\u00A0TRAF2174,\u00C2\u00A0177.\u00C2\u00A0\u00C2\u00A0 Alternatively,\u00C2\u00A0 Complex\u00C2\u00A0 II\u00C2\u00A0 can\u00C2\u00A0 form,\u00C2\u00A0 which\u00C2\u00A0 contains\u00C2\u00A0 the\u00C2\u00A0 DISC\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 5.2.2.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 therefore\u00C2\u00A0 contains\u00C2\u00A0 procaspase\u00E2\u0080\u00908,\u00C2\u00A0 which\u00C2\u00A0 upon\u00C2\u00A0 cleavage\u00C2\u00A0 to\u00C2\u00A0 caspase\u00E2\u0080\u00908,\u00C2\u00A0 induces\u00C2\u00A0 apoptosis\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 extrinsic\u00C2\u00A0pathway.\u00C2\u00A0\u00C2\u00A0Complex\u00C2\u00A0II\u00C2\u00A0forms\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0where\u00C2\u00A0Complex\u00C2\u00A0I\u00C2\u00A0activates\u00C2\u00A0JNK\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0sustained\u00C2\u00A0 manner.\u00C2\u00A0 \u00C2\u00A0JNK\u00C2\u00A0 inhibits\u00C2\u00A0cFLIP,\u00C2\u00A0which\u00C2\u00A0 itself\u00C2\u00A0 inhibits\u00C2\u00A0the\u00C2\u00A0cleavage\u00C2\u00A0of\u00C2\u00A0procaspase\u00E2\u0080\u00908\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0DISC/Complex\u00C2\u00A0 II174,\u00C2\u00A0177.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0Complex\u00C2\u00A0 I\u00C2\u00A0 remains\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0while\u00C2\u00A0 clathrin\u00E2\u0080\u0090dependent\u00C2\u00A0 endocytosis\u00C2\u00A0 of\u00C2\u00A0 Complex\u00C2\u00A0 II\u00C2\u00A0 is\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 proapoptotic\u00C2\u00A0 signaling,\u00C2\u00A0 although\u00C2\u00A0 endocytosis\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0cell\u00E2\u0080\u0090type\u00C2\u00A0dependent175.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0124\u00C2\u00A0\u00C2\u00A0 Many\u00C2\u00A0 reports\u00C2\u00A0 have\u00C2\u00A0 demonstrated\u00C2\u00A0 that\u00C2\u00A0 Complex\u00C2\u00A0 I\u00C2\u00A0 efficiently\u00C2\u00A0 activates\u00C2\u00A0 NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0 only\u00C2\u00A0 when\u00C2\u00A0 present\u00C2\u00A0 in\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0 (see\u00C2\u00A0below).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0because\u00C2\u00A0 the\u00C2\u00A0necessary\u00C2\u00A0post\u00E2\u0080\u0090translational\u00C2\u00A0 modifications\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0activation\u00C2\u00A0can\u00C2\u00A0only\u00C2\u00A0take\u00C2\u00A0place\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0raft\u00C2\u00A0microenvironment174,\u00C2\u00A0177.\u00C2\u00A0\u00C2\u00A0 Outside\u00C2\u00A0of\u00C2\u00A0 rafts,\u00C2\u00A0Complex\u00C2\u00A0 I\u00C2\u00A0 therefore\u00C2\u00A0 activates\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0poorly,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0proapoptotic\u00C2\u00A0Complex\u00C2\u00A0 II\u00C2\u00A0 is\u00C2\u00A0 formed\u00C2\u00A0instead198.\u00C2\u00A0\u00C2\u00A0 6.2.2\u00C2\u00A0 Plasma\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 is\u00C2\u00A0 an\u00C2\u00A0 extremely\u00C2\u00A0 heterogeneous\u00C2\u00A0 environment.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 contrast\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 familiar\u00C2\u00A0\u00E2\u0080\u009Cfluid\u00C2\u00A0mosaic\u00E2\u0080\u009D\u00C2\u00A0model\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane199,\u00C2\u00A0the\u00C2\u00A0 local\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0proteins\u00C2\u00A0and\u00C2\u00A0 other\u00C2\u00A0 molecular\u00C2\u00A0 constituents\u00C2\u00A0 vary\u00C2\u00A0 considerably\u00C2\u00A0 across\u00C2\u00A0 the\u00C2\u00A0 membrane\u00C2\u00A0 surface200.\u00C2\u00A0 \u00C2\u00A0 Normal\u00C2\u00A0 cell\u00C2\u00A0 signaling\u00C2\u00A0events\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0 these\u00C2\u00A0variations\u00C2\u00A0 in\u00C2\u00A0 local\u00C2\u00A0protein\u00C2\u00A0concentrations,\u00C2\u00A0and\u00C2\u00A0disruption\u00C2\u00A0of\u00C2\u00A0 this\u00C2\u00A0 regulation\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 factor\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 pathogenesis\u00C2\u00A0 of\u00C2\u00A0many\u00C2\u00A0 diseases\u00C2\u00A0 including\u00C2\u00A0 cancer201\u00E2\u0080\u0090204.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 non\u00E2\u0080\u0090 random\u00C2\u00A0 arrangements\u00C2\u00A0 are\u00C2\u00A0 maintained\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 complex\u00C2\u00A0 interplay\u00C2\u00A0 that\u00C2\u00A0 separates\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 into\u00C2\u00A0 various\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 domains\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 clathrin\u00E2\u0080\u0090coated\u00C2\u00A0 pits,\u00C2\u00A0 caveolae,\u00C2\u00A0 and\u00C2\u00A0membrane\u00C2\u00A0 rafts205.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Plasma\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0are\u00C2\u00A0small\u00C2\u00A0(10\u00E2\u0080\u0093200\u00C2\u00A0nm)\u00C2\u00A0domains,\u00C2\u00A0enriched\u00C2\u00A0in\u00C2\u00A0cholesterol\u00C2\u00A0(Chol)\u00C2\u00A0and\u00C2\u00A0 sphingolipids,\u00C2\u00A0that\u00C2\u00A0compartmentalize\u00C2\u00A0cellular\u00C2\u00A0processes206.\u00C2\u00A0\u00C2\u00A0They\u00C2\u00A0are\u00C2\u00A0unstable\u00C2\u00A0and\u00C2\u00A0transient\u00C2\u00A0on\u00C2\u00A0their\u00C2\u00A0 own,\u00C2\u00A0but\u00C2\u00A0if\u00C2\u00A0stabilizing\u00C2\u00A0interactions\u00C2\u00A0arise,\u00C2\u00A0they\u00C2\u00A0aggregate\u00C2\u00A0into\u00C2\u00A0larger\u00C2\u00A0platforms207,\u00C2\u00A0and\u00C2\u00A0these\u00C2\u00A0platforms\u00C2\u00A0 play\u00C2\u00A0significant\u00C2\u00A0roles\u00C2\u00A0in\u00C2\u00A0cell\u00C2\u00A0signaling108,\u00C2\u00A0208.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 For\u00C2\u00A0example,\u00C2\u00A0 rafts\u00C2\u00A0and\u00C2\u00A0platforms\u00C2\u00A0 confine\u00C2\u00A0 specific\u00C2\u00A0proteins\u00C2\u00A0 to\u00C2\u00A0 small\u00C2\u00A0 regions\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0 membrane,\u00C2\u00A0and\u00C2\u00A0only\u00C2\u00A0proteins\u00C2\u00A0within\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0raft\u00C2\u00A0or\u00C2\u00A0platform\u00C2\u00A0may\u00C2\u00A0 interact\u00C2\u00A0with\u00C2\u00A0each\u00C2\u00A0other.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 limits\u00C2\u00A0the\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0proteins\u00C2\u00A0with\u00C2\u00A0which\u00C2\u00A0a\u00C2\u00A0raft\u00E2\u0080\u0090associated\u00C2\u00A0protein\u00C2\u00A0may\u00C2\u00A0interact209.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0addition,\u00C2\u00A0 membrane\u00C2\u00A0rafts\u00C2\u00A0are\u00C2\u00A0slightly\u00C2\u00A0thicker\u00C2\u00A0and\u00C2\u00A0more\u00C2\u00A0tightly\u00C2\u00A0packed\u00C2\u00A0than\u00C2\u00A0the\u00C2\u00A0contiguous\u00C2\u00A0fluid\u00C2\u00A0phase,\u00C2\u00A0which\u00C2\u00A0 may\u00C2\u00A0 impact\u00C2\u00A0protein\u00C2\u00A0conformation\u00C2\u00A0and\u00C2\u00A0thus\u00C2\u00A0function210.\u00C2\u00A0 \u00C2\u00A0Such\u00C2\u00A0spatial\u00C2\u00A0and\u00C2\u00A0temporal\u00C2\u00A0modulations\u00C2\u00A0of\u00C2\u00A0 local\u00C2\u00A0protein\u00C2\u00A0concentrations\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0play\u00C2\u00A0key\u00C2\u00A0roles\u00C2\u00A0in\u00C2\u00A0signal\u00C2\u00A0transduction,\u00C2\u00A0including,\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0125\u00C2\u00A0\u00C2\u00A0 for\u00C2\u00A0 example,\u00C2\u00A0 those\u00C2\u00A0 of\u00C2\u00A0 IgE\u00C2\u00A0 Fc\u00C2\u00A0 receptor\u00C2\u00A0 I\u00C2\u00A0 (Fc\u00CE\u00B5RI)\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 T\u00E2\u0080\u0090cell\u00C2\u00A0 antigen\u00C2\u00A0 receptor211\u00E2\u0080\u0090213,\u00C2\u00A0 as\u00C2\u00A0well\u00C2\u00A0 as\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 (see\u00C2\u00A0below).\u00C2\u00A0 \u00C2\u00A0Because\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 localization\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0protein\u00C2\u00A0often\u00C2\u00A0dictates\u00C2\u00A0 the\u00C2\u00A0 signals\u00C2\u00A0it\u00C2\u00A0transmits,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0surprising\u00C2\u00A0that\u00C2\u00A0such\u00C2\u00A0information\u00C2\u00A0is\u00C2\u00A0usually\u00C2\u00A0not\u00C2\u00A0specified\u00C2\u00A0in\u00C2\u00A0traditional\u00C2\u00A0linear\u00C2\u00A0 signaling\u00C2\u00A0pathways.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0protein\u00C2\u00A0 composition\u00C2\u00A0of\u00C2\u00A0 rafts\u00C2\u00A0and\u00C2\u00A0 signaling\u00C2\u00A0platforms\u00C2\u00A0 is\u00C2\u00A0highly\u00C2\u00A0dynamic214\u00E2\u0080\u0090217.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 raft\u00C2\u00A0 affinity\u00C2\u00A0of\u00C2\u00A0 a\u00C2\u00A0 given\u00C2\u00A0membrane\u00C2\u00A0protein\u00C2\u00A0 is\u00C2\u00A0 affected\u00C2\u00A0by\u00C2\u00A0 (and\u00C2\u00A0 affects)\u00C2\u00A0 the\u00C2\u00A0 formation\u00C2\u00A0of\u00C2\u00A0 stabilized\u00C2\u00A0 raft\u00C2\u00A0 platforms\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0lipid\u00E2\u0080\u0090lipid,\u00C2\u00A0protein\u00E2\u0080\u0090protein,\u00C2\u00A0and\u00C2\u00A0lipid\u00E2\u0080\u0090protein\u00C2\u00A0interactions207.\u00C2\u00A0 \u00C2\u00A0One\u00C2\u00A0 important\u00C2\u00A0 example\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0when\u00C2\u00A0 a\u00C2\u00A0moderately\u00C2\u00A0 raft\u00E2\u0080\u0090associating\u00C2\u00A0 protein\u00C2\u00A0 becomes\u00C2\u00A0 oligomerized\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 result\u00C2\u00A0 of\u00C2\u00A0 crosslinking,\u00C2\u00A0 its\u00C2\u00A0raft\u00C2\u00A0affinity\u00C2\u00A0usually\u00C2\u00A0 increases\u00C2\u00A0or\u00C2\u00A0decreases108,\u00C2\u00A0218\u00E2\u0080\u0090220.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0model\u00C2\u00A0 membrane\u00C2\u00A0systems221,\u00C2\u00A0222\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0live\u00C2\u00A0cells223,\u00C2\u00A0224.\u00C2\u00A0\u00C2\u00A0Clearly\u00C2\u00A0this\u00C2\u00A0has\u00C2\u00A0implications\u00C2\u00A0with\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0 constructs,\u00C2\u00A0which\u00C2\u00A0hypercrosslink\u00C2\u00A0 their\u00C2\u00A0 target\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane;\u00C2\u00A0 it\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0 that\u00C2\u00A0 hypercrosslinking\u00C2\u00A0would\u00C2\u00A0modulate\u00C2\u00A0the\u00C2\u00A0raft\u00C2\u00A0affinity\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0target.\u00C2\u00A0 In\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0many\u00C2\u00A0studies\u00C2\u00A0have\u00C2\u00A0documented\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0raft\u00C2\u00A0affinity\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0 increases\u00C2\u00A0as\u00C2\u00A0 early\u00C2\u00A0as\u00C2\u00A015\u00C2\u00A0min\u00C2\u00A0following\u00C2\u00A0Rtx\u00C2\u00A0treatment74,\u00C2\u00A0168,\u00C2\u00A0169.\u00C2\u00A0\u00C2\u00A0Rtx\u00C2\u00A0induces\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0CD20+\u00C2\u00A0lymphoma\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0 in\u00C2\u00A0culture\u00C2\u00A0only\u00C2\u00A0when\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0crosslinked\u00C2\u00A0with\u00C2\u00A0 reagents\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A02oAb\u00C2\u00A0 (Sections\u00C2\u00A01.4.3\u00C2\u00A0and\u00C2\u00A01.6.1),\u00C2\u00A0and\u00C2\u00A0 crosslinking\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0modulation\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0raft\u00C2\u00A0affinity;\u00C2\u00A0some\u00C2\u00A0reports\u00C2\u00A0have\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A0 apoptosis\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0occur\u00C2\u00A0after\u00C2\u00A0Rtx/2oAb\u00C2\u00A0 treatment\u00C2\u00A0 if\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0are\u00C2\u00A0disrupted\u00C2\u00A0 through\u00C2\u00A0Chol\u00C2\u00A0 depletion74.\u00C2\u00A0\u00C2\u00A0 With\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 relationship\u00C2\u00A0between\u00C2\u00A0 rafts\u00C2\u00A0 and\u00C2\u00A0CD120a,\u00C2\u00A0disrupting\u00C2\u00A0 rafts\u00C2\u00A0by\u00C2\u00A0depleting\u00C2\u00A0 cells\u00C2\u00A0 of\u00C2\u00A0 Chol\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 numerous\u00C2\u00A0 instances\u00C2\u00A0 to\u00C2\u00A0 switch\u00C2\u00A0 antiapoptotic\u00C2\u00A0 CD120a\u00C2\u00A0 signaling\u00C2\u00A0 (Complex\u00C2\u00A0 I)\u00C2\u00A0 to\u00C2\u00A0proapoptotic\u00C2\u00A0 signaling\u00C2\u00A0 (Complex\u00C2\u00A0 II).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0Chol\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 is\u00C2\u00A0 therefore\u00C2\u00A0 one\u00C2\u00A0 factor\u00C2\u00A0 that\u00C2\u00A0 influences\u00C2\u00A0 the\u00C2\u00A0 dual\u00C2\u00A0 nature\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 signaling174,\u00C2\u00A0 175,\u00C2\u00A0 177,\u00C2\u00A0 196\u00E2\u0080\u0090198.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 signaling\u00C2\u00A0of\u00C2\u00A0CD95\u00C2\u00A0 (Fas)\u00C2\u00A0has\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0rafts,\u00C2\u00A0but\u00C2\u00A0unlike\u00C2\u00A0CD120a,\u00C2\u00A0CD95\u00C2\u00A0triggers\u00C2\u00A0 apoptosis\u00C2\u00A0when\u00C2\u00A0 it\u00C2\u00A0 translocates\u00C2\u00A0 into\u00C2\u00A0 (rather\u00C2\u00A0 than\u00C2\u00A0 out\u00C2\u00A0 of)\u00C2\u00A0 rafts186.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 interplay\u00C2\u00A0 between\u00C2\u00A0 CD20,\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0126\u00C2\u00A0\u00C2\u00A0 CD120a,\u00C2\u00A0and\u00C2\u00A0rafts\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0may\u00C2\u00A0therefore\u00C2\u00A0give\u00C2\u00A0clues\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0 6.2.3\u00C2\u00A0 Studying\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0 6.2.3.1\u00C2\u00A0\u00C2\u00A0Detergent\u00C2\u00ADresistant\u00C2\u00A0membranes\u00C2\u00A0 The\u00C2\u00A0predominant\u00C2\u00A0biochemical\u00C2\u00A0means\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0potential\u00C2\u00A0affinity\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0protein\u00C2\u00A0to\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0rafts\u00C2\u00A0is\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0detergent\u00E2\u0080\u0090resistant\u00C2\u00A0membranes\u00C2\u00A0(DRMs).\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0was\u00C2\u00A0originally\u00C2\u00A0 observed\u00C2\u00A0 that\u00C2\u00A0 raft\u00C2\u00A0 constituents\u00C2\u00A0were\u00C2\u00A0 insoluble\u00C2\u00A0 in\u00C2\u00A0 cold\u00C2\u00A0 nonionic\u00C2\u00A0 detergents\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 Triton\u00C2\u00A0 X\u00E2\u0080\u0090100\u00C2\u00A0 (TX100),\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0resulting\u00C2\u00A0DRM\u00C2\u00A0fractions\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0isolated\u00C2\u00A0by\u00C2\u00A0flotation\u00C2\u00A0using\u00C2\u00A0equilibrium\u00C2\u00A0density\u00C2\u00A0 gradient\u00C2\u00A0centrifugation225.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0was\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0detergent\u00C2\u00A0resistance\u00C2\u00A0of\u00C2\u00A0proteins\u00C2\u00A0in\u00C2\u00A0DRMs\u00C2\u00A0was\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0 Chol\u00C2\u00A0content226,\u00C2\u00A0so\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0hypothesized\u00C2\u00A0that\u00C2\u00A0DRMs\u00C2\u00A0were\u00C2\u00A0equivalent\u00C2\u00A0to\u00C2\u00A0membrane\u00C2\u00A0rafts.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0became\u00C2\u00A0clear,\u00C2\u00A0however,\u00C2\u00A0that\u00C2\u00A0DRMs\u00C2\u00A0are\u00C2\u00A0in\u00C2\u00A0fact\u00C2\u00A0the\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0aggregation\u00C2\u00A0process\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0 not\u00C2\u00A0 fully\u00C2\u00A0 understood227\u00E2\u0080\u0090229.\u00C2\u00A0 \u00C2\u00A0Model\u00C2\u00A0membrane\u00C2\u00A0 studies\u00C2\u00A0have\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0 although\u00C2\u00A0 raft\u00C2\u00A0 lipids\u00C2\u00A0 resist\u00C2\u00A0 solubilization\u00C2\u00A0by\u00C2\u00A0detergent230,\u00C2\u00A0231,\u00C2\u00A0the\u00C2\u00A0detergent\u00C2\u00A0can\u00C2\u00A0also\u00C2\u00A0artificially\u00C2\u00A0 induce\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0rafts232.\u00C2\u00A0\u00C2\u00A0 DRM\u00C2\u00A0analysis\u00C2\u00A0has\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0highly\u00C2\u00A0cell\u00C2\u00A0type\u00E2\u0080\u0090dependent233.\u00C2\u00A0\u00C2\u00A0Although\u00C2\u00A0useful\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0tool\u00C2\u00A0to\u00C2\u00A0 initially\u00C2\u00A0 assess\u00C2\u00A0 raft\u00C2\u00A0 affinity,\u00C2\u00A0 DRMs\u00C2\u00A0 do\u00C2\u00A0 not\u00C2\u00A0 reflect\u00C2\u00A0 membrane\u00C2\u00A0 organization\u00C2\u00A0 under\u00C2\u00A0 physiological\u00C2\u00A0 conditions\u00C2\u00A0and\u00C2\u00A0therefore\u00C2\u00A0cannot\u00C2\u00A0be\u00C2\u00A0equated\u00C2\u00A0with\u00C2\u00A0rafts228.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Although\u00C2\u00A0 detergent\u00C2\u00A0 solubility\u00C2\u00A0 and\u00C2\u00A0 insolubility\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 strict\u00C2\u00A0 criteria\u00C2\u00A0 in\u00C2\u00A0 themselves,\u00C2\u00A0 the\u00C2\u00A0 usefulness\u00C2\u00A0of\u00C2\u00A0DRM\u00C2\u00A0analysis\u00C2\u00A0 lies\u00C2\u00A0 in\u00C2\u00A0observations\u00C2\u00A0of\u00C2\u00A0 changes\u00C2\u00A0 in\u00C2\u00A0DRM\u00C2\u00A0association\u00C2\u00A0upon\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0 physiologically\u00C2\u00A0 relevant\u00C2\u00A0 stimuli234.\u00C2\u00A0 \u00C2\u00A0 One\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 first\u00C2\u00A0 such\u00C2\u00A0 demonstrations\u00C2\u00A0 was\u00C2\u00A0 in\u00C2\u00A0 studies\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 allergic\u00C2\u00A0 immune\u00C2\u00A0 response,\u00C2\u00A0where\u00C2\u00A0 the\u00C2\u00A0multivalent\u00C2\u00A0 ligand\u00C2\u00A0 IgE\u00C2\u00A0 crosslinked\u00C2\u00A0 the\u00C2\u00A0Fc\u00CE\u00B5RI\u00C2\u00A0 receptor,\u00C2\u00A0which\u00C2\u00A0 became\u00C2\u00A0detergent\u00C2\u00A0 insoluble\u00C2\u00A0 along\u00C2\u00A0with\u00C2\u00A0 its\u00C2\u00A0downstream\u00C2\u00A0 effectors235.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0DRMs\u00C2\u00A0 therefore\u00C2\u00A0 represents\u00C2\u00A0a\u00C2\u00A0preliminary\u00C2\u00A0means\u00C2\u00A0of\u00C2\u00A0assessing\u00C2\u00A0raft\u00C2\u00A0affinity;\u00C2\u00A0differences\u00C2\u00A0 in\u00C2\u00A0detergent\u00C2\u00A0solubility\u00C2\u00A0before\u00C2\u00A0 and\u00C2\u00A0after\u00C2\u00A0biological\u00C2\u00A0stimuli\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0basis\u00C2\u00A0for\u00C2\u00A0raft\u00C2\u00A0affinity234.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0127\u00C2\u00A0\u00C2\u00A0 Alternative\u00C2\u00A0 approaches\u00C2\u00A0 to\u00C2\u00A0 study\u00C2\u00A0 rafts\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0described\u00C2\u00A0 including\u00C2\u00A0detergent\u00C2\u00A0 resistance\u00C2\u00A0 analysis\u00C2\u00A0on\u00C2\u00A0intact\u00C2\u00A0cells\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0cell\u00C2\u00A0lysate\u00C2\u00A0after\u00C2\u00A0ultracentrifugation236\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0preparation\u00C2\u00A0of\u00C2\u00A0 fractions\u00C2\u00A0enriched\u00C2\u00A0in\u00C2\u00A0raft\u00C2\u00A0constituents\u00C2\u00A0without\u00C2\u00A0the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0detergents237.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0common\u00C2\u00A0way\u00C2\u00A0to\u00C2\u00A0support\u00C2\u00A0 DRM\u00C2\u00A0data\u00C2\u00A0 is\u00C2\u00A0with\u00C2\u00A0confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy\u00C2\u00A0by\u00C2\u00A0observing\u00C2\u00A0 the\u00C2\u00A0colocalization\u00C2\u00A0 between\u00C2\u00A0 a\u00C2\u00A0 protein\u00C2\u00A0 of\u00C2\u00A0 interest\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A0 constituent\u00C2\u00A0 known\u00C2\u00A0 to\u00C2\u00A0 associate\u00C2\u00A0 constitutively\u00C2\u00A0with\u00C2\u00A0 rafts.\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0 frequently\u00C2\u00A0 employed\u00C2\u00A0 raft\u00C2\u00A0marker\u00C2\u00A0 is\u00C2\u00A0 ganglioside\u00C2\u00A0 GM1,\u00C2\u00A0which\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 detected\u00C2\u00A0with\u00C2\u00A0 fluorescently\u00C2\u00A0 labeled\u00C2\u00A0cholera\u00C2\u00A0toxin\u00C2\u00A0B\u00C2\u00A0subunit\u00C2\u00A0(CTX)224.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0further\u00C2\u00A0study\u00C2\u00A0the\u00C2\u00A0raft\u00C2\u00A0affinity\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0protein,\u00C2\u00A0data\u00C2\u00A0obtained\u00C2\u00A0 using\u00C2\u00A0DRMs\u00C2\u00A0and/or\u00C2\u00A0microscopy\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0examined\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0modulated\u00C2\u00A0Chol\u00C2\u00A0content\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 plasma\u00C2\u00A0membrane.\u00C2\u00A0\u00C2\u00A0Addition\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0new\u00C2\u00A0rafts,\u00C2\u00A0while\u00C2\u00A0decreasing\u00C2\u00A0the\u00C2\u00A0Chol\u00C2\u00A0 content\u00C2\u00A0disrupts\u00C2\u00A0raft\u00C2\u00A0integrity238.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 6.2.3.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0Manipulation\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0cholesterol\u00C2\u00A0content\u00C2\u00A0 MBCD\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0reagent\u00C2\u00A0commonly\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0remove\u00C2\u00A0Chol\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane201,\u00C2\u00A0238.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0 cyclic\u00C2\u00A0 oligosaccharide\u00C2\u00A0 with\u00C2\u00A0 hydrophilic\u00C2\u00A0 external\u00C2\u00A0 faces\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A0 hydrophobic\u00C2\u00A0 internal\u00C2\u00A0 cavity.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 mechanism\u00C2\u00A0by\u00C2\u00A0which\u00C2\u00A0it\u00C2\u00A0efficiently\u00C2\u00A0removes\u00C2\u00A0Chol\u00C2\u00A0is\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0its\u00C2\u00A0ability\u00C2\u00A0to\u00C2\u00A0lower\u00C2\u00A0the\u00C2\u00A0activation\u00C2\u00A0energy\u00C2\u00A0 for\u00C2\u00A0 Chol\u00C2\u00A0 efflux;\u00C2\u00A0 this\u00C2\u00A0 is\u00C2\u00A0 done\u00C2\u00A0 by\u00C2\u00A0 allowing\u00C2\u00A0 the\u00C2\u00A0 Chol\u00C2\u00A0 molecule\u00C2\u00A0 to\u00C2\u00A0 incorporate\u00C2\u00A0 directly\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 hydrophobic\u00C2\u00A0cavity\u00C2\u00A0without\u00C2\u00A0having\u00C2\u00A0to\u00C2\u00A0pass\u00C2\u00A0through\u00C2\u00A0an\u00C2\u00A0 intermediate\u00C2\u00A0aqueous\u00C2\u00A0phase.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0also\u00C2\u00A0this\u00C2\u00A0 property\u00C2\u00A0that\u00C2\u00A0allows\u00C2\u00A0MBCD\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0an\u00C2\u00A0efficient\u00C2\u00A0donor\u00C2\u00A0of\u00C2\u00A0Chol,\u00C2\u00A0so\u00C2\u00A0by\u00C2\u00A0employing\u00C2\u00A0MBCD\u00C2\u00A0complexed\u00C2\u00A0with\u00C2\u00A0 Chol\u00C2\u00A0 (MBCD/Chol),\u00C2\u00A0cellular\u00C2\u00A0Chol\u00C2\u00A0content\u00C2\u00A0 can\u00C2\u00A0be\u00C2\u00A0enriched239.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0understood\u00C2\u00A0 that\u00C2\u00A0MBCD\u00C2\u00A0disrupts\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0 by\u00C2\u00A0 removal\u00C2\u00A0 of\u00C2\u00A0 Chol,\u00C2\u00A0 but\u00C2\u00A0 some\u00C2\u00A0 caution\u00C2\u00A0must\u00C2\u00A0 be\u00C2\u00A0 observed\u00C2\u00A0 because\u00C2\u00A0 other\u00C2\u00A0 cellular\u00C2\u00A0effects\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0observed\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0MBCD\u00C2\u00A0 treatment\u00C2\u00A0 that\u00C2\u00A0are\u00C2\u00A0unrelated\u00C2\u00A0 to\u00C2\u00A0raft\u00C2\u00A0disruption,\u00C2\u00A0 such\u00C2\u00A0as\u00C2\u00A0disruption\u00C2\u00A0of\u00C2\u00A0exocytosis,\u00C2\u00A0blockage\u00C2\u00A0of\u00C2\u00A0clathrin\u00E2\u0080\u0090coated\u00C2\u00A0endocytic\u00C2\u00A0vesicles,\u00C2\u00A0and\u00C2\u00A0disruption\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 actin\u00C2\u00A0cytoskeleton210.\u00C2\u00A0 \u00C2\u00A0It\u00C2\u00A0 is\u00C2\u00A0therefore\u00C2\u00A0 important\u00C2\u00A0to\u00C2\u00A0use\u00C2\u00A0more\u00C2\u00A0than\u00C2\u00A0one\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0depletion\u00C2\u00A0to\u00C2\u00A0 verify\u00C2\u00A0results\u00C2\u00A0obtained\u00C2\u00A0with\u00C2\u00A0MBCD.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0128\u00C2\u00A0\u00C2\u00A0 Other\u00C2\u00A0methods\u00C2\u00A0of\u00C2\u00A0disrupting\u00C2\u00A0 rafts\u00C2\u00A0by\u00C2\u00A0Chol\u00C2\u00A0 interference\u00C2\u00A0 include\u00C2\u00A0 sequestration\u00C2\u00A0using\u00C2\u00A0 filipin,\u00C2\u00A0 nystatin\u00C2\u00A0or\u00C2\u00A0amphotericin,\u00C2\u00A0or\u00C2\u00A0inhibition\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0synthesis\u00C2\u00A0using\u00C2\u00A03\u00E2\u0080\u0090hydroxy\u00E2\u0080\u00903\u00E2\u0080\u0090methylglutaryl\u00C2\u00A0coenzyme\u00C2\u00A0 A\u00C2\u00A0 (HMG\u00E2\u0080\u0090CoA)\u00C2\u00A0 reductase\u00C2\u00A0 inhibitors,\u00C2\u00A0 also\u00C2\u00A0 known\u00C2\u00A0 as\u00C2\u00A0 statins240.\u00C2\u00A0 \u00C2\u00A0 For\u00C2\u00A0 example,\u00C2\u00A0 simvastatin\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 drug\u00C2\u00A0 prescribed\u00C2\u00A0 for\u00C2\u00A0 lowering\u00C2\u00A0 total\u00C2\u00A0Chol\u00C2\u00A0 levels\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0 control\u00C2\u00A0hypercholesterolemia\u00C2\u00A0and\u00C2\u00A0 to\u00C2\u00A0prevent\u00C2\u00A0 atherosclerosis.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 was\u00C2\u00A0 originally\u00C2\u00A0 marketed\u00C2\u00A0 under\u00C2\u00A0 the\u00C2\u00A0 trade\u00C2\u00A0 name\u00C2\u00A0 Zocor\u00C2\u00A0 but\u00C2\u00A0 is\u00C2\u00A0 now\u00C2\u00A0 available\u00C2\u00A0 generically\u00C2\u00A0 in\u00C2\u00A0most\u00C2\u00A0countries241.\u00C2\u00A0 \u00C2\u00A0Simvastatin\u00C2\u00A0 is\u00C2\u00A0also\u00C2\u00A0used\u00C2\u00A0 to\u00C2\u00A0 inhibit\u00C2\u00A0Chol\u00C2\u00A0synthesis\u00C2\u00A0 in\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0vitro.\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0disruption\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0by\u00C2\u00A0decreasing\u00C2\u00A0total\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0Chol\u00C2\u00A0 levels240,\u00C2\u00A0242,\u00C2\u00A0243.\u00C2\u00A0 6.2.4\u00C2\u00A0 Can\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0involve\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0vivo?\u00C2\u00A0 Chapter\u00C2\u00A05\u00C2\u00A0 illustrated\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 involves\u00C2\u00A0apoptosis\u00C2\u00A0resulting\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0 for\u00C2\u00A0different\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 constructs\u00C2\u00A0 (Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090MS),\u00C2\u00A0 suggesting\u00C2\u00A0 that\u00C2\u00A0 CD20\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 was\u00C2\u00A0 responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0enhanced\u00C2\u00A0therapeutic\u00C2\u00A0effects\u00C2\u00A0as\u00C2\u00A0opposed\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0liposomal\u00C2\u00A0or\u00C2\u00A0MS\u00C2\u00A0component\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 formulation.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0chapter\u00C2\u00A0extends\u00C2\u00A0 these\u00C2\u00A0 findings\u00C2\u00A0 to\u00C2\u00A0 investigate\u00C2\u00A0whether\u00C2\u00A0a\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0 mechanism\u00C2\u00A0 could\u00C2\u00A0occur\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0after\u00C2\u00A0normal\u00C2\u00A0Rtx\u00C2\u00A0 therapy\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0by\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090 expressing\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 immune\u00C2\u00A0system.\u00C2\u00A0 \u00C2\u00A0Such\u00C2\u00A0a\u00C2\u00A0mechanism\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0demonstrated\u00C2\u00A0 in\u00C2\u00A0 vivo,\u00C2\u00A0 and\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 clear\u00C2\u00A0whether\u00C2\u00A0 Fc\u00CE\u00B3R\u00C2\u00A0 on\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0would\u00C2\u00A0 be\u00C2\u00A0 capable\u00C2\u00A0 of\u00C2\u00A0 producing\u00C2\u00A0 sufficient\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 direct\u00C2\u00A0 therapeutic\u00C2\u00A0 effects.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 known,\u00C2\u00A0 however,\u00C2\u00A0 that\u00C2\u00A0 adequate\u00C2\u00A0 hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0is\u00C2\u00A0achieved\u00C2\u00A0by\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0ADCC\u00C2\u00A0(Sections\u00C2\u00A01.4.2\u00C2\u00A0&\u00C2\u00A02.2.2).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0known\u00C2\u00A0function\u00C2\u00A0and\u00C2\u00A0biology\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0(Section\u00C2\u00A06.2.1),\u00C2\u00A0the\u00C2\u00A0experiments\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0 chapter\u00C2\u00A0 also\u00C2\u00A0examine\u00C2\u00A0how\u00C2\u00A0modulation\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0Chol\u00C2\u00A0 content\u00C2\u00A0of\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 influences\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0increased\u00C2\u00A0and\u00C2\u00A0decreased\u00C2\u00A0 plasma\u00E2\u0080\u0090membrane\u00C2\u00A0Chol\u00C2\u00A0concentrations,\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0CD120a\u00C2\u00A0detergent\u00C2\u00A0resistance\u00C2\u00A0 and\u00C2\u00A0membrane\u00C2\u00A0 raft\u00C2\u00A0 localization\u00C2\u00A0 are\u00C2\u00A0 characterized\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0129\u00C2\u00A0\u00C2\u00A0 experiments\u00C2\u00A0 help\u00C2\u00A0 define\u00C2\u00A0 cases\u00C2\u00A0 where\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 more\u00C2\u00A0 or\u00C2\u00A0 less\u00C2\u00A0 sensitive\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00C2\u00A0 treatment,\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0of\u00C2\u00A0particular\u00C2\u00A0interest\u00C2\u00A0because\u00C2\u00A0clinical\u00C2\u00A0resistance\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0well\u00C2\u00A0understood139,\u00C2\u00A0 140.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 6.3\u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0methods\u00C2\u00A0 6.3.1\u00C2\u00A0 Materials\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0 \u00C2\u00A0 Alexa\u00C2\u00A0 Fluor\u00C2\u00A0488\u00E2\u0080\u0090labeled\u00C2\u00A0 cholera\u00C2\u00A0 toxin\u00C2\u00A0 subunit\u00C2\u00A0B\u00C2\u00A0 (A488\u00E2\u0080\u0090CTX),\u00C2\u00A0Texas\u00C2\u00A0Red\u00E2\u0080\u0090labeled\u00C2\u00A0phalloidin,\u00C2\u00A0 Annexin\u00E2\u0080\u0090V\u00C2\u00A0binding\u00C2\u00A0buffer,\u00C2\u00A0 recombinant\u00C2\u00A0human\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00C2\u00A0 labelled\u00C2\u00A0with\u00C2\u00A0 fluorescein\u00C2\u00A0 isothiocyanate\u00C2\u00A0 (Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC),\u00C2\u00A0 an\u00C2\u00A0 Alexa\u00C2\u00A0 Fluor\u00C2\u00A0 568\u00C2\u00A0 (A568)\u00C2\u00A0 protein\u00C2\u00A0 labeling\u00C2\u00A0 kit,\u00C2\u00A0 and\u00C2\u00A0 an\u00C2\u00A0 Alexa\u00C2\u00A0 Fluor\u00C2\u00A0 647\u00C2\u00A0 (A647)\u00C2\u00A0 monoclonal\u00C2\u00A0Ab\u00C2\u00A0labeling\u00C2\u00A0kit\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Invitrogen\u00C2\u00A0(Burlington\u00C2\u00A0ON,\u00C2\u00A0Canada).\u00C2\u00A0\u00C2\u00A0Alexa\u00C2\u00A0Fluor\u00C2\u00A0647\u00C2\u00A0 (A647)\u00E2\u0080\u0090labeled\u00C2\u00A0 Abs\u00C2\u00A0 against\u00C2\u00A0 CD120a\u00C2\u00A0 (A647\u00E2\u0080\u0090CD120a)\u00C2\u00A0 and\u00C2\u00A0 an\u00C2\u00A0 A647\u00E2\u0080\u0090labeled\u00C2\u00A0 mouse\u00C2\u00A0 IgG2a\u00C2\u00A0 negative\u00C2\u00A0 control\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0 from\u00C2\u00A0 AbD\u00C2\u00A0 Serotec\u00C2\u00A0 (Kidlington,\u00C2\u00A0 UK).\u00C2\u00A0 \u00C2\u00A0 Streptavidin\u00E2\u0080\u0090coated\u00C2\u00A0 polystyrene\u00C2\u00A0 microspheres\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0diameter\u00C2\u00A0of\u00C2\u00A0100\u00C2\u00A0nm\u00C2\u00A0(streptavidin\u00E2\u0080\u0090MS)\u00C2\u00A0were\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0Bangs\u00C2\u00A0Laboratories\u00C2\u00A0 (Fishers\u00C2\u00A0 IN,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0goat\u00C2\u00A0anti\u00E2\u0080\u0090human\u00E2\u0080\u0090Fc\u00CE\u00B3\u00E2\u0080\u0090fragment\u00C2\u00A0secondary\u00C2\u00A0Ab\u00C2\u00A0 (2oAb)\u00C2\u00A0with\u00C2\u00A0no\u00C2\u00A0reactivity\u00C2\u00A0toward\u00C2\u00A0 the\u00C2\u00A0Fab\u00C2\u00A0region\u00C2\u00A0and\u00C2\u00A0minimal\u00C2\u00A0reactivity\u00C2\u00A0toward\u00C2\u00A0Fc\u00C2\u00A0regions\u00C2\u00A0of\u00C2\u00A0IgG\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0species\u00C2\u00A0was\u00C2\u00A0purchased\u00C2\u00A0from\u00C2\u00A0 Jackson\u00C2\u00A0 ImmunoResearch\u00C2\u00A0 (West\u00C2\u00A0 Grove\u00C2\u00A0 PA,\u00C2\u00A0 USA).\u00C2\u00A0 \u00C2\u00A0 Human\u00C2\u00A0 peripheral\u00C2\u00A0 blood\u00C2\u00A0 mononuclear\u00C2\u00A0 cells\u00C2\u00A0 (PBMCs)\u00C2\u00A0 from\u00C2\u00A0healthy\u00C2\u00A0donors,\u00C2\u00A0CTL\u00E2\u0080\u0090Anti\u00E2\u0080\u0090Aggregate\u00E2\u0080\u0090Wash\u00C2\u00A0 supplement,\u00C2\u00A0 and\u00C2\u00A0CTL\u00E2\u0080\u0090Test\u00C2\u00A0medium\u00C2\u00A0were\u00C2\u00A0 purchased\u00C2\u00A0 from\u00C2\u00A0Cellular\u00C2\u00A0 Technology\u00C2\u00A0 Ltd.\u00C2\u00A0 (Cleveland\u00C2\u00A0OH,\u00C2\u00A0USA).\u00C2\u00A0 \u00C2\u00A0Unless\u00C2\u00A0noted\u00C2\u00A0otherwise,\u00C2\u00A0 all\u00C2\u00A0other\u00C2\u00A0 reagents\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0Sigma\u00E2\u0080\u0090Aldrich\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0highest\u00C2\u00A0quality\u00C2\u00A0available.\u00C2\u00A0 \u00C2\u00A0 Ramos\u00C2\u00A0and\u00C2\u00A0Z138\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0were\u00C2\u00A0acquired\u00C2\u00A0and\u00C2\u00A0maintained\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A04.3.2.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0130\u00C2\u00A0\u00C2\u00A0 6.3.2\u00C2\u00A0 Preparation\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00ADLNP\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00ADMS\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0methodology\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0and\u00C2\u00A0characterize\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 with\u00C2\u00A0valences\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0204.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0Two\u00C2\u00A0separate\u00C2\u00A0batches\u00C2\u00A0of\u00C2\u00A0four\u00C2\u00A0valences\u00C2\u00A0each\u00C2\u00A0were\u00C2\u00A0prepared.\u00C2\u00A0 \u00C2\u00A0Please\u00C2\u00A0see\u00C2\u00A0 Section\u00C2\u00A05.3.8\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0procedure\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0Rtx\u00E2\u0080\u0090MS.\u00C2\u00A0 6.3.3\u00C2\u00A0 Depletion\u00C2\u00A0and\u00C2\u00A0augmentation\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0cholesterol\u00C2\u00A0levels\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 deplete\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 of\u00C2\u00A0 Chol\u00C2\u00A0 using\u00C2\u00A0MBCD,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 different\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0MBCD\u00C2\u00A0for\u00C2\u00A0times\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0the\u00C2\u00A0toxicity\u00C2\u00A0of\u00C2\u00A0MBCD\u00C2\u00A0in\u00C2\u00A0each\u00C2\u00A0cell\u00C2\u00A0 line\u00C2\u00A0used.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0a\u00C2\u00A0final\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A00.2%\u00C2\u00A0MBCD\u00C2\u00A0(w/v)\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0toxic\u00C2\u00A0but\u00C2\u00A0concentrations\u00C2\u00A0 of\u00C2\u00A00.3%\u00C2\u00A0and\u00C2\u00A0higher\u00C2\u00A0were.\u00C2\u00A0\u00C2\u00A0MBCD\u00C2\u00A0treatment\u00C2\u00A0at\u00C2\u00A0concentrations\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A00.3%\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0 time\u00C2\u00A0 as\u00C2\u00A0 treatment\u00C2\u00A0 with\u00C2\u00A0 Rtx,\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 or\u00C2\u00A0 controls.\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 insert\u00C2\u00A0 additional\u00C2\u00A0 Chol\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0complexes\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0Chol\u00E2\u0080\u0090loaded\u00C2\u00A0MBCD\u00C2\u00A0(MBCD/Chol).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0 complexes\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0as\u00C2\u00A0previously\u00C2\u00A0described239\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0stock\u00C2\u00A0solution\u00C2\u00A0containing\u00C2\u00A03.5\u00C2\u00A0mM\u00C2\u00A0Chol\u00C2\u00A0 and\u00C2\u00A03.4%\u00C2\u00A0MBCD,\u00C2\u00A0representing\u00C2\u00A0a\u00C2\u00A025:1\u00C2\u00A0mass\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0MBCD\u00C2\u00A0to\u00C2\u00A0Chol.\u00C2\u00A0\u00C2\u00A0MBCD/Chol\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 way\u00C2\u00A0 as\u00C2\u00A0 MBCD.\u00C2\u00A0 \u00C2\u00A0 Reported\u00C2\u00A0 values\u00C2\u00A0 of\u00C2\u00A0 MBCD/Chol\u00C2\u00A0 added\u00C2\u00A0 are\u00C2\u00A0 with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 concentration\u00C2\u00A0of\u00C2\u00A0MBCD.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 reduce\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 Chol\u00C2\u00A0 through\u00C2\u00A0 inhibition\u00C2\u00A0 of\u00C2\u00A0 Chol\u00C2\u00A0 synthesis\u00C2\u00A0 using\u00C2\u00A0 simvastatin,\u00C2\u00A0a\u00C2\u00A0titration\u00C2\u00A0of\u00C2\u00A0simvastatin\u00C2\u00A0was\u00C2\u00A0performed\u00C2\u00A0in\u00C2\u00A0each\u00C2\u00A0cell\u00C2\u00A0line\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0its\u00C2\u00A0 toxicity.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0simvastatin\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0toxic\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5M,\u00C2\u00A0but\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A030\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0and\u00C2\u00A0 above,\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0 found\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0cytotoxic\u00C2\u00A0after\u00C2\u00A0a\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0 incubation\u00C2\u00A0 time.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0same\u00C2\u00A0way\u00C2\u00A0as\u00C2\u00A0MBCD\u00C2\u00A0and\u00C2\u00A0 MBCD/Chol,\u00C2\u00A0 simvastatin\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0or\u00C2\u00A0 controls,\u00C2\u00A0and\u00C2\u00A0 incubated\u00C2\u00A0 for\u00C2\u00A0up\u00C2\u00A0 to\u00C2\u00A016\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0 Analyses\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0previous\u00C2\u00A0sections.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0131\u00C2\u00A0\u00C2\u00A0 6.3.4\u00C2\u00A0 Use\u00C2\u00A0of\u00C2\u00A0flow\u00C2\u00A0cytometry\u00C2\u00A0to\u00C2\u00A0determine\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0or\u00C2\u00A0expression\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0cell\u00C2\u00ADsurface\u00C2\u00A0proteins\u00C2\u00A0 \u00C2\u00A0 Please\u00C2\u00A0see\u00C2\u00A0Sections\u00C2\u00A05.3.5\u00E2\u0080\u00935.3.6;\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0procedures\u00C2\u00A0were\u00C2\u00A0used.\u00C2\u00A0 6.3.5\u00C2\u00A0 Assay\u00C2\u00A0 for\u00C2\u00A0 quantifying\u00C2\u00A0 the\u00C2\u00A0 detergent\u00C2\u00A0 resistance\u00C2\u00A0 of\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00AD associated\u00C2\u00A0proteins\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0previously\u00C2\u00A0described\u00C2\u00A0method\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0quantify\u00C2\u00A0the\u00C2\u00A0degree\u00C2\u00A0to\u00C2\u00A0which\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 proteins\u00C2\u00A0 are\u00C2\u00A0 resistant\u00C2\u00A0 to\u00C2\u00A0 solubilization\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 nonionic\u00C2\u00A0 surfactant\u00C2\u00A0 TX100236.\u00C2\u00A0 \u00C2\u00A0 Briefly,\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0Rtx,\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0 and\u00C2\u00A0 stained\u00C2\u00A0with\u00C2\u00A0 a\u00C2\u00A0 fluorescently\u00C2\u00A0 tagged\u00C2\u00A0Ab\u00C2\u00A0directed\u00C2\u00A0 against\u00C2\u00A0 a\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 constituent\u00C2\u00A0 for\u00C2\u00A0which\u00C2\u00A0 the\u00C2\u00A0detergent\u00C2\u00A0 solubility\u00C2\u00A0was\u00C2\u00A0being\u00C2\u00A0measured\u00C2\u00A0 (treatment\u00C2\u00A0 and\u00C2\u00A0 staining\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0previous\u00C2\u00A0 sections).\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0 cold\u00C2\u00A0 PBSB,\u00C2\u00A0and\u00C2\u00A0mean\u00C2\u00A0fluorescence\u00C2\u00A0 intensities\u00C2\u00A0(MFIs)\u00C2\u00A0of\u00C2\u00A0 labeled\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0triplicate\u00C2\u00A0before\u00C2\u00A0 (MFIlab)\u00C2\u00A0 and\u00C2\u00A0 after\u00C2\u00A0 (MFIlab+TX100)\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 cold\u00C2\u00A0 TX100\u00C2\u00A0 at\u00C2\u00A0 a\u00C2\u00A0 concentration\u00C2\u00A0 of\u00C2\u00A0 0.2%\u00C2\u00A0 for\u00C2\u00A0 5\u00C2\u00A0min.\u00C2\u00A0\u00C2\u00A0 Autofluorescence\u00C2\u00A0 of\u00C2\u00A0 unlabeled\u00C2\u00A0 cells\u00C2\u00A0 was\u00C2\u00A0 also\u00C2\u00A0measured\u00C2\u00A0 before\u00C2\u00A0 (MFIauto)\u00C2\u00A0 and\u00C2\u00A0 after\u00C2\u00A0 (MFIauto+TX100)\u00C2\u00A0 TX100\u00C2\u00A0treatment.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0quantity\u00C2\u00A0defined\u00C2\u00A0as\u00C2\u00A0detergent\u00C2\u00A0resistance\u00C2\u00A0(DR)\u00C2\u00A0was\u00C2\u00A0then\u00C2\u00A0calculated\u00C2\u00A0as\u00C2\u00A0follows:\u00C2\u00A0\u00C2\u00A0 DR\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0=\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0(MFIlab+TX100\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0MFIauto+TX100)\u00C2\u00A0/\u00C2\u00A0(MFIlab\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0MFIauto)\u00C2\u00A0 The\u00C2\u00A0method\u00C2\u00A0was\u00C2\u00A0verified\u00C2\u00A0using\u00C2\u00A0GM1\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0positive\u00C2\u00A0control\u00C2\u00A0 (using\u00C2\u00A0A488\u00E2\u0080\u0090CTX)\u00C2\u00A0and\u00C2\u00A0CD95\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 negative\u00C2\u00A0control\u00C2\u00A0 (using\u00C2\u00A0A647\u00E2\u0080\u0090CD95).\u00C2\u00A0 \u00C2\u00A0GM1\u00C2\u00A0 is\u00C2\u00A0generally\u00C2\u00A0regarded\u00C2\u00A0as\u00C2\u00A0constitutively\u00C2\u00A0raft\u00E2\u0080\u0090associated201\u00C2\u00A0 while\u00C2\u00A0CD95\u00C2\u00A0is\u00C2\u00A0non\u00E2\u0080\u0090raft\u00E2\u0080\u0090associated\u00C2\u00A0in\u00C2\u00A0unstimulated\u00C2\u00A0cells198,\u00C2\u00A0244.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0positive\u00C2\u00A0control\u00C2\u00A0showed\u00C2\u00A0DR\u00C2\u00A0values\u00C2\u00A0 approximately\u00C2\u00A0equal\u00C2\u00A0 to\u00C2\u00A01\u00C2\u00A0while\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0negative\u00C2\u00A0 control,\u00C2\u00A0DR\u00C2\u00A0<\u00C2\u00A00.35\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0 shown).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 same\u00C2\u00A0 measurements\u00C2\u00A0were\u00C2\u00A0performed\u00C2\u00A0after\u00C2\u00A0Chol\u00C2\u00A0depletion\u00C2\u00A0using\u00C2\u00A00.2%\u00C2\u00A0MBCD\u00C2\u00A0for\u00C2\u00A016\u00C2\u00A0h,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 positive\u00C2\u00A0control\u00C2\u00A0dropped\u00C2\u00A0to\u00C2\u00A00.54\u00C2\u00A0or\u00C2\u00A0lower\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0negative\u00C2\u00A0control\u00C2\u00A0was\u00C2\u00A0unaffected\u00C2\u00A0(not\u00C2\u00A0 shown).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0experiments\u00C2\u00A0verified\u00C2\u00A0that\u00C2\u00A0DR\u00C2\u00A0values\u00C2\u00A0close\u00C2\u00A0to\u00C2\u00A01\u00C2\u00A0represent\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0proteins\u00C2\u00A0 (or\u00C2\u00A0 lipids)\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 detergent\u00E2\u0080\u0090resistant,\u00C2\u00A0 while\u00C2\u00A0 the\u00C2\u00A0 detergent\u00C2\u00A0 solubility\u00C2\u00A0 increases\u00C2\u00A0 as\u00C2\u00A0 DR\u00C2\u00A0 values\u00C2\u00A0 decrease.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0132\u00C2\u00A0\u00C2\u00A0 6.3.6\u00C2\u00A0 Confocal\u00C2\u00A0laser\u00C2\u00ADscanning\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0and\u00C2\u00A0imaged\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0procedure\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A05.3.7.\u00C2\u00A0 The\u00C2\u00A0 labeling\u00C2\u00A0of\u00C2\u00A0GM1/plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0with\u00C2\u00A0A488\u00E2\u0080\u0090CTX\u00C2\u00A0was\u00C2\u00A0 carried\u00C2\u00A0out\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0way\u00C2\u00A0as\u00C2\u00A0 A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 staining,\u00C2\u00A0 but\u00C2\u00A0 the\u00C2\u00A0 staining\u00C2\u00A0 of\u00C2\u00A0 F\u00E2\u0080\u0090actin\u00C2\u00A0 staining\u00C2\u00A0 using\u00C2\u00A0 Texas\u00C2\u00A0 Red\u00C2\u00A0 phalloidin,\u00C2\u00A0 required\u00C2\u00A0 additional\u00C2\u00A0steps.\u00C2\u00A0\u00C2\u00A0F\u00E2\u0080\u0090actin\u00C2\u00A0labeling\u00C2\u00A0was\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0immediately\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0step\u00C2\u00A0where\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0fixed\u00C2\u00A0 with\u00C2\u00A0paraformaldehyde.\u00C2\u00A0\u00C2\u00A0Cover\u00C2\u00A0slips\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0and\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0permeabilized\u00C2\u00A0with\u00C2\u00A075\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0 of\u00C2\u00A00.5%\u00C2\u00A0Triton\u00C2\u00A0X\u00E2\u0080\u0090100\u00C2\u00A0(TX100)\u00C2\u00A0 in\u00C2\u00A0HBS\u00C2\u00A0for\u00C2\u00A05\u00C2\u00A0min;\u00C2\u00A0samples\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0again\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0then\u00C2\u00A0a\u00C2\u00A025\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0 drop\u00C2\u00A0of\u00C2\u00A0PBSB\u00C2\u00A0containing\u00C2\u00A00.25\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A00.2\u00C2\u00A0U/\u00C2\u00B5L\u00C2\u00A0Texas\u00C2\u00A0Red\u00C2\u00A0phalloidin\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0for\u00C2\u00A020\u00C2\u00A0min\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0 stain\u00C2\u00A0F\u00E2\u0080\u0090actin,\u00C2\u00A0then\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0HBS.\u00C2\u00A0\u00C2\u00A0Sample\u00C2\u00A0preparation,\u00C2\u00A0image\u00C2\u00A0acquisition,\u00C2\u00A0and\u00C2\u00A0analysis\u00C2\u00A0proceeded\u00C2\u00A0 as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A05.3.7.\u00C2\u00A0 6.3.7\u00C2\u00A0 Experiments\u00C2\u00A0using\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0A568\u00C2\u00ADRtx\u00C2\u00A0 followed\u00C2\u00A0 by\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0or\u00C2\u00A02oAb\u00C2\u00ADMS\u00C2\u00A0 \u00C2\u00A0 Samples\u00C2\u00A0of\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0way\u00C2\u00A0as\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A05.3.8,\u00C2\u00A0 with\u00C2\u00A0Rtx\u00C2\u00A0being\u00C2\u00A0replaced\u00C2\u00A0with\u00C2\u00A02oAb.\u00C2\u00A0\u00C2\u00A0 A568\u00E2\u0080\u0090labeled\u00C2\u00A0Rtx\u00C2\u00A0 (A568\u00E2\u0080\u0090Rtx)\u00C2\u00A0was\u00C2\u00A0prepared\u00C2\u00A0using\u00C2\u00A0an\u00C2\u00A0A568\u00C2\u00A0protein\u00C2\u00A0 labeling\u00C2\u00A0kit\u00C2\u00A0according\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0experiments\u00C2\u00A0 involving\u00C2\u00A02oAb\u00E2\u0080\u0090MS,\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0pretreated\u00C2\u00A0 with\u00C2\u00A0saturating\u00C2\u00A0levels\u00C2\u00A0(see\u00C2\u00A0Figure\u00C2\u00A04.1)\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0(20\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0per\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium)\u00C2\u00A0 for\u00C2\u00A01\u00C2\u00A0h\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0medium\u00C2\u00A0to\u00C2\u00A0remove\u00C2\u00A0unbound\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0A568\u00E2\u0080\u0090Rtx,\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0 subsequently\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0or\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0for\u00C2\u00A0different\u00C2\u00A0times,\u00C2\u00A0depending\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0experiment.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0133\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A06.3.8\u00C2\u00A0 Coculture\u00C2\u00A0 of\u00C2\u00A0 treated\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 human\u00C2\u00A0 peripheral\u00C2\u00A0 blood\u00C2\u00A0 mononuclear\u00C2\u00A0cells\u00C2\u00A0 \u00C2\u00A0 A\u00C2\u00A0sample\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0labeled\u00C2\u00A0with\u00C2\u00A0A647\u00C2\u00A0using\u00C2\u00A0an\u00C2\u00A0A647\u00C2\u00A0monoclonal\u00C2\u00A0Ab\u00C2\u00A0labeling\u00C2\u00A0kit\u00C2\u00A0according\u00C2\u00A0 to\u00C2\u00A0the\u00C2\u00A0manufacturer\u00E2\u0080\u0099s\u00C2\u00A0 instructions\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0A647\u00E2\u0080\u0090Rtx.\u00C2\u00A0 \u00C2\u00A0A647\u00E2\u0080\u0090Rtx\u00C2\u00A0 induced\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0elevated\u00C2\u00A0 CD120a\u00C2\u00A0expression\u00C2\u00A0upon\u00C2\u00A0hypercrosslinking\u00C2\u00A0with\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0 to\u00C2\u00A0an\u00C2\u00A0equivalent\u00C2\u00A0extent\u00C2\u00A0as\u00C2\u00A0unlabeled\u00C2\u00A0Rtx\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0at\u00C2\u00A05.0\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0cells/mL\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A020\u00C2\u00A0\u00C2\u00B5g/mL\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0A647\u00E2\u0080\u0090Rtx\u00C2\u00A0 (see\u00C2\u00A0below)\u00C2\u00A0 or\u00C2\u00A0 an\u00C2\u00A0 equivalent\u00C2\u00A0 volume\u00C2\u00A0 of\u00C2\u00A0HBS\u00C2\u00A0 for\u00C2\u00A0 1\u00C2\u00A0h\u00C2\u00A0 at\u00C2\u00A0 37\u00C2\u00A0 oC.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0washed\u00C2\u00A0with\u00C2\u00A0CTL\u00E2\u0080\u0090Test\u00C2\u00A0 medium\u00C2\u00A0to\u00C2\u00A0remove\u00C2\u00A0unbound\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0A647\u00E2\u0080\u0090Rtx,\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0resuspended\u00C2\u00A0 in\u00C2\u00A0CTL\u00E2\u0080\u0090Test\u00C2\u00A0medium\u00C2\u00A0at\u00C2\u00A02.0\u00C2\u00A0\u00C3\u0097\u00C2\u00A0 106\u00C2\u00A0 cells/mL.\u00C2\u00A0 \u00C2\u00A0 Cryopreserved\u00C2\u00A0 human\u00C2\u00A0 PBMCs\u00C2\u00A0 were\u00C2\u00A0 thawed\u00C2\u00A0 using\u00C2\u00A0 CTL\u00E2\u0080\u0090Anti\u00E2\u0080\u0090Aggregate\u00E2\u0080\u0090Wash\u00C2\u00A0 supplement\u00C2\u00A0according\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0provider\u00E2\u0080\u0099s\u00C2\u00A0 instructions,\u00C2\u00A0and\u00C2\u00A0were\u00C2\u00A0resuspended\u00C2\u00A0 in\u00C2\u00A0CTL\u00E2\u0080\u0090Test\u00C2\u00A0medium\u00C2\u00A0at\u00C2\u00A0 4.0\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0cells/mL.\u00C2\u00A0\u00C2\u00A0Viability\u00C2\u00A0was\u00C2\u00A0>98%\u00C2\u00A0as\u00C2\u00A0assessed\u00C2\u00A0by\u00C2\u00A0trypan\u00C2\u00A0blue\u00C2\u00A0exclusion.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Appropriate\u00C2\u00A0volumes\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0combined\u00C2\u00A0to\u00C2\u00A0obtain\u00C2\u00A0different\u00C2\u00A0ratios\u00C2\u00A0of\u00C2\u00A0Ramos:PBMC\u00C2\u00A0cells\u00C2\u00A0 from\u00C2\u00A01:0\u00C2\u00A0to\u00C2\u00A01:8,\u00C2\u00A0and\u00C2\u00A0CTL\u00E2\u0080\u0090Test\u00C2\u00A0medium\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0to\u00C2\u00A0bring\u00C2\u00A0the\u00C2\u00A0final\u00C2\u00A0cell\u00C2\u00A0concentration\u00C2\u00A0to\u00C2\u00A01.0\u00C2\u00A0\u00C3\u0097\u00C2\u00A0106\u00C2\u00A0 cells/mL.\u00C2\u00A0 \u00C2\u00A0Co\u00E2\u0080\u0090incubation\u00C2\u00A0at\u00C2\u00A037\u00C2\u00A0oC\u00C2\u00A0was\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0proceed\u00C2\u00A0 for\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0 for\u00C2\u00A0measurement\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0or\u00C2\u00A0 apoptosis\u00C2\u00A0levels.\u00C2\u00A0\u00C2\u00A0Previous\u00C2\u00A0experiments\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0there\u00C2\u00A0were\u00C2\u00A0no\u00C2\u00A0differences\u00C2\u00A0between\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0 grown\u00C2\u00A0 in\u00C2\u00A0 complete\u00C2\u00A0 culture\u00C2\u00A0medium\u00C2\u00A0 (Section\u00C2\u00A0 4.3.2)\u00C2\u00A0 or\u00C2\u00A0 in\u00C2\u00A0 CTL\u00E2\u0080\u0090Test\u00C2\u00A0Medium\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0or\u00C2\u00A0apoptosis\u00C2\u00A0after\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Different\u00C2\u00A0 triple\u00E2\u0080\u0090staining\u00C2\u00A0 strategies\u00C2\u00A0were\u00C2\u00A0 employed\u00C2\u00A0 for\u00C2\u00A0 analysis,\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 all\u00C2\u00A0 cases,\u00C2\u00A0 positive\u00C2\u00A0 controls\u00C2\u00A0 consisted\u00C2\u00A0 of\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(158)\u00C2\u00A0 and\u00C2\u00A0 single\u00E2\u0080\u0090stained\u00C2\u00A0 with\u00C2\u00A0 each\u00C2\u00A0 fluorophore\u00C2\u00A0 and\u00C2\u00A0dual\u00E2\u0080\u0090stained\u00C2\u00A0 in\u00C2\u00A0 every\u00C2\u00A0 combination\u00C2\u00A0 (to\u00C2\u00A0 set\u00C2\u00A0 compensation)\u00C2\u00A0 and\u00C2\u00A0 triple\u00E2\u0080\u0090stained.\u00C2\u00A0 \u00C2\u00A0An\u00C2\u00A0 additional\u00C2\u00A0positive\u00C2\u00A0control\u00C2\u00A0for\u00C2\u00A0apoptosis\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A04.0\u00C2\u00A0\u00C2\u00B5M\u00C2\u00A0camptothecin\u00C2\u00A0 for\u00C2\u00A04\u00C2\u00A0h\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0employed.\u00C2\u00A0 \u00C2\u00A0Negative\u00C2\u00A0controls\u00C2\u00A0consisted\u00C2\u00A0of\u00C2\u00A0PBMCs\u00C2\u00A0only\u00C2\u00A0 (no\u00C2\u00A0Rtx),\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0only\u00C2\u00A0 (with\u00C2\u00A0and\u00C2\u00A0without\u00C2\u00A0Rtx),\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0 combination\u00C2\u00A0 stained\u00C2\u00A0with\u00C2\u00A0an\u00C2\u00A0A647\u00E2\u0080\u0090labeled\u00C2\u00A0negative\u00C2\u00A0 isotype\u00C2\u00A0control\u00C2\u00A0 (for\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0measurements).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0134\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0identify\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0PBMCs,\u00C2\u00A0the\u00C2\u00A0following\u00C2\u00A0preliminary\u00C2\u00A0experiments\u00C2\u00A0were\u00C2\u00A0carried\u00C2\u00A0 out.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0apoptosis\u00C2\u00A0measurements,\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0A647\u00E2\u0080\u0090Rtx,\u00C2\u00A0and\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0 co\u00E2\u0080\u0090incubation,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC\u00C2\u00A0and\u00C2\u00A0PI\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A04.3.8.\u00C2\u00A0 \u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 identified\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0A647\u00E2\u0080\u0090Rtx+\u00C2\u00A0 population,\u00C2\u00A0 and\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0within\u00C2\u00A0 this\u00C2\u00A0 population\u00C2\u00A0 were\u00C2\u00A0measured.\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0 the\u00C2\u00A0basis\u00C2\u00A0of\u00C2\u00A0 this\u00C2\u00A0population\u00C2\u00A0 identified\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0SSC\u00C2\u00A0versus\u00C2\u00A0FSC\u00C2\u00A0plot,\u00C2\u00A0apoptosis\u00C2\u00A0 levels\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 instead\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090Rtx,\u00C2\u00A0and\u00C2\u00A0excellent\u00C2\u00A0agreement\u00C2\u00A0was\u00C2\u00A0 obtained\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0 two\u00C2\u00A0 values\u00C2\u00A0 (within\u00C2\u00A03%;\u00C2\u00A0data\u00C2\u00A0not\u00C2\u00A0 shown).\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0negative\u00C2\u00A0 control\u00C2\u00A0 consisting\u00C2\u00A0of\u00C2\u00A0 PBMCs\u00C2\u00A0only\u00C2\u00A0revealed\u00C2\u00A0that\u00C2\u00A097%\u00C2\u00A0of\u00C2\u00A0PBMCs\u00C2\u00A0were\u00C2\u00A0excluded\u00C2\u00A0from\u00C2\u00A0this\u00C2\u00A0area.\u00C2\u00A0 For\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 CD120a,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 stained\u00C2\u00A0with\u00C2\u00A0 FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx\u00C2\u00A0 and\u00C2\u00A0A647\u00E2\u0080\u0090 CD120a\u00C2\u00A0before\u00C2\u00A0being\u00C2\u00A0 resuspended\u00C2\u00A0 in\u00C2\u00A0PBSB\u00C2\u00A0containing\u00C2\u00A0PI\u00C2\u00A0 (using\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0procedure\u00C2\u00A0as\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A0 5.3.6).\u00C2\u00A0 \u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 identified\u00C2\u00A0as\u00C2\u00A0FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx+,\u00C2\u00A0and\u00C2\u00A0within\u00C2\u00A0this\u00C2\u00A0population,\u00C2\u00A0A647\u00E2\u0080\u0090 CD120a\u00C2\u00A0 fluorescence\u00C2\u00A0 intensity\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0PI\u00E2\u0080\u0093\u00C2\u00A0population\u00C2\u00A0to\u00C2\u00A0quantify\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0 \u00C2\u00A0Using\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 strategy\u00C2\u00A0as\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0apoptosis\u00C2\u00A0measurements,\u00C2\u00A0a\u00C2\u00A0 sample\u00C2\u00A0of\u00C2\u00A0 cells\u00C2\u00A0 stained\u00C2\u00A0 only\u00C2\u00A0 with\u00C2\u00A0 PI\u00C2\u00A0 and\u00C2\u00A0 A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 showed\u00C2\u00A0 very\u00C2\u00A0 similar\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 mean\u00C2\u00A0 fluorescence\u00C2\u00A0 intensities\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0as\u00C2\u00A0the\u00C2\u00A0cells\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0FITC\u00E2\u0080\u0090anti\u00E2\u0080\u0090Rtx\u00C2\u00A0(within\u00C2\u00A05%;\u00C2\u00A0data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 These\u00C2\u00A0experiments\u00C2\u00A0 revealed\u00C2\u00A0 that\u00C2\u00A0gating\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0basis\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0defined\u00C2\u00A0 region\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0side\u00C2\u00A0scatter\u00C2\u00A0 (SSC)\u00C2\u00A0versus\u00C2\u00A0forward\u00C2\u00A0scatter\u00C2\u00A0(FSC)\u00C2\u00A0plot\u00C2\u00A0was\u00C2\u00A0an\u00C2\u00A0effective\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0identifying\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 mixture\u00C2\u00A0of\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 therefore\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 and\u00C2\u00A0 cocultured\u00C2\u00A0with\u00C2\u00A0PBMCs\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0above\u00C2\u00A0for\u00C2\u00A016\u00C2\u00A0h,\u00C2\u00A0then\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC,\u00C2\u00A0PI,\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090CD120a.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0before,\u00C2\u00A097%\u00C2\u00A0 of\u00C2\u00A0 PBMCs\u00C2\u00A0were\u00C2\u00A0 excluded\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 defined\u00C2\u00A0 area.\u00C2\u00A0 \u00C2\u00A0 Apoptosis\u00C2\u00A0 and\u00C2\u00A0 CD120a\u00C2\u00A0 levels\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0 simultaneously\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0cells\u00C2\u00A0(similar\u00C2\u00A0to\u00C2\u00A0Section\u00C2\u00A05.3.7)\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0either\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0or\u00C2\u00A0 with\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Triplicates\u00C2\u00A0were\u00C2\u00A0prepared\u00C2\u00A0for\u00C2\u00A0every\u00C2\u00A0cocultured\u00C2\u00A0sample.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0135\u00C2\u00A0\u00C2\u00A0 6.4\u00C2\u00A0\u00C2\u00A0 Results\u00C2\u00A0 6.4.1\u00C2\u00A0 Apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0cholesterol\u00C2\u00ADdependent\u00C2\u00A0 The\u00C2\u00A0biology\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0interesting\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0sense\u00C2\u00A0that\u00C2\u00A0this\u00C2\u00A0receptor\u00C2\u00A0can\u00C2\u00A0serve\u00C2\u00A0either\u00C2\u00A0an\u00C2\u00A0anti\u00E2\u0080\u0090 apoptotic\u00C2\u00A0 role\u00C2\u00A0 or\u00C2\u00A0 a\u00C2\u00A0 pro\u00E2\u0080\u0090apoptotic\u00C2\u00A0 one,\u00C2\u00A0 depending\u00C2\u00A0 on\u00C2\u00A0 whether\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 localized\u00C2\u00A0 in\u00C2\u00A0 Chol\u00E2\u0080\u0090dependent\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0 (Sections\u00C2\u00A06.2.1\u00C2\u00A0&\u00C2\u00A06.2.2).\u00C2\u00A0 \u00C2\u00A0Since\u00C2\u00A0CD120a\u00C2\u00A0was\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 involved\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx,\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0 examined\u00C2\u00A0 under\u00C2\u00A0 conditions\u00C2\u00A0 of\u00C2\u00A0 decreased\u00C2\u00A0 or\u00C2\u00A0 increased\u00C2\u00A0 concentrations\u00C2\u00A0 of\u00C2\u00A0 Chol\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 is\u00C2\u00A0because\u00C2\u00A0a\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0that\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0as\u00C2\u00A0 illustrated\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0previous\u00C2\u00A0chapter,\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0amplified\u00C2\u00A0or\u00C2\u00A0attenuated\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0 decreased\u00C2\u00A0or\u00C2\u00A0increased\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0 Using\u00C2\u00A0an\u00C2\u00A0established\u00C2\u00A0procedure\u00C2\u00A0(Section\u00C2\u00A06.3.5),\u00C2\u00A0we\u00C2\u00A0measured\u00C2\u00A0the\u00C2\u00A0detergent\u00C2\u00A0resistance\u00C2\u00A0(DR)\u00C2\u00A0 of\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0after\u00C2\u00A0different\u00C2\u00A0treatments\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0decreased\u00C2\u00A0or\u00C2\u00A0 increased\u00C2\u00A0 Chol\u00C2\u00A0 concentrations.\u00C2\u00A0 \u00C2\u00A0 Controls\u00C2\u00A0 to\u00C2\u00A0 verify\u00C2\u00A0 the\u00C2\u00A0 DR\u00C2\u00A0 measurements\u00C2\u00A0 were\u00C2\u00A0 performed\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A06.3.5.\u00C2\u00A0 \u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 incubated\u00C2\u00A0 for\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0 MBCD\u00C2\u00A0or\u00C2\u00A0MBCD/Chol\u00C2\u00A0along\u00C2\u00A0with\u00C2\u00A0HBS,\u00C2\u00A0Rtx,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(152),\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0and\u00C2\u00A0these\u00C2\u00A0data\u00C2\u00A0are\u00C2\u00A0illustrated\u00C2\u00A0in\u00C2\u00A0 Figure\u00C2\u00A06.1.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0general,\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0data\u00C2\u00A0support\u00C2\u00A0the\u00C2\u00A0Chol\u00C2\u00A0manipulation\u00C2\u00A0that\u00C2\u00A0occurred;\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 (gray\u00C2\u00A0bars)\u00C2\u00A0decreased\u00C2\u00A0as\u00C2\u00A0more\u00C2\u00A0Chol\u00C2\u00A0was\u00C2\u00A0removed\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0membrane\u00C2\u00A0with\u00C2\u00A0MBCD,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0 CD120a\u00C2\u00A0increased\u00C2\u00A0or\u00C2\u00A0stayed\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0when\u00C2\u00A0Chol\u00C2\u00A0was\u00C2\u00A0added.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0136\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A06.1\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0manipulation\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0Chol\u00C2\u00A0content\u00C2\u00A0using\u00C2\u00A0MBCD\u00C2\u00A0or\u00C2\u00A0MBCD/Chol\u00C2\u00A0significantly\u00C2\u00A0 impacts\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0modulate\u00C2\u00A0the\u00C2\u00A0Chol\u00C2\u00A0content\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0the\u00C2\u00A0indicated\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0MBCD\u00C2\u00A0or\u00C2\u00A0MBCD/Chol\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0simultaneously\u00C2\u00A0with\u00C2\u00A0 HBS\u00C2\u00A0or\u00C2\u00A0Rtx\u00C2\u00A0(A),\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(152)\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0(B).\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0all\u00C2\u00A0cases,\u00C2\u00A0a\u00C2\u00A010\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0Rtx\u00C2\u00A0dose\u00C2\u00A0was\u00C2\u00A0given\u00C2\u00A0to\u00C2\u00A02.5\u00C2\u00A0\u00C3\u0097\u00C2\u00A0105\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0500\u00C2\u00A0\u00C2\u00B5L\u00C2\u00A0medium,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0detergent\u00C2\u00A0 resistance\u00C2\u00A0 (DR)\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0after\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0 in\u00C2\u00A0samples\u00C2\u00A0obtained\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0population\u00C2\u00A0of\u00C2\u00A0treated\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0samples\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(152)\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0elevated\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 Chol\u00C2\u00A0 levels\u00C2\u00A0 from\u00C2\u00A0 added\u00C2\u00A0 Chol/MBCD\u00C2\u00A0 inhibited\u00C2\u00A0 the\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 that\u00C2\u00A0 occurred\u00C2\u00A0 under\u00C2\u00A0 normal\u00C2\u00A0 Chol\u00C2\u00A0 levels.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0same\u00C2\u00A0relative\u00C2\u00A0scales\u00C2\u00A0are\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0(A)\u00C2\u00A0and\u00C2\u00A0(B).\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 0 0.4 0.8 1.2 1.6 2 \u00E2\u0080\u0093 \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + \u00E2\u0080\u0093 \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + Re la ti ve \u00C2\u00A0va lu es [MBCD]\u00C2\u00A0/\u00C2\u00A0%\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 DR\u00C2\u00A0(CD120a) Early\u00C2\u00A0apop. HBS Rtx 0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A00.3\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A00\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0.3 A Chol 0 0.4 0.8 1.2 1.6 2 \u00E2\u0080\u0093 \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + \u00E2\u0080\u0093 \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + Re la ti ve \u00C2\u00A0va lu es [MBCD]\u00C2\u00A0/\u00C2\u00A0%\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 DR\u00C2\u00A0(CD120a) Early\u00C2\u00A0apop. Rtx\u00E2\u0080\u0090LNP(152) Rtx\u00E2\u0080\u0090MS 0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A00.3\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A00\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0.2\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 0.3 B Chol \u00E2\u0080\u00A2\u00C2\u00A0137\u00C2\u00A0\u00C2\u00A0 Figure\u00C2\u00A0 6.1A\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0 after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0 or\u00C2\u00A0 Rtx,\u00C2\u00A0 a\u00C2\u00A0 concentration\u00C2\u00A0 of\u00C2\u00A0 0.2%\u00C2\u00A0MBCD\u00C2\u00A0 reduced\u00C2\u00A0 the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 to\u00C2\u00A065%\u00C2\u00A0 (HBS)\u00C2\u00A0or\u00C2\u00A072%\u00C2\u00A0 (Rtx)\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0value\u00C2\u00A0measured\u00C2\u00A0under\u00C2\u00A0normal\u00C2\u00A0Chol\u00C2\u00A0 concentrations\u00C2\u00A0 (p\u00C2\u00A0 <\u00C2\u00A0 0.05\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 cases).\u00C2\u00A0 \u00C2\u00A0 Note\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0of\u00C2\u00A0HBS\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0quite\u00C2\u00A0 low,\u00C2\u00A0particularly\u00C2\u00A0when\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0(Figure\u00C2\u00A05.2).\u00C2\u00A0\u00C2\u00A0Figure\u00C2\u00A06.1A\u00C2\u00A0also\u00C2\u00A0demonstrates\u00C2\u00A0that\u00C2\u00A0both\u00C2\u00A0HBS\u00C2\u00A0and\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0showed\u00C2\u00A0 similar\u00C2\u00A0profiles\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 relationship\u00C2\u00A0between\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0under\u00C2\u00A0 different\u00C2\u00A0 Chol\u00C2\u00A0 concentrations;\u00C2\u00A0 specifically,\u00C2\u00A0 that\u00C2\u00A0 increased\u00C2\u00A0 apoptosis\u00C2\u00A0 occurred\u00C2\u00A0 under\u00C2\u00A0 conditions\u00C2\u00A0 of\u00C2\u00A0 decreased\u00C2\u00A0CD120a\u00C2\u00A0DR.\u00C2\u00A0 \u00C2\u00A0 Importantly,\u00C2\u00A0 these\u00C2\u00A0data\u00C2\u00A0 illustrate\u00C2\u00A0 that\u00C2\u00A0a\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A00.3%\u00C2\u00A0MBCD\u00C2\u00A0was\u00C2\u00A0 toxic\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0experiments,\u00C2\u00A0as\u00C2\u00A0shown\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0large\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0apoptosis\u00C2\u00A0 observed\u00C2\u00A0 from\u00C2\u00A00.2%\u00C2\u00A0 to\u00C2\u00A00.3%\u00C2\u00A0MBCD\u00C2\u00A0 in\u00C2\u00A0HBS\u00E2\u0080\u0090treated\u00C2\u00A0 cells\u00C2\u00A0 (6.8%\u00C2\u00A0 to\u00C2\u00A033%;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.0001).\u00C2\u00A0 \u00C2\u00A0 Insertion\u00C2\u00A0of\u00C2\u00A0 additional\u00C2\u00A0Chol\u00C2\u00A0into\u00C2\u00A0the\u00C2\u00A0membrane,\u00C2\u00A0however,\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0toxic\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0conditions\u00C2\u00A0tested,\u00C2\u00A0since\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0apoptosis\u00C2\u00A0remained\u00C2\u00A0low\u00C2\u00A0(or\u00C2\u00A0were\u00C2\u00A0reduced)\u00C2\u00A0after\u00C2\u00A0all\u00C2\u00A0MBCD/Chol\u00C2\u00A0treatments.\u00C2\u00A0 Under\u00C2\u00A0equivalent\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0manipulation,\u00C2\u00A0Figure\u00C2\u00A06.1B\u00C2\u00A0shows\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 and\u00C2\u00A0 CD120a\u00C2\u00A0 DR\u00C2\u00A0measured\u00C2\u00A0 after\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(152)\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090MS.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 data\u00C2\u00A0 illustrate,\u00C2\u00A0like\u00C2\u00A0Figure\u00C2\u00A06.1A,\u00C2\u00A0that\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0generally\u00C2\u00A0higher\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0was\u00C2\u00A0reduced.\u00C2\u00A0\u00C2\u00A0 One\u00C2\u00A0exception\u00C2\u00A0involves\u00C2\u00A0the\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(152)\u00C2\u00A0treatment\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.1B.\u00C2\u00A0\u00C2\u00A0When\u00C2\u00A0comparing\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 absence\u00C2\u00A0and\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0MBCD,\u00C2\u00A0the\u00C2\u00A00%\u00C2\u00A0and\u00C2\u00A00.2%\u00C2\u00A0MBCD\u00C2\u00A0treatments\u00C2\u00A0produced\u00C2\u00A0equivalent\u00C2\u00A0data\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A0 0.05)\u00C2\u00A0but\u00C2\u00A0both\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0were\u00C2\u00A0significantly\u00C2\u00A0reduced\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A00.3%\u00C2\u00A0 MBCD\u00C2\u00A0 sample\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 0%\u00C2\u00A0 sample\u00C2\u00A0 (p\u00C2\u00A0 <\u00C2\u00A0 0.05\u00C2\u00A0 for\u00C2\u00A0 both\u00C2\u00A0 values).\u00C2\u00A0 \u00C2\u00A0 Separate\u00C2\u00A0 experiments\u00C2\u00A0 revealed\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0pretreatment\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0with\u00C2\u00A00.3%\u00C2\u00A0MBCD\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0an\u00C2\u00A0abolishment\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0construct\u00C2\u00A0to\u00C2\u00A0 induce\u00C2\u00A0apoptosis,\u00C2\u00A0most\u00C2\u00A0 likely\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0disruption\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0structure\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0LNP,\u00C2\u00A0which\u00C2\u00A0 contains\u00C2\u00A0Chol.\u00C2\u00A0 \u00C2\u00A0Pretreatment\u00C2\u00A0with\u00C2\u00A00.3%\u00C2\u00A0MBCD/Chol\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0affect\u00C2\u00A0the\u00C2\u00A0apoptotic\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Equivalent\u00C2\u00A0experiments\u00C2\u00A0were\u00C2\u00A0therefore\u00C2\u00A0performed\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0construct\u00C2\u00A0 that\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0contain\u00C2\u00A0Chol,\u00C2\u00A0and\u00C2\u00A0as\u00C2\u00A0opposed\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(152)\u00C2\u00A0treatment,\u00C2\u00A0the\u00C2\u00A0data\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0right\u00E2\u0080\u0090hand\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0138\u00C2\u00A0\u00C2\u00A0 side\u00C2\u00A0of\u00C2\u00A0Figure\u00C2\u00A06.1B\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A00%,\u00C2\u00A00.2%,\u00C2\u00A0and\u00C2\u00A00.3%\u00C2\u00A0MBCD\u00C2\u00A0treatment\u00C2\u00A0resulted\u00C2\u00A0 in\u00C2\u00A0equivalent\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 apoptosis\u00C2\u00A0and\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 (p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0 for\u00C2\u00A0all\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0parameter).\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0even\u00C2\u00A0 though\u00C2\u00A0 0.3%\u00C2\u00A0MBCD\u00C2\u00A0was\u00C2\u00A0 toxic\u00C2\u00A0 to\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0multivalent\u00C2\u00A0 Rtx,\u00C2\u00A0 no\u00C2\u00A0 further\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0 apoptosis\u00C2\u00A0(or\u00C2\u00A0decrease\u00C2\u00A0in\u00C2\u00A0CD120a\u00C2\u00A0DR)\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0 constructs\u00C2\u00A0tested.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0most\u00C2\u00A0important\u00C2\u00A0observation\u00C2\u00A0from\u00C2\u00A0Figure\u00C2\u00A06.1B\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0increased\u00C2\u00A0Chol\u00C2\u00A0 content\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0 the\u00C2\u00A0apoptotic\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0was\u00C2\u00A0strikingly\u00C2\u00A0reduced.\u00C2\u00A0\u00C2\u00A0 For\u00C2\u00A0the\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(152)\u00C2\u00A0treatment,\u00C2\u00A0a\u00C2\u00A0comparison\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A00%\u00C2\u00A0and\u00C2\u00A00.3%\u00C2\u00A0MBCD/Chol\u00C2\u00A0concentrations\u00C2\u00A0shows\u00C2\u00A0 that\u00C2\u00A0 apoptosis\u00C2\u00A0 was\u00C2\u00A0 reduced\u00C2\u00A0 from\u00C2\u00A0 34%\u00C2\u00A0 to\u00C2\u00A0 7.9%\u00C2\u00A0 (p\u00C2\u00A0 <\u00C2\u00A0 0.0001)\u00C2\u00A0 while\u00C2\u00A0 the\u00C2\u00A0 relative\u00C2\u00A0 DR\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 increased\u00C2\u00A0from\u00C2\u00A00.61\u00C2\u00A0to\u00C2\u00A00.79\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0same\u00C2\u00A0MBCD/Choltreatments\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0 reduced\u00C2\u00A0apoptosis\u00C2\u00A0from\u00C2\u00A022%\u00C2\u00A0to\u00C2\u00A07.5%\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.0005)\u00C2\u00A0and\u00C2\u00A0 increased\u00C2\u00A0relative\u00C2\u00A0DR\u00C2\u00A0from\u00C2\u00A00.73\u00C2\u00A0to\u00C2\u00A00.92\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A0 0.05).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0both\u00C2\u00A0cases,\u00C2\u00A0the\u00C2\u00A0 level\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A00.2%\u00C2\u00A0or\u00C2\u00A00.3%\u00C2\u00A0 MBCD/Chol\u00C2\u00A0was\u00C2\u00A0equivalent\u00C2\u00A0to\u00C2\u00A0that\u00C2\u00A0which\u00C2\u00A0was\u00C2\u00A0measured\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0HBS\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0 MBCD\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0in\u00C2\u00A0all\u00C2\u00A0cases).\u00C2\u00A0 To\u00C2\u00A0 further\u00C2\u00A0 investigate\u00C2\u00A0 the\u00C2\u00A0 phenomenon\u00C2\u00A0 where\u00C2\u00A0 increased\u00C2\u00A0 Chol\u00C2\u00A0 content\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 rescues\u00C2\u00A0 cells\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 apoptosis\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0multivalent\u00C2\u00A0 Rtx,\u00C2\u00A0 confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy\u00C2\u00A0images\u00C2\u00A0were\u00C2\u00A0obtained\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0 of\u00C2\u00A0normal\u00C2\u00A0or\u00C2\u00A0increased\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0Chol\u00C2\u00A0content.\u00C2\u00A0\u00C2\u00A0Figure\u00C2\u00A06.2\u00C2\u00A0shows\u00C2\u00A0representative\u00C2\u00A0images\u00C2\u00A0of\u00C2\u00A0 Ramos\u00C2\u00A0cells\u00C2\u00A0obtained\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment;\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0stained\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A06.3.6.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0blue\u00C2\u00A0 signal\u00C2\u00A0represents\u00C2\u00A0staining\u00C2\u00A0by\u00C2\u00A0A488\u00E2\u0080\u0090CTX,\u00C2\u00A0which\u00C2\u00A0stains\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0raft\u00C2\u00A0marker\u00C2\u00A0ganglioside\u00C2\u00A0 GM1,\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0red\u00C2\u00A0signal\u00C2\u00A0represents\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0staining.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0two\u00C2\u00A0signals\u00C2\u00A0are\u00C2\u00A0shown\u00C2\u00A0on\u00C2\u00A0their\u00C2\u00A0 own\u00C2\u00A0 (first\u00C2\u00A0and\u00C2\u00A0 second\u00C2\u00A0panels\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 left)\u00C2\u00A0and\u00C2\u00A0merged\u00C2\u00A0 (third\u00C2\u00A0panels).\u00C2\u00A0 \u00C2\u00A0Colocalization\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0 blue\u00C2\u00A0and\u00C2\u00A0red\u00C2\u00A0signals\u00C2\u00A0indicates\u00C2\u00A0CD120a\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0139\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A06.2\u00C2\u00A0\u00C2\u00A0Cells\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0rescued\u00C2\u00A0from\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0increasing\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0Chol\u00C2\u00A0content\u00C2\u00A0show\u00C2\u00A0elevated\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0raft\u00E2\u0080\u0090associated\u00C2\u00A0CD120a.\u00C2\u00A0\u00C2\u00A0 Representative\u00C2\u00A0 images\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0either\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0 (A)\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0 +0.2%\u00C2\u00A0MBCD/Chol\u00C2\u00A0(B)\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0conditions\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.1.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A06.3.6,\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0stained\u00C2\u00A0before\u00C2\u00A0 permeabilization\u00C2\u00A0with\u00C2\u00A0A488\u00E2\u0080\u0090CTX\u00C2\u00A0(blue;\u00C2\u00A0stains\u00C2\u00A0ganglioside\u00C2\u00A0GM1,\u00C2\u00A0a\u00C2\u00A0membrane\u00C2\u00A0raft\u00C2\u00A0marker)\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0(red),\u00C2\u00A0and\u00C2\u00A0after\u00C2\u00A0 permeabilization\u00C2\u00A0with\u00C2\u00A0Texas\u00C2\u00A0Red\u00E2\u0080\u0090labeled\u00C2\u00A0phalloidin\u00C2\u00A0(green;\u00C2\u00A0stains\u00C2\u00A0the\u00C2\u00A0actin\u00C2\u00A0cytoskeleton).\u00C2\u00A0 \u00C2\u00A0Panels,\u00C2\u00A0from\u00C2\u00A0 left\u00C2\u00A0to\u00C2\u00A0right,\u00C2\u00A0are:\u00C2\u00A0 ganglioside\u00C2\u00A0GM1\u00C2\u00A0staining\u00C2\u00A0only,\u00C2\u00A0CD120a\u00C2\u00A0staining\u00C2\u00A0only,\u00C2\u00A0a\u00C2\u00A0merge\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0ganglioside\u00C2\u00A0GM1\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0signals,\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0merge\u00C2\u00A0of\u00C2\u00A0 all\u00C2\u00A0three\u00C2\u00A0signals.\u00C2\u00A0 \u00C2\u00A0 When\u00C2\u00A0comparing\u00C2\u00A0the\u00C2\u00A0membrane\u00C2\u00A0raft\u00C2\u00A0association\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0between\u00C2\u00A0Figure\u00C2\u00A06.2A\u00C2\u00A0and\u00C2\u00A06.2B,\u00C2\u00A0it\u00C2\u00A0 is\u00C2\u00A0 evident\u00C2\u00A0 that\u00C2\u00A0 CD120a\u00C2\u00A0 was\u00C2\u00A0 almost\u00C2\u00A0 exclusively\u00C2\u00A0 found\u00C2\u00A0 in\u00C2\u00A0 membrane\u00C2\u00A0 rafts\u00C2\u00A0 under\u00C2\u00A0 conditions\u00C2\u00A0 of\u00C2\u00A0 increased\u00C2\u00A0Chol\u00C2\u00A0 (Figure\u00C2\u00A06.2B),\u00C2\u00A0while\u00C2\u00A0a\u00C2\u00A0greater\u00C2\u00A0 fraction\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0was\u00C2\u00A0 found\u00C2\u00A0outside\u00C2\u00A0of\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0under\u00C2\u00A0normal\u00C2\u00A0Chol\u00C2\u00A0concentrations\u00C2\u00A0(Figure\u00C2\u00A06.2A).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0same\u00C2\u00A0result\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0across\u00C2\u00A0a\u00C2\u00A0large\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0 images.\u00C2\u00A0 \u00C2\u00A0 Evidence\u00C2\u00A0 of\u00C2\u00A0 higher\u00C2\u00A0 plasma\u00E2\u0080\u0090membrane\u00C2\u00A0 Chol\u00C2\u00A0 concentrations\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 6.2B\u00C2\u00A0 is\u00C2\u00A0 provided\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0greater\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0covered\u00C2\u00A0by\u00C2\u00A0rafts\u00C2\u00A0(blue\u00C2\u00A0signal)\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0 Figure\u00C2\u00A06.2A.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 rightmost\u00C2\u00A0panels\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 6.2\u00C2\u00A0 show\u00C2\u00A0 the\u00C2\u00A0A488\u00E2\u0080\u0090CTX\u00C2\u00A0 and\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 signals\u00C2\u00A0merged\u00C2\u00A0 with\u00C2\u00A0 the\u00C2\u00A0 signal\u00C2\u00A0 from\u00C2\u00A0 Texas\u00C2\u00A0 Red\u00C2\u00A0 phalloidin,\u00C2\u00A0which\u00C2\u00A0 stains\u00C2\u00A0 F\u00E2\u0080\u0090actin.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 illustrated\u00C2\u00A0 F\u00E2\u0080\u0090actin\u00C2\u00A0 staining\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0\u00C2\u00A0+0.2%\u00C2\u00A0MBCD/Chol A B Rtx\u00E2\u0080\u0090LNP(165) \u00E2\u0080\u00A2\u00C2\u00A0140\u00C2\u00A0\u00C2\u00A0 pattern\u00C2\u00A0consisting\u00C2\u00A0of\u00C2\u00A0wavy\u00C2\u00A0ring\u00E2\u0080\u0090like\u00C2\u00A0structures\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0outer\u00C2\u00A0rim\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0 is\u00C2\u00A0characteristic\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 cortical\u00C2\u00A0actin\u00C2\u00A0cytoskeleton\u00C2\u00A0and\u00C2\u00A0 is\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0other\u00C2\u00A0reports245.\u00C2\u00A0 \u00C2\u00A0F\u00E2\u0080\u0090actin\u00C2\u00A0staining\u00C2\u00A0was\u00C2\u00A0 less\u00C2\u00A0 intense\u00C2\u00A0 and\u00C2\u00A0 less\u00C2\u00A0uniform\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 surface\u00C2\u00A0of\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(165)\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0MBCD/Chol\u00C2\u00A0 compared\u00C2\u00A0to\u00C2\u00A0in\u00C2\u00A0its\u00C2\u00A0presence.\u00C2\u00A0\u00C2\u00A0F\u00E2\u0080\u0090actin\u00C2\u00A0staining\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0completely\u00C2\u00A0absent\u00C2\u00A0in\u00C2\u00A0necrotic\u00C2\u00A0cells,\u00C2\u00A0which\u00C2\u00A0only\u00C2\u00A0 showed\u00C2\u00A0high\u00C2\u00A0 intracellular\u00C2\u00A0 staining\u00C2\u00A0of\u00C2\u00A0A488\u00E2\u0080\u0090CTX\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090CD120a,\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0Figure\u00C2\u00A05.3\u00C2\u00A0 (not\u00C2\u00A0 shown).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0F\u00E2\u0080\u0090actin\u00C2\u00A0staining\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.2\u00C2\u00A0confirms\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0observed\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0rafts\u00C2\u00A0are\u00C2\u00A0present\u00C2\u00A0 on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface,\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0considered\u00C2\u00A0either\u00C2\u00A0viable\u00C2\u00A0or\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0using\u00C2\u00A0 the\u00C2\u00A0Annexin\u00E2\u0080\u0090V/PI\u00C2\u00A0apoptosis\u00C2\u00A0assay.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 6.4.2\u00C2\u00A0 Inhibition\u00C2\u00A0of\u00C2\u00A0cholesterol\u00C2\u00A0synthesis\u00C2\u00A0using\u00C2\u00A0simvastatin\u00C2\u00A0sensitizes\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00AD LNP\u00C2\u00ADinduced\u00C2\u00A0apoptosis\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 MBCD\u00C2\u00A0is\u00C2\u00A0widely\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0deplete\u00C2\u00A0Chol\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0disruption\u00C2\u00A0 of\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts,\u00C2\u00A0 but\u00C2\u00A0MBCD\u00C2\u00A0 is\u00C2\u00A0 known\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 secondary\u00C2\u00A0 effects\u00C2\u00A0 on\u00C2\u00A0 some\u00C2\u00A0 cells,\u00C2\u00A0 as\u00C2\u00A0 described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A06.2.3.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0an\u00C2\u00A0alternative\u00C2\u00A0means\u00C2\u00A0of\u00C2\u00A0reducing\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0Chol\u00C2\u00A0levels,\u00C2\u00A0the\u00C2\u00A0 HMG\u00E2\u0080\u0090CoA\u00C2\u00A0reductase\u00C2\u00A0 inhibitor\u00C2\u00A0simvastatin\u00C2\u00A0was\u00C2\u00A0employed,\u00C2\u00A0which\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0disrupt\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0 rafts240,\u00C2\u00A0242,\u00C2\u00A0243.\u00C2\u00A0 \u00C2\u00A0Unlike\u00C2\u00A0MBCD,\u00C2\u00A0which\u00C2\u00A0 removes\u00C2\u00A0Chol\u00C2\u00A0 from\u00C2\u00A0 lipid\u00C2\u00A0bilayers,\u00C2\u00A0simvastatin\u00C2\u00A0 is\u00C2\u00A0an\u00C2\u00A0 inhibitor\u00C2\u00A0 of\u00C2\u00A0 Chol\u00C2\u00A0 synthesis.\u00C2\u00A0 \u00C2\u00A0 As\u00C2\u00A0 opposed\u00C2\u00A0 to\u00C2\u00A0 MBCD,\u00C2\u00A0 therefore,\u00C2\u00A0 pretreatment\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 with\u00C2\u00A0 simvastatin\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0(30\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0for\u00C2\u00A016\u00C2\u00A0h)\u00C2\u00A0had\u00C2\u00A0no\u00C2\u00A0effect\u00C2\u00A0on\u00C2\u00A0its\u00C2\u00A0apoptotic\u00C2\u00A0potency\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0 shown).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 behavior\u00C2\u00A0 of\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0 with\u00C2\u00A0 simvastatin\u00C2\u00A0 along\u00C2\u00A0 with\u00C2\u00A0 Rtx\u00C2\u00A0 or\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 was\u00C2\u00A0 therefore\u00C2\u00A0examined,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0resulting\u00C2\u00A0data\u00C2\u00A0 is\u00C2\u00A0summarized\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A06.3.\u00C2\u00A0 \u00C2\u00A0As\u00C2\u00A0was\u00C2\u00A0the\u00C2\u00A0case\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A0 6.1,\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0 obtained\u00C2\u00A0 for\u00C2\u00A0HBS\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00C2\u00A0 treatment\u00C2\u00A0were\u00C2\u00A0 very\u00C2\u00A0 similar.\u00C2\u00A0 \u00C2\u00A0 Both\u00C2\u00A0 of\u00C2\u00A0 these\u00C2\u00A0 treatments\u00C2\u00A0 indicate\u00C2\u00A0that\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0experiments,\u00C2\u00A0a\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0simvastatin\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0toxic,\u00C2\u00A0 while\u00C2\u00A0a\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A030\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0on\u00C2\u00A0 its\u00C2\u00A0own.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0HBS\u00E2\u0080\u0090 treated\u00C2\u00A0 samples,\u00C2\u00A05.3%\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A010\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0 simvastatin,\u00C2\u00A0while\u00C2\u00A0 this\u00C2\u00A0 percentage\u00C2\u00A0increased\u00C2\u00A0to\u00C2\u00A012%\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A030\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0dose\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.05).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0was\u00C2\u00A0also\u00C2\u00A0observed\u00C2\u00A0to\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0141\u00C2\u00A0\u00C2\u00A0 decrease\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A030\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A0simvastatin,\u00C2\u00A0but\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0above,\u00C2\u00A0the\u00C2\u00A0significance\u00C2\u00A0 of\u00C2\u00A0this\u00C2\u00A0decrease\u00C2\u00A0is\u00C2\u00A0unclear\u00C2\u00A0given\u00C2\u00A0the\u00C2\u00A0very\u00C2\u00A0low\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0in\u00C2\u00A0HBS\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 Rtx\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0(Figure\u00C2\u00A05.2).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A06.3\u00C2\u00A0\u00C2\u00A0Simvastatin\u00C2\u00A0decreases\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0sensitizes\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0underwent\u00C2\u00A0 incubation\u00C2\u00A0with\u00C2\u00A0 simvastatin\u00C2\u00A0at\u00C2\u00A0different\u00C2\u00A0doses\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0 study\u00C2\u00A0 the\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0 reduced\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0Chol\u00C2\u00A0content\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0to\u00C2\u00A0 induce\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0(gray\u00C2\u00A0bars)\u00C2\u00A0and\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0 apoptosis\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0after\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0and\u00C2\u00A0are\u00C2\u00A0shown\u00C2\u00A0for\u00C2\u00A0each\u00C2\u00A0treatment.\u00C2\u00A0 \u00C2\u00A0 When\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP(150)\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 presence\u00C2\u00A0 of\u00C2\u00A0 simvastatin\u00C2\u00A0 at\u00C2\u00A0 doses\u00C2\u00A0 that\u00C2\u00A0 were\u00C2\u00A0not\u00C2\u00A0toxic\u00C2\u00A0to\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0the\u00C2\u00A0right\u00E2\u0080\u0090hand\u00C2\u00A0side\u00C2\u00A0of\u00C2\u00A0Figure\u00C2\u00A06.3\u00C2\u00A0shows\u00C2\u00A0a\u00C2\u00A0dose\u00E2\u0080\u0090dependent\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0 early\u00C2\u00A0apoptosis\u00C2\u00A0alongside\u00C2\u00A0a\u00C2\u00A0decrease\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 (now\u00C2\u00A0present\u00C2\u00A0at\u00C2\u00A0elevated\u00C2\u00A0 levels\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 upregulation\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP(150)).\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0a\u00C2\u00A0dose\u00C2\u00A0of\u00C2\u00A03\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0simvastatin,\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A028%\u00C2\u00A0 of\u00C2\u00A0 cells,\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 21%\u00C2\u00A0 at\u00C2\u00A0 0\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0 (p\u00C2\u00A0 <\u00C2\u00A0 0.05);\u00C2\u00A0 the\u00C2\u00A0 CD120a\u00C2\u00A0DR\u00C2\u00A0 values\u00C2\u00A0 at\u00C2\u00A0 these\u00C2\u00A0 two\u00C2\u00A0 doses\u00C2\u00A0were\u00C2\u00A0 equivalent,\u00C2\u00A0however\u00C2\u00A0 (p\u00C2\u00A0 >\u00C2\u00A00.05).\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0 significant\u00C2\u00A0decrease\u00C2\u00A0 in\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0 at\u00C2\u00A010\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0 simvastatin;\u00C2\u00A0 the\u00C2\u00A0DR\u00C2\u00A0 at\u00C2\u00A010\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0was\u00C2\u00A00.55,\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A00.73\u00C2\u00A0 at\u00C2\u00A03\u00C2\u00A0 \u00CE\u00BCM\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.001),\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 level\u00C2\u00A0of\u00C2\u00A0 apoptosis\u00C2\u00A0at\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0simvastatin\u00C2\u00A0increased\u00C2\u00A0to\u00C2\u00A047%\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.0001\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A03\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0dose).\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0terms\u00C2\u00A0 of\u00C2\u00A0both\u00C2\u00A0parameters,\u00C2\u00A0the\u00C2\u00A010\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0and\u00C2\u00A030\u00C2\u00A0\u00CE\u00BCM\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0simvastatin\u00C2\u00A0produced\u00C2\u00A0equivalent\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP(150)\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0(p\u00C2\u00A0>\u00C2\u00A00.05).\u00C2\u00A0\u00C2\u00A0Overall,\u00C2\u00A0the\u00C2\u00A0simvastatin\u00C2\u00A0experiments\u00C2\u00A0corroborate\u00C2\u00A0the\u00C2\u00A0MBCD\u00C2\u00A0data\u00C2\u00A0 0 1 2 3 0 3 10 30 0 3 10 30 0 3 10 30 Re la ti ve \u00C2\u00A0va lu es [Simvastatin]\u00C2\u00A0/\u00C2\u00A0\u00CE\u00BCM DR\u00C2\u00A0(CD120a) Early\u00C2\u00A0apop. Rtx\u00E2\u0080\u0090LNP(150)RtxHBS \u00E2\u0080\u00A2\u00C2\u00A0142\u00C2\u00A0\u00C2\u00A0 which\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 increases\u00C2\u00A0 in\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0Chol\u00C2\u00A0 inhibit\u00C2\u00A0 apoptosis\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0 Rtx,\u00C2\u00A0 and\u00C2\u00A0 decreases\u00C2\u00A0 in\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 Chol\u00C2\u00A0 sensitize\u00C2\u00A0 the\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 6.4.3\u00C2\u00A0 A\u00C2\u00A0 model\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 that\u00C2\u00A0 occurs\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 after\u00C2\u00A0 normal\u00C2\u00A0 Rtx\u00C2\u00A0 therapy\u00C2\u00A0 The\u00C2\u00A0data\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A05\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0Sections\u00C2\u00A06.4.1\u00C2\u00A0&\u00C2\u00A06.4.2\u00C2\u00A0establish\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0 involves\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0Chol\u00C2\u00A0 levels\u00C2\u00A0 influence\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0 multivalent\u00C2\u00A0Rtx\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0experiments\u00C2\u00A0would\u00C2\u00A0have\u00C2\u00A0more\u00C2\u00A0clinical\u00C2\u00A0relevance\u00C2\u00A0 if\u00C2\u00A0evidence\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0provided\u00C2\u00A0for\u00C2\u00A0this\u00C2\u00A0mechanism\u00C2\u00A0occurring\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0upon\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0by\u00C2\u00A0 Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0(Section\u00C2\u00A06.2.4).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0first\u00C2\u00A0model\u00C2\u00A0the\u00C2\u00A0interaction\u00C2\u00A0between\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0on\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0lymphoma\u00C2\u00A0cells,\u00C2\u00A0a\u00C2\u00A0 2oAb\u00C2\u00A0specific\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0human\u00C2\u00A0IgGs\u00C2\u00A0was\u00C2\u00A0employed.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0specificity\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A02oAb\u00C2\u00A0paralleled\u00C2\u00A0 that\u00C2\u00A0of\u00C2\u00A0Fc\u00CE\u00B3R.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0enable\u00C2\u00A0hypercrosslinking,\u00C2\u00A0the\u00C2\u00A02oAb\u00C2\u00A0was\u00C2\u00A0coupled\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0polystyrene\u00C2\u00A0MS\u00C2\u00A0to\u00C2\u00A0 create\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0 (Section\u00C2\u00A06.3.7).\u00C2\u00A0 \u00C2\u00A0 Lymphoma\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 saturated\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0after\u00C2\u00A0washing\u00C2\u00A0 the\u00C2\u00A0 cells,\u00C2\u00A0 the\u00C2\u00A02oAb\u00C2\u00A0was\u00C2\u00A0either\u00C2\u00A0added\u00C2\u00A0on\u00C2\u00A0 its\u00C2\u00A0own,\u00C2\u00A0producing\u00C2\u00A0a\u00C2\u00A0 low\u00C2\u00A0degree\u00C2\u00A0of\u00C2\u00A0 crosslinking,\u00C2\u00A0or\u00C2\u00A0added\u00C2\u00A0as\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS,\u00C2\u00A0enabling\u00C2\u00A0hypercrosslinking.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090pretreated\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0was\u00C2\u00A0first\u00C2\u00A0assessed\u00C2\u00A0 in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0added.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0data\u00C2\u00A0is\u00C2\u00A0summarized\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.4A;\u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0showed\u00C2\u00A0low\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0(3.3%)\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0treatment,\u00C2\u00A0while\u00C2\u00A0 maximum\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0(39%)\u00C2\u00A0occurred\u00C2\u00A0when\u00C2\u00A0masses\u00C2\u00A0of\u00C2\u00A08\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0and\u00C2\u00A0 above\u00C2\u00A0were\u00C2\u00A0given\u00C2\u00A0 (p\u00C2\u00A0<\u00C2\u00A00.0001).\u00C2\u00A0 \u00C2\u00A0From\u00C2\u00A08\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0to\u00C2\u00A016\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A02oAb\u00E2\u0080\u0090MS),\u00C2\u00A0the\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0cells\u00C2\u00A0 dropped\u00C2\u00A0from\u00C2\u00A039%\u00C2\u00A0to\u00C2\u00A014%\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.001).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0decreased\u00C2\u00A0viable\u00C2\u00A0fraction\u00C2\u00A0corresponded\u00C2\u00A0to\u00C2\u00A0an\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0percentage\u00C2\u00A0of\u00C2\u00A0necrotic\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0at\u00C2\u00A0doses\u00C2\u00A0above\u00C2\u00A016\u00C2\u00A0\u00C2\u00B5g,\u00C2\u00A0the\u00C2\u00A0necrotic\u00C2\u00A0fraction\u00C2\u00A0was\u00C2\u00A0so\u00C2\u00A0dominant\u00C2\u00A0 that\u00C2\u00A0very\u00C2\u00A0few\u00C2\u00A0viable\u00C2\u00A0or\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0(not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0parallel\u00C2\u00A0earlier\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0143\u00C2\u00A0\u00C2\u00A0 results\u00C2\u00A0 from\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 and\u00C2\u00A0 Rtx\u00E2\u0080\u0090MS\u00C2\u00A0 which\u00C2\u00A0 showed\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 hypercrosslinking,\u00C2\u00A0 and\u00C2\u00A0 significantly\u00C2\u00A0 higher\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 upon\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 via\u00C2\u00A0 a\u00C2\u00A0 multivalent\u00C2\u00A0interaction.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A06.4\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0model\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0that\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0by\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090bearing\u00C2\u00A0effector\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0 (A)\u00C2\u00A0The\u00C2\u00A0percentages\u00C2\u00A0of\u00C2\u00A0viable\u00C2\u00A0and\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0subjecting\u00C2\u00A01.25\u00C2\u00A0\u00EF\u0082\u00B4\u00C2\u00A0105\u00C2\u00A0Rtx\u00E2\u0080\u0090presaturated\u00C2\u00A0 Ramos\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0250\u00C2\u00A0\u00CE\u00BCL\u00C2\u00A0medium\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0indicated\u00C2\u00A0masses\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A02oAb\u00E2\u0080\u0090MS).\u00C2\u00A0\u00C2\u00A0(B)\u00C2\u00A0Samples\u00C2\u00A0were\u00C2\u00A0presaturated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0 (A),\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0treatments\u00C2\u00A0shown.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0(white\u00C2\u00A0bars)\u00C2\u00A0 and\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0(black\u00C2\u00A0bars)\u00C2\u00A0were\u00C2\u00A0measured\u00C2\u00A0in\u00C2\u00A0each\u00C2\u00A0case\u00C2\u00A0on\u00C2\u00A0cells\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0treated\u00C2\u00A0sample.\u00C2\u00A0\u00C2\u00A0+1\u00C2\u00A0refers\u00C2\u00A0to\u00C2\u00A0 the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0 instead\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0+2\u00C2\u00A0 indicates\u00C2\u00A0that\u00C2\u00A0streptavidin\u00E2\u0080\u0090MS\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0together\u00C2\u00A0with\u00C2\u00A0unbiotinylated\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 0 25 50 75 100 0 4 8 12 16 20 % \u00C2\u00A0of \u00C2\u00A0to ta l\u00C2\u00A0ce ll\u00C2\u00A0p op ul at io n Mass\u00C2\u00A0of\u00C2\u00A0added\u00C2\u00A02oAb\u00C2\u00A0 (in\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0/\u00C2\u00A0\u00CE\u00BCg viable e.\u00C2\u00A0apoptotic A 0 4 8 12 53 57 + +\u00C2\u00B9 +\u00C2\u00B2 \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + Re la ti ve \u00C2\u00A0va lu es Mass\u00C2\u00A0of\u00C2\u00A0added\u00C2\u00A02oAb\u00C2\u00A0/\u00C2\u00A0\u00CE\u00BCg CD120a\u00C2\u00A0exp. E.\u00C2\u00A0apoptotic Rtx 0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 16 32\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A01\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A04\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 16 2oAb\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A02oAb\u00E2\u0080\u0090MSB \u00E2\u0080\u00A2\u00C2\u00A0144\u00C2\u00A0\u00C2\u00A0 Experiments\u00C2\u00A0 were\u00C2\u00A0 next\u00C2\u00A0 carried\u00C2\u00A0 out\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0 whether\u00C2\u00A0 the\u00C2\u00A0 apoptosis\u00C2\u00A0 observed\u00C2\u00A0 after\u00C2\u00A0 hypercrosslinking\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0occurs\u00C2\u00A0alongside\u00C2\u00A0upregulated\u00C2\u00A0CD120a\u00C2\u00A0 levels,\u00C2\u00A0as\u00C2\u00A0was\u00C2\u00A0the\u00C2\u00A0case\u00C2\u00A0 with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090MS.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0total\u00C2\u00A0of\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0different\u00C2\u00A0amounts\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0or\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00E2\u0080\u0090presaturated\u00C2\u00A0 cells,\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 early\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0 plasma\u00E2\u0080\u0090membrane\u00C2\u00A0 CD120a\u00C2\u00A0 were\u00C2\u00A0 measured.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0resulting\u00C2\u00A0data\u00C2\u00A0is\u00C2\u00A0summarized\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.4B,\u00C2\u00A0which\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 pretreatment,\u00C2\u00A0 the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0or\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0on\u00C2\u00A0 their\u00C2\u00A0own\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A0concentrations\u00C2\u00A0 tested\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 induce\u00C2\u00A0apoptosis\u00C2\u00A0after\u00C2\u00A012\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0contrast,\u00C2\u00A0mild\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0apoptosis\u00C2\u00A0occurred\u00C2\u00A0when\u00C2\u00A02oAb\u00C2\u00A0was\u00C2\u00A0added\u00C2\u00A0 to\u00C2\u00A0Rtx\u00E2\u0080\u0090pretreated\u00C2\u00A0 cells\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 cells\u00C2\u00A0not\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx;\u00C2\u00A0 for\u00C2\u00A0example,\u00C2\u00A0when\u00C2\u00A032\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0 were\u00C2\u00A0added,\u00C2\u00A0there\u00C2\u00A0was\u00C2\u00A0a\u00C2\u00A01.6\u00E2\u0080\u0090fold\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0(MFI\u00C2\u00A0after\u00C2\u00A0 subtracting\u00C2\u00A0negative\u00C2\u00A0 isotype\u00C2\u00A0control\u00C2\u00A0 increased\u00C2\u00A0from\u00C2\u00A020\u00C2\u00A0to\u00C2\u00A033;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.005)\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A01.7\u00E2\u0080\u0090fold\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0 early\u00C2\u00A0apoptosis\u00C2\u00A0(6.9%\u00C2\u00A0to\u00C2\u00A012%;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.001)\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0in\u00C2\u00A0its\u00C2\u00A0absence.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 When\u00C2\u00A0 significantly\u00C2\u00A0 lower\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0as\u00C2\u00A02oAb\u00E2\u0080\u0090MS,\u00C2\u00A0however,\u00C2\u00A0a\u00C2\u00A0considerable\u00C2\u00A0 upregulation\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0was\u00C2\u00A0 observed\u00C2\u00A0 alongside\u00C2\u00A0 significantly\u00C2\u00A0 elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis,\u00C2\u00A0only\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0when\u00C2\u00A016\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0were\u00C2\u00A0 added,\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0 increased\u00C2\u00A013\u00E2\u0080\u0090fold\u00C2\u00A0(3.1%\u00C2\u00A0to\u00C2\u00A039%;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.0005)\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0MFI\u00C2\u00A0observed\u00C2\u00A0 for\u00C2\u00A0CD120a\u00C2\u00A0 increased\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0factor\u00C2\u00A0of\u00C2\u00A046\u00C2\u00A0(from\u00C2\u00A046\u00C2\u00A0to\u00C2\u00A02130;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.0001)\u00C2\u00A0 in\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0pretreated\u00C2\u00A0 with\u00C2\u00A0Rtx\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0pretreated.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation,\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0 presaturated\u00C2\u00A0 cells\u00C2\u00A0with\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0highest\u00C2\u00A0measured\u00C2\u00A0upregulation\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0methods\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0studied\u00C2\u00A0(Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090MS).\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0apoptosis\u00C2\u00A0 was\u00C2\u00A04.9\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0(8.0%\u00C2\u00A0to\u00C2\u00A039%;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.0005)\u00C2\u00A0at\u00C2\u00A016\u00C2\u00A0\u00C2\u00B5g\u00C2\u00A0of\u00C2\u00A0added\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A01\u00C2\u00A0 \u00C2\u00B5g,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0MFI\u00C2\u00A0 corresponding\u00C2\u00A0 to\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0was\u00C2\u00A0 higher\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 factor\u00C2\u00A0 of\u00C2\u00A0 25\u00C2\u00A0 (from\u00C2\u00A0 84\u00C2\u00A0 to\u00C2\u00A0 2130;\u00C2\u00A0 p\u00C2\u00A0 <\u00C2\u00A0 0.0001).\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 controls\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 6.4B\u00C2\u00A0 (+1\u00C2\u00A0Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0 and\u00C2\u00A0 +2\u00C2\u00A0 streptavidin\u00E2\u0080\u0090MS\u00C2\u00A0 +Rtx)\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 neither\u00C2\u00A0 Rtx\u00E2\u0080\u0090biotin\u00C2\u00A0 nor\u00C2\u00A0 streptavidin\u00E2\u0080\u0090MS\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 own\u00C2\u00A0 were\u00C2\u00A0 responsible\u00C2\u00A0for\u00C2\u00A0any\u00C2\u00A0observed\u00C2\u00A0effects\u00C2\u00A0since\u00C2\u00A0all\u00C2\u00A0measured\u00C2\u00A0values\u00C2\u00A0were\u00C2\u00A0equal\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0cells\u00C2\u00A0 treated\u00C2\u00A0only\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 (p\u00C2\u00A0 >\u00C2\u00A00.05\u00C2\u00A0 in\u00C2\u00A0 all\u00C2\u00A0 cases).\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0 further\u00C2\u00A0 support\u00C2\u00A0 the\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0145\u00C2\u00A0\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0observed\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0and\u00C2\u00A0 they\u00C2\u00A0also\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0 drives\u00C2\u00A0 this\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 since\u00C2\u00A0 different\u00C2\u00A0 methods\u00C2\u00A0 of\u00C2\u00A0 hypercrosslinking\u00C2\u00A0produced\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0result.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 6.4.4\u00C2\u00A0 CD120a\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0excluded\u00C2\u00A0 from\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0 is\u00C2\u00A0colocalized\u00C2\u00A0with\u00C2\u00A0 hypercrosslinked\u00C2\u00A0Rtx\u00C2\u00ADenriched\u00C2\u00A0patches\u00C2\u00A0 To\u00C2\u00A0 further\u00C2\u00A0 study\u00C2\u00A0 the\u00C2\u00A0 relationship\u00C2\u00A0 between\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 and\u00C2\u00A0 CD120a\u00C2\u00A0 upregulation,\u00C2\u00A0 confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy\u00C2\u00A0 was\u00C2\u00A0 employed\u00C2\u00A0 to\u00C2\u00A0 investigate\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 organization\u00C2\u00A0 of\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 Rtx\u00C2\u00A0 with\u00C2\u00A0 respect\u00C2\u00A0 to\u00C2\u00A0 CD120a\u00C2\u00A0 and\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0rafts.\u00C2\u00A0\u00C2\u00A0To\u00C2\u00A0visualize\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface,\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0labeled\u00C2\u00A0with\u00C2\u00A0A568\u00C2\u00A0(Section\u00C2\u00A06.3.7).\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0was\u00C2\u00A0first\u00C2\u00A0verified\u00C2\u00A0that\u00C2\u00A0A568\u00E2\u0080\u0090Rtx,\u00C2\u00A0when\u00C2\u00A0hypercrosslinked\u00C2\u00A0using\u00C2\u00A02oAb\u00E2\u0080\u0090MS,\u00C2\u00A0 induced\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0 same\u00C2\u00A0way\u00C2\u00A0as\u00C2\u00A0unlabeled\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Figure\u00C2\u00A06.5A\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0after\u00C2\u00A0a\u00C2\u00A024\u00C2\u00A0h\u00C2\u00A0incubation,\u00C2\u00A020\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0of\u00C2\u00A0added\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS)\u00C2\u00A0on\u00C2\u00A0 its\u00C2\u00A0own\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0 toxic\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0 (top\u00C2\u00A0 left\u00C2\u00A0panel),\u00C2\u00A0while\u00C2\u00A0a\u00C2\u00A0mass\u00C2\u00A0of\u00C2\u00A050\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 cytotoxic\u00C2\u00A0effects\u00C2\u00A0 in\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0 (top\u00C2\u00A0 right).\u00C2\u00A0 \u00C2\u00A0Treatment\u00C2\u00A0with\u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0on\u00C2\u00A0 its\u00C2\u00A0own\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0(bottom\u00C2\u00A0 left),\u00C2\u00A0while\u00C2\u00A0cells\u00C2\u00A0presaturated\u00C2\u00A0with\u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0and\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A020\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0of\u00C2\u00A0 2oAb\u00C2\u00A0 (in\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0 (bottom\u00C2\u00A0right)\u00C2\u00A0underwent\u00C2\u00A0apoptosis\u00C2\u00A0at\u00C2\u00A0 levels\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0 those\u00C2\u00A0when\u00C2\u00A0using\u00C2\u00A0 unlabeled\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.4A.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.5A\u00C2\u00A0therefore\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0A568\u00C2\u00A0labeling\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0 significantly\u00C2\u00A0affect\u00C2\u00A0the\u00C2\u00A0function\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0they\u00C2\u00A0also\u00C2\u00A0confirm\u00C2\u00A0that\u00C2\u00A020\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0 toxic\u00C2\u00A0to\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0under\u00C2\u00A0the\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0experiments.\u00C2\u00A0 To\u00C2\u00A0 obtain\u00C2\u00A0 confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0 microscopy\u00C2\u00A0 images,\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 presaturated\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A06.4.3\u00C2\u00A0with\u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0instead\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0 or\u00C2\u00A0an\u00C2\u00A0equivalent\u00C2\u00A0volume\u00C2\u00A0of\u00C2\u00A0HBS.\u00C2\u00A0 \u00C2\u00A0At\u00C2\u00A0a\u00C2\u00A0 time\u00C2\u00A0point\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0 the\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A02oAb\u00E2\u0080\u0090MS,\u00C2\u00A0 cells\u00C2\u00A0were\u00C2\u00A0 stained\u00C2\u00A0without\u00C2\u00A0permeabilization\u00C2\u00A0with\u00C2\u00A0A488\u00E2\u0080\u0090CTX\u00C2\u00A0(to\u00C2\u00A0 label\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts)\u00C2\u00A0and\u00C2\u00A0with\u00C2\u00A0A647\u00E2\u0080\u0090 CD120a.\u00C2\u00A0\u00C2\u00A0Representative\u00C2\u00A0images\u00C2\u00A0are\u00C2\u00A0exhibited\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.5B\u00C2\u00A0&\u00C2\u00A0C.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0top\u00C2\u00A0two\u00C2\u00A0panels\u00C2\u00A0in\u00C2\u00A0each\u00C2\u00A0case\u00C2\u00A0 show\u00C2\u00A0the\u00C2\u00A0staining\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0(red)\u00C2\u00A0and\u00C2\u00A0A488\u00E2\u0080\u0090CTX\u00C2\u00A0(blue)\u00C2\u00A0on\u00C2\u00A0their\u00C2\u00A0own\u00C2\u00A0before\u00C2\u00A0being\u00C2\u00A0merged\u00C2\u00A0 in\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0146\u00C2\u00A0\u00C2\u00A0 the\u00C2\u00A0 third\u00C2\u00A0 panels\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 top.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 fourth\u00C2\u00A0 panel\u00C2\u00A0 shows\u00C2\u00A0 staining\u00C2\u00A0 of\u00C2\u00A0 A568\u00E2\u0080\u0090Rtx\u00C2\u00A0 (green)\u00C2\u00A0while\u00C2\u00A0 the\u00C2\u00A0 bottommost\u00C2\u00A0panel\u00C2\u00A0contains\u00C2\u00A0a\u00C2\u00A0merge\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0 three\u00C2\u00A0signals.\u00C2\u00A0 \u00C2\u00A0When\u00C2\u00A0comparing\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS\u00C2\u00A0(Figure\u00C2\u00A06.5B)\u00C2\u00A0to\u00C2\u00A0those\u00C2\u00A0that\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0(Figure\u00C2\u00A06.5C),\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0clear\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0 absence\u00C2\u00A0of\u00C2\u00A02oAb\u00E2\u0080\u0090MS,\u00C2\u00A0 the\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 is\u00C2\u00A0 significantly\u00C2\u00A0 lower\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0distributions\u00C2\u00A0of\u00C2\u00A0 A568\u00E2\u0080\u0090Rtx\u00C2\u00A0and\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0are\u00C2\u00A0more\u00C2\u00A0uniform\u00C2\u00A0across\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cells.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A06.5\u00C2\u00A0\u00C2\u00A0Membrane\u00C2\u00A0raft\u00E2\u0080\u0090associated\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0excluded\u00C2\u00A0from\u00C2\u00A0regions\u00C2\u00A0enriched\u00C2\u00A0in\u00C2\u00A0hypercrosslinked\u00C2\u00A0Rtx/CD20,\u00C2\u00A0and\u00C2\u00A0non\u00E2\u0080\u0090 raft\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0colocalized\u00C2\u00A0with\u00C2\u00A0hypercrosslinked\u00C2\u00A0Rtx/CD20.\u00C2\u00A0\u00C2\u00A0 (A)\u00C2\u00A0 Verification\u00C2\u00A0 that\u00C2\u00A0 labeling\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0with\u00C2\u00A0 A568\u00C2\u00A0 did\u00C2\u00A0 not\u00C2\u00A0 affect\u00C2\u00A0 its\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 upon\u00C2\u00A0 hypercrosslinking\u00C2\u00A0with\u00C2\u00A02oAb\u00E2\u0080\u0090MS.\u00C2\u00A0\u00C2\u00A0(Continued\u00C2\u00A0on\u00C2\u00A0next\u00C2\u00A0page.)\u00C2\u00A0 \u00C2\u00A0 92.6% 3.7% 3.7% 66.3% 17.2% 16.4% 91.0% 4.9% 4.0% 13.1% 57.2% 29.6% 20\u00C2\u00A0\u00CE\u00BCg 2oAb\u00E2\u0080\u0090MS only 50\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS only A568\u00E2\u0080\u0090Rtx only A568\u00E2\u0080\u0090Rtx +20\u00C2\u00A0\u00CE\u00BCg 2oAb\u00E2\u0080\u0090MS 100 101 102 103 104 104 103 102 101 100 Fl uo re sc en ce \u00C2\u00A0int en si ty \u00C2\u00A0\u00C2\u00A0(P I) Fluorescence\u00C2\u00A0intensity\u00C2\u00A0 \u00C2\u00A0(Annexin\u00E2\u0080\u0090V\u00E2\u0080\u0090FITC) 100 101 102 103 104 104 103 102 101 100 A \u00E2\u0080\u00A2\u00C2\u00A0147\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A0 6.5\u00C2\u00A0 \u00C2\u00A0 (Continued\u00C2\u00A0 from\u00C2\u00A0 previous\u00C2\u00A0 page.)\u00C2\u00A0 (B\u00C2\u00A0&\u00C2\u00A0 C)\u00C2\u00A0 Confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy\u00C2\u00A0 images\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0only\u00C2\u00A0(B)\u00C2\u00A0or\u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A020\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0of\u00C2\u00A02oAb\u00C2\u00A0(in\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0(C)\u00C2\u00A0for\u00C2\u00A012\u00C2\u00A0h.\u00C2\u00A0 \u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0 localization\u00C2\u00A0 is\u00C2\u00A0 represented\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0green\u00C2\u00A0signal;\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0also\u00C2\u00A0stained\u00C2\u00A0with\u00C2\u00A0A488\u00E2\u0080\u0090CTX\u00C2\u00A0to\u00C2\u00A0 label\u00C2\u00A0membrane\u00C2\u00A0rafts\u00C2\u00A0(blue)\u00C2\u00A0and\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0 (red).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 (C),\u00C2\u00A0 CD120a\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 raft\u00E2\u0080\u0090associated\u00C2\u00A0 (purple\u00C2\u00A0 regions\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 third\u00C2\u00A0 panel\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 top)\u00C2\u00A0 is\u00C2\u00A0 excluded\u00C2\u00A0 from\u00C2\u00A0 hypercrosslinked\u00C2\u00A0Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0regions\u00C2\u00A0(green),\u00C2\u00A0while\u00C2\u00A0non\u00E2\u0080\u0090raft\u00C2\u00A0associated\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0colocalized\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0regions.\u00C2\u00A0 B A568\u00E2\u0080\u0090Rtx\u00C2\u00A0only C A568\u00E2\u0080\u0090Rtx\u00C2\u00A0\u00C2\u00A0+20\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A02oAb\u00E2\u0080\u0090MS \u00E2\u0080\u00A2\u00C2\u00A0148\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 third\u00C2\u00A0panels\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 top\u00C2\u00A0specifically\u00C2\u00A0show\u00C2\u00A0 the\u00C2\u00A0regions\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 that\u00C2\u00A0are\u00C2\u00A0associated\u00C2\u00A0 with\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0 (purple)\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 regions\u00C2\u00A0 that\u00C2\u00A0 are\u00C2\u00A0 non\u00E2\u0080\u0090raft\u00E2\u0080\u0090associated\u00C2\u00A0 (red).\u00C2\u00A0 \u00C2\u00A0When\u00C2\u00A0 examining\u00C2\u00A0the\u00C2\u00A0distribution\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0purple\u00C2\u00A0signal\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0that\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0green\u00C2\u00A0one\u00C2\u00A0(A568\u00E2\u0080\u0090Rtx),\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 clear\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A06.5C\u00C2\u00A0 that\u00C2\u00A0 there\u00C2\u00A0 is\u00C2\u00A0 low\u00C2\u00A0 colocalization\u00C2\u00A0between\u00C2\u00A0 the\u00C2\u00A0purple\u00C2\u00A0 and\u00C2\u00A0 green\u00C2\u00A0 regions.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 implies\u00C2\u00A0 that\u00C2\u00A0 raft\u00E2\u0080\u0090associated\u00C2\u00A0 CD120\u00C2\u00A0 does\u00C2\u00A0 not\u00C2\u00A0 colocalize\u00C2\u00A0with\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 non\u00E2\u0080\u0090raft\u00C2\u00A0 CD120a\u00C2\u00A0 (red\u00C2\u00A0 signal\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 third\u00C2\u00A0 panel)\u00C2\u00A0 is\u00C2\u00A0 almost\u00C2\u00A0 exclusively\u00C2\u00A0 colocalized\u00C2\u00A0 with\u00C2\u00A0 hypercrosslinked\u00C2\u00A0A568\u00E2\u0080\u0090Rtx\u00C2\u00A0 (green)\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A06.5C.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 fact,\u00C2\u00A0 the\u00C2\u00A0green\u00C2\u00A0and\u00C2\u00A0blue\u00C2\u00A0 signals\u00C2\u00A0 show\u00C2\u00A0a\u00C2\u00A0 low\u00C2\u00A0 degree\u00C2\u00A0 of\u00C2\u00A0 colocalization,\u00C2\u00A0 indicating\u00C2\u00A0 that\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 A568\u00E2\u0080\u0090Rtx\u00C2\u00A0 is\u00C2\u00A0 excluded\u00C2\u00A0 from\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 rafts.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 same\u00C2\u00A0 trends\u00C2\u00A0were\u00C2\u00A0observed\u00C2\u00A0 across\u00C2\u00A0 a\u00C2\u00A0 large\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 samples\u00C2\u00A0obtained\u00C2\u00A0 under\u00C2\u00A0a\u00C2\u00A0variety\u00C2\u00A0of\u00C2\u00A0conditions.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Together,\u00C2\u00A0these\u00C2\u00A0data\u00C2\u00A0imply\u00C2\u00A0that\u00C2\u00A0Rtx/CD20,\u00C2\u00A0when\u00C2\u00A0hypercrosslinked\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface,\u00C2\u00A0forms\u00C2\u00A0 large\u00C2\u00A0patches\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0excluded\u00C2\u00A0from\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts.\u00C2\u00A0\u00C2\u00A0They\u00C2\u00A0also\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0CD120a\u00C2\u00A0present\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 (after\u00C2\u00A0 upregulation\u00C2\u00A0 resulting\u00C2\u00A0 from\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 Rtx/CD20)\u00C2\u00A0 forms\u00C2\u00A0 different\u00C2\u00A0domains,\u00C2\u00A0some\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0 raft\u00E2\u0080\u0090associated\u00C2\u00A0and\u00C2\u00A0some\u00C2\u00A0of\u00C2\u00A0which\u00C2\u00A0are\u00C2\u00A0not.\u00C2\u00A0 \u00C2\u00A0Raft\u00E2\u0080\u0090associated\u00C2\u00A0 CD120a\u00C2\u00A0is\u00C2\u00A0almost\u00C2\u00A0exclusively\u00C2\u00A0excluded\u00C2\u00A0from\u00C2\u00A0Rtx\u00E2\u0080\u0090enriched\u00C2\u00A0patches,\u00C2\u00A0while\u00C2\u00A0non\u00E2\u0080\u0090raft\u00E2\u0080\u0090associated\u00C2\u00A0CD120a\u00C2\u00A0 is\u00C2\u00A0 largely\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0hypercrosslinked\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0suggests\u00C2\u00A0 that\u00C2\u00A0Rtx\u00C2\u00A0hypercrosslinking\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0formation\u00C2\u00A0of\u00C2\u00A0non\u00E2\u0080\u0090raft\u00C2\u00A0patches\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0proapoptotic\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0localized\u00C2\u00A0following\u00C2\u00A0its\u00C2\u00A0 upregulation.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figures\u00C2\u00A0 6.4\u00C2\u00A0 &\u00C2\u00A0 6.5,\u00C2\u00A0 taken\u00C2\u00A0 together,\u00C2\u00A0 show\u00C2\u00A0 that\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 using\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0significant\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a.\u00C2\u00A0 \u00C2\u00A0Since\u00C2\u00A0the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0served\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 model\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0of\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090coated\u00C2\u00A0 lymphoma\u00C2\u00A0cells,\u00C2\u00A0 these\u00C2\u00A0data\u00C2\u00A0support\u00C2\u00A0 the\u00C2\u00A0 hypothesis\u00C2\u00A0 that\u00C2\u00A0 this\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 occurs\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 after\u00C2\u00A0 administration\u00C2\u00A0 of\u00C2\u00A0 regular\u00C2\u00A0 bivalent\u00C2\u00A0Rtx\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Rtx/CD20\u00C2\u00A0by\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0cells.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0149\u00C2\u00A0\u00C2\u00A0 6.4.5\u00C2\u00A0 Effector\u00C2\u00A0 cells\u00C2\u00A0 induce\u00C2\u00A0 elevated\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 only\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0 determine\u00C2\u00A0 if\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 themselves\u00C2\u00A0 are\u00C2\u00A0 capable\u00C2\u00A0 of\u00C2\u00A0 directly\u00C2\u00A0 inducing\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 through\u00C2\u00A0 a\u00C2\u00A0mechanism\u00C2\u00A0 that\u00C2\u00A0 depends\u00C2\u00A0 on\u00C2\u00A0 CD120a,\u00C2\u00A0 experiments\u00C2\u00A0 were\u00C2\u00A0 carried\u00C2\u00A0 out\u00C2\u00A0 where\u00C2\u00A0 Ramos\u00C2\u00A0 cells\u00C2\u00A0 pretreated\u00C2\u00A0with\u00C2\u00A0 either\u00C2\u00A0 Rtx\u00C2\u00A0 or\u00C2\u00A0HBS\u00C2\u00A0were\u00C2\u00A0 co\u00E2\u0080\u0090incubated\u00C2\u00A0with\u00C2\u00A0 human\u00C2\u00A0 peripheral\u00C2\u00A0 blood\u00C2\u00A0mononuclear\u00C2\u00A0cells\u00C2\u00A0(PBMCs)\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0healthy\u00C2\u00A0donors.\u00C2\u00A0\u00C2\u00A0PBMCs\u00C2\u00A0are\u00C2\u00A0rich\u00C2\u00A0in\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0 effector\u00C2\u00A0cells\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0monocytes246\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0subpopulation\u00C2\u00A0of\u00C2\u00A0these\u00C2\u00A0cells\u00C2\u00A0would\u00C2\u00A0therefore\u00C2\u00A0 be\u00C2\u00A0expected\u00C2\u00A0to\u00C2\u00A0hypercrosslink\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0treated\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0manner\u00C2\u00A0similar\u00C2\u00A0to\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS\u00C2\u00A0in\u00C2\u00A0Sections\u00C2\u00A06.4.3\u00C2\u00A0and\u00C2\u00A06.4.4.\u00C2\u00A0 A\u00C2\u00A0 detailed\u00C2\u00A0 description\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 strategies\u00C2\u00A0 employed\u00C2\u00A0 to\u00C2\u00A0 detect\u00C2\u00A0 expression\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 and\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0 is\u00C2\u00A0provided\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A06.3.8.\u00C2\u00A0 \u00C2\u00A0After\u00C2\u00A0pretreatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0or\u00C2\u00A0HBS,\u00C2\u00A0PBMCs\u00C2\u00A0 were\u00C2\u00A0added\u00C2\u00A0in\u00C2\u00A0different\u00C2\u00A0excesses\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0coincubation\u00C2\u00A0was\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0proceed\u00C2\u00A0for\u00C2\u00A016\u00C2\u00A0h.\u00C2\u00A0\u00C2\u00A0 Levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD120a\u00C2\u00A0were\u00C2\u00A0then\u00C2\u00A0measured\u00C2\u00A0only\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0Ramos\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0resulting\u00C2\u00A0data\u00C2\u00A0is\u00C2\u00A0shown\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A06.6.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0figure\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0PBMCs\u00C2\u00A0were\u00C2\u00A0 capable\u00C2\u00A0of\u00C2\u00A0inducing\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0depended\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0ratio\u00C2\u00A0of\u00C2\u00A0 PBMCs\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0While\u00C2\u00A08.9%\u00C2\u00A0of\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 in\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0at\u00C2\u00A0 the\u00C2\u00A01:1\u00C2\u00A0Ramos:PBMC\u00C2\u00A0ratio,\u00C2\u00A0this\u00C2\u00A0value\u00C2\u00A0increased\u00C2\u00A0to\u00C2\u00A043%\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A01:8\u00C2\u00A0ratio\u00C2\u00A0(p\u00C2\u00A0<\u00C2\u00A00.0001).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 absence\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0however,\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0PBMC\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis\u00C2\u00A0occurred\u00C2\u00A0via\u00C2\u00A0a\u00C2\u00A0mechanism\u00C2\u00A0that\u00C2\u00A0was\u00C2\u00A0 CD120a\u00E2\u0080\u0090independent\u00C2\u00A0because\u00C2\u00A0as\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 increased\u00C2\u00A0at\u00C2\u00A0 ratios\u00C2\u00A0of\u00C2\u00A01:1\u00C2\u00A0and\u00C2\u00A0above,\u00C2\u00A0 the\u00C2\u00A0 expression\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 remained\u00C2\u00A0 constant\u00C2\u00A0 (p\u00C2\u00A0>\u00C2\u00A00.05\u00C2\u00A0between\u00C2\u00A0all\u00C2\u00A0pairs\u00C2\u00A0of\u00C2\u00A0 treatments).\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0150\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Figure\u00C2\u00A06.6\u00C2\u00A0 \u00C2\u00A0Human\u00C2\u00A0PBMCs\u00C2\u00A0 induce\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0non\u00E2\u0080\u0090pretreated\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0 PBMCs\u00C2\u00A0from\u00C2\u00A0healthy\u00C2\u00A0donors\u00C2\u00A0were\u00C2\u00A0coincubated\u00C2\u00A0with\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0either\u00C2\u00A0HBS\u00C2\u00A0or\u00C2\u00A0saturating\u00C2\u00A0levels\u00C2\u00A0 of\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 Ramos\u00C2\u00A0 cells,\u00C2\u00A0 both\u00C2\u00A0 the\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 level\u00C2\u00A0 of\u00C2\u00A0 early\u00C2\u00A0 apoptosis\u00C2\u00A0were\u00C2\u00A0 measured\u00C2\u00A016\u00C2\u00A0h\u00C2\u00A0after\u00C2\u00A0addition\u00C2\u00A0of\u00C2\u00A0PBMCs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 most\u00C2\u00A0 important\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 6.6\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0 every\u00C2\u00A0 ratio\u00C2\u00A0 tested\u00C2\u00A0 showed\u00C2\u00A0 significantly\u00C2\u00A0 elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 early\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 samples\u00C2\u00A0 containing\u00C2\u00A0Rtx\u00E2\u0080\u0090pretreated\u00C2\u00A0Ramos\u00C2\u00A0 cells\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0 These\u00C2\u00A0 elevated\u00C2\u00A0 apoptosis\u00C2\u00A0 levels\u00C2\u00A0were\u00C2\u00A0 accompanied\u00C2\u00A0by\u00C2\u00A0 yet\u00C2\u00A0 more\u00C2\u00A0pronounced\u00C2\u00A0increases\u00C2\u00A0in\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD120a\u00C2\u00A0expression.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A01:1\u00C2\u00A0ratio,\u00C2\u00A0 there\u00C2\u00A0was\u00C2\u00A0a\u00C2\u00A02.0\u00E2\u0080\u0090fold\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0(8.9%\u00C2\u00A0to\u00C2\u00A018%;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.0001)\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A01.3\u00E2\u0080\u0090fold\u00C2\u00A0increase\u00C2\u00A0in\u00C2\u00A0 the\u00C2\u00A0MFI\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00E2\u0080\u0090associated\u00C2\u00A0A647\u00E2\u0080\u0090CD120a\u00C2\u00A0(203\u00C2\u00A0to\u00C2\u00A0269;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.01)\u00C2\u00A0 in\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0 Rtx\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A0HBS.\u00C2\u00A0\u00C2\u00A0At\u00C2\u00A0the\u00C2\u00A01:8\u00C2\u00A0ratio,\u00C2\u00A0early\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A01.3\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0(43%\u00C2\u00A0to\u00C2\u00A058%;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.001)\u00C2\u00A0 while\u00C2\u00A0CD120a\u00C2\u00A0levels\u00C2\u00A0were\u00C2\u00A03.4\u00E2\u0080\u0090fold\u00C2\u00A0higher\u00C2\u00A0(increase\u00C2\u00A0in\u00C2\u00A0MFI\u00C2\u00A0from\u00C2\u00A0202\u00C2\u00A0to\u00C2\u00A0694;\u00C2\u00A0p\u00C2\u00A0<\u00C2\u00A00.0001).\u00C2\u00A0\u00C2\u00A0 These\u00C2\u00A0experiments\u00C2\u00A0 therefore\u00C2\u00A0confirm\u00C2\u00A0 that\u00C2\u00A0circulating\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0 are\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0inducing\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0of\u00C2\u00A0target\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0 containing\u00C2\u00A0bound\u00C2\u00A0Rtx,\u00C2\u00A0resulting\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0these\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0imply\u00C2\u00A0 that\u00C2\u00A0a\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0involving\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0 bivalent\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Others\u00C2\u00A0have\u00C2\u00A0postulated\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0may\u00C2\u00A0actually\u00C2\u00A0be\u00C2\u00A0responsible\u00C2\u00A0 0 10 20 30 \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + \u00E2\u0080\u0093 + Re la ti ve \u00C2\u00A0va lu es Ramos:PBMC CD120a\u00C2\u00A0exp. E.\u00C2\u00A0apoptotic Rtx 1:0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A01:1\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 1:4\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A01:8 \u00E2\u0080\u00A2\u00C2\u00A0151\u00C2\u00A0\u00C2\u00A0 for\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0therapeutic\u00C2\u00A0activity\u00C2\u00A0observed\u00C2\u00A0from\u00C2\u00A0Rtx77,\u00C2\u00A0but\u00C2\u00A0until\u00C2\u00A0now,\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0 mechanism\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0undefined.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 6.5\u00C2\u00A0 Discussion\u00C2\u00A0and\u00C2\u00A0conclusions\u00C2\u00A0 6.5.1\u00C2\u00A0 Discussion\u00C2\u00A0 The\u00C2\u00A0 results\u00C2\u00A0 from\u00C2\u00A0 this\u00C2\u00A0 chapter\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 a\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0 involving\u00C2\u00A0 upregulation\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0apoptosis\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0after\u00C2\u00A0normal\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0therapy.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0stated\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A0 5.4.1,\u00C2\u00A0 the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0been\u00C2\u00A0definitively\u00C2\u00A0 shown,\u00C2\u00A0but\u00C2\u00A0here\u00C2\u00A0we\u00C2\u00A0have\u00C2\u00A0 demonstrated\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 expressed\u00C2\u00A0dramatically\u00C2\u00A0 elevated\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0CD120a\u00C2\u00A0 and\u00C2\u00A0apoptosis\u00C2\u00A0when\u00C2\u00A0 the\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0was\u00C2\u00A0hypercrosslinked\u00C2\u00A0with\u00C2\u00A02oAb\u00E2\u0080\u0090MS,\u00C2\u00A0compared\u00C2\u00A0 to\u00C2\u00A0 only\u00C2\u00A0modest\u00C2\u00A0 increases\u00C2\u00A0 at\u00C2\u00A0 higher\u00C2\u00A0 doses\u00C2\u00A0 of\u00C2\u00A0 2oAb\u00C2\u00A0 (Figure\u00C2\u00A0 6.4).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 hypercrosslinking\u00C2\u00A0modeled\u00C2\u00A0 the\u00C2\u00A0 multivalent\u00C2\u00A0 interaction\u00C2\u00A0 that\u00C2\u00A0 occurs\u00C2\u00A0when\u00C2\u00A0 Rtx\u00E2\u0080\u0090coated\u00C2\u00A0 cells\u00C2\u00A0 interact\u00C2\u00A0with\u00C2\u00A0 Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0 immune\u00C2\u00A0 effector\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0vivo,\u00C2\u00A0lending\u00C2\u00A0support\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 involving\u00C2\u00A0upregulation\u00C2\u00A0 of\u00C2\u00A0CD120a.\u00C2\u00A0\u00C2\u00A0We\u00C2\u00A0subsequently\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0also\u00C2\u00A0occurred\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090 coated\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0when\u00C2\u00A0 they\u00C2\u00A0were\u00C2\u00A0 allowed\u00C2\u00A0 to\u00C2\u00A0 interact\u00C2\u00A0with\u00C2\u00A0 human\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 PBMC\u00C2\u00A0 samples\u00C2\u00A0from\u00C2\u00A0healthy\u00C2\u00A0donors\u00C2\u00A0(Figure\u00C2\u00A06.6).\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0strongly\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0CD120a\u00E2\u0080\u0090mediated\u00C2\u00A0apoptosis\u00C2\u00A0is\u00C2\u00A0 capable\u00C2\u00A0of\u00C2\u00A0occurring\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0bivalent\u00C2\u00A0Rtx.\u00C2\u00A0 These\u00C2\u00A0 studies\u00C2\u00A0 also\u00C2\u00A0 demonstrate\u00C2\u00A0 that\u00C2\u00A0 increasing\u00C2\u00A0 the\u00C2\u00A0 concentration\u00C2\u00A0 of\u00C2\u00A0 Chol\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0of\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0 rescues\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0 from\u00C2\u00A0apoptosis\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0 (Figure\u00C2\u00A0 6.1).\u00C2\u00A0 \u00C2\u00A0Insertion\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0resulted\u00C2\u00A0 in\u00C2\u00A0an\u00C2\u00A0 increase\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0 increased\u00C2\u00A0colocalization\u00C2\u00A0of\u00C2\u00A0 CD120a\u00C2\u00A0 in\u00C2\u00A0GM1\u00E2\u0080\u0090positive\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0 (Figures\u00C2\u00A06.1\u00C2\u00A0&\u00C2\u00A06.2).\u00C2\u00A0 \u00C2\u00A0Depletion\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 increased\u00C2\u00A0 detergent\u00C2\u00A0 solubility\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 (Figures\u00C2\u00A0 6.1\u00C2\u00A0&\u00C2\u00A0 6.3)\u00C2\u00A0 and\u00C2\u00A0 sensitized\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0(Figure\u00C2\u00A06.3).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0level\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0 by\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0 is\u00C2\u00A0 therefore\u00C2\u00A0dependent\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0concentration\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane.\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0152\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 consistent\u00C2\u00A0 with\u00C2\u00A0 a\u00C2\u00A0 CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 since\u00C2\u00A0 raft\u00E2\u0080\u0090associated\u00C2\u00A0 CD120a\u00C2\u00A0 elicits\u00C2\u00A0 antiapoptotic\u00C2\u00A0 signals,\u00C2\u00A0 as\u00C2\u00A0 opposed\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 non\u00E2\u0080\u0090raft\u00E2\u0080\u0090associated\u00C2\u00A0 form,\u00C2\u00A0 which\u00C2\u00A0 is\u00C2\u00A0 proapoptotic\u00C2\u00A0(Sections\u00C2\u00A06.2.1\u00C2\u00A0&\u00C2\u00A06.2.2).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0measure\u00C2\u00A0 the\u00C2\u00A0 resistance\u00C2\u00A0 of\u00C2\u00A0 proteins\u00C2\u00A0 to\u00C2\u00A0 solubilization\u00C2\u00A0 in\u00C2\u00A0 detergent,\u00C2\u00A0 a\u00C2\u00A0method\u00C2\u00A0 making\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00C2\u00A0was\u00C2\u00A0 employed\u00C2\u00A0which\u00C2\u00A0 allowed\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 quantification\u00C2\u00A0 of\u00C2\u00A0DR\u00C2\u00A0 values\u00C2\u00A0 (Section\u00C2\u00A0 6.3.5).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 method\u00C2\u00A0 is\u00C2\u00A0 less\u00C2\u00A0 commonly\u00C2\u00A0 employed\u00C2\u00A0 than\u00C2\u00A0 equilibrium\u00C2\u00A0 density\u00C2\u00A0 gradient\u00C2\u00A0 ultracentrifugation\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A0western\u00C2\u00A0blotting\u00C2\u00A0of\u00C2\u00A0fractions\u00C2\u00A0along\u00C2\u00A0the\u00C2\u00A0gradient\u00C2\u00A0(described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A0 6.2.3.1).\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 these\u00C2\u00A0 experiments,\u00C2\u00A0however,\u00C2\u00A0 employing\u00C2\u00A0 the\u00C2\u00A0 traditional\u00C2\u00A0ultracentrifugation\u00C2\u00A0 technique\u00C2\u00A0 was\u00C2\u00A0not\u00C2\u00A0possible\u00C2\u00A0because\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 very\u00C2\u00A0 large\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0 cells\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0each\u00C2\u00A0 sample\u00C2\u00A0 (1\u00E2\u0080\u00935\u00C2\u00A0\u00EF\u0082\u00B4\u00C2\u00A0108\u00C2\u00A0 cells/sample)47,\u00C2\u00A0247,\u00C2\u00A0248.\u00C2\u00A0 \u00C2\u00A0Cells\u00C2\u00A0were\u00C2\u00A0 typically\u00C2\u00A0given\u00C2\u00A040\u00C2\u00A0\u00CE\u00BCg\u00C2\u00A0Rtx/106\u00C2\u00A0cells\u00C2\u00A0 to\u00C2\u00A0achieve\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0(Figure\u00C2\u00A04.6)\u00C2\u00A0and\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0(Figure\u00C2\u00A05.4);\u00C2\u00A0this\u00C2\u00A0dose\u00C2\u00A0corresponds\u00C2\u00A0to\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0 4\u00C2\u00A0mg\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0(in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP)\u00C2\u00A0per\u00C2\u00A0ultracentrifugation\u00C2\u00A0sample.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 is\u00C2\u00A0an\u00C2\u00A0extremely\u00C2\u00A0 large\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP;\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0approximately\u00C2\u00A0equal\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0mass\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0present\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0samples\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0prepared\u00C2\u00A0 using\u00C2\u00A0one\u00C2\u00A0 iteration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A03.\u00C2\u00A0 \u00C2\u00A0Producing\u00C2\u00A0the\u00C2\u00A0minimum\u00C2\u00A0amount\u00C2\u00A0of\u00C2\u00A0material\u00C2\u00A0 necessary\u00C2\u00A0 for\u00C2\u00A0 ultracentrifugation\u00C2\u00A0 experiments\u00C2\u00A0 using\u00C2\u00A0 this\u00C2\u00A0 methodology\u00C2\u00A0 (or\u00C2\u00A0 the\u00C2\u00A0 previous\u00C2\u00A0 method\u00C2\u00A0 employed\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A02.3.2)\u00C2\u00A0was\u00C2\u00A0 therefore\u00C2\u00A0not\u00C2\u00A0possible\u00C2\u00A0 in\u00C2\u00A0practical\u00C2\u00A0 terms.\u00C2\u00A0 \u00C2\u00A0Developing\u00C2\u00A0a\u00C2\u00A0scaled\u00C2\u00A0up\u00C2\u00A0 procedure\u00C2\u00A0 for\u00C2\u00A0preparing\u00C2\u00A0precise\u00C2\u00A0valences,\u00C2\u00A0or\u00C2\u00A0an\u00C2\u00A0altogether\u00C2\u00A0different\u00C2\u00A0procedure,\u00C2\u00A0would\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 required.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 flow\u00C2\u00A0 cytometry\u00E2\u0080\u0090based\u00C2\u00A0method\u00C2\u00A0used\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 chapter\u00C2\u00A0provides\u00C2\u00A0 a\u00C2\u00A0 suitable\u00C2\u00A0 alternative\u00C2\u00A0 to\u00C2\u00A0 density\u00C2\u00A0 gradient\u00C2\u00A0 ultracentrifugation,\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 data\u00C2\u00A0 was\u00C2\u00A0 corroborated\u00C2\u00A0 by\u00C2\u00A0 confocal\u00C2\u00A0 laser\u00E2\u0080\u0090scanning\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy\u00C2\u00A0 images\u00C2\u00A0 (Figures\u00C2\u00A0 6.2\u00C2\u00A0&\u00C2\u00A0 6.5).\u00C2\u00A0 \u00C2\u00A0Others\u00C2\u00A0 have\u00C2\u00A0 used\u00C2\u00A0 an\u00C2\u00A0 identical\u00C2\u00A0 approach\u00C2\u00A0 to\u00C2\u00A0 determine\u00C2\u00A0membrane\u00C2\u00A0raft\u00C2\u00A0affinity\u00C2\u00A0when\u00C2\u00A0ultracentrifugation\u00C2\u00A0 is\u00C2\u00A0 impractical\u00C2\u00A0or\u00C2\u00A0 impossible249,\u00C2\u00A0250,\u00C2\u00A0or\u00C2\u00A0to\u00C2\u00A0 verify\u00C2\u00A0western\u00C2\u00A0blots\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0fractions\u00C2\u00A0after\u00C2\u00A0ultracentrifugation251.\u00C2\u00A0 \u00C2\u00A0Simons\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 have\u00C2\u00A0 pointed\u00C2\u00A0 out\u00C2\u00A0 that\u00C2\u00A0 a\u00C2\u00A0 common\u00C2\u00A0 feature\u00C2\u00A0 of\u00C2\u00A0 density\u00C2\u00A0 gradient\u00C2\u00A0 ultracentrifugation\u00C2\u00A0 DR\u00C2\u00A0 analyses\u00C2\u00A0 is\u00C2\u00A0 variation\u00C2\u00A0 in\u00C2\u00A0 results,\u00C2\u00A0 even\u00C2\u00A0 between\u00C2\u00A0 replicates\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0153\u00C2\u00A0\u00C2\u00A0 experiment,\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0major\u00C2\u00A0weakness\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0technique234.\u00C2\u00A0\u00C2\u00A0They\u00C2\u00A0also\u00C2\u00A0point\u00C2\u00A0out\u00C2\u00A0that\u00C2\u00A0DR\u00C2\u00A0is\u00C2\u00A0only\u00C2\u00A0a\u00C2\u00A0 starting\u00C2\u00A0point\u00C2\u00A0for\u00C2\u00A0studying\u00C2\u00A0raft\u00C2\u00A0association,\u00C2\u00A0and\u00C2\u00A0that\u00C2\u00A0changes\u00C2\u00A0 in\u00C2\u00A0DR\u00C2\u00A0 in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0biological\u00C2\u00A0stimuli\u00C2\u00A0 are\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0useful\u00C2\u00A0indicators\u00C2\u00A0of\u00C2\u00A0membrane\u00C2\u00A0raft\u00C2\u00A0involvement\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0response.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0strong\u00C2\u00A0 DR\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0protein\u00C2\u00A0 in\u00C2\u00A0 itself\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0provide\u00C2\u00A0much\u00C2\u00A0 information\u00C2\u00A0pertaining\u00C2\u00A0 to\u00C2\u00A0raft\u00C2\u00A0association,\u00C2\u00A0but\u00C2\u00A0 if\u00C2\u00A0a\u00C2\u00A0 stimulus\u00C2\u00A0 causes\u00C2\u00A0 the\u00C2\u00A0protein\u00C2\u00A0 to\u00C2\u00A0undergo\u00C2\u00A0a\u00C2\u00A0decrease\u00C2\u00A0 in\u00C2\u00A0DR,\u00C2\u00A0a\u00C2\u00A0movement\u00C2\u00A0out\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0stimulus\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0assigned234.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0data\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A06.1\u00C2\u00A0&\u00C2\u00A06.3\u00C2\u00A0employ\u00C2\u00A0this\u00C2\u00A0differential\u00C2\u00A0 approach\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0DR\u00C2\u00A0data,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0changes\u00C2\u00A0in\u00C2\u00A0DR\u00C2\u00A0measured\u00C2\u00A0in\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0 treatments\u00C2\u00A0are\u00C2\u00A0supported\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0confocal\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0images\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A06.2\u00C2\u00A0and\u00C2\u00A06.5.\u00C2\u00A0 The\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0chapter\u00C2\u00A0deviate\u00C2\u00A0from\u00C2\u00A0those\u00C2\u00A0reported\u00C2\u00A0by\u00C2\u00A0many\u00C2\u00A0other\u00C2\u00A0groups\u00C2\u00A0that\u00C2\u00A0show\u00C2\u00A0that\u00C2\u00A0 CD20\u00C2\u00A0 is\u00C2\u00A0 found\u00C2\u00A0 outside\u00C2\u00A0 of\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 rafts\u00C2\u00A0 in\u00C2\u00A0 different\u00C2\u00A0 lymphoma\u00C2\u00A0 cell\u00C2\u00A0 lines,\u00C2\u00A0 but\u00C2\u00A0 upon\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Rtx,\u00C2\u00A0 CD20\u00C2\u00A0 translocates\u00C2\u00A0 to\u00C2\u00A0membrane\u00C2\u00A0 rafts74,\u00C2\u00A0168,\u00C2\u00A0169.\u00C2\u00A0 \u00C2\u00A0Using\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 approach\u00C2\u00A0 to\u00C2\u00A0 quantify\u00C2\u00A0DR\u00C2\u00A0as\u00C2\u00A0in\u00C2\u00A0Figures\u00C2\u00A06.1\u00C2\u00A0&\u00C2\u00A06.3,\u00C2\u00A0we\u00C2\u00A0employed\u00C2\u00A0A647\u00E2\u0080\u0090pCD20\u00C2\u00A0(Section\u00C2\u00A04.3.6)\u00C2\u00A0to\u00C2\u00A0measure\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0 CD20,\u00C2\u00A0and\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0no\u00C2\u00A0changes\u00C2\u00A0 in\u00C2\u00A0DR\u00C2\u00A0values\u00C2\u00A0for\u00C2\u00A0CD20\u00C2\u00A0(~0.6)\u00C2\u00A0before\u00C2\u00A0and\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0 and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 (data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0 \u00C2\u00A0Confocal\u00C2\u00A0 fluorescence\u00C2\u00A0microscopy\u00C2\u00A0 images\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0 those\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0 Figures\u00C2\u00A06.5\u00C2\u00A0also\u00C2\u00A0indicate\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0(and\u00C2\u00A0therefore\u00C2\u00A0CD20)\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0colocalize\u00C2\u00A0with\u00C2\u00A0GM1,\u00C2\u00A0 a\u00C2\u00A0membrane\u00C2\u00A0raft\u00C2\u00A0marker.\u00C2\u00A0\u00C2\u00A0Most\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0studies\u00C2\u00A0indicating\u00C2\u00A0increased\u00C2\u00A0raft\u00C2\u00A0association\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0after\u00C2\u00A0Rtx\u00C2\u00A0 treatment\u00C2\u00A0employed\u00C2\u00A0density\u00C2\u00A0gradient\u00C2\u00A0ultracentrifugation\u00C2\u00A0and\u00C2\u00A0western\u00C2\u00A0blotting,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0explain\u00C2\u00A0 the\u00C2\u00A0difference\u00C2\u00A0 in\u00C2\u00A0part\u00C2\u00A0due\u00C2\u00A0the\u00C2\u00A0variability\u00C2\u00A0of\u00C2\u00A0such\u00C2\u00A0results,\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0above.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0differences\u00C2\u00A0may\u00C2\u00A0 also\u00C2\u00A0be\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0that\u00C2\u00A0most\u00C2\u00A0previous\u00C2\u00A0studies\u00C2\u00A0have\u00C2\u00A0employed\u00C2\u00A0a\u00C2\u00A02oAb,\u00C2\u00A0which\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0moderate\u00C2\u00A0 crosslinking\u00C2\u00A0 of\u00C2\u00A0 Rtx/CD20,\u00C2\u00A0 as\u00C2\u00A0 opposed\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0more\u00C2\u00A0 substantial\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 provided\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 multivalent\u00C2\u00A0 constructs\u00C2\u00A0 used\u00C2\u00A0 here.\u00C2\u00A0 \u00C2\u00A0 Different\u00C2\u00A0 degrees\u00C2\u00A0 of\u00C2\u00A0 hypercrosslinking\u00C2\u00A0would\u00C2\u00A0 be\u00C2\u00A0 expected\u00C2\u00A0 to\u00C2\u00A0 modulate\u00C2\u00A0raft\u00C2\u00A0behavior\u00C2\u00A0to\u00C2\u00A0different\u00C2\u00A0extents\u00C2\u00A0(Section\u00C2\u00A06.2.2).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 One\u00C2\u00A0study\u00C2\u00A0in\u00C2\u00A0particular\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0by\u00C2\u00A0depleting\u00C2\u00A0Chol\u00C2\u00A0using\u00C2\u00A0MBCD\u00C2\u00A0concentrations\u00C2\u00A0similar\u00C2\u00A0to\u00C2\u00A0 those\u00C2\u00A0 employed\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 chapter,\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 apoptosis\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00C2\u00A0 and\u00C2\u00A0 a\u00C2\u00A02oAb\u00C2\u00A0decreased\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 MBCD\u00C2\u00A0 concentration\u00C2\u00A0 was\u00C2\u00A0 increased74.\u00C2\u00A0 \u00C2\u00A0 Our\u00C2\u00A0 results,\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 other\u00C2\u00A0 hand,\u00C2\u00A0 show\u00C2\u00A0 that\u00C2\u00A0 insertion\u00C2\u00A0 of\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0154\u00C2\u00A0\u00C2\u00A0 additional\u00C2\u00A0 Chol\u00C2\u00A0 into\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane\u00C2\u00A0 using\u00C2\u00A0 MBCD/Chol\u00C2\u00A0 abolishes\u00C2\u00A0 apoptosis,\u00C2\u00A0 while\u00C2\u00A0 Chol\u00C2\u00A0 depletion\u00C2\u00A0 using\u00C2\u00A0 MBCD,\u00C2\u00A0 and\u00C2\u00A0 particularly\u00C2\u00A0 simvastatin,\u00C2\u00A0 sensitize\u00C2\u00A0 cells\u00C2\u00A0 to\u00C2\u00A0 apoptosis\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx.\u00C2\u00A0 \u00C2\u00A0One\u00C2\u00A0 possibility\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 different\u00C2\u00A0 results\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 that\u00C2\u00A0MBCD\u00C2\u00A0 interfered\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A02oAb\u00C2\u00A0 to\u00C2\u00A0hypercrosslink\u00C2\u00A0Rtx\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 study\u00C2\u00A0of\u00C2\u00A0 Janas\u00C2\u00A0et\u00C2\u00A0al.,\u00C2\u00A0an\u00C2\u00A0event\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 required\u00C2\u00A0 for\u00C2\u00A0 apoptosis\u00C2\u00A0(Sections\u00C2\u00A01.4.3\u00C2\u00A0and\u00C2\u00A01.6.1).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 One\u00C2\u00A0 other\u00C2\u00A0way\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 results\u00C2\u00A0 here\u00C2\u00A0 differ\u00C2\u00A0 from\u00C2\u00A0 those\u00C2\u00A0 in\u00C2\u00A0 other\u00C2\u00A0 reports\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0 apoptotic\u00C2\u00A0 CD120a\u00C2\u00A0signaling\u00C2\u00A0 is\u00C2\u00A0generally\u00C2\u00A0considered\u00C2\u00A0 to\u00C2\u00A0 require\u00C2\u00A0 internalization\u00C2\u00A0of\u00C2\u00A0activated\u00C2\u00A0CD120a\u00E2\u0080\u0090containing\u00C2\u00A0 Complex\u00C2\u00A0II\u00C2\u00A0by\u00C2\u00A0clathrin\u00E2\u0080\u0090dependent\u00C2\u00A0endocytosis\u00C2\u00A0(see\u00C2\u00A0Section\u00C2\u00A06.2.1).\u00C2\u00A0\u00C2\u00A0Our\u00C2\u00A0results\u00C2\u00A0show\u00C2\u00A0no\u00C2\u00A0evidence\u00C2\u00A0of\u00C2\u00A0 CD120a\u00C2\u00A0internalization\u00C2\u00A0preceding\u00C2\u00A0apoptosis,\u00C2\u00A0including\u00C2\u00A0confocal\u00C2\u00A0fluorescence\u00C2\u00A0microscopy\u00C2\u00A0images\u00C2\u00A0that\u00C2\u00A0 were\u00C2\u00A0 obtained\u00C2\u00A0where\u00C2\u00A0 CD120a\u00C2\u00A0 staining\u00C2\u00A0 occurred\u00C2\u00A0 after\u00C2\u00A0 permeabilization\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0 (not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0Moreover,\u00C2\u00A0we\u00C2\u00A0employed\u00C2\u00A0chlorpromazine\u00C2\u00A0treatment,\u00C2\u00A0which\u00C2\u00A0inhibits\u00C2\u00A0clathrin\u00E2\u0080\u0090dependent\u00C2\u00A0 apoptosis,\u00C2\u00A0at\u00C2\u00A0subcytotoxic\u00C2\u00A0doses\u00C2\u00A0in\u00C2\u00A0conjunction\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0and\u00C2\u00A0we\u00C2\u00A0found\u00C2\u00A0no\u00C2\u00A0differences\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0induced\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0chlorpromazine\u00C2\u00A0(data\u00C2\u00A0not\u00C2\u00A0shown).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0 requirement\u00C2\u00A0 for\u00C2\u00A0endocytosis\u00C2\u00A0may\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0universal\u00C2\u00A0 to\u00C2\u00A0all\u00C2\u00A0cell\u00C2\u00A0 types175,\u00C2\u00A0 so\u00C2\u00A0apoptosis\u00C2\u00A0via\u00C2\u00A0Complex\u00C2\u00A0 II\u00C2\u00A0 may\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0required\u00C2\u00A0in\u00C2\u00A0Ramos\u00C2\u00A0cells.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0Other\u00C2\u00A0death\u00C2\u00A0receptors,\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0DR4\u00C2\u00A0and\u00C2\u00A0DR5,\u00C2\u00A0have\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0 shown\u00C2\u00A0to\u00C2\u00A0not\u00C2\u00A0require\u00C2\u00A0endocytosis\u00C2\u00A0before\u00C2\u00A0initiating\u00C2\u00A0apoptosis87.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 chapter\u00C2\u00A0 have\u00C2\u00A0 demonstrated\u00C2\u00A0 that\u00C2\u00A0 a\u00C2\u00A0 CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0is\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0occurring\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0after\u00C2\u00A0normal\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 by\u00C2\u00A0 Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0 They\u00C2\u00A0 also\u00C2\u00A0 showed\u00C2\u00A0 that\u00C2\u00A0 increased\u00C2\u00A0 cellular\u00C2\u00A0 Chol\u00C2\u00A0 content\u00C2\u00A0 abolished\u00C2\u00A0 multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis,\u00C2\u00A0while\u00C2\u00A0lower\u00C2\u00A0levels\u00C2\u00A0sensitized\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0apoptosis.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0synthesis\u00C2\u00A0of\u00C2\u00A0 these\u00C2\u00A0two\u00C2\u00A0results\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0improvements\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0 achieved\u00C2\u00A0with\u00C2\u00A0concurrent\u00C2\u00A0Chol\u00E2\u0080\u0090lowering\u00C2\u00A0therapies\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0simvastatin\u00C2\u00A0or\u00C2\u00A0other\u00C2\u00A0statins.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0utility\u00C2\u00A0of\u00C2\u00A0 such\u00C2\u00A0an\u00C2\u00A0approach\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0investigated\u00C2\u00A0using\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0lymphoma\u00C2\u00A0xenograft\u00C2\u00A0models\u00C2\u00A0to\u00C2\u00A0study\u00C2\u00A0the\u00C2\u00A0efficacy\u00C2\u00A0 of\u00C2\u00A0such\u00C2\u00A0treatment\u00C2\u00A0strategies\u00C2\u00A0and\u00C2\u00A0to\u00C2\u00A0further\u00C2\u00A0investigate\u00C2\u00A0the\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Section\u00C2\u00A07.4.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0155\u00C2\u00A0\u00C2\u00A0 6.5.2\u00C2\u00A0 Conclusions\u00C2\u00A0 Our\u00C2\u00A0 results\u00C2\u00A0 have\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 serves\u00C2\u00A0 to\u00C2\u00A0 upregulate\u00C2\u00A0 CD120a\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane,\u00C2\u00A0where\u00C2\u00A0it\u00C2\u00A0elicits\u00C2\u00A0proapoptotic\u00C2\u00A0signaling\u00C2\u00A0when\u00C2\u00A0localized\u00C2\u00A0outside\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts.\u00C2\u00A0\u00C2\u00A0 Insertion\u00C2\u00A0of\u00C2\u00A0additional\u00C2\u00A0Chol\u00C2\u00A0 into\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0resulted\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0translocation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0to\u00C2\u00A0 rafts\u00C2\u00A0and\u00C2\u00A0abolishment\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis,\u00C2\u00A0while\u00C2\u00A0depletion\u00C2\u00A0of\u00C2\u00A0Chol\u00C2\u00A0sensitized\u00C2\u00A0cells\u00C2\u00A0 to\u00C2\u00A0apoptosis,\u00C2\u00A0indicating\u00C2\u00A0that\u00C2\u00A0apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0is\u00C2\u00A0Chol\u00E2\u0080\u0090dependent.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0model\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0of\u00C2\u00A0 Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0effector\u00C2\u00A0 cells\u00C2\u00A0with\u00C2\u00A0Ab\u00E2\u0080\u0090coated\u00C2\u00A0 tumor\u00C2\u00A0 cells,\u00C2\u00A0 Fc\u00C2\u00A0 region\u00E2\u0080\u0090specific\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0 induced\u00C2\u00A0 significantly\u00C2\u00A0elevated\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0only\u00C2\u00A0 when\u00C2\u00A0 the\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0pretreated\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 same\u00C2\u00A0effect\u00C2\u00A0was\u00C2\u00A0observed\u00C2\u00A0when\u00C2\u00A0Ramos\u00C2\u00A0cells\u00C2\u00A0were\u00C2\u00A0 cocultured\u00C2\u00A0with\u00C2\u00A0samples\u00C2\u00A0of\u00C2\u00A0PBMCs,\u00C2\u00A0which\u00C2\u00A0contain\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0 that\u00C2\u00A0express\u00C2\u00A0Fc\u00CE\u00B3Rs.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0suggests\u00C2\u00A0 that\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0capable\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0Rtx\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0lymphoma\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 vivo,\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0 increased\u00C2\u00A0 apoptosis\u00C2\u00A0 resulting\u00C2\u00A0 from\u00C2\u00A0 CD120a\u00C2\u00A0 upregulation\u00C2\u00A0may\u00C2\u00A0 constitute\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0that\u00C2\u00A0occurs\u00C2\u00A0upon\u00C2\u00A0regular\u00C2\u00A0Rtx\u00C2\u00A0therapy.\u00C2\u00A0 \u00C2\u00A0Together,\u00C2\u00A0this\u00C2\u00A0data\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0 Rtx\u00C2\u00A0 efficacy\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 improved\u00C2\u00A0 through\u00C2\u00A0 concurrent\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0 Chol\u00E2\u0080\u0090lowering\u00C2\u00A0 agents\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0statins.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0156\u00C2\u00A0\u00C2\u00A0 7\u00C2\u00A0 Discussion\u00C2\u00A0 and\u00C2\u00A0 conclusions:\u00C2\u00A0 Antibody\u00C2\u00ADlipid\u00C2\u00A0 nanoparticles\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 promising\u00C2\u00A0tool\u00C2\u00A0in\u00C2\u00A0cancer\u00C2\u00A0drug\u00C2\u00A0development\u00C2\u00A0 7.1\u00C2\u00A0 Recapitulation\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0work\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0dissertation\u00C2\u00A0outlines\u00C2\u00A0a\u00C2\u00A0complete\u00C2\u00A0methodology\u00C2\u00A0that\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0 discover\u00C2\u00A0 new\u00C2\u00A0 drugs\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 therapeutic\u00C2\u00A0 monoclonal\u00C2\u00A0 Abs\u00C2\u00A0 and\u00C2\u00A0 develop\u00C2\u00A0 such\u00C2\u00A0 therapies\u00C2\u00A0 to\u00C2\u00A0 an\u00C2\u00A0 advanced\u00C2\u00A0preclinical\u00C2\u00A0stage,\u00C2\u00A0including\u00C2\u00A0an\u00C2\u00A0elucidation\u00C2\u00A0of\u00C2\u00A0their\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0methodology\u00C2\u00A0 makes\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs,\u00C2\u00A0a\u00C2\u00A0type\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0construct\u00C2\u00A0that\u00C2\u00A0employs\u00C2\u00A0a\u00C2\u00A0liposome\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0 nanoscale\u00C2\u00A0scaffold\u00C2\u00A0 for\u00C2\u00A0bridging\u00C2\u00A0many\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0 together.\u00C2\u00A0 \u00C2\u00A0Therapeutic\u00C2\u00A0Abs\u00C2\u00A0 that\u00C2\u00A0show\u00C2\u00A0minimal\u00C2\u00A0 activity\u00C2\u00A0 as\u00C2\u00A0 bivalent\u00C2\u00A0molecules\u00C2\u00A0may\u00C2\u00A0 show\u00C2\u00A0 enhanced\u00C2\u00A0 therapeutic\u00C2\u00A0 activity\u00C2\u00A0 as\u00C2\u00A0multivalent\u00C2\u00A0 constructs,\u00C2\u00A0 enabling\u00C2\u00A0 their\u00C2\u00A0 further\u00C2\u00A0 development\u00C2\u00A0 as\u00C2\u00A0multivalent\u00C2\u00A0 Abs\u00C2\u00A0 of\u00C2\u00A0 defined\u00C2\u00A0 (optimal)\u00C2\u00A0 valence,\u00C2\u00A0 rather\u00C2\u00A0 than\u00C2\u00A0 bivalent\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0proof\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0concept\u00C2\u00A0was\u00C2\u00A0illustrated\u00C2\u00A0using\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0case\u00C2\u00A0with\u00C2\u00A0other\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Abs\u00C2\u00A0directed\u00C2\u00A0against\u00C2\u00A0different\u00C2\u00A0targets,\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 exhibited\u00C2\u00A0 enhanced\u00C2\u00A0 activity\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx;\u00C2\u00A0 as\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 2,\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 induced\u00C2\u00A0 higher\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis,\u00C2\u00A0ADCC,\u00C2\u00A0and\u00C2\u00A0CDC\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0equal\u00C2\u00A0doses\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0 further\u00C2\u00A0 understand\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs,\u00C2\u00A0 a\u00C2\u00A0 new\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 preparation\u00C2\u00A0 methodology\u00C2\u00A0was\u00C2\u00A0developed\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A03\u00C2\u00A0which\u00C2\u00A0allowed\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0creation\u00C2\u00A0of\u00C2\u00A0many\u00C2\u00A0valences\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090 LNP\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0Using\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0prepared\u00C2\u00A0with\u00C2\u00A0this\u00C2\u00A0methodology,\u00C2\u00A0Chapter\u00C2\u00A04\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0 apoptosis\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0depended\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 valence,\u00C2\u00A0 and\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0 exhibited\u00C2\u00A0 unique\u00C2\u00A0biological\u00C2\u00A0properties\u00C2\u00A0consistent\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0notion\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0higher\u00C2\u00A0valences\u00C2\u00A0can\u00C2\u00A0hypercrosslink\u00C2\u00A0 CD20\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0more\u00C2\u00A0appreciably\u00C2\u00A0and\u00C2\u00A0form\u00C2\u00A0Rtx/CD20\u00C2\u00A0clusters\u00C2\u00A0that\u00C2\u00A0may\u00C2\u00A0play\u00C2\u00A0a\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0 apoptosis.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0improved\u00C2\u00A0methodology\u00C2\u00A0was\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0study\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0in\u00C2\u00A0 Chapter\u00C2\u00A0 5,\u00C2\u00A0which\u00C2\u00A0 uncovered\u00C2\u00A0 a\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 involving\u00C2\u00A0 the\u00C2\u00A0 valence\u00E2\u0080\u0090dependent\u00C2\u00A0 upregulation\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0subsequent\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0caspase\u00E2\u0080\u00908,\u00C2\u00A0resulting\u00C2\u00A0 in\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0Moreover,\u00C2\u00A0 this\u00C2\u00A0chapter\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0which\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0contain\u00C2\u00A0liposomes,\u00C2\u00A0also\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0through\u00C2\u00A0a\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0157\u00C2\u00A0\u00C2\u00A0 CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action,\u00C2\u00A0 illustrating\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 liposome\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 apoptosis\u00C2\u00A0induced\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0Chapter\u00C2\u00A06\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0 was\u00C2\u00A0 Chol\u00E2\u0080\u0090dependent,\u00C2\u00A0 with\u00C2\u00A0 lower\u00C2\u00A0 levels\u00C2\u00A0 promoting\u00C2\u00A0 apoptosis\u00C2\u00A0 and\u00C2\u00A0 higher\u00C2\u00A0 levels\u00C2\u00A0 inhibiting\u00C2\u00A0 it,\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0known\u00C2\u00A0Chol\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0 signaling.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 final\u00C2\u00A0chapter\u00C2\u00A0also\u00C2\u00A0 showed\u00C2\u00A0 that\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 on\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 by\u00C2\u00A0 Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0 immune\u00C2\u00A0 effector\u00C2\u00A0 cells\u00C2\u00A0 is\u00C2\u00A0 sufficient\u00C2\u00A0to\u00C2\u00A0upregulate\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0 induce\u00C2\u00A0apoptosis,\u00C2\u00A0suggesting\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0direct\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0occurs\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0after\u00C2\u00A0normal\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0therapy.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0constitutes\u00C2\u00A0a\u00C2\u00A0novel\u00C2\u00A0direct\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0which\u00C2\u00A0so\u00C2\u00A0far\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0undefined.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 7.2\u00C2\u00A0 Discussion\u00C2\u00A0concerning\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0\u00C2\u00A0 7.2.1\u00C2\u00A0 Strengths\u00C2\u00A0and\u00C2\u00A0weaknesses\u00C2\u00A0of\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0tool\u00C2\u00A0for\u00C2\u00A0developing\u00C2\u00A0new\u00C2\u00A0drugs\u00C2\u00A0 The\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0as\u00C2\u00A0 liposomal\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0 is\u00C2\u00A0a\u00C2\u00A0unique\u00C2\u00A0approach\u00C2\u00A0to\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0 liposome\u00C2\u00A0to\u00C2\u00A0enhance\u00C2\u00A0the\u00C2\u00A0activity\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0drug\u00C2\u00A0that\u00C2\u00A0is\u00C2\u00A0present\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0outside,\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0the\u00C2\u00A0inside,\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0liposome.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0one\u00C2\u00A0feature\u00C2\u00A0that\u00C2\u00A0distinguishes\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0from\u00C2\u00A0immunoliposomes,\u00C2\u00A0as\u00C2\u00A0discussed\u00C2\u00A0in\u00C2\u00A0 Section\u00C2\u00A01.2.\u00C2\u00A0\u00C2\u00A0However,\u00C2\u00A0the\u00C2\u00A0most\u00C2\u00A0important\u00C2\u00A0difference\u00C2\u00A0between\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0and\u00C2\u00A0immunoliposomes,\u00C2\u00A0the\u00C2\u00A0 evidence\u00C2\u00A0for\u00C2\u00A0which\u00C2\u00A0is\u00C2\u00A0provided\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0dissertation,\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0while\u00C2\u00A0immunoliposomes\u00C2\u00A0are\u00C2\u00A0the\u00C2\u00A0final\u00C2\u00A0drug\u00C2\u00A0 product\u00C2\u00A0 themselves,\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0are\u00C2\u00A0not;\u00C2\u00A0 instead,\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0are\u00C2\u00A0a\u00C2\u00A0 tool\u00C2\u00A0 for\u00C2\u00A0optimizing\u00C2\u00A0and\u00C2\u00A0studying\u00C2\u00A0 the\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0Their\u00C2\u00A0use\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0particularly\u00C2\u00A0advantageous\u00C2\u00A0 when\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0straightforward\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0many\u00C2\u00A0valences\u00C2\u00A0using\u00C2\u00A0other\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0formulations,\u00C2\u00A0as\u00C2\u00A0is\u00C2\u00A0the\u00C2\u00A0 case\u00C2\u00A0with\u00C2\u00A0gold\u00C2\u00A0nanoparticles\u00C2\u00A0 for\u00C2\u00A0example96,\u00C2\u00A0or\u00C2\u00A0when\u00C2\u00A0 the\u00C2\u00A0multivalent\u00C2\u00A0Ab\u00C2\u00A0 is\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 included\u00C2\u00A0 in\u00C2\u00A0more\u00C2\u00A0 complex\u00C2\u00A0systems\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0multifunctional\u00C2\u00A0therapeutic\u00C2\u00A0nanoparticles167,\u00C2\u00A0where\u00C2\u00A0 it\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0desirable\u00C2\u00A0 to\u00C2\u00A0study\u00C2\u00A0Ab\u00C2\u00A0multivalency\u00C2\u00A0separately\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0rest\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0system.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Another\u00C2\u00A0 strength\u00C2\u00A0 of\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0 is\u00C2\u00A0 that\u00C2\u00A0 valences\u00C2\u00A0 as\u00C2\u00A0 high\u00C2\u00A0 as\u00C2\u00A0 250\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 reproducibly\u00C2\u00A0 prepared\u00C2\u00A0 using\u00C2\u00A0 the\u00C2\u00A0methodology\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 3.\u00C2\u00A0 \u00C2\u00A0Although\u00C2\u00A0 other\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 probably\u00C2\u00A0 employed\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0158\u00C2\u00A0\u00C2\u00A0 valences\u00C2\u00A0 in\u00C2\u00A0this\u00C2\u00A0range\u00C2\u00A0as\u00C2\u00A0described\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A01.6,\u00C2\u00A0the\u00C2\u00A0actual\u00C2\u00A0valences\u00C2\u00A0were\u00C2\u00A0never\u00C2\u00A0defined,\u00C2\u00A0possibly\u00C2\u00A0 because\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0was\u00C2\u00A0heterogeneous,\u00C2\u00A0suitable\u00C2\u00A0assays\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0available,\u00C2\u00A0or\u00C2\u00A0it\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0necessary\u00C2\u00A0 to\u00C2\u00A0measure\u00C2\u00A0 the\u00C2\u00A0 valence.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 studies\u00C2\u00A0 where\u00C2\u00A0 the\u00C2\u00A0 valence\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 defined\u00C2\u00A0 for\u00C2\u00A0 a\u00C2\u00A0multivalent\u00C2\u00A0 Ab\u00C2\u00A0 construct,\u00C2\u00A0 it\u00C2\u00A0generally\u00C2\u00A0has\u00C2\u00A0not\u00C2\u00A0exceeded\u00C2\u00A010\u00C2\u00A0(Section\u00C2\u00A01.6.3).\u00C2\u00A0 \u00C2\u00A0A\u00C2\u00A0range\u00C2\u00A0of\u00C2\u00A0specific\u00C2\u00A0valences\u00C2\u00A0up\u00C2\u00A0to\u00C2\u00A0250\u00C2\u00A0 can\u00C2\u00A0now\u00C2\u00A0be\u00C2\u00A0reproducibly\u00C2\u00A0prepared\u00C2\u00A0with\u00C2\u00A0high\u00C2\u00A0start\u00E2\u0080\u0090to\u00E2\u0080\u0090finish\u00C2\u00A0yields\u00C2\u00A0of\u00C2\u00A0coupled\u00C2\u00A0Ab\u00C2\u00A0(80%).\u00C2\u00A0 The\u00C2\u00A0principal\u00C2\u00A0weakness\u00C2\u00A0of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0is\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0constructs\u00C2\u00A0resulting\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0in\u00C2\u00A0 Chapter\u00C2\u00A03\u00C2\u00A0are\u00C2\u00A0likely\u00C2\u00A0not\u00C2\u00A0suitable\u00C2\u00A0for\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0studies.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0because\u00C2\u00A0the\u00C2\u00A0Neut\u00C2\u00A0molecules\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 coupling\u00C2\u00A0process\u00C2\u00A0are\u00C2\u00A0proteins\u00C2\u00A0and\u00C2\u00A0there\u00C2\u00A0therefore\u00C2\u00A0potentially\u00C2\u00A0antigenic.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 information\u00C2\u00A0obtained\u00C2\u00A0 using\u00C2\u00A0these\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0(optimal\u00C2\u00A0valence\u00C2\u00A0and\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action),\u00C2\u00A0however,\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0translated\u00C2\u00A0into\u00C2\u00A0a\u00C2\u00A0 variety\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0constructs92\u00E2\u0080\u009096,\u00C2\u00A0167.\u00C2\u00A0\u00C2\u00A0If\u00C2\u00A0it\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0desirable\u00C2\u00A0to\u00C2\u00A0study\u00C2\u00A0a\u00C2\u00A0liposomal\u00C2\u00A0multivalent\u00C2\u00A0 Ab\u00C2\u00A0construct\u00C2\u00A0in\u00C2\u00A0vivo,\u00C2\u00A0an\u00C2\u00A0alternative\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0preparing\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0would\u00C2\u00A0have\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0employed,\u00C2\u00A0such\u00C2\u00A0 as\u00C2\u00A0that\u00C2\u00A0used\u00C2\u00A0 in\u00C2\u00A0Chapter\u00C2\u00A02\u00C2\u00A0which\u00C2\u00A0uses\u00C2\u00A0covalent\u00C2\u00A0coupling.\u00C2\u00A0 \u00C2\u00A0Section\u00C2\u00A03.2.1\u00C2\u00A0described\u00C2\u00A0the\u00C2\u00A0difficulties\u00C2\u00A0 in\u00C2\u00A0 terms\u00C2\u00A0of\u00C2\u00A0yield,\u00C2\u00A0reproducibility,\u00C2\u00A0and\u00C2\u00A0control\u00C2\u00A0over\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0such\u00C2\u00A0methods,\u00C2\u00A0so\u00C2\u00A0they\u00C2\u00A0would\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0 suitable\u00C2\u00A0for\u00C2\u00A0studies\u00C2\u00A0on\u00C2\u00A0multivalency\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0outset,\u00C2\u00A0but\u00C2\u00A0they\u00C2\u00A0may\u00C2\u00A0potentially\u00C2\u00A0be\u00C2\u00A0used\u00C2\u00A0to\u00C2\u00A0prepare\u00C2\u00A0a\u00C2\u00A0 single\u00C2\u00A0valence\u00C2\u00A0for\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0studies.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 7.2.2\u00C2\u00A0 The\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0whole\u00C2\u00A0Abs\u00C2\u00A0in\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0versus\u00C2\u00A0Ab\u00C2\u00A0fragments\u00C2\u00A0 If\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 target\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 surface\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 cancer\u00C2\u00A0 cell\u00C2\u00A0 is\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 improved\u00C2\u00A0 therapeutic\u00C2\u00A0effects,\u00C2\u00A0 then\u00C2\u00A0 it\u00C2\u00A0may\u00C2\u00A0appear\u00C2\u00A0advantageous\u00C2\u00A0 to\u00C2\u00A0employ\u00C2\u00A0Ab\u00C2\u00A0 fragments\u00C2\u00A0 (such\u00C2\u00A0as\u00C2\u00A0 Fab\u00C2\u00A0regions)\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0the\u00C2\u00A0whole\u00C2\u00A0IgG.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0this\u00C2\u00A0way,\u00C2\u00A0defined\u00C2\u00A0chemical\u00C2\u00A0species\u00C2\u00A0could\u00C2\u00A0more\u00C2\u00A0easily\u00C2\u00A0be\u00C2\u00A0 prepared\u00C2\u00A0which\u00C2\u00A0could\u00C2\u00A0 then\u00C2\u00A0be\u00C2\u00A0coupled\u00C2\u00A0 to\u00C2\u00A0 liposomes;\u00C2\u00A0 this\u00C2\u00A0 is\u00C2\u00A0 the\u00C2\u00A0approach\u00C2\u00A0used\u00C2\u00A0by\u00C2\u00A0Nellis\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 for\u00C2\u00A0 preparing\u00C2\u00A0anti\u00E2\u0080\u0090ErbB2\u00C2\u00A0 immunoliposomes252,\u00C2\u00A0253.\u00C2\u00A0 \u00C2\u00A0Using\u00C2\u00A0Fab\u00C2\u00A0 fragments\u00C2\u00A0 for\u00C2\u00A0preparing\u00C2\u00A0multivalent\u00C2\u00A0Abs,\u00C2\u00A0 however,\u00C2\u00A0would\u00C2\u00A0completely\u00C2\u00A0eliminate\u00C2\u00A0the\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Abs\u00C2\u00A0(ADCC\u00C2\u00A0and\u00C2\u00A0CDC),\u00C2\u00A0 which\u00C2\u00A0are\u00C2\u00A0important\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0overall\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0(Section\u00C2\u00A01.4).\u00C2\u00A0\u00C2\u00A0Chapter\u00C2\u00A02\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0 the\u00C2\u00A0indirect\u00C2\u00A0mechanisms\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0preserved\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0but\u00C2\u00A0enhanced.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0was\u00C2\u00A0explained\u00C2\u00A0by\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0159\u00C2\u00A0\u00C2\u00A0 the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 processes\u00C2\u00A0 of\u00C2\u00A0 ADCC\u00C2\u00A0 and\u00C2\u00A0 CDC\u00C2\u00A0 themselves\u00C2\u00A0 result\u00C2\u00A0 from\u00C2\u00A0 multivalent\u00C2\u00A0 interactions\u00C2\u00A0 involving\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region;\u00C2\u00A0these\u00C2\u00A0interactions\u00C2\u00A0are\u00C2\u00A0facilitated\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0is\u00C2\u00A0multivalent.\u00C2\u00A0\u00C2\u00A0ADCC\u00C2\u00A0and\u00C2\u00A0CDC\u00C2\u00A0 are\u00C2\u00A0common\u00C2\u00A0mechanisms\u00C2\u00A0to\u00C2\u00A0all\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0(Sections\u00C2\u00A01.4.1\u00C2\u00A0&\u00C2\u00A01.4.2),\u00C2\u00A0so\u00C2\u00A0the\u00C2\u00A0augmentation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0observed\u00C2\u00A0using\u00C2\u00A0other\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0methodology\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 3\u00C2\u00A0 utilizes\u00C2\u00A0 biotinylation\u00C2\u00A0 on\u00C2\u00A0 a\u00C2\u00A0NIMAC\u00C2\u00A0 column,\u00C2\u00A0 and\u00C2\u00A0 under\u00C2\u00A0 the\u00C2\u00A0 conditions\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0employed,\u00C2\u00A0between\u00C2\u00A0one\u00C2\u00A0and\u00C2\u00A0two\u00C2\u00A0biotin\u00C2\u00A0groups\u00C2\u00A0were\u00C2\u00A0added\u00C2\u00A0per\u00C2\u00A0Ab.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0 biotinylation\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0Ab,\u00C2\u00A0however,\u00C2\u00A0 is\u00C2\u00A0not\u00C2\u00A0homogeneous.\u00C2\u00A0 \u00C2\u00A0On\u00C2\u00A0some\u00C2\u00A0Ab\u00C2\u00A0molecules,\u00C2\u00A0 it\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0present\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 Fab\u00C2\u00A0 region,\u00C2\u00A0 which\u00C2\u00A0 may\u00C2\u00A0 negatively\u00C2\u00A0 affect\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 target\u00C2\u00A0 and\u00C2\u00A0 therefore\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 mechanism;\u00C2\u00A0on\u00C2\u00A0other\u00C2\u00A0molecules,\u00C2\u00A0a\u00C2\u00A0site\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0biotinylated,\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0affect\u00C2\u00A0the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0nonhomogeneous\u00C2\u00A0biotinylation\u00C2\u00A0constitutes\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0advantage\u00C2\u00A0when\u00C2\u00A0 considering\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0overall\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 consists\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0combination\u00C2\u00A0of\u00C2\u00A0direct\u00C2\u00A0and\u00C2\u00A0 indirect\u00C2\u00A0 factors;\u00C2\u00A0one\u00C2\u00A0could\u00C2\u00A0 imagine\u00C2\u00A0that\u00C2\u00A0a\u00C2\u00A0single\u00C2\u00A0biotinylation\u00C2\u00A0site\u00C2\u00A0on\u00C2\u00A0either\u00C2\u00A0the\u00C2\u00A0Fab\u00C2\u00A0or\u00C2\u00A0Fc\u00C2\u00A0would\u00C2\u00A0reduce\u00C2\u00A0or\u00C2\u00A0 eliminate\u00C2\u00A0one\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0contributing\u00C2\u00A0mechanisms.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0 appears\u00C2\u00A0 to\u00C2\u00A0 have\u00C2\u00A0 occurred,\u00C2\u00A0 for\u00C2\u00A0 example,\u00C2\u00A0 with\u00C2\u00A0 a\u00C2\u00A0multivalent\u00C2\u00A0 Ab\u00C2\u00A0 that\u00C2\u00A0 uses\u00C2\u00A0 a\u00C2\u00A0 specific\u00C2\u00A0 configuration\u00C2\u00A0of\u00C2\u00A0six\u00C2\u00A0Fab\u00C2\u00A0regions\u00C2\u00A0and\u00C2\u00A0one\u00C2\u00A0Fc\u00C2\u00A0region.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0configuration\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0complete\u00C2\u00A0abolition\u00C2\u00A0 of\u00C2\u00A0all\u00C2\u00A0CDC\u00C2\u00A0activity,\u00C2\u00A0possibly\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0point\u00C2\u00A0of\u00C2\u00A0attachment\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0Fc\u00C2\u00A0 region\u00C2\u00A0 interfering\u00C2\u00A0with\u00C2\u00A0CDC\u00C2\u00A0 processes\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0C1q\u00C2\u00A0binding90.\u00C2\u00A0\u00C2\u00A0By\u00C2\u00A0having\u00C2\u00A0many\u00C2\u00A0different\u00C2\u00A0biotinylation\u00C2\u00A0sites\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0in\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP,\u00C2\u00A0 some\u00C2\u00A0 individual\u00C2\u00A0molecules\u00C2\u00A0may\u00C2\u00A0 show\u00C2\u00A0 decreased\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 target\u00C2\u00A0 (biotinylation\u00C2\u00A0 in\u00C2\u00A0 Fab\u00C2\u00A0 region)\u00C2\u00A0 or\u00C2\u00A0 decreases\u00C2\u00A0 ADCC\u00C2\u00A0 or\u00C2\u00A0 CDC\u00C2\u00A0 (Fc\u00C2\u00A0 region);\u00C2\u00A0 these\u00C2\u00A0 processes,\u00C2\u00A0 however,\u00C2\u00A0 rely\u00C2\u00A0 on\u00C2\u00A0 multivalent\u00C2\u00A0 interactions\u00C2\u00A0 that\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 facilitated\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 neighboring\u00C2\u00A0 Abs\u00C2\u00A0 in\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP,\u00C2\u00A0 resulting\u00C2\u00A0 not\u00C2\u00A0 only\u00C2\u00A0 in\u00C2\u00A0 compensation\u00C2\u00A0but\u00C2\u00A0improvement\u00C2\u00A0in\u00C2\u00A0therapeutic\u00C2\u00A0responses.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 For\u00C2\u00A0example,\u00C2\u00A0Section\u00C2\u00A01.4.1\u00C2\u00A0described\u00C2\u00A0how\u00C2\u00A0C1q\u00C2\u00A0is\u00C2\u00A0a\u00C2\u00A0hexavalent\u00C2\u00A0ligand\u00C2\u00A0requiring\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0 several\u00C2\u00A0Fc\u00C2\u00A0regions\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0initiate\u00C2\u00A0CDC;\u00C2\u00A0if\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0one\u00C2\u00A0Ab\u00C2\u00A0in\u00C2\u00A0Ab\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0unable\u00C2\u00A0to\u00C2\u00A0bind\u00C2\u00A0C1q\u00C2\u00A0 due\u00C2\u00A0to\u00C2\u00A0 interference\u00C2\u00A0from\u00C2\u00A0a\u00C2\u00A0specific\u00C2\u00A0biotinylation\u00C2\u00A0site,\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0other\u00C2\u00A0unaffected\u00C2\u00A0Abs\u00C2\u00A0 would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0close\u00C2\u00A0by\u00C2\u00A0for\u00C2\u00A0binding,\u00C2\u00A0allowing\u00C2\u00A0CDC\u00C2\u00A0to\u00C2\u00A0progress.\u00C2\u00A0 \u00C2\u00A0It\u00C2\u00A0therefore\u00C2\u00A0appears\u00C2\u00A0that\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0160\u00C2\u00A0\u00C2\u00A0 having\u00C2\u00A0many\u00C2\u00A0sites\u00C2\u00A0of\u00C2\u00A0biotinylation\u00C2\u00A0 in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 is\u00C2\u00A0advantageous\u00C2\u00A0 to\u00C2\u00A0preserving\u00C2\u00A0all\u00C2\u00A0 the\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0 action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0construct.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 7.2.3\u00C2\u00A0 Relevance\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0studied\u00C2\u00A0in\u00C2\u00A0 vitro\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0absence\u00C2\u00A0of\u00C2\u00A0hypercrosslinking\u00C2\u00A0 Different\u00C2\u00A0strategies\u00C2\u00A0for\u00C2\u00A0creating\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0were\u00C2\u00A0employed\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0dissertation,\u00C2\u00A0including\u00C2\u00A0 the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP,\u00C2\u00A0Rtx\u00E2\u0080\u0090MS,\u00C2\u00A0Rtx\u00C2\u00A0+\u00C2\u00A02oAb,\u00C2\u00A0and\u00C2\u00A0Rtx\u00C2\u00A0+\u00C2\u00A02oAb\u00E2\u0080\u0090MS.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0treatment\u00C2\u00A0that\u00C2\u00A0provided\u00C2\u00A0maximum\u00C2\u00A0 therapeutic\u00C2\u00A0efficacy\u00C2\u00A0(levels\u00C2\u00A0of\u00C2\u00A0plasma\u00E2\u0080\u0090membrane\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0apoptosis)\u00C2\u00A0was\u00C2\u00A0hypercrosslinking\u00C2\u00A0with\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS\u00C2\u00A0following\u00C2\u00A0Rtx\u00C2\u00A0treatment\u00C2\u00A0(Figure\u00C2\u00A06.4B).\u00C2\u00A0\u00C2\u00A0These\u00C2\u00A0levels\u00C2\u00A0were\u00C2\u00A0higher\u00C2\u00A0than\u00C2\u00A0those\u00C2\u00A0observed\u00C2\u00A0after\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0of\u00C2\u00A0high\u00C2\u00A0 valence\u00C2\u00A0 (above\u00C2\u00A0100;\u00C2\u00A0 Figure\u00C2\u00A04.6)\u00C2\u00A0or\u00C2\u00A0Rtx\u00E2\u0080\u0090MS\u00C2\u00A0 (Figure\u00C2\u00A05.6),\u00C2\u00A0 and\u00C2\u00A0 they\u00C2\u00A0 were\u00C2\u00A0especially\u00C2\u00A0higher\u00C2\u00A0than\u00C2\u00A0cells\u00C2\u00A0treated\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0followed\u00C2\u00A0by\u00C2\u00A02oAb\u00C2\u00A0(Figure\u00C2\u00A06.4B).\u00C2\u00A0\u00C2\u00A0Note\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A0 6.4A\u00C2\u00A0(24\u00C2\u00A0h\u00C2\u00A0time\u00C2\u00A0point),\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0cells\u00C2\u00A0had\u00C2\u00A0become\u00C2\u00A0necrotic;\u00C2\u00A0Figure\u00C2\u00A06.4B\u00C2\u00A0provides\u00C2\u00A0data\u00C2\u00A0 at\u00C2\u00A012\u00C2\u00A0h\u00C2\u00A0post\u00E2\u0080\u0090treatment,\u00C2\u00A0when\u00C2\u00A0the\u00C2\u00A0necrotic\u00C2\u00A0fraction\u00C2\u00A0was\u00C2\u00A0much\u00C2\u00A0lower.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS\u00C2\u00A0 acted\u00C2\u00A0 on\u00C2\u00A0 cells\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0 already\u00C2\u00A0 saturated\u00C2\u00A0with\u00C2\u00A0 Rtx,\u00C2\u00A0 but\u00C2\u00A0 Figure\u00C2\u00A0 4.1\u00C2\u00A0 shows\u00C2\u00A0 that\u00C2\u00A0 saturating\u00C2\u00A0cells\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0still\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0significant\u00C2\u00A0unbound\u00C2\u00A0CD20\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0large\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0 Rtx\u00C2\u00A0 that\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0bound\u00C2\u00A0 to\u00C2\u00A0CD20.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 lack\u00C2\u00A0of\u00C2\u00A0 saturation\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0 could\u00C2\u00A0be\u00C2\u00A0due\u00C2\u00A0 to\u00C2\u00A0a\u00C2\u00A0 steric\u00C2\u00A0effect\u00C2\u00A0 where\u00C2\u00A0 the\u00C2\u00A0 liposomal\u00C2\u00A0 component\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 takes\u00C2\u00A0 up\u00C2\u00A0 too\u00C2\u00A0much\u00C2\u00A0 space\u00C2\u00A0 to\u00C2\u00A0 allow\u00C2\u00A0 sufficient\u00C2\u00A0 Rtx\u00C2\u00A0 to\u00C2\u00A0 saturate\u00C2\u00A0 all\u00C2\u00A0 CD20\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface.\u00C2\u00A0 \u00C2\u00A0However,\u00C2\u00A0 because\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS\u00C2\u00A0 acted\u00C2\u00A0 on\u00C2\u00A0 Rtx\u00E2\u0080\u0090saturated\u00C2\u00A0 cells,\u00C2\u00A0 higher\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 CD20\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 achieved\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane,\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 more\u00C2\u00A0 significant\u00C2\u00A0 CD120a\u00C2\u00A0 upregulation\u00C2\u00A0 and\u00C2\u00A0 apoptosis\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 saturation\u00C2\u00A0 of\u00C2\u00A0 cells\u00C2\u00A0with\u00C2\u00A0 Rtx,\u00C2\u00A0which\u00C2\u00A0 allows\u00C2\u00A0 for\u00C2\u00A0 high\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 clustered\u00C2\u00A0 CD20,\u00C2\u00A0 can\u00C2\u00A0 also\u00C2\u00A0 be\u00C2\u00A0 used\u00C2\u00A0 to\u00C2\u00A0 explain\u00C2\u00A0why\u00C2\u00A0 the\u00C2\u00A0hypercrosslinking\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0can\u00C2\u00A0produce\u00C2\u00A0a\u00C2\u00A0 strong\u00C2\u00A0apoptotic\u00C2\u00A0 signal\u00C2\u00A0 (Figure\u00C2\u00A06.6).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Importantly,\u00C2\u00A0 although\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 2oAb\u00C2\u00A0 is\u00C2\u00A0 a\u00C2\u00A0 common\u00C2\u00A0 method\u00C2\u00A0 to\u00C2\u00A0 assess\u00C2\u00A0 the\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 crosslinking\u00C2\u00A0a\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0in\u00C2\u00A0vitro7,\u00C2\u00A074\u00E2\u0080\u009076,\u00C2\u00A0Figure\u00C2\u00A06.4B\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A02oAb\u00E2\u0080\u0090MS\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A02oAb\u00C2\u00A0alone\u00C2\u00A0was\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0161\u00C2\u00A0\u00C2\u00A0 able\u00C2\u00A0 to\u00C2\u00A0 provide\u00C2\u00A0 a\u00C2\u00A0multivalent\u00C2\u00A0 interaction\u00C2\u00A0 that\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 sufficient\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0when\u00C2\u00A0 testing\u00C2\u00A0 the\u00C2\u00A0crosslinking\u00C2\u00A0of\u00C2\u00A0 therapeutic\u00C2\u00A0Abs,\u00C2\u00A0 using\u00C2\u00A0a\u00C2\u00A02oAb\u00C2\u00A0may\u00C2\u00A0not\u00C2\u00A0provide\u00C2\u00A0the\u00C2\u00A0necessary\u00C2\u00A0degree\u00C2\u00A0of\u00C2\u00A0crosslinking\u00C2\u00A0to\u00C2\u00A0induce\u00C2\u00A0meaningful\u00C2\u00A0therapeutic\u00C2\u00A0 effects,\u00C2\u00A0and\u00C2\u00A0a\u00C2\u00A0multivalent\u00C2\u00A02oAb\u00C2\u00A0construct\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0appropriate.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0 known\u00C2\u00A0 that\u00C2\u00A0 ADCC\u00C2\u00A0 occurs\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 result\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 binding\u00C2\u00A0 of\u00C2\u00A0 therapeutic\u00C2\u00A0 Abs\u00C2\u00A0 like\u00C2\u00A0 Rtx\u00C2\u00A0 to\u00C2\u00A0 activating\u00C2\u00A0Fc\u00CE\u00B3Rs\u00C2\u00A0on\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0immune\u00C2\u00A0system.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0causes\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 activating\u00C2\u00A0 Fc\u00CE\u00B3R\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 effector\u00C2\u00A0 cell,\u00C2\u00A0 which\u00C2\u00A0 triggers\u00C2\u00A0 the\u00C2\u00A0 ADCC\u00C2\u00A0 response\u00C2\u00A0 (Section\u00C2\u00A01.4.2).\u00C2\u00A0\u00C2\u00A0With\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0Chapter\u00C2\u00A06,\u00C2\u00A0it\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0argued\u00C2\u00A0that\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0necessarily\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0which\u00C2\u00A0 causes\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0Fc\u00CE\u00B3R,\u00C2\u00A0but\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0mutually\u00C2\u00A0hypercrosslink\u00C2\u00A0each\u00C2\u00A0other,\u00C2\u00A0 which\u00C2\u00A0in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0would\u00C2\u00A0result\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0in\u00C2\u00A0ADCC\u00C2\u00A0but\u00C2\u00A0also\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0 vivo.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Moreover,\u00C2\u00A0as\u00C2\u00A0discussed\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A01.4.2,\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0granulocytes\u00C2\u00A0express\u00C2\u00A0only\u00C2\u00A0activating\u00C2\u00A0 Fc\u00CE\u00B3Rs,\u00C2\u00A0while\u00C2\u00A0other\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0 including\u00C2\u00A0macrophages\u00C2\u00A0also\u00C2\u00A0express\u00C2\u00A0 inhibitory\u00C2\u00A0Fc\u00CE\u00B3Rs,\u00C2\u00A0which\u00C2\u00A0upon\u00C2\u00A0 hypercrosslinking,\u00C2\u00A0 negatively\u00C2\u00A0 regulate\u00C2\u00A0 ADCC.\u00C2\u00A0 \u00C2\u00A0We\u00C2\u00A0 demonstrated\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 6\u00C2\u00A0 that\u00C2\u00A0 2oAb\u00E2\u0080\u0090MS\u00C2\u00A0 and\u00C2\u00A0 effector\u00C2\u00A0cells\u00C2\u00A0had\u00C2\u00A0similar\u00C2\u00A0effects\u00C2\u00A0on\u00C2\u00A0Rtx\u00E2\u0080\u0090treated\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0terms\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0 and\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis,\u00C2\u00A0 indicating\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0hypercrosslinking\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0 influence\u00C2\u00A0 the\u00C2\u00A0therapeutic\u00C2\u00A0response.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0implies\u00C2\u00A0that\u00C2\u00A0in\u00C2\u00A0vivo,\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0resulting\u00C2\u00A0from\u00C2\u00A0 hypercrosslinking\u00C2\u00A0should\u00C2\u00A0not\u00C2\u00A0depend\u00C2\u00A0on\u00C2\u00A0whether\u00C2\u00A0the\u00C2\u00A0particular\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0that\u00C2\u00A0 is\u00C2\u00A0hypercrosslinked\u00C2\u00A0 is\u00C2\u00A0an\u00C2\u00A0 activating\u00C2\u00A0or\u00C2\u00A0inhibitory\u00C2\u00A0receptor,\u00C2\u00A0because\u00C2\u00A0Rtx\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0hypercrosslinked\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0result.\u00C2\u00A0\u00C2\u00A0Inhibitory\u00C2\u00A0Fc\u00CE\u00B3Rs\u00C2\u00A0 could\u00C2\u00A0therefore\u00C2\u00A0be\u00C2\u00A0responsible\u00C2\u00A0 for\u00C2\u00A0 inducing\u00C2\u00A0direct\u00C2\u00A0apoptosis\u00C2\u00A0as\u00C2\u00A0well\u00C2\u00A0as\u00C2\u00A0activating\u00C2\u00A0Fc\u00CE\u00B3Rs.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0 is\u00C2\u00A0also\u00C2\u00A0 conceivable\u00C2\u00A0that\u00C2\u00A0responses\u00C2\u00A0of\u00C2\u00A0bivalent\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0possibly\u00C2\u00A0other\u00C2\u00A0Abs\u00C2\u00A0that\u00C2\u00A0were\u00C2\u00A0thought\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0entirely\u00C2\u00A0 the\u00C2\u00A0result\u00C2\u00A0of\u00C2\u00A0ADCC\u00C2\u00A0(such\u00C2\u00A0as\u00C2\u00A0those\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.3C\u00C2\u00A0&\u00C2\u00A0D)\u00C2\u00A0could\u00C2\u00A0have\u00C2\u00A0actually\u00C2\u00A0been\u00C2\u00A0due,\u00C2\u00A0at\u00C2\u00A0least\u00C2\u00A0in\u00C2\u00A0part,\u00C2\u00A0 to\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0162\u00C2\u00A0\u00C2\u00A0 7.2.4\u00C2\u00A0 The\u00C2\u00A0nature\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 interaction\u00C2\u00A0between\u00C2\u00A0Ab\u00C2\u00ADLNPs\u00C2\u00A0and\u00C2\u00A0 the\u00C2\u00A0 surface\u00C2\u00A0of\u00C2\u00A0 target\u00C2\u00A0 cells\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0 Ab\u00E2\u0080\u0090LNPs,\u00C2\u00A0 as\u00C2\u00A0 in\u00C2\u00A0 immunoliposomes,\u00C2\u00A0 the\u00C2\u00A0 Ab\u00C2\u00A0molecules\u00C2\u00A0 are\u00C2\u00A0 not\u00C2\u00A0 rigidly\u00C2\u00A0 held\u00C2\u00A0 in\u00C2\u00A0 place,\u00C2\u00A0 but\u00C2\u00A0 diffuse\u00C2\u00A0around\u00C2\u00A0the\u00C2\u00A0liposome254.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0property\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0particularly\u00C2\u00A0advantageous\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0 efficacy\u00C2\u00A0 of\u00C2\u00A0Ab\u00E2\u0080\u0090LNPs\u00C2\u00A0 because\u00C2\u00A0 it\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 that\u00C2\u00A0 rigid\u00C2\u00A0multivalent\u00C2\u00A0 constructs\u00C2\u00A0 exhibit\u00C2\u00A0 variable\u00C2\u00A0 activation\u00C2\u00A0or\u00C2\u00A0inhibition\u00C2\u00A0against\u00C2\u00A0targets\u00C2\u00A0depending\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0spacing\u00C2\u00A0between\u00C2\u00A0the\u00C2\u00A0different\u00C2\u00A0binding\u00C2\u00A0sites\u00C2\u00A0 on\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0 ligand102.\u00C2\u00A0 \u00C2\u00A0This\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0by\u00C2\u00A0being\u00C2\u00A0allowed\u00C2\u00A0to\u00C2\u00A0diffuse,\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0molecules\u00C2\u00A0can\u00C2\u00A0 arrange\u00C2\u00A0in\u00C2\u00A0lowest\u00E2\u0080\u0090energy\u00C2\u00A0(most\u00C2\u00A0stable)\u00C2\u00A0configuration\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Ab/target\u00C2\u00A0complex.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 On\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0since\u00C2\u00A0Figure\u00C2\u00A04.1\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0not\u00C2\u00A0all\u00C2\u00A0Ab\u00C2\u00A0 is\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0target\u00C2\u00A0 in\u00C2\u00A0these\u00C2\u00A0 multivalent\u00C2\u00A0 complexes,\u00C2\u00A0 an\u00C2\u00A0 even\u00C2\u00A0more\u00C2\u00A0 efficient\u00C2\u00A0 use\u00C2\u00A0 of\u00C2\u00A0Ab\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 a\u00C2\u00A0 near\u00E2\u0080\u0090planar\u00C2\u00A0 nanometer\u00E2\u0080\u0090scale\u00C2\u00A0 configuration\u00C2\u00A0with\u00C2\u00A0Ab\u00C2\u00A0only\u00C2\u00A0on\u00C2\u00A0one\u00C2\u00A0side\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0plane.\u00C2\u00A0\u00C2\u00A0After\u00C2\u00A0treatment\u00C2\u00A0of\u00C2\u00A0target\u00C2\u00A0cells,\u00C2\u00A0no\u00C2\u00A0unbound\u00C2\u00A0Ab\u00C2\u00A0 would\u00C2\u00A0 be\u00C2\u00A0 present,\u00C2\u00A0 allowing\u00C2\u00A0 all\u00C2\u00A0 Ab\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 engaged\u00C2\u00A0 in\u00C2\u00A0 hypercrosslinking.\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0would\u00C2\u00A0 be\u00C2\u00A0 possible\u00C2\u00A0 to\u00C2\u00A0 synthesize\u00C2\u00A0 different\u00C2\u00A0 sizes\u00C2\u00A0 and\u00C2\u00A0 intermolecular\u00C2\u00A0 spacings\u00C2\u00A0 of\u00C2\u00A0 such\u00C2\u00A0 constructs102,\u00C2\u00A0 or\u00C2\u00A0 possibly\u00C2\u00A0 create\u00C2\u00A0 constructs\u00C2\u00A0where\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0rigidly\u00C2\u00A0held\u00C2\u00A0in\u00C2\u00A0place\u00C2\u00A0using\u00C2\u00A0PEG\u00C2\u00A0linkers\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0those\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs.\u00C2\u00A0\u00C2\u00A0 Although\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0construct\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0enhanced,\u00C2\u00A0it\u00C2\u00A0is\u00C2\u00A0 unclear\u00C2\u00A0 how\u00C2\u00A0 this\u00C2\u00A0would\u00C2\u00A0 affect\u00C2\u00A0 the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms.\u00C2\u00A0 \u00C2\u00A0 To\u00C2\u00A0 preserve\u00C2\u00A0 all\u00C2\u00A0mechanisms\u00C2\u00A0 of\u00C2\u00A0 action,\u00C2\u00A0 whole\u00C2\u00A0IgG\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0required\u00C2\u00A0(not\u00C2\u00A0fragments)\u00C2\u00A0and\u00C2\u00A0variation\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0point\u00C2\u00A0of\u00C2\u00A0attachment\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0linker\u00C2\u00A0 would\u00C2\u00A0also\u00C2\u00A0be\u00C2\u00A0necessary,\u00C2\u00A0as\u00C2\u00A0discussed\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0previous\u00C2\u00A0section.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 dynamics\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 association\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0with\u00C2\u00A0 target\u00C2\u00A0 cells,\u00C2\u00A0 there\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 parallels\u00C2\u00A0between\u00C2\u00A0with\u00C2\u00A0other\u00C2\u00A0types\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0ligands\u00C2\u00A0in\u00C2\u00A0nature\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0carbohydrates\u00C2\u00A0and\u00C2\u00A0DNA.\u00C2\u00A0\u00C2\u00A0 As\u00C2\u00A0 outlined\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 1.6,\u00C2\u00A0multivalent\u00C2\u00A0 ligands\u00C2\u00A0 show\u00C2\u00A0 decreased\u00C2\u00A0 dissociation\u00C2\u00A0 rates\u00C2\u00A0 from\u00C2\u00A0 receptors\u00C2\u00A0 compared\u00C2\u00A0 to\u00C2\u00A0 univalent\u00C2\u00A0 ones\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 recapture\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 ligand\u00C2\u00A0 before\u00C2\u00A0 complete\u00C2\u00A0 dissociation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 ligand\u00E2\u0080\u0090receptor\u00C2\u00A0 complex.\u00C2\u00A0 \u00C2\u00A0 Evidence\u00C2\u00A0 suggests\u00C2\u00A0 that\u00C2\u00A0 some\u00C2\u00A0DNA\u00C2\u00A0 regulatory\u00C2\u00A0 proteins\u00C2\u00A0 bind\u00C2\u00A0 to\u00C2\u00A0DNA\u00C2\u00A0 at\u00C2\u00A0 nonspecific\u00C2\u00A0base\u00C2\u00A0pair\u00C2\u00A0sites\u00C2\u00A0and\u00C2\u00A0then\u00C2\u00A0through\u00C2\u00A0Brownian\u00C2\u00A0motion\u00C2\u00A0diffuse\u00C2\u00A0along\u00C2\u00A0the\u00C2\u00A0backbone\u00C2\u00A0until\u00C2\u00A0they\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0163\u00C2\u00A0\u00C2\u00A0 reach\u00C2\u00A0a\u00C2\u00A0high\u00E2\u0080\u0090affinity\u00C2\u00A0site.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0\u00E2\u0080\u009Cbind\u00C2\u00A0and\u00C2\u00A0slide\u00E2\u0080\u009D\u00C2\u00A0mechanism\u00C2\u00A0has\u00C2\u00A0also\u00C2\u00A0been\u00C2\u00A0observed\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0interaction\u00C2\u00A0 between\u00C2\u00A0multivalent\u00C2\u00A0carbohydrates\u00C2\u00A0and\u00C2\u00A0lectins100.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0hypothesized\u00C2\u00A0that\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0initial\u00C2\u00A0univalent\u00C2\u00A0interaction\u00C2\u00A0between\u00C2\u00A0an\u00C2\u00A0Ab\u00C2\u00A0molecule\u00C2\u00A0in\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 surface\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 target\u00C2\u00A0 cell,\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 \u00E2\u0080\u009Crecaptures\u00E2\u0080\u009D\u00C2\u00A0 another\u00C2\u00A0Ab\u00C2\u00A0molecule\u00C2\u00A0 before\u00C2\u00A0 (or\u00C2\u00A0 during)\u00C2\u00A0dissociation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0molecule.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0constant\u00C2\u00A0association\u00C2\u00A0and\u00C2\u00A0dissociation\u00C2\u00A0allows\u00C2\u00A0the\u00C2\u00A0Ab\u00E2\u0080\u0090 LNP\u00C2\u00A0to\u00C2\u00A0essentially\u00C2\u00A0roll\u00C2\u00A0across\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0until\u00C2\u00A0a\u00C2\u00A0higher\u00E2\u0080\u0090affinity\u00C2\u00A0interaction\u00C2\u00A0is\u00C2\u00A0formed,\u00C2\u00A0such\u00C2\u00A0 as\u00C2\u00A0a\u00C2\u00A0cluster\u00C2\u00A0of\u00C2\u00A0target\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0on\u00C2\u00A0a\u00C2\u00A0 length\u00C2\u00A0scale\u00C2\u00A0similar\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0diameter\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0Ab\u00E2\u0080\u0090 LNP.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0more\u00C2\u00A0stable\u00C2\u00A0multivalent\u00C2\u00A0 interactions\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0 responsible\u00C2\u00A0 for\u00C2\u00A0 inducing\u00C2\u00A0 the\u00C2\u00A0 therapeutic\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0 Abs,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 those\u00C2\u00A0 illustrated\u00C2\u00A0 in\u00C2\u00A0 Figure\u00C2\u00A0 6.5\u00C2\u00A0 where\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 Rtx\u00C2\u00A0 colocalized\u00C2\u00A0with\u00C2\u00A0CD120a\u00C2\u00A0outside\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 7.3\u00C2\u00A0 Discussion\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 7.3.1\u00C2\u00A0 The\u00C2\u00A0issue\u00C2\u00A0of\u00C2\u00A0rapid\u00C2\u00A0clearance\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0Rtx\u00C2\u00A0remains\u00C2\u00A0unresolved\u00C2\u00A0 In\u00C2\u00A0Chapter\u00C2\u00A02,\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0shown\u00C2\u00A0 that\u00C2\u00A0even\u00C2\u00A0 though\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0showed\u00C2\u00A0significantly\u00C2\u00A0higher\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0 apoptosis,\u00C2\u00A0ADCC,\u00C2\u00A0and\u00C2\u00A0CDC,\u00C2\u00A0the\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0and\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0were\u00C2\u00A0equivalent\u00C2\u00A0(Figure\u00C2\u00A02.5).\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0 was\u00C2\u00A0explained\u00C2\u00A0by\u00C2\u00A0pharmacokinetic\u00C2\u00A0studies\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.6\u00C2\u00A0that\u00C2\u00A0showed\u00C2\u00A0rapid\u00C2\u00A0clearance\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0from\u00C2\u00A0 the\u00C2\u00A0 circulation.\u00C2\u00A0 \u00C2\u00A0 Efficacy\u00C2\u00A0 and\u00C2\u00A0 pharmacokinetics\u00C2\u00A0 studies\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 performed\u00C2\u00A0 on\u00C2\u00A0 other\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 Abs,\u00C2\u00A0 including\u00C2\u00A0 a\u00C2\u00A0 hexavalent\u00C2\u00A0 construct\u00C2\u00A0 based\u00C2\u00A0 on\u00C2\u00A0 veltuzumab\u00C2\u00A0 (a\u00C2\u00A0 humanized\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0Ab)90.\u00C2\u00A0\u00C2\u00A0Compared\u00C2\u00A0to\u00C2\u00A0veltuzumab,\u00C2\u00A0the\u00C2\u00A0hexavalent\u00C2\u00A0Ab\u00C2\u00A0induced\u00C2\u00A0higher\u00C2\u00A0apoptosis\u00C2\u00A0and\u00C2\u00A0similar\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0ADCC,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0CDC\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0hexavalent\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0completely\u00C2\u00A0abolished.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0contrasts\u00C2\u00A0to\u00C2\u00A0Rtx\u00E2\u0080\u0090 LNP,\u00C2\u00A0which\u00C2\u00A0showed\u00C2\u00A0enhanced\u00C2\u00A0CDC\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0Rtx,\u00C2\u00A0and\u00C2\u00A0which\u00C2\u00A0may\u00C2\u00A0have\u00C2\u00A0resulted\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0intact\u00C2\u00A0 Fc\u00C2\u00A0regions\u00C2\u00A0in\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0enabling\u00C2\u00A0multivalent\u00C2\u00A0C1q\u00C2\u00A0binding.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0highlights\u00C2\u00A0the\u00C2\u00A0advantage\u00C2\u00A0of\u00C2\u00A0using\u00C2\u00A0entire\u00C2\u00A0 IgG\u00C2\u00A0molecules\u00C2\u00A0in\u00C2\u00A0multivalent\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0constructs\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0to\u00C2\u00A0preserve\u00C2\u00A0the\u00C2\u00A0favorable\u00C2\u00A0effects\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 in\u00C2\u00A0vivo.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0hexavalent\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0not\u00C2\u00A0 found\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0164\u00C2\u00A0\u00C2\u00A0 superior\u00C2\u00A0to\u00C2\u00A0that\u00C2\u00A0of\u00C2\u00A0veltuzumab\u00C2\u00A0except\u00C2\u00A0at\u00C2\u00A0very\u00C2\u00A0high\u00C2\u00A0doses,\u00C2\u00A0and\u00C2\u00A0this\u00C2\u00A0effect\u00C2\u00A0was\u00C2\u00A0attributed\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0 that\u00C2\u00A0 it\u00C2\u00A0was\u00C2\u00A0cleared\u00C2\u00A0 from\u00C2\u00A0the\u00C2\u00A0circulation\u00C2\u00A0at\u00C2\u00A0 least\u00C2\u00A04.5\u00C2\u00A0times\u00C2\u00A0 faster\u00C2\u00A0 than\u00C2\u00A0 the\u00C2\u00A0bivalent\u00C2\u00A0Ab.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0data\u00C2\u00A0 mirror\u00C2\u00A0 those\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 current\u00C2\u00A0 study,\u00C2\u00A0where\u00C2\u00A0 in\u00C2\u00A0 both\u00C2\u00A0 cases\u00C2\u00A0 the\u00C2\u00A0 enhanced\u00C2\u00A0 effects\u00C2\u00A0 from\u00C2\u00A0multivalency\u00C2\u00A0 observed\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0are\u00C2\u00A0countered\u00C2\u00A0by\u00C2\u00A0decreases\u00C2\u00A0in\u00C2\u00A0circulation\u00C2\u00A0lifetime.\u00C2\u00A0 On\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0hand,\u00C2\u00A0Zhang\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0have\u00C2\u00A0demonstrated\u00C2\u00A0more\u00C2\u00A0favorable\u00C2\u00A0pharmacokinetics\u00C2\u00A0and\u00C2\u00A0 efficacy\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0Rtx\u00C2\u00A0polymer\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0~10\u00C2\u00A0(ref.\u00C2\u00A075).\u00C2\u00A0 \u00C2\u00A0Although\u00C2\u00A0they\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0examine\u00C2\u00A0ADCC\u00C2\u00A0or\u00C2\u00A0 CDC,\u00C2\u00A0they\u00C2\u00A0found\u00C2\u00A0that\u00C2\u00A0decavalent\u00C2\u00A0Rtx\u00C2\u00A0 induced\u00C2\u00A0apoptosis\u00C2\u00A0more\u00C2\u00A0effectively\u00C2\u00A0 in\u00C2\u00A0ten\u00C2\u00A0different\u00C2\u00A0CD20+\u00C2\u00A0cell\u00C2\u00A0 lines,\u00C2\u00A0and\u00C2\u00A0also\u00C2\u00A0exhibited\u00C2\u00A0a\u00C2\u00A0half\u00E2\u0080\u0090life\u00C2\u00A0if\u00C2\u00A0144\u00C2\u00A0h,\u00C2\u00A0compared\u00C2\u00A0to\u00C2\u00A096\u00C2\u00A0h\u00C2\u00A0for\u00C2\u00A0free\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0decavalent\u00C2\u00A0construct\u00C2\u00A0 also\u00C2\u00A0exhibited\u00C2\u00A0 superior\u00C2\u00A0efficacy\u00C2\u00A0 in\u00C2\u00A0Raji\u00C2\u00A0 tumor\u00E2\u0080\u0090bearing\u00C2\u00A0nude\u00C2\u00A0mice.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 reasons\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 increase\u00C2\u00A0 in\u00C2\u00A0 circulation\u00C2\u00A0 lifetime\u00C2\u00A0of\u00C2\u00A0decavalent\u00C2\u00A0Rtx\u00C2\u00A0versus\u00C2\u00A0 the\u00C2\u00A0decrease\u00C2\u00A0 for\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0are\u00C2\u00A0unclear,\u00C2\u00A0but\u00C2\u00A0one\u00C2\u00A0reason\u00C2\u00A0 may\u00C2\u00A0be\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0coupling\u00C2\u00A0method\u00C2\u00A0used\u00C2\u00A0may\u00C2\u00A0affect\u00C2\u00A0pharmacokinetic\u00C2\u00A0characteristics\u00C2\u00A0and,\u00C2\u00A0obviously,\u00C2\u00A0 the\u00C2\u00A0size\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0constructs\u00C2\u00A0are\u00C2\u00A0remarkably\u00C2\u00A0different.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0study,\u00C2\u00A0however,\u00C2\u00A0suggests\u00C2\u00A0that\u00C2\u00A0if\u00C2\u00A0 the\u00C2\u00A0 pharmacokinetics\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 can\u00C2\u00A0 be\u00C2\u00A0 improved,\u00C2\u00A0 the\u00C2\u00A0 enhanced\u00C2\u00A0 effects\u00C2\u00A0 from\u00C2\u00A0 both\u00C2\u00A0 indirect\u00C2\u00A0 and\u00C2\u00A0 direct\u00C2\u00A0 factors\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0manifested\u00C2\u00A0 in\u00C2\u00A0vivo.\u00C2\u00A0 Importantly,\u00C2\u00A0the\u00C2\u00A0 fact\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0exhibits\u00C2\u00A0such\u00C2\u00A0a\u00C2\u00A0short\u00C2\u00A0 residence\u00C2\u00A0 time\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 circulation\u00C2\u00A0yet\u00C2\u00A0exhibit\u00C2\u00A0an\u00C2\u00A0efficacy\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0equivalent\u00C2\u00A0 to\u00C2\u00A0 free\u00C2\u00A0Rtx\u00C2\u00A0 reflects\u00C2\u00A0 the\u00C2\u00A0 enhanced\u00C2\u00A0potency\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 The\u00C2\u00A0rapid\u00C2\u00A0clearance\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0was\u00C2\u00A0surprising\u00C2\u00A0because\u00C2\u00A0previous\u00C2\u00A0studies\u00C2\u00A0on\u00C2\u00A0Trz\u00E2\u0080\u0090LNP\u00C2\u00A0showed\u00C2\u00A0 improvements\u00C2\u00A0in\u00C2\u00A0circulation\u00C2\u00A0lifetime\u00C2\u00A0and\u00C2\u00A0other\u00C2\u00A0pharmacokinetic\u00C2\u00A0parameters\u00C2\u00A0(Table\u00C2\u00A01.1).\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0results\u00C2\u00A0 using\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0imply\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0rapid\u00C2\u00A0clearance\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Ab\u00C2\u00A0employed\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0the\u00C2\u00A0size\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0constructs\u00C2\u00A0or\u00C2\u00A0the\u00C2\u00A0liposomal\u00C2\u00A0component\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0formulation.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0may\u00C2\u00A0still\u00C2\u00A0be\u00C2\u00A0possible,\u00C2\u00A0however,\u00C2\u00A0to\u00C2\u00A0 improve\u00C2\u00A0 the\u00C2\u00A0 pharmacokinetics\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 by\u00C2\u00A0 employing\u00C2\u00A0 a\u00C2\u00A0 non\u00E2\u0080\u0090liposomal\u00C2\u00A0 construct.\u00C2\u00A0\u00C2\u00A0 Chapters\u00C2\u00A05\u00C2\u00A0&\u00C2\u00A06\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0enhanced\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0contribution\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0 liposome,\u00C2\u00A0because\u00C2\u00A0other\u00C2\u00A0means\u00C2\u00A0of\u00C2\u00A0achieving\u00C2\u00A0multivalency\u00C2\u00A0(Rtx\u00E2\u0080\u0090MS,\u00C2\u00A02oAb\u00E2\u0080\u0090MS)\u00C2\u00A0resulted\u00C2\u00A0in\u00C2\u00A0therapeutic\u00C2\u00A0 effects\u00C2\u00A0 through\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action.\u00C2\u00A0 \u00C2\u00A0Different\u00C2\u00A0 types\u00C2\u00A0of\u00C2\u00A0 constructs\u00C2\u00A0 that\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 explored\u00C2\u00A0 include\u00C2\u00A0 polymeric\u00C2\u00A0 nanoparticles93,\u00C2\u00A0 rotaxanes92,\u00C2\u00A0 gold\u00C2\u00A0 nanoparticles96,\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0165\u00C2\u00A0\u00C2\u00A0 dendrimers94,\u00C2\u00A0 95,\u00C2\u00A0 or\u00C2\u00A0 multifunctional\u00C2\u00A0 nanoparticles167.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 issue\u00C2\u00A0 of\u00C2\u00A0 rapid\u00C2\u00A0 clearance\u00C2\u00A0 may\u00C2\u00A0 also\u00C2\u00A0 be\u00C2\u00A0 resolved\u00C2\u00A0through\u00C2\u00A0the\u00C2\u00A0selection\u00C2\u00A0of\u00C2\u00A0an\u00C2\u00A0alternative\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0Ab\u00C2\u00A0such\u00C2\u00A0as\u00C2\u00A0those\u00C2\u00A0 listed\u00C2\u00A0 in\u00C2\u00A0Table\u00C2\u00A01.2,\u00C2\u00A0but\u00C2\u00A0 an\u00C2\u00A0alternative\u00C2\u00A0Ab\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0to\u00C2\u00A0possess\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0and\u00C2\u00A0different\u00C2\u00A0 relative\u00C2\u00A0 contributions\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 and\u00C2\u00A0 indirect\u00C2\u00A0 mechanisms,\u00C2\u00A0 as\u00C2\u00A0 discussed\u00C2\u00A0 in\u00C2\u00A0 Section\u00C2\u00A0 1.4.\u00C2\u00A0 \u00C2\u00A0 An\u00C2\u00A0 alternative\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0Ab\u00C2\u00A0would\u00C2\u00A0therefore\u00C2\u00A0require\u00C2\u00A0re\u00E2\u0080\u0090initiation\u00C2\u00A0of\u00C2\u00A0mechanistic\u00C2\u00A0studies.\u00C2\u00A0\u00C2\u00A0 7.3.2\u00C2\u00A0 Local\u00C2\u00A0 concentrations\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0 membrane,\u00C2\u00A0 and\u00C2\u00A0 not\u00C2\u00A0 the\u00C2\u00A0 number\u00C2\u00A0 of\u00C2\u00A0Rtx\u00C2\u00ADCD20\u00C2\u00A0 interactions,\u00C2\u00A0 determine\u00C2\u00A0 the\u00C2\u00A0 level\u00C2\u00A0 of\u00C2\u00A0 apoptosis\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0 multivalent\u00C2\u00A0Rtx\u00C2\u00A0 \u00C2\u00A0 Section\u00C2\u00A0 7.2.3\u00C2\u00A0 described\u00C2\u00A0 how\u00C2\u00A0 the\u00C2\u00A0 various\u00C2\u00A0 types\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 employed\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 dissertation\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0different\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0 cells.\u00C2\u00A0\u00C2\u00A0 Bivalent\u00C2\u00A0Rtx\u00C2\u00A0showed\u00C2\u00A0the\u00C2\u00A0lowest\u00C2\u00A0efficacy\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0although\u00C2\u00A0it\u00C2\u00A0saturated\u00C2\u00A0all\u00C2\u00A0CD20\u00C2\u00A0molecules\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0 surface,\u00C2\u00A0while\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0bound\u00C2\u00A0only\u00C2\u00A0to\u00C2\u00A0a\u00C2\u00A0fraction\u00C2\u00A0of\u00C2\u00A0surface\u00C2\u00A0CD20,\u00C2\u00A0formed\u00C2\u00A0domains,\u00C2\u00A0and\u00C2\u00A0showed\u00C2\u00A0much\u00C2\u00A0 higher\u00C2\u00A0 efficacy\u00C2\u00A0 (Figures\u00C2\u00A0 4.1,\u00C2\u00A0 4.2,\u00C2\u00A0 &\u00C2\u00A0 4.6).\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 treatment\u00C2\u00A0 also\u00C2\u00A0 resulted\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 fraction\u00C2\u00A0 of\u00C2\u00A0 Rtx\u00C2\u00A0 molecules\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0 not\u00C2\u00A0 bound\u00C2\u00A0 to\u00C2\u00A0CD20,\u00C2\u00A0but\u00C2\u00A0 remained\u00C2\u00A0 associated\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0because\u00C2\u00A0other\u00C2\u00A0Rtx\u00C2\u00A0 molecules\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0same\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0were\u00C2\u00A0bound\u00C2\u00A0to\u00C2\u00A0CD20\u00C2\u00A0(Figure\u00C2\u00A04.1).\u00C2\u00A0 \u00C2\u00A0Together,\u00C2\u00A0this\u00C2\u00A0 indicates\u00C2\u00A0that\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 not\u00C2\u00A0 the\u00C2\u00A0direct\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0 to\u00C2\u00A0CD20\u00C2\u00A0 that\u00C2\u00A0 causes\u00C2\u00A0apoptosis;\u00C2\u00A0otherwise\u00C2\u00A0Rtx\u00C2\u00A0would\u00C2\u00A0have\u00C2\u00A0exhibited\u00C2\u00A0 higher\u00C2\u00A0efficacy\u00C2\u00A0than\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0 \u00C2\u00A0Rather,\u00C2\u00A0high\u00C2\u00A0 local\u00C2\u00A0CD20\u00C2\u00A0densities\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane,\u00C2\u00A0 induced\u00C2\u00A0 through\u00C2\u00A0hypercrosslinking\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0construct,\u00C2\u00A0appear\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0 increased\u00C2\u00A0 efficacies.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 supported\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0valence\u00E2\u0080\u0090dependent\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 shown\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A04.6.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0 higher\u00C2\u00A0 valences\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 are\u00C2\u00A0 capable\u00C2\u00A0of\u00C2\u00A0more\u00C2\u00A0 substantial\u00C2\u00A0hypercrosslinking\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0within\u00C2\u00A0 the\u00C2\u00A0 same\u00C2\u00A0 area\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 plasma\u00C2\u00A0membrane,\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 greater\u00C2\u00A0 local\u00C2\u00A0 CD20\u00C2\u00A0 concentrations.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 also\u00C2\u00A0 explains\u00C2\u00A0why\u00C2\u00A0CD20\u00C2\u00A0expression\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0observed\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0independent\u00C2\u00A0of\u00C2\u00A0sensitivity\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0therapy255;\u00C2\u00A0 higher\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0bound\u00C2\u00A0Rtx\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0necessarily\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0greater\u00C2\u00A0Rtx\u00C2\u00A0sensitivity.\u00C2\u00A0\u00C2\u00A0A\u00C2\u00A0better\u00C2\u00A0indicator\u00C2\u00A0of\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0166\u00C2\u00A0\u00C2\u00A0 sensitivity\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00C2\u00A0 treatment\u00C2\u00A0 may\u00C2\u00A0 be\u00C2\u00A0 an\u00C2\u00A0 assessment\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 to\u00C2\u00A0 undergo\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 (discussed\u00C2\u00A0 further\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A07.3.4).\u00C2\u00A0 \u00C2\u00A0By\u00C2\u00A0applying\u00C2\u00A0 the\u00C2\u00A0methodology\u00C2\u00A0 to\u00C2\u00A0other\u00C2\u00A0Abs,\u00C2\u00A0 particularly\u00C2\u00A0those\u00C2\u00A0against\u00C2\u00A0targets\u00C2\u00A0known\u00C2\u00A0to\u00C2\u00A0function\u00C2\u00A0through\u00C2\u00A0hypercrosslinking,\u00C2\u00A0it\u00C2\u00A0will\u00C2\u00A0become\u00C2\u00A0clear\u00C2\u00A0 whether\u00C2\u00A0this\u00C2\u00A0same\u00C2\u00A0phenomenon\u00C2\u00A0occurs\u00C2\u00A0with\u00C2\u00A0other\u00C2\u00A0Abs\u00C2\u00A0(Section\u00C2\u00A07.4).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 It\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0noted\u00C2\u00A0that\u00C2\u00A0one\u00C2\u00A0potential\u00C2\u00A0weakness\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A04.1\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Abs\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0employed.\u00C2\u00A0 \u00C2\u00A0Based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0discussion\u00C2\u00A0 in\u00C2\u00A0Section\u00C2\u00A07.2.4,\u00C2\u00A0 if\u00C2\u00A0the\u00C2\u00A0 interaction\u00C2\u00A0between\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 and\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface\u00C2\u00A0 is\u00C2\u00A0highly\u00C2\u00A0dynamic\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0translocating\u00C2\u00A0across\u00C2\u00A0the\u00C2\u00A0surface\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0cell,\u00C2\u00A0the\u00C2\u00A0 binding\u00C2\u00A0of\u00C2\u00A0A647\u00E2\u0080\u0090mCD20\u00C2\u00A0on\u00C2\u00A0unoccupied\u00C2\u00A0CD20\u00C2\u00A0molecules\u00C2\u00A0at\u00C2\u00A0any\u00C2\u00A0instant\u00C2\u00A0could\u00C2\u00A0cause\u00C2\u00A0those\u00C2\u00A0molecules\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0unavailable\u00C2\u00A0for\u00C2\u00A0further\u00C2\u00A0interactions\u00C2\u00A0with\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0may\u00C2\u00A0eventually\u00C2\u00A0cause\u00C2\u00A0more\u00C2\u00A0staining\u00C2\u00A0of\u00C2\u00A0 unbound\u00C2\u00A0CD20\u00C2\u00A0then\u00C2\u00A0was\u00C2\u00A0actually\u00C2\u00A0present\u00C2\u00A0at\u00C2\u00A0any\u00C2\u00A0 instantaneous\u00C2\u00A0 time\u00C2\u00A0point.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0results\u00C2\u00A0would\u00C2\u00A0still\u00C2\u00A0 indicate\u00C2\u00A0 that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0occupy\u00C2\u00A0 all\u00C2\u00A0 available\u00C2\u00A0CD20\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 surface,\u00C2\u00A0 consistent\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0 current\u00C2\u00A0interpretation\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0data,\u00C2\u00A0but\u00C2\u00A0the\u00C2\u00A0levels\u00C2\u00A0of\u00C2\u00A0unoccupied\u00C2\u00A0CD20\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0overestimated.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 7.3.3\u00C2\u00A0 The\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0may\u00C2\u00A0not\u00C2\u00A0exclusively\u00C2\u00A0involve\u00C2\u00A0CD120a,\u00C2\u00A0 but\u00C2\u00A0also\u00C2\u00A0other\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0and\u00C2\u00A0signaling\u00C2\u00A0molecules\u00C2\u00A0 \u00C2\u00A0 Cell\u00C2\u00A0signaling,\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0general\u00C2\u00A0sense,\u00C2\u00A0encompasses\u00C2\u00A0greater\u00C2\u00A0complexities\u00C2\u00A0than\u00C2\u00A0linear\u00C2\u00A0transduction\u00C2\u00A0 pathways\u00C2\u00A0with\u00C2\u00A0clearly\u00C2\u00A0defined\u00C2\u00A0endpoints256.\u00C2\u00A0\u00C2\u00A0Rather,\u00C2\u00A0a\u00C2\u00A0cell\u00C2\u00A0contains\u00C2\u00A0a\u00C2\u00A0network\u00C2\u00A0of\u00C2\u00A0signaling\u00C2\u00A0cascades\u00C2\u00A0 that\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0envisaged\u00C2\u00A0as\u00C2\u00A0a\u00C2\u00A0spider\u00C2\u00A0web,\u00C2\u00A0where\u00C2\u00A0plucking\u00C2\u00A0one\u00C2\u00A0fiber\u00C2\u00A0results\u00C2\u00A0 in\u00C2\u00A0small\u00C2\u00A0reverberations\u00C2\u00A0at\u00C2\u00A0 distant\u00C2\u00A0sites\u00C2\u00A0which\u00C2\u00A0determine\u00C2\u00A0different\u00C2\u00A0biological\u00C2\u00A0outcomes256,\u00C2\u00A0257.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0this\u00C2\u00A0sense,\u00C2\u00A0 it\u00C2\u00A0 is\u00C2\u00A0 important\u00C2\u00A0to\u00C2\u00A0 note\u00C2\u00A0that\u00C2\u00A0CD120a\u00C2\u00A0upregulation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0extrinsic\u00C2\u00A0apoptosis\u00C2\u00A0pathway\u00C2\u00A0(Figure\u00C2\u00A05.1)\u00C2\u00A0 likely\u00C2\u00A0 cannot\u00C2\u00A0explain\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0itself.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0particularly\u00C2\u00A0true\u00C2\u00A0in\u00C2\u00A0light\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0fact\u00C2\u00A0that\u00C2\u00A0essentially\u00C2\u00A0all\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0showed\u00C2\u00A0elevated\u00C2\u00A0expression\u00C2\u00A0after\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0treatment,\u00C2\u00A0 particularly\u00C2\u00A0CD95\u00C2\u00A0and\u00C2\u00A0DR5\u00C2\u00A0 in\u00C2\u00A0addition\u00C2\u00A0to\u00C2\u00A0CD120a\u00C2\u00A0(Section\u00C2\u00A05.4.2).\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0direct\u00C2\u00A0 induction\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0 by\u00C2\u00A0 hypercrosslinked\u00C2\u00A0 Rtx\u00C2\u00A0 therefore\u00C2\u00A0 appears\u00C2\u00A0 to\u00C2\u00A0 involve\u00C2\u00A0 a\u00C2\u00A0 coordinated\u00C2\u00A0 network\u00C2\u00A0 of\u00C2\u00A0 many\u00C2\u00A0 death\u00C2\u00A0 receptors\u00C2\u00A0that\u00C2\u00A0ultimately\u00C2\u00A0induce\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0target\u00C2\u00A0cells,\u00C2\u00A0and\u00C2\u00A0from\u00C2\u00A0the\u00C2\u00A0experiments\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0167\u00C2\u00A0\u00C2\u00A0 dissertation,\u00C2\u00A0CD120a\u00C2\u00A0appears\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0the\u00C2\u00A0major\u00C2\u00A0player\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0network.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0remains\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0seen\u00C2\u00A0if\u00C2\u00A0this\u00C2\u00A0is\u00C2\u00A0 the\u00C2\u00A0case\u00C2\u00A0in\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0other\u00C2\u00A0than\u00C2\u00A0those\u00C2\u00A0employed\u00C2\u00A0here,\u00C2\u00A0or\u00C2\u00A0in\u00C2\u00A0primary\u00C2\u00A0cells\u00C2\u00A0from\u00C2\u00A0xenograft\u00C2\u00A0models\u00C2\u00A0or\u00C2\u00A0 from\u00C2\u00A0humans.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0 spite\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 strong\u00C2\u00A0 evidence\u00C2\u00A0 in\u00C2\u00A0 support\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0 mechanism\u00C2\u00A0 at\u00C2\u00A0 the\u00C2\u00A0 beginning\u00C2\u00A0of\u00C2\u00A0Section\u00C2\u00A05.5.1,\u00C2\u00A0a\u00C2\u00A0knockdown\u00C2\u00A0approach\u00C2\u00A0or\u00C2\u00A0a\u00C2\u00A0CD120a\u00E2\u0080\u0090specific\u00C2\u00A0antagonistic\u00C2\u00A0antibody258\u00C2\u00A0may\u00C2\u00A0 further\u00C2\u00A0yield\u00C2\u00A0 results\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0overall\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0on\u00C2\u00A0apoptosis.\u00C2\u00A0 \u00C2\u00A0CD120a\u00C2\u00A0 is\u00C2\u00A0nearly\u00C2\u00A0always\u00C2\u00A0 expressed\u00C2\u00A0at\u00C2\u00A0 very\u00C2\u00A0 low\u00C2\u00A0 levels174,\u00C2\u00A0 so\u00C2\u00A0 strategies\u00C2\u00A0aimed\u00C2\u00A0at\u00C2\u00A0 inhibiting\u00C2\u00A0 its\u00C2\u00A0upregulation\u00C2\u00A0by\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0may\u00C2\u00A0 yield\u00C2\u00A0 important\u00C2\u00A0 information\u00C2\u00A0on\u00C2\u00A0whether\u00C2\u00A0CD120a\u00C2\u00A0 is\u00C2\u00A0necessary\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0 Rtx,\u00C2\u00A0and\u00C2\u00A0also\u00C2\u00A0whether\u00C2\u00A0the\u00C2\u00A0other\u00C2\u00A0death\u00C2\u00A0receptors\u00C2\u00A0can\u00C2\u00A0compensate\u00C2\u00A0for\u00C2\u00A0CD120a\u00C2\u00A0in\u00C2\u00A0its\u00C2\u00A0absence.\u00C2\u00A0 7.3.4\u00C2\u00A0 CD120a\u00C2\u00A0expression\u00C2\u00A0levels\u00C2\u00A0or\u00C2\u00A0mutation\u00C2\u00A0status\u00C2\u00A0as\u00C2\u00A0predictive\u00C2\u00A0markers\u00C2\u00A0for\u00C2\u00A0Rtx\u00C2\u00A0 response\u00C2\u00A0 \u00C2\u00A0There\u00C2\u00A0is\u00C2\u00A0significant\u00C2\u00A0interest\u00C2\u00A0in\u00C2\u00A0identifying\u00C2\u00A0molecular\u00C2\u00A0markers\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0predictive\u00C2\u00A0of\u00C2\u00A0response\u00C2\u00A0 to\u00C2\u00A0Rtx\u00C2\u00A0because\u00C2\u00A0resistance\u00C2\u00A0to\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0well\u00C2\u00A0understood139,\u00C2\u00A0140.\u00C2\u00A0\u00C2\u00A0As\u00C2\u00A0mentioned\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0previous\u00C2\u00A0 section,\u00C2\u00A0CD120a\u00C2\u00A0 is\u00C2\u00A0normally\u00C2\u00A0expressed\u00C2\u00A0at\u00C2\u00A0constitutively\u00C2\u00A0 low\u00C2\u00A0 levels\u00C2\u00A0 in\u00C2\u00A0all\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0human\u00C2\u00A0body174,\u00C2\u00A0 177.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 Rtx,\u00C2\u00A0 it\u00C2\u00A0 appears\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0multivalent\u00C2\u00A0 Rtx\u00C2\u00A0 to\u00C2\u00A0 upregulate\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0enhanced\u00C2\u00A0therapeutic\u00C2\u00A0effects.\u00C2\u00A0\u00C2\u00A0Specifically,\u00C2\u00A0as\u00C2\u00A0illustrated\u00C2\u00A0in\u00C2\u00A0 Figures\u00C2\u00A06.1,\u00C2\u00A06.2,\u00C2\u00A0&\u00C2\u00A06.5,\u00C2\u00A0the\u00C2\u00A0majority\u00C2\u00A0of\u00C2\u00A0upregulated\u00C2\u00A0CD120a\u00C2\u00A0is\u00C2\u00A0localized\u00C2\u00A0outside\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 constitutes\u00C2\u00A0 the\u00C2\u00A0 apoptosis\u00E2\u0080\u0090inducing\u00C2\u00A0 form\u00C2\u00A0 of\u00C2\u00A0 CD120a,\u00C2\u00A0 as\u00C2\u00A0 opposed\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 antiapoptotic\u00C2\u00A0 form,\u00C2\u00A0which\u00C2\u00A0involves\u00C2\u00A0association\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0with\u00C2\u00A0rafts\u00C2\u00A0(Section\u00C2\u00A06.2.1).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0 would\u00C2\u00A0 be\u00C2\u00A0 expected\u00C2\u00A0 that\u00C2\u00A0 oncogenic\u00C2\u00A0 cells,\u00C2\u00A0 in\u00C2\u00A0 order\u00C2\u00A0 to\u00C2\u00A0 survive,\u00C2\u00A0 would\u00C2\u00A0 express\u00C2\u00A0 the\u00C2\u00A0 raft\u00E2\u0080\u0090 associated,\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB\u00E2\u0080\u0090activating\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0CD120a.\u00C2\u00A0 \u00C2\u00A0In\u00C2\u00A0this\u00C2\u00A0sense,\u00C2\u00A0measuring\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0may\u00C2\u00A0not\u00C2\u00A0 be\u00C2\u00A0a\u00C2\u00A0good\u00C2\u00A0predictive\u00C2\u00A0marker\u00C2\u00A0for\u00C2\u00A0Rtx\u00C2\u00A0response\u00C2\u00A0because\u00C2\u00A0our\u00C2\u00A0results\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0upregulation\u00C2\u00A0of\u00C2\u00A0 non\u00E2\u0080\u0090raft\u00E2\u0080\u0090associated\u00C2\u00A0 CD120a\u00C2\u00A0 drives\u00C2\u00A0 the\u00C2\u00A0 direct\u00C2\u00A0 induction\u00C2\u00A0 of\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 results\u00C2\u00A0 in\u00C2\u00A0 Chapter\u00C2\u00A0 6\u00C2\u00A0 (particularly\u00C2\u00A0 Figure\u00C2\u00A0 6.3),\u00C2\u00A0 however,\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 if\u00C2\u00A0 lymphoma\u00C2\u00A0 cells\u00C2\u00A0 express\u00C2\u00A0 high\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 CD120a,\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0168\u00C2\u00A0\u00C2\u00A0 which\u00C2\u00A0 would\u00C2\u00A0 be\u00C2\u00A0 expected\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 raft\u00E2\u0080\u0090associated,\u00C2\u00A0 that\u00C2\u00A0 the\u00C2\u00A0 patient\u00C2\u00A0 may\u00C2\u00A0 show\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 Chol\u00C2\u00A0 depletion\u00C2\u00A0 therapy\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 statin\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 Such\u00C2\u00A0 a\u00C2\u00A0 strategy\u00C2\u00A0 may\u00C2\u00A0 lower\u00C2\u00A0 the\u00C2\u00A0 levels\u00C2\u00A0 of\u00C2\u00A0 plasma\u00E2\u0080\u0090 membrane\u00C2\u00A0Chol\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0cause\u00C2\u00A0CD120a\u00C2\u00A0to\u00C2\u00A0translocate\u00C2\u00A0outside\u00C2\u00A0of\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 rafts,\u00C2\u00A0which\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0apoptosis\u00C2\u00A0 in\u00C2\u00A0 these\u00C2\u00A0 cells.\u00C2\u00A0 \u00C2\u00A0 Section\u00C2\u00A07.3.5\u00C2\u00A0discusses\u00C2\u00A0 statin\u00C2\u00A0 treatment\u00C2\u00A0in\u00C2\u00A0conjunction\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0therapy.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Since\u00C2\u00A0 total\u00C2\u00A0CD120a\u00C2\u00A0 levels\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0appear\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0a\u00C2\u00A0good\u00C2\u00A0marker\u00C2\u00A0 for\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0Rtx,\u00C2\u00A0there\u00C2\u00A0 is\u00C2\u00A0 also\u00C2\u00A0the\u00C2\u00A0possibility\u00C2\u00A0of\u00C2\u00A0assessing\u00C2\u00A0the\u00C2\u00A0mutation\u00C2\u00A0status\u00C2\u00A0of\u00C2\u00A0CD120a.\u00C2\u00A0\u00C2\u00A0Mutations\u00C2\u00A0may\u00C2\u00A0influence\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 apoptosis,\u00C2\u00A0 affecting\u00C2\u00A0 the\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 Rtx\u00C2\u00A0 treatment.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 most\u00C2\u00A0 common\u00C2\u00A0 mutations\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 gene\u00C2\u00A0 encoding\u00C2\u00A0 CD120a\u00C2\u00A0 occur\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 group\u00C2\u00A0 of\u00C2\u00A0 autoinflammatory\u00C2\u00A0 disorders\u00C2\u00A0 called\u00C2\u00A0 TNFR\u00E2\u0080\u0090associated\u00C2\u00A0 periodic\u00C2\u00A0 syndrome\u00C2\u00A0 (TRAPS)259,\u00C2\u00A0 260.\u00C2\u00A0 \u00C2\u00A0 Symptoms\u00C2\u00A0 of\u00C2\u00A0 TRAPS\u00C2\u00A0 consist\u00C2\u00A0 of\u00C2\u00A0 seemingly\u00C2\u00A0 unprovoked\u00C2\u00A0 inflammatory\u00C2\u00A0 attacks\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 impaired\u00C2\u00A0 control\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 innate\u00C2\u00A0 immune\u00C2\u00A0 system,\u00C2\u00A0 but\u00C2\u00A0 autoantibodies\u00C2\u00A0or\u00C2\u00A0antigen\u00E2\u0080\u0090specific\u00C2\u00A0T\u00E2\u0080\u0090cells\u00C2\u00A0(the\u00C2\u00A0usual\u00C2\u00A0markers\u00C2\u00A0of\u00C2\u00A0autoimmune\u00C2\u00A0disease)\u00C2\u00A0are\u00C2\u00A0absent261.\u00C2\u00A0\u00C2\u00A0 Mutations\u00C2\u00A0in\u00C2\u00A0CD120a\u00C2\u00A0that\u00C2\u00A0cause\u00C2\u00A0TRAPS\u00C2\u00A0occur\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0extracellular\u00C2\u00A0domain,\u00C2\u00A0and\u00C2\u00A0it\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A0 these\u00C2\u00A0mutations\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0cause\u00C2\u00A0constitutive\u00C2\u00A0or\u00C2\u00A0enhanced\u00C2\u00A0CD120a\u00C2\u00A0activation,\u00C2\u00A0but\u00C2\u00A0 they\u00C2\u00A0may\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 defective\u00C2\u00A0apoptosis,\u00C2\u00A0allowing\u00C2\u00A0increased\u00C2\u00A0survival\u00C2\u00A0of\u00C2\u00A0proinflammatory\u00C2\u00A0cells262.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 Apart\u00C2\u00A0from\u00C2\u00A0those\u00C2\u00A0that\u00C2\u00A0are\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0TRAPS,\u00C2\u00A0however,\u00C2\u00A0CD120a\u00C2\u00A0mutations\u00C2\u00A0are\u00C2\u00A0relatively\u00C2\u00A0 rare,\u00C2\u00A0 including\u00C2\u00A0 in\u00C2\u00A0cancer263;\u00C2\u00A0this\u00C2\u00A0 is\u00C2\u00A0 in\u00C2\u00A0contrast\u00C2\u00A0to\u00C2\u00A0CD95,\u00C2\u00A0DR4,\u00C2\u00A0and\u00C2\u00A0DR5,\u00C2\u00A0where\u00C2\u00A0mutations\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0 identified\u00C2\u00A0 in\u00C2\u00A0many\u00C2\u00A0cancer\u00C2\u00A0types\u00C2\u00A0 including\u00C2\u00A0different\u00C2\u00A0 lymphomas262,\u00C2\u00A0264.\u00C2\u00A0 \u00C2\u00A0 In\u00C2\u00A0an\u00C2\u00A0effort\u00C2\u00A0to\u00C2\u00A0 identify\u00C2\u00A0novel\u00C2\u00A0 lymphoma\u00E2\u0080\u0090associated\u00C2\u00A0 mutations\u00C2\u00A0 in\u00C2\u00A0 CD120a,\u00C2\u00A0 its\u00C2\u00A0 mutation\u00C2\u00A0 status\u00C2\u00A0 was\u00C2\u00A0 queried\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 database\u00C2\u00A0 of\u00C2\u00A0 hundreds\u00C2\u00A0of\u00C2\u00A0diffuse\u00C2\u00A0 large\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0 lymphoma\u00C2\u00A0 (DLBCL)\u00C2\u00A0biopsies\u00C2\u00A0which\u00C2\u00A0underwent\u00C2\u00A0genome\u00C2\u00A0sequencing\u00C2\u00A0 (Dr.\u00C2\u00A0 Randy\u00C2\u00A0Gascoyne,\u00C2\u00A0 personal\u00C2\u00A0 communication).\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 analysis\u00C2\u00A0 yielded\u00C2\u00A0 the\u00C2\u00A0 result\u00C2\u00A0 that\u00C2\u00A0 no\u00C2\u00A0 specific\u00C2\u00A0 mutations\u00C2\u00A0in\u00C2\u00A0CD120a\u00C2\u00A0were\u00C2\u00A0found\u00C2\u00A0across\u00C2\u00A0a\u00C2\u00A0statistically\u00C2\u00A0significant\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0samples,\u00C2\u00A0confirming\u00C2\u00A0the\u00C2\u00A0 lack\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0mutations\u00C2\u00A0 observed\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 and\u00C2\u00A0 in\u00C2\u00A0 diseases\u00C2\u00A0 other\u00C2\u00A0 than\u00C2\u00A0 TRAPS.\u00C2\u00A0 \u00C2\u00A0 Like\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 levels,\u00C2\u00A0 therefore,\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0 status\u00C2\u00A0does\u00C2\u00A0not\u00C2\u00A0 appear\u00C2\u00A0 to\u00C2\u00A0be\u00C2\u00A0 a\u00C2\u00A0 good\u00C2\u00A0marker\u00C2\u00A0 for\u00C2\u00A0 clinical\u00C2\u00A0Rtx\u00C2\u00A0response.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0169\u00C2\u00A0\u00C2\u00A0 It\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0more\u00C2\u00A0promising\u00C2\u00A0to\u00C2\u00A0look\u00C2\u00A0beyond\u00C2\u00A0CD120a\u00C2\u00A0to\u00C2\u00A0other\u00C2\u00A0proteins\u00C2\u00A0that\u00C2\u00A0interact\u00C2\u00A0with\u00C2\u00A0it;\u00C2\u00A0for\u00C2\u00A0 example,\u00C2\u00A0 mutations\u00C2\u00A0 in\u00C2\u00A0 proteins\u00C2\u00A0 that\u00C2\u00A0 associate\u00C2\u00A0 with\u00C2\u00A0 CD120a\u00C2\u00A0 and\u00C2\u00A0 which\u00C2\u00A0 may\u00C2\u00A0 contribute\u00C2\u00A0 to\u00C2\u00A0 oncogenesis\u00C2\u00A0 have\u00C2\u00A0 been\u00C2\u00A0 identified,\u00C2\u00A0 such\u00C2\u00A0 as\u00C2\u00A0 mutations\u00C2\u00A0 in\u00C2\u00A0 TRADD,\u00C2\u00A0 which\u00C2\u00A0 forms\u00C2\u00A0 the\u00C2\u00A0 DISC\u00C2\u00A0 with\u00C2\u00A0 CD120a265,\u00C2\u00A0266.\u00C2\u00A0\u00C2\u00A0Another\u00C2\u00A0possibility\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0to\u00C2\u00A0assess\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0healthy\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0effector\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 a\u00C2\u00A0 patient\u00C2\u00A0 to\u00C2\u00A0 engage\u00C2\u00A0 in\u00C2\u00A0 hypercrosslinking,\u00C2\u00A0 because\u00C2\u00A0 hypercrosslinking\u00C2\u00A0 was\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 be\u00C2\u00A0 important\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0(Figures\u00C2\u00A06.4\u00E2\u0080\u00936.6).\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0can\u00C2\u00A0be\u00C2\u00A0assessed\u00C2\u00A0not\u00C2\u00A0only\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0expression\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0Fc\u00CE\u00B3Rs\u00C2\u00A0on\u00C2\u00A0effector\u00C2\u00A0cells,\u00C2\u00A0but\u00C2\u00A0also\u00C2\u00A0by\u00C2\u00A0 the\u00C2\u00A0 total\u00C2\u00A0counts\u00C2\u00A0of\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0 cells;\u00C2\u00A0both\u00C2\u00A0higher\u00C2\u00A0average\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0higher\u00C2\u00A0 total\u00C2\u00A0 cell\u00C2\u00A0 counts\u00C2\u00A0would\u00C2\u00A0be\u00C2\u00A0expected\u00C2\u00A0 to\u00C2\u00A0produce\u00C2\u00A0 enhanced\u00C2\u00A0Rtx\u00C2\u00A0hypercrosslinking.\u00C2\u00A0 \u00C2\u00A0Another\u00C2\u00A0possibility\u00C2\u00A0may\u00C2\u00A0be\u00C2\u00A0 to\u00C2\u00A0develop\u00C2\u00A0an\u00C2\u00A0assay\u00C2\u00A0 to\u00C2\u00A0measure\u00C2\u00A0 the\u00C2\u00A0 affinity\u00C2\u00A0of\u00C2\u00A0Fc\u00CE\u00B3Rs\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0therapeutic\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0This\u00C2\u00A0is\u00C2\u00A0in\u00C2\u00A0light\u00C2\u00A0of\u00C2\u00A0studies\u00C2\u00A0that\u00C2\u00A0have\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A0 certain\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0dimorphisms\u00C2\u00A0result\u00C2\u00A0in\u00C2\u00A0greater\u00C2\u00A0affinity\u00C2\u00A0interaction\u00C2\u00A0between\u00C2\u00A0Fc\u00CE\u00B3R\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0Fc\u00C2\u00A0region\u00C2\u00A0of\u00C2\u00A0IgG\u00C2\u00A0 (Section\u00C2\u00A02.2.2);\u00C2\u00A0greater\u00E2\u0080\u0090affinity\u00C2\u00A0 Fc\u00CE\u00B3R\u00E2\u0080\u0093Fc\u00C2\u00A0 interactions\u00C2\u00A0would\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0more\u00C2\u00A0 stable\u00C2\u00A0hypercrosslinked\u00C2\u00A0 Rtx/CD20\u00C2\u00A0 clusters\u00C2\u00A0 and\u00C2\u00A0 may\u00C2\u00A0 result\u00C2\u00A0 in\u00C2\u00A0 enhanced\u00C2\u00A0 efficacy\u00C2\u00A0 due\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0 direct\u00C2\u00A0 mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0described\u00C2\u00A0in\u00C2\u00A0Chapters\u00C2\u00A05\u00C2\u00A0&\u00C2\u00A06\u00C2\u00A0(refs.\u00C2\u00A0122,\u00C2\u00A0123).\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A07.3.5\u00C2\u00A0 Statins\u00C2\u00A0in\u00C2\u00A0combination\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0 Chapter\u00C2\u00A06\u00C2\u00A0shows\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0hypercrosslinking\u00C2\u00A0provided\u00C2\u00A0by\u00C2\u00A0immune\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0sufficient\u00C2\u00A0to\u00C2\u00A0 induce\u00C2\u00A0 CD120a\u00C2\u00A0 upregulation\u00C2\u00A0 and\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 is\u00C2\u00A0 strong\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 CD120a\u00E2\u0080\u0090mediated\u00C2\u00A0 cell\u00C2\u00A0 death\u00C2\u00A0 occurs\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 direct\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 of\u00C2\u00A0 regular\u00C2\u00A0 bivalent\u00C2\u00A0 Rtx\u00C2\u00A0 therapy\u00C2\u00A0 in\u00C2\u00A0 vivo.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 final\u00C2\u00A0 chapter\u00C2\u00A0also\u00C2\u00A0examines\u00C2\u00A0the\u00C2\u00A0Chol\u00C2\u00A0dependence\u00C2\u00A0of\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0induced\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0light\u00C2\u00A0of\u00C2\u00A0this\u00C2\u00A0mechanism\u00C2\u00A0 since\u00C2\u00A0CD120a\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0produce\u00C2\u00A0proapoptotic\u00C2\u00A0signals\u00C2\u00A0under\u00C2\u00A0conditions\u00C2\u00A0of\u00C2\u00A0reduced\u00C2\u00A0plasma\u00C2\u00A0 membrane\u00C2\u00A0Chol\u00C2\u00A0(Sections\u00C2\u00A06.2.1\u00C2\u00A0&\u00C2\u00A06.2.2).\u00C2\u00A0 \u00C2\u00A0It\u00C2\u00A0was\u00C2\u00A0shown\u00C2\u00A0 in\u00C2\u00A0Figures\u00C2\u00A06.1\u00C2\u00A0&\u00C2\u00A06.2\u00C2\u00A0that\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00C2\u00A0 increased\u00C2\u00A0 the\u00C2\u00A0detergent\u00C2\u00A0solubility\u00C2\u00A0of\u00C2\u00A0CD120a,\u00C2\u00A0but\u00C2\u00A0when\u00C2\u00A0Chol\u00C2\u00A0was\u00C2\u00A0 reinserted\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0using\u00C2\u00A0 MBCD/Chol,\u00C2\u00A0 the\u00C2\u00A0 DR\u00C2\u00A0 and\u00C2\u00A0 raft\u00C2\u00A0 association\u00C2\u00A0 of\u00C2\u00A0 CD120a\u00C2\u00A0 increased,\u00C2\u00A0 and\u00C2\u00A0 cells\u00C2\u00A0 were\u00C2\u00A0 rescued\u00C2\u00A0 from\u00C2\u00A0 apoptosis.\u00C2\u00A0 \u00C2\u00A0Simvastatin\u00C2\u00A0treatment\u00C2\u00A0also\u00C2\u00A0decreased\u00C2\u00A0the\u00C2\u00A0DR\u00C2\u00A0of\u00C2\u00A0CD120a\u00C2\u00A0and\u00C2\u00A0sensitized\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0170\u00C2\u00A0\u00C2\u00A0 to\u00C2\u00A0Rtx\u00E2\u0080\u0090LNP\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis\u00C2\u00A0 (Figure\u00C2\u00A06.3).\u00C2\u00A0 \u00C2\u00A0Along\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0PBMC\u00C2\u00A0experiments\u00C2\u00A0 in\u00C2\u00A0Figure\u00C2\u00A06.6\u00C2\u00A0which\u00C2\u00A0 supported\u00C2\u00A0 an\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism,\u00C2\u00A0 these\u00C2\u00A0 data\u00C2\u00A0 suggest\u00C2\u00A0 that\u00C2\u00A0 concurrent\u00C2\u00A0 statin\u00C2\u00A0 treatment\u00C2\u00A0with\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0may\u00C2\u00A0increase\u00C2\u00A0the\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0dual\u00C2\u00A0raft\u00E2\u0080\u0090dependent\u00C2\u00A0nature\u00C2\u00A0 of\u00C2\u00A0CD120a\u00C2\u00A0signaling.\u00C2\u00A0 Statins\u00C2\u00A0 have\u00C2\u00A0 shown\u00C2\u00A0 anticancer\u00C2\u00A0 activity\u00C2\u00A0 in\u00C2\u00A0 leukemia,\u00C2\u00A0 lymphoma,\u00C2\u00A0 colorectal\u00C2\u00A0 cancer,\u00C2\u00A0 breast\u00C2\u00A0 cancer,\u00C2\u00A0 lung\u00C2\u00A0 cancer,\u00C2\u00A0 prostate\u00C2\u00A0 cancer,\u00C2\u00A0 and\u00C2\u00A0 pancreatic\u00C2\u00A0 cancer267.\u00C2\u00A0 \u00C2\u00A0 Many\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 action\u00C2\u00A0 responsible\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0effects\u00C2\u00A0have\u00C2\u00A0been\u00C2\u00A0described,\u00C2\u00A0including\u00C2\u00A0the\u00C2\u00A0ability\u00C2\u00A0of\u00C2\u00A0statins\u00C2\u00A0to\u00C2\u00A0inhibit\u00C2\u00A0 antiapoptotic\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0signaling.\u00C2\u00A0\u00C2\u00A0Reduced\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0NF\u00E2\u0080\u0090\u00CE\u00BAB\u00C2\u00A0resulting\u00C2\u00A0from\u00C2\u00A0simvastatin\u00C2\u00A0treatment\u00C2\u00A0has\u00C2\u00A0 been\u00C2\u00A0 demonstrated\u00C2\u00A0 in\u00C2\u00A0 Epstein\u00E2\u0080\u0090Barr\u00C2\u00A0 virus\u00E2\u0080\u0090associated\u00C2\u00A0 lymphomas268\u00C2\u00A0 and\u00C2\u00A0 cancer\u00E2\u0080\u0090associated\u00C2\u00A0 osteoclastogenesis269,\u00C2\u00A0 and\u00C2\u00A0 it\u00C2\u00A0 occurred\u00C2\u00A0 alongside\u00C2\u00A0 increased\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 human\u00C2\u00A0myeloid\u00C2\u00A0 leukemia\u00C2\u00A0 cells270\u00C2\u00A0and\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0cells,\u00C2\u00A0where\u00C2\u00A0 the\u00C2\u00A0apoptosis\u00C2\u00A0was\u00C2\u00A0 induced\u00C2\u00A0 in\u00C2\u00A0response\u00C2\u00A0 to\u00C2\u00A0TNF\u00E2\u0080\u0090\u00CE\u00B1271.\u00C2\u00A0 \u00C2\u00A0Statins\u00C2\u00A0 have\u00C2\u00A0 also\u00C2\u00A0 been\u00C2\u00A0 shown\u00C2\u00A0 to\u00C2\u00A0 induce\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 ovarian\u00C2\u00A0 cancer\u00C2\u00A0 cells272,\u00C2\u00A0 to\u00C2\u00A0 inhibit\u00C2\u00A0 the\u00C2\u00A0 cell\u00C2\u00A0 growth,\u00C2\u00A0 migration,\u00C2\u00A0and\u00C2\u00A0 invasion\u00C2\u00A0of\u00C2\u00A0melanoma\u00C2\u00A0 cells273,\u00C2\u00A0and\u00C2\u00A0 to\u00C2\u00A0help\u00C2\u00A0overcome\u00C2\u00A0drug\u00C2\u00A0 resistance\u00C2\u00A0 in\u00C2\u00A0 refractory\u00C2\u00A0 multiple\u00C2\u00A0myeloma\u00C2\u00A0in\u00C2\u00A0a\u00C2\u00A0Phase\u00C2\u00A0II\u00C2\u00A0trial274.\u00C2\u00A0\u00C2\u00A0Finally,\u00C2\u00A0many\u00C2\u00A0studies\u00C2\u00A0have\u00C2\u00A0associated\u00C2\u00A0statin\u00C2\u00A0use\u00C2\u00A0with\u00C2\u00A0reduced\u00C2\u00A0 risk\u00C2\u00A0of\u00C2\u00A0cancer.\u00C2\u00A0\u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0a\u00C2\u00A0European\u00C2\u00A0case\u00E2\u0080\u0090control\u00C2\u00A0study\u00C2\u00A0across\u00C2\u00A022\u00C2\u00A0centers\u00C2\u00A0demonstrated\u00C2\u00A0an\u00C2\u00A0odds\u00C2\u00A0 ratio\u00C2\u00A0of\u00C2\u00A00.61\u00C2\u00A0 for\u00C2\u00A0regular\u00C2\u00A0statin\u00C2\u00A0use\u00C2\u00A0with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0non\u00E2\u0080\u0090use,\u00C2\u00A0representing\u00C2\u00A0a\u00C2\u00A0significant\u00C2\u00A0reduction\u00C2\u00A0 in\u00C2\u00A0 overall\u00C2\u00A0lymphoma\u00C2\u00A0risk\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0statin\u00C2\u00A0treatment275.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 There\u00C2\u00A0 is\u00C2\u00A0 some\u00C2\u00A0 evidence\u00C2\u00A0 that\u00C2\u00A0 statin\u00C2\u00A0 use\u00C2\u00A0 may\u00C2\u00A0 directly\u00C2\u00A0 inhibit\u00C2\u00A0 Rtx\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 CD20\u00C2\u00A0 and,\u00C2\u00A0 therefore,\u00C2\u00A0the\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0Inhibition\u00C2\u00A0was\u00C2\u00A0shown\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0conformational\u00C2\u00A0changes\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 CD20\u00C2\u00A0molecule\u00C2\u00A0during\u00C2\u00A0statin\u00C2\u00A0treatment,\u00C2\u00A0and\u00C2\u00A0B\u00C2\u00A0cells\u00C2\u00A0obtained\u00C2\u00A0from\u00C2\u00A0patients\u00C2\u00A0on\u00C2\u00A0Rtx\u00C2\u00A0therapy\u00C2\u00A0showed\u00C2\u00A0 lower\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0binding276.\u00C2\u00A0 \u00C2\u00A0Although\u00C2\u00A0these\u00C2\u00A0studies\u00C2\u00A0raised\u00C2\u00A0concerns\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0statins\u00C2\u00A0during\u00C2\u00A0 Rtx\u00C2\u00A0treatment,\u00C2\u00A0there\u00C2\u00A0is\u00C2\u00A0no\u00C2\u00A0consensus\u00C2\u00A0on\u00C2\u00A0whether\u00C2\u00A0or\u00C2\u00A0not\u00C2\u00A0to\u00C2\u00A0stop\u00C2\u00A0statin\u00C2\u00A0treatment\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0initiation\u00C2\u00A0of\u00C2\u00A0 lymphoma\u00C2\u00A0 therapy\u00C2\u00A0 containing\u00C2\u00A0Rtx277.\u00C2\u00A0 \u00C2\u00A0One\u00C2\u00A0 study\u00C2\u00A0 that\u00C2\u00A0 addressed\u00C2\u00A0 this\u00C2\u00A0 issue\u00C2\u00A0 examined\u00C2\u00A0Rtx\u00C2\u00A0 efficacy\u00C2\u00A0 with\u00C2\u00A0respect\u00C2\u00A0to\u00C2\u00A0statin\u00C2\u00A0use\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0outcome\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0cohort\u00C2\u00A0of\u00C2\u00A0prospectively\u00C2\u00A0observed\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0newly\u00C2\u00A0 diagnosed\u00C2\u00A0DLBCL\u00C2\u00A0and\u00C2\u00A0follicular\u00C2\u00A0 lymphoma\u00C2\u00A0(FL).\u00C2\u00A0 \u00C2\u00A0It\u00C2\u00A0was\u00C2\u00A0shown\u00C2\u00A0that\u00C2\u00A0statin\u00C2\u00A0use\u00C2\u00A0had\u00C2\u00A0no\u00C2\u00A0 impact\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0171\u00C2\u00A0\u00C2\u00A0 overall\u00C2\u00A0response\u00C2\u00A0rate,\u00C2\u00A0overall\u00C2\u00A0survival,\u00C2\u00A0or\u00C2\u00A0event\u00E2\u0080\u0090free\u00C2\u00A0survival\u00C2\u00A0in\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0DLBCL,\u00C2\u00A0but\u00C2\u00A0statin\u00C2\u00A0use\u00C2\u00A0at\u00C2\u00A0 diagnosis\u00C2\u00A0was\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0improved\u00C2\u00A0event\u00E2\u0080\u0090free\u00C2\u00A0survival\u00C2\u00A0in\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0FL,\u00C2\u00A0including\u00C2\u00A0subgroups\u00C2\u00A0 treated\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0Rtx\u00E2\u0080\u0090containing\u00C2\u00A0regimen.\u00C2\u00A0\u00C2\u00A0Statins\u00C2\u00A0therefore\u00C2\u00A0did\u00C2\u00A0not\u00C2\u00A0adversely\u00C2\u00A0affect\u00C2\u00A0outcome\u00C2\u00A0in\u00C2\u00A0DLBCL\u00C2\u00A0 and\u00C2\u00A0FL,\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0apparent\u00C2\u00A0benefit\u00C2\u00A0of\u00C2\u00A0statin\u00C2\u00A0therapy\u00C2\u00A0on\u00C2\u00A0FL\u00C2\u00A0outcome\u00C2\u00A0requires\u00C2\u00A0further\u00C2\u00A0studies277.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 At\u00C2\u00A0least\u00C2\u00A0in\u00C2\u00A0some\u00C2\u00A0cases,\u00C2\u00A0the\u00C2\u00A0anticancer\u00C2\u00A0effect\u00C2\u00A0of\u00C2\u00A0statins\u00C2\u00A0is\u00C2\u00A0related\u00C2\u00A0to\u00C2\u00A0lowering\u00C2\u00A0the\u00C2\u00A0area\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 plasma\u00C2\u00A0membrane\u00C2\u00A0covered\u00C2\u00A0with\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0rafts267.\u00C2\u00A0 \u00C2\u00A0For\u00C2\u00A0example,\u00C2\u00A0Chol\u00C2\u00A0targeting\u00C2\u00A0has\u00C2\u00A0been\u00C2\u00A0 shown\u00C2\u00A0to\u00C2\u00A0alter\u00C2\u00A0lipid\u00C2\u00A0raft\u00C2\u00A0composition\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0survival\u00C2\u00A0in\u00C2\u00A0prostate\u00C2\u00A0cancer\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0xenografts.\u00C2\u00A0\u00C2\u00A0Both\u00C2\u00A0 in\u00C2\u00A0vitro\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0experiments\u00C2\u00A0showed\u00C2\u00A0that\u00C2\u00A0simvastatin\u00C2\u00A0induced\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0prostate\u00C2\u00A0cancer\u00C2\u00A0cells\u00C2\u00A0 by\u00C2\u00A0 inhibiting\u00C2\u00A0 Akt\u00C2\u00A0 pathway\u00C2\u00A0 signaling,\u00C2\u00A0 and\u00C2\u00A0 replenishing\u00C2\u00A0 cell\u00C2\u00A0membranes\u00C2\u00A0with\u00C2\u00A0 Chol\u00C2\u00A0 reversed\u00C2\u00A0 these\u00C2\u00A0 inhibitory\u00C2\u00A0 and\u00C2\u00A0 apoptotic\u00C2\u00A0 effects243.\u00C2\u00A0 \u00C2\u00A0 This\u00C2\u00A0 large\u00C2\u00A0 body\u00C2\u00A0 of\u00C2\u00A0 evidence\u00C2\u00A0 concerning\u00C2\u00A0 statin\u00C2\u00A0 use\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 treatment\u00C2\u00A0 lends\u00C2\u00A0 credibility\u00C2\u00A0 to\u00C2\u00A0 future\u00C2\u00A0 studies\u00C2\u00A0 on\u00C2\u00A0 the\u00C2\u00A0 ability\u00C2\u00A0 of\u00C2\u00A0 statins\u00C2\u00A0 to\u00C2\u00A0 improve\u00C2\u00A0 the\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0 therapeutic\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0through\u00C2\u00A0a\u00C2\u00A0mechanism\u00C2\u00A0that\u00C2\u00A0involves\u00C2\u00A0CD120a.\u00C2\u00A0 7.4\u00C2\u00A0 Future\u00C2\u00A0work\u00C2\u00A0 In\u00C2\u00A0an\u00C2\u00A0effort\u00C2\u00A0 to\u00C2\u00A0 further\u00C2\u00A0understand\u00C2\u00A0 the\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx,\u00C2\u00A0 the\u00C2\u00A0results\u00C2\u00A0 from\u00C2\u00A0 Chapter\u00C2\u00A06\u00C2\u00A0suggest\u00C2\u00A0that\u00C2\u00A0an\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0study\u00C2\u00A0in\u00C2\u00A0lymphoma\u00C2\u00A0xenograft\u00C2\u00A0models\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0in\u00C2\u00A0order\u00C2\u00A0 to\u00C2\u00A0determine\u00C2\u00A0whether\u00C2\u00A0changes\u00C2\u00A0 in\u00C2\u00A0CD120a\u00C2\u00A0expression\u00C2\u00A0 levels\u00C2\u00A0 in\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0are\u00C2\u00A0observed\u00C2\u00A0before\u00C2\u00A0 and\u00C2\u00A0after\u00C2\u00A0treatment\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0\u00C2\u00A0The\u00C2\u00A0same\u00C2\u00A0two\u00C2\u00A0models\u00C2\u00A0as\u00C2\u00A0used\u00C2\u00A0in\u00C2\u00A0Figure\u00C2\u00A02.1B\u00C2\u00A0&\u00C2\u00A0C\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0employed;\u00C2\u00A0 the\u00C2\u00A0JVM2\u00C2\u00A0model\u00C2\u00A0was\u00C2\u00A0Rtx\u00E2\u0080\u0090insensitive\u00C2\u00A0while\u00C2\u00A0the\u00C2\u00A0Z138\u00C2\u00A0model\u00C2\u00A0was\u00C2\u00A0Rtx\u00E2\u0080\u0090sensitive.\u00C2\u00A0\u00C2\u00A0Based\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0 Chapter\u00C2\u00A06,\u00C2\u00A0it\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0established\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0animals\u00C2\u00A0have\u00C2\u00A0functioning\u00C2\u00A0Fc\u00CE\u00B3R\u00E2\u0080\u0090expressing\u00C2\u00A0effector\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0order\u00C2\u00A0for\u00C2\u00A0hypercrosslinking\u00C2\u00A0to\u00C2\u00A0occur.\u00C2\u00A0 \u00C2\u00A0These\u00C2\u00A0experiments\u00C2\u00A0could\u00C2\u00A0be\u00C2\u00A0carried\u00C2\u00A0out\u00C2\u00A0alongside\u00C2\u00A0a\u00C2\u00A0study\u00C2\u00A0 which\u00C2\u00A0examines\u00C2\u00A0the\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0Rtx\u00C2\u00A0in\u00C2\u00A0conjunction\u00C2\u00A0with\u00C2\u00A0statin\u00C2\u00A0therapy,\u00C2\u00A0and\u00C2\u00A0if\u00C2\u00A0possible,\u00C2\u00A0differences\u00C2\u00A0in\u00C2\u00A0 efficacy\u00C2\u00A0 could\u00C2\u00A0 be\u00C2\u00A0 examined\u00C2\u00A0 alongside\u00C2\u00A0 CD120a\u00C2\u00A0 expression\u00C2\u00A0 to\u00C2\u00A0 support\u00C2\u00A0 or\u00C2\u00A0 discredit\u00C2\u00A0 the\u00C2\u00A0 hypothesis\u00C2\u00A0 regarding\u00C2\u00A0a\u00C2\u00A0CD120a\u00E2\u0080\u0090dependent\u00C2\u00A0direct\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0Rtx.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0172\u00C2\u00A0\u00C2\u00A0 In\u00C2\u00A0 terms\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 Ab\u00E2\u0080\u0090LNP\u00C2\u00A0methodology\u00C2\u00A0 presented\u00C2\u00A0 in\u00C2\u00A0 this\u00C2\u00A0 dissertation,\u00C2\u00A0 the\u00C2\u00A0most\u00C2\u00A0 important\u00C2\u00A0 future\u00C2\u00A0work\u00C2\u00A0involves\u00C2\u00A0its\u00C2\u00A0application\u00C2\u00A0to\u00C2\u00A0other\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0in\u00C2\u00A0an\u00C2\u00A0effort\u00C2\u00A0to\u00C2\u00A0discover\u00C2\u00A0new\u00C2\u00A0multivalent\u00C2\u00A0 drugs.\u00C2\u00A0\u00C2\u00A0In\u00C2\u00A0industrial\u00C2\u00A0settings,\u00C2\u00A0a\u00C2\u00A0large\u00C2\u00A0number\u00C2\u00A0of\u00C2\u00A0potential\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0candidates\u00C2\u00A0do\u00C2\u00A0not\u00C2\u00A0progress\u00C2\u00A0 past\u00C2\u00A0initial\u00C2\u00A0stages\u00C2\u00A0of\u00C2\u00A0development\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0their\u00C2\u00A0marginal\u00C2\u00A0activity\u00C2\u00A0as\u00C2\u00A0bivalent\u00C2\u00A0molecules.\u00C2\u00A0\u00C2\u00A0It\u00C2\u00A0is\u00C2\u00A0possible,\u00C2\u00A0 however,\u00C2\u00A0 that\u00C2\u00A0 creating\u00C2\u00A0multivalent\u00C2\u00A0 constructs\u00C2\u00A0 from\u00C2\u00A0 these\u00C2\u00A0Abs\u00C2\u00A0 could\u00C2\u00A0 lead\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0discovery\u00C2\u00A0of\u00C2\u00A0new\u00C2\u00A0 responses\u00C2\u00A0 that\u00C2\u00A0were\u00C2\u00A0not\u00C2\u00A0observed\u00C2\u00A0when\u00C2\u00A0 the\u00C2\u00A0Ab\u00C2\u00A0was\u00C2\u00A0bivalent,\u00C2\u00A0as\u00C2\u00A0was\u00C2\u00A0 the\u00C2\u00A0case\u00C2\u00A0with\u00C2\u00A0Rtx.\u00C2\u00A0 \u00C2\u00A0The\u00C2\u00A0new\u00C2\u00A0 drugs\u00C2\u00A0could\u00C2\u00A0then\u00C2\u00A0be\u00C2\u00A0developed\u00C2\u00A0as\u00C2\u00A0multivalent,\u00C2\u00A0rather\u00C2\u00A0than\u00C2\u00A0bivalent,\u00C2\u00A0Abs.\u00C2\u00A0\u00C2\u00A0\u00C2\u00A0 To\u00C2\u00A0work\u00C2\u00A0toward\u00C2\u00A0a\u00C2\u00A0system\u00C2\u00A0for\u00C2\u00A0applying\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0dissertation\u00C2\u00A0to\u00C2\u00A0other\u00C2\u00A0Abs,\u00C2\u00A0the\u00C2\u00A0 creation\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 business\u00C2\u00A0 plan\u00C2\u00A0 has\u00C2\u00A0 been\u00C2\u00A0 underway\u00C2\u00A0with\u00C2\u00A0 two\u00C2\u00A0 partners,\u00C2\u00A0 Joel\u00C2\u00A0 Jaffe\u00C2\u00A0 and\u00C2\u00A0Meir\u00C2\u00A0Deutsch,\u00C2\u00A0 under\u00C2\u00A0 the\u00C2\u00A0 auspices\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 British\u00C2\u00A0 Columbia\u00C2\u00A0 Innovation\u00C2\u00A0 Council.\u00C2\u00A0 \u00C2\u00A0 The\u00C2\u00A0 process\u00C2\u00A0 would\u00C2\u00A0 involve\u00C2\u00A0 partnering\u00C2\u00A0with\u00C2\u00A0academic\u00C2\u00A0laboratories\u00C2\u00A0or\u00C2\u00A0companies\u00C2\u00A0engaged\u00C2\u00A0in\u00C2\u00A0therapeutic\u00C2\u00A0antibody\u00C2\u00A0development,\u00C2\u00A0 who\u00C2\u00A0would\u00C2\u00A0provide\u00C2\u00A0\u00E2\u0080\u009Cdiscarded\u00E2\u0080\u009D\u00C2\u00A0Abs\u00C2\u00A0for\u00C2\u00A0testing.\u00C2\u00A0\u00C2\u00A0Besides\u00C2\u00A0showing\u00C2\u00A0low\u00C2\u00A0bivalent\u00C2\u00A0activity,\u00C2\u00A0the\u00C2\u00A0Abs\u00C2\u00A0to\u00C2\u00A0be\u00C2\u00A0 studied\u00C2\u00A0 should\u00C2\u00A0be\u00C2\u00A0at\u00C2\u00A0an\u00C2\u00A0early\u00C2\u00A0 stage\u00C2\u00A0of\u00C2\u00A0development\u00C2\u00A0and\u00C2\u00A0 they\u00C2\u00A0 should\u00C2\u00A0 ideally\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0 target\u00C2\u00A0 that\u00C2\u00A0 is\u00C2\u00A0 known\u00C2\u00A0 to\u00C2\u00A0 produce\u00C2\u00A0 therapeutic\u00C2\u00A0 responses\u00C2\u00A0 through\u00C2\u00A0 clustering.\u00C2\u00A0 \u00C2\u00A0 Such\u00C2\u00A0 a\u00C2\u00A0 strategy\u00C2\u00A0would\u00C2\u00A0 expand\u00C2\u00A0 the\u00C2\u00A0 proof\u00E2\u0080\u0090of\u00E2\u0080\u0090concept\u00C2\u00A0provided\u00C2\u00A0using\u00C2\u00A0Rtx\u00E2\u0080\u0090LNPs\u00C2\u00A0and\u00C2\u00A0may\u00C2\u00A0lead\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0generation\u00C2\u00A0of\u00C2\u00A0new\u00C2\u00A0cancer\u00C2\u00A0medicines.\u00C2\u00A0 7.5\u00C2\u00A0 Conclusions\u00C2\u00A0 This\u00C2\u00A0proof\u00E2\u0080\u0090of\u00E2\u0080\u0090concept\u00C2\u00A0has\u00C2\u00A0established\u00C2\u00A0that\u00C2\u00A0the\u00C2\u00A0methodology\u00C2\u00A0outlined\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0dissertation,\u00C2\u00A0in\u00C2\u00A0 its\u00C2\u00A0application\u00C2\u00A0to\u00C2\u00A0other\u00C2\u00A0Abs\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0context\u00C2\u00A0of\u00C2\u00A0drug\u00C2\u00A0development,\u00C2\u00A0can\u00C2\u00A0serve\u00C2\u00A0to:\u00C2\u00A0\u00C2\u00A0 (1) Identify\u00C2\u00A0 therapeutic\u00C2\u00A0 Abs\u00C2\u00A0 that\u00C2\u00A0 show\u00C2\u00A0 enhanced\u00C2\u00A0 activities\u00C2\u00A0 when\u00C2\u00A0 multivalent\u00C2\u00A0 rather\u00C2\u00A0 than\u00C2\u00A0 bivalent\u00C2\u00A0(Chapter\u00C2\u00A02);\u00C2\u00A0 (2) Determine\u00C2\u00A0the\u00C2\u00A0optimal\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0Abs\u00C2\u00A0that\u00C2\u00A0benefit\u00C2\u00A0from\u00C2\u00A0multivalency\u00C2\u00A0(Chapter\u00C2\u00A04);\u00C2\u00A0 (3) Decipher\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0new\u00C2\u00A0multivalent\u00C2\u00A0drug\u00C2\u00A0(Chapters\u00C2\u00A02,\u00C2\u00A04,\u00C2\u00A05,\u00C2\u00A0&\u00C2\u00A06);\u00C2\u00A0 (4) Use\u00C2\u00A0the\u00C2\u00A0above\u00C2\u00A0 information\u00C2\u00A0to\u00C2\u00A0develop\u00C2\u00A0non\u00E2\u0080\u0090liposomal\u00C2\u00A0formulations\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0multivalent\u00C2\u00A0Ab,\u00C2\u00A0 if\u00C2\u00A0 required\u00C2\u00A0(Chapters\u00C2\u00A05\u00C2\u00A0&\u00C2\u00A06).\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0173\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 Although\u00C2\u00A0the\u00C2\u00A0valence\u00C2\u00A0of\u00C2\u00A0all\u00C2\u00A0approved\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0is\u00C2\u00A0currently\u00C2\u00A0two,\u00C2\u00A0the\u00C2\u00A0results\u00C2\u00A0in\u00C2\u00A0this\u00C2\u00A0dissertation\u00C2\u00A0 suggest\u00C2\u00A0that\u00C2\u00A0every\u00C2\u00A0therapeutic\u00C2\u00A0Ab\u00C2\u00A0may\u00C2\u00A0have\u00C2\u00A0a\u00C2\u00A0different\u00C2\u00A0valence\u00C2\u00A0where\u00C2\u00A0 it\u00C2\u00A0shows\u00C2\u00A0 its\u00C2\u00A0highest\u00C2\u00A0activity.\u00C2\u00A0\u00C2\u00A0 This\u00C2\u00A0new\u00C2\u00A0concept\u00C2\u00A0should\u00C2\u00A0be\u00C2\u00A0included\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0toolbox\u00C2\u00A0for\u00C2\u00A0creating\u00C2\u00A0therapeutic\u00C2\u00A0Abs\u00C2\u00A0for\u00C2\u00A0treating\u00C2\u00A0cancer.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0174\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0References\u00C2\u00A0 \u00C2\u00A0 1.\u00C2\u00A0 Weiner\u00C2\u00A0 LM,\u00C2\u00A0 Surana\u00C2\u00A0 R,\u00C2\u00A0 Wang\u00C2\u00A0 S.\u00C2\u00A0 Monoclonal\u00C2\u00A0 antibodies:\u00C2\u00A0 Versatile\u00C2\u00A0 platforms\u00C2\u00A0 for\u00C2\u00A0 cancer\u00C2\u00A0 immunotherapy.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Immunol\u00C2\u00A02010\u00C2\u00A0May;10(5):317\u00E2\u0080\u009027.\u00C2\u00A0 \u00C2\u00A0 2.\u00C2\u00A0 Harris\u00C2\u00A0 M.\u00C2\u00A0 Monoclonal\u00C2\u00A0 antibodies\u00C2\u00A0 as\u00C2\u00A0 therapeutic\u00C2\u00A0 agents\u00C2\u00A0 for\u00C2\u00A0 cancer.\u00C2\u00A0 Lancet\u00C2\u00A0 Oncol\u00C2\u00A0 2004\u00C2\u00A0 May;5(5):292\u00E2\u0080\u0090302.\u00C2\u00A0 \u00C2\u00A0 3.\u00C2\u00A0 Allen\u00C2\u00A0 TM,\u00C2\u00A0 Cullis\u00C2\u00A0 PR.\u00C2\u00A0 Drug\u00C2\u00A0 delivery\u00C2\u00A0 systems:\u00C2\u00A0 Entering\u00C2\u00A0 the\u00C2\u00A0 mainstream.\u00C2\u00A0 Science\u00C2\u00A0 2004\u00C2\u00A0 Mar\u00C2\u00A0 19;303(5665):1818\u00E2\u0080\u009022.\u00C2\u00A0 \u00C2\u00A0 4.\u00C2\u00A0Heath\u00C2\u00A0TD,\u00C2\u00A0Fraley\u00C2\u00A0RT,\u00C2\u00A0Papahdjopoulos\u00C2\u00A0D.\u00C2\u00A0Antibody\u00C2\u00A0targeting\u00C2\u00A0of\u00C2\u00A0liposomes:\u00C2\u00A0Cell\u00C2\u00A0specificity\u00C2\u00A0obtained\u00C2\u00A0 by\u00C2\u00A0conjugation\u00C2\u00A0of\u00C2\u00A0F(ab')2\u00C2\u00A0to\u00C2\u00A0vesicle\u00C2\u00A0surface.\u00C2\u00A0Science\u00C2\u00A01980\u00C2\u00A0Oct\u00C2\u00A031;210(4469):539\u00E2\u0080\u009041.\u00C2\u00A0 \u00C2\u00A0 5.\u00C2\u00A0 Zhang\u00C2\u00A0 L,\u00C2\u00A0 Gu\u00C2\u00A0 FX,\u00C2\u00A0 Chan\u00C2\u00A0 JM,\u00C2\u00A0 Wang\u00C2\u00A0 AZ,\u00C2\u00A0 Langer\u00C2\u00A0 RS,\u00C2\u00A0 Farokhzad\u00C2\u00A0 OC.\u00C2\u00A0 Nanoparticles\u00C2\u00A0 in\u00C2\u00A0 medicine:\u00C2\u00A0 Therapeutic\u00C2\u00A0applications\u00C2\u00A0and\u00C2\u00A0developments.\u00C2\u00A0Clin\u00C2\u00A0Pharmacol\u00C2\u00A0Ther\u00C2\u00A02008\u00C2\u00A0May;83(5):761\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 6.\u00C2\u00A0Park\u00C2\u00A0JW,\u00C2\u00A0Benz\u00C2\u00A0CC,\u00C2\u00A0Martin\u00C2\u00A0FJ.\u00C2\u00A0Future\u00C2\u00A0directions\u00C2\u00A0of\u00C2\u00A0 liposome\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 immunoliposome\u00E2\u0080\u0090based\u00C2\u00A0cancer\u00C2\u00A0 therapeutics.\u00C2\u00A0Semin\u00C2\u00A0Oncol\u00C2\u00A02004\u00C2\u00A0Dec;31(6\u00C2\u00A0Suppl\u00C2\u00A013):196\u00E2\u0080\u0090205.\u00C2\u00A0 \u00C2\u00A0 7.\u00C2\u00A0Chiu\u00C2\u00A0GN,\u00C2\u00A0Edwards\u00C2\u00A0LA,\u00C2\u00A0Kapanen\u00C2\u00A0AI,\u00C2\u00A0Malinen\u00C2\u00A0MM,\u00C2\u00A0Dragowska\u00C2\u00A0WH,\u00C2\u00A0Warburton\u00C2\u00A0C,\u00C2\u00A0Chikh\u00C2\u00A0GG,\u00C2\u00A0Fang\u00C2\u00A0KY,\u00C2\u00A0 Tan\u00C2\u00A0 S,\u00C2\u00A0 Sy\u00C2\u00A0 J,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0Modulation\u00C2\u00A0 of\u00C2\u00A0 cancer\u00C2\u00A0 cell\u00C2\u00A0 survival\u00C2\u00A0 pathways\u00C2\u00A0 using\u00C2\u00A0multivalent\u00C2\u00A0 liposomal\u00C2\u00A0 therapeutic\u00C2\u00A0antibody\u00C2\u00A0constructs.\u00C2\u00A0Mol\u00C2\u00A0Cancer\u00C2\u00A0Ther\u00C2\u00A02007\u00C2\u00A0Mar;6(3):844\u00E2\u0080\u009055.\u00C2\u00A0 \u00C2\u00A0 8.\u00C2\u00A0Drummond\u00C2\u00A0DC,\u00C2\u00A0Noble\u00C2\u00A0CO,\u00C2\u00A0Hayes\u00C2\u00A0ME,\u00C2\u00A0Park\u00C2\u00A0 JW,\u00C2\u00A0Kirpotin\u00C2\u00A0DB.\u00C2\u00A0Pharmacokinetics\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0vivo\u00C2\u00A0drug\u00C2\u00A0 release\u00C2\u00A0rates\u00C2\u00A0in\u00C2\u00A0liposomal\u00C2\u00A0nanocarrier\u00C2\u00A0development.\u00C2\u00A0J\u00C2\u00A0Pharm\u00C2\u00A0Sci\u00C2\u00A02008\u00C2\u00A0Nov;97(11):4696\u00E2\u0080\u0090740.\u00C2\u00A0 \u00C2\u00A0 9.\u00C2\u00A0 Slingerland\u00C2\u00A0M,\u00C2\u00A0Guchelaar\u00C2\u00A0H,\u00C2\u00A0Gelderblom\u00C2\u00A0H.\u00C2\u00A0 Liposomal\u00C2\u00A0drug\u00C2\u00A0 formulations\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 therapy:\u00C2\u00A015\u00C2\u00A0 years\u00C2\u00A0along\u00C2\u00A0the\u00C2\u00A0road.\u00C2\u00A0Drug\u00C2\u00A0Discov\u00C2\u00A0Today\u00C2\u00A02012\u00C2\u00A0Feb;17(3\u00E2\u0080\u00904):160\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0 10.\u00C2\u00A0Heidel\u00C2\u00A0 JD,\u00C2\u00A0Davis\u00C2\u00A0ME.\u00C2\u00A0Clinical\u00C2\u00A0developments\u00C2\u00A0 in\u00C2\u00A0nanotechnology\u00C2\u00A0 for\u00C2\u00A0 cancer\u00C2\u00A0 therapy.\u00C2\u00A0Pharm\u00C2\u00A0Res\u00C2\u00A0 2011\u00C2\u00A0Feb;28(2):187\u00E2\u0080\u009099.\u00C2\u00A0 \u00C2\u00A0 11.\u00C2\u00A0Park\u00C2\u00A0 JW,\u00C2\u00A0Hong\u00C2\u00A0K,\u00C2\u00A0Kirpotin\u00C2\u00A0DB,\u00C2\u00A0Colbern\u00C2\u00A0G,\u00C2\u00A0Shalaby\u00C2\u00A0R,\u00C2\u00A0Baselga\u00C2\u00A0 J,\u00C2\u00A0Shao\u00C2\u00A0Y,\u00C2\u00A0Nielsen\u00C2\u00A0UB,\u00C2\u00A0Marks\u00C2\u00A0 JD,\u00C2\u00A0 Moore\u00C2\u00A0 D,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Anti\u00E2\u0080\u0090HER2\u00C2\u00A0 immunoliposomes:\u00C2\u00A0 Enhanced\u00C2\u00A0 efficacy\u00C2\u00A0 attributable\u00C2\u00A0 to\u00C2\u00A0 targeted\u00C2\u00A0 delivery.\u00C2\u00A0Clin\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02002\u00C2\u00A0Apr;8(4):1172\u00E2\u0080\u009081.\u00C2\u00A0 \u00C2\u00A0 12.\u00C2\u00A0Mamot\u00C2\u00A0C,\u00C2\u00A0Drummond\u00C2\u00A0DC,\u00C2\u00A0Noble\u00C2\u00A0CO,\u00C2\u00A0Kallab\u00C2\u00A0V,\u00C2\u00A0Guo\u00C2\u00A0Z,\u00C2\u00A0Hong\u00C2\u00A0K,\u00C2\u00A0Kirpotin\u00C2\u00A0DB,\u00C2\u00A0Park\u00C2\u00A0JW.\u00C2\u00A0Epidermal\u00C2\u00A0 growth\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00E2\u0080\u0090targeted\u00C2\u00A0 immunoliposomes\u00C2\u00A0 significantly\u00C2\u00A0 enhance\u00C2\u00A0 the\u00C2\u00A0 efficacy\u00C2\u00A0 of\u00C2\u00A0 multiple\u00C2\u00A0anticancer\u00C2\u00A0drugs\u00C2\u00A0in\u00C2\u00A0vivo.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02005\u00C2\u00A0Dec\u00C2\u00A015;65(24):11631\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 13.\u00C2\u00A0Kirpotin\u00C2\u00A0D,\u00C2\u00A0Park\u00C2\u00A0JW,\u00C2\u00A0Hong\u00C2\u00A0K,\u00C2\u00A0Zalipsky\u00C2\u00A0S,\u00C2\u00A0Li\u00C2\u00A0WL,\u00C2\u00A0Carter\u00C2\u00A0P,\u00C2\u00A0Benz\u00C2\u00A0CC,\u00C2\u00A0Papahadjopoulos\u00C2\u00A0D.\u00C2\u00A0Sterically\u00C2\u00A0 stabilized\u00C2\u00A0anti\u00E2\u0080\u0090HER2\u00C2\u00A0 immunoliposomes:\u00C2\u00A0Design\u00C2\u00A0and\u00C2\u00A0 targeting\u00C2\u00A0 to\u00C2\u00A0human\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 vitro.\u00C2\u00A0Biochemistry\u00C2\u00A01997\u00C2\u00A0Jan\u00C2\u00A07;36(1):66\u00E2\u0080\u009075.\u00C2\u00A0 \u00C2\u00A0 14.\u00C2\u00A0Omelyanenko\u00C2\u00A0V,\u00C2\u00A0Kopeckova\u00C2\u00A0P,\u00C2\u00A0Gentry\u00C2\u00A0C,\u00C2\u00A0Shiah\u00C2\u00A0JG,\u00C2\u00A0Kopecek\u00C2\u00A0J.\u00C2\u00A0HPMA\u00C2\u00A0copolymer\u00E2\u0080\u0090anticancer\u00C2\u00A0drug\u00E2\u0080\u0090 OV\u00E2\u0080\u0090TL16\u00C2\u00A0antibody\u00C2\u00A0conjugates.\u00C2\u00A01.\u00C2\u00A0Influence\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0synthesis\u00C2\u00A0on\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0affinity\u00C2\u00A0to\u00C2\u00A0 OVCAR\u00E2\u0080\u00903\u00C2\u00A0ovarian\u00C2\u00A0carcinoma\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0vitro.\u00C2\u00A0J\u00C2\u00A0Drug\u00C2\u00A0Target\u00C2\u00A01996;3(5):357\u00E2\u0080\u009073.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0175\u00C2\u00A0\u00C2\u00A0 15.\u00C2\u00A0 Johnson\u00C2\u00A0RN,\u00C2\u00A0Kopeckova\u00C2\u00A0P,\u00C2\u00A0Kopecek\u00C2\u00A0 J.\u00C2\u00A0Synthesis\u00C2\u00A0and\u00C2\u00A0evaluation\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0branched\u00C2\u00A0HPMA\u00C2\u00A0 copolymer\u00E2\u0080\u0090fab'\u00C2\u00A0 conjugates\u00C2\u00A0 targeted\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 B\u00E2\u0080\u0090cell\u00C2\u00A0 antigen\u00C2\u00A0 CD20.\u00C2\u00A0 Bioconjug\u00C2\u00A0 Chem\u00C2\u00A0 2009\u00C2\u00A0 Jan;20(1):129\u00E2\u0080\u009037.\u00C2\u00A0 \u00C2\u00A0 16.\u00C2\u00A0Popov\u00C2\u00A0J,\u00C2\u00A0Kapanen\u00C2\u00A0AI,\u00C2\u00A0Turner\u00C2\u00A0C,\u00C2\u00A0Ng\u00C2\u00A0R,\u00C2\u00A0Tucker\u00C2\u00A0C,\u00C2\u00A0Chiu\u00C2\u00A0G,\u00C2\u00A0Klasa\u00C2\u00A0R,\u00C2\u00A0Bally\u00C2\u00A0MB,\u00C2\u00A0Chikh\u00C2\u00A0G.\u00C2\u00A0Multivalent\u00C2\u00A0 rituximab\u00C2\u00A0 lipid\u00C2\u00A0nanoparticles\u00C2\u00A0as\u00C2\u00A0 improved\u00C2\u00A0 lymphoma\u00C2\u00A0therapies:\u00C2\u00A0 Indirect\u00C2\u00A0mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0 and\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0activity.\u00C2\u00A0Nanomedicine\u00C2\u00A02011\u00C2\u00A0Nov;6(9):1575\u00E2\u0080\u009091.\u00C2\u00A0 \u00C2\u00A0 17.\u00C2\u00A0Mufamadi\u00C2\u00A0MS,\u00C2\u00A0Pillay\u00C2\u00A0V,\u00C2\u00A0Choonara\u00C2\u00A0YE,\u00C2\u00A0Du\u00C2\u00A0Toit\u00C2\u00A0LC,\u00C2\u00A0Modi\u00C2\u00A0G,\u00C2\u00A0Naidoo\u00C2\u00A0D,\u00C2\u00A0Ndesendo\u00C2\u00A0VMK.\u00C2\u00A0A\u00C2\u00A0review\u00C2\u00A0on\u00C2\u00A0 composite\u00C2\u00A0 liposomal\u00C2\u00A0 technologies\u00C2\u00A0 for\u00C2\u00A0 specialized\u00C2\u00A0 drug\u00C2\u00A0 delivery.\u00C2\u00A0 J\u00C2\u00A0 Drug\u00C2\u00A0 Deliv\u00C2\u00A0 2011\u00C2\u00A0 Feb\u00C2\u00A0 8;2011(Article\u00C2\u00A0ID\u00C2\u00A0939851):19\u00C2\u00A0pgs.\u00C2\u00A0 \u00C2\u00A0 18.\u00C2\u00A0 Lian\u00C2\u00A0T,\u00C2\u00A0Ho\u00C2\u00A0RJ.\u00C2\u00A0Trends\u00C2\u00A0and\u00C2\u00A0developments\u00C2\u00A0 in\u00C2\u00A0 liposome\u00C2\u00A0drug\u00C2\u00A0delivery\u00C2\u00A0 systems.\u00C2\u00A0 J\u00C2\u00A0Pharm\u00C2\u00A0Sci\u00C2\u00A02001\u00C2\u00A0 Jun;90(6):667\u00E2\u0080\u009080.\u00C2\u00A0 \u00C2\u00A0 19.\u00C2\u00A0Chonn\u00C2\u00A0A,\u00C2\u00A0Semple\u00C2\u00A0SC,\u00C2\u00A0Cullis\u00C2\u00A0PR.\u00C2\u00A0Association\u00C2\u00A0of\u00C2\u00A0blood\u00C2\u00A0proteins\u00C2\u00A0with\u00C2\u00A0large\u00C2\u00A0unilamellar\u00C2\u00A0liposomes\u00C2\u00A0in\u00C2\u00A0 vivo.\u00C2\u00A0Relation\u00C2\u00A0to\u00C2\u00A0circulation\u00C2\u00A0lifetimes.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A01992\u00C2\u00A0Sep\u00C2\u00A015;267(26):18759\u00E2\u0080\u009065.\u00C2\u00A0 \u00C2\u00A0 20.\u00C2\u00A0Strebhardt\u00C2\u00A0K,\u00C2\u00A0Ullrich\u00C2\u00A0A.\u00C2\u00A0Paul\u00C2\u00A0ehrlich's\u00C2\u00A0magic\u00C2\u00A0bullet\u00C2\u00A0concept:\u00C2\u00A0100\u00C2\u00A0years\u00C2\u00A0of\u00C2\u00A0progress.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Cancer\u00C2\u00A0 2008\u00C2\u00A0Jun;8(6):473\u00E2\u0080\u009080.\u00C2\u00A0 \u00C2\u00A0 21.\u00C2\u00A0 Kohler\u00C2\u00A0 G,\u00C2\u00A0 Milstein\u00C2\u00A0 C.\u00C2\u00A0 Continuous\u00C2\u00A0 cultures\u00C2\u00A0 of\u00C2\u00A0 fused\u00C2\u00A0 cells\u00C2\u00A0 secreting\u00C2\u00A0 antibody\u00C2\u00A0 of\u00C2\u00A0 predefined\u00C2\u00A0 specificity.\u00C2\u00A0Nature\u00C2\u00A01975\u00C2\u00A0Aug\u00C2\u00A07;256(5517):495\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 22.\u00C2\u00A0 Boulianne\u00C2\u00A0 GL,\u00C2\u00A0 Hozumi\u00C2\u00A0 N,\u00C2\u00A0 Shulman\u00C2\u00A0 MJ.\u00C2\u00A0 Production\u00C2\u00A0 of\u00C2\u00A0 functional\u00C2\u00A0 chimaeric\u00C2\u00A0 mouse/human\u00C2\u00A0 antibody.\u00C2\u00A0Nature\u00C2\u00A01984\u00C2\u00A0Dec\u00C2\u00A013\u00E2\u0080\u009019;312(5995):643\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0 23.\u00C2\u00A0Riechmann\u00C2\u00A0L,\u00C2\u00A0Clark\u00C2\u00A0M,\u00C2\u00A0Waldmann\u00C2\u00A0H,\u00C2\u00A0Winter\u00C2\u00A0G.\u00C2\u00A0Reshaping\u00C2\u00A0human\u00C2\u00A0antibodies\u00C2\u00A0for\u00C2\u00A0therapy.\u00C2\u00A0Nature\u00C2\u00A0 1988\u00C2\u00A0Mar\u00C2\u00A024;332(6162):323\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 24.\u00C2\u00A0Weiner\u00C2\u00A0LM,\u00C2\u00A0Belldegrun\u00C2\u00A0AS,\u00C2\u00A0Crawford\u00C2\u00A0 J,\u00C2\u00A0Tolcher\u00C2\u00A0AW,\u00C2\u00A0Lockbaum\u00C2\u00A0P,\u00C2\u00A0Arends\u00C2\u00A0RH,\u00C2\u00A0Navale\u00C2\u00A0L,\u00C2\u00A0Amado\u00C2\u00A0 RG,\u00C2\u00A0Schwab\u00C2\u00A0G,\u00C2\u00A0Figlin\u00C2\u00A0RA.\u00C2\u00A0Dose\u00C2\u00A0and\u00C2\u00A0schedule\u00C2\u00A0study\u00C2\u00A0of\u00C2\u00A0panitumumab\u00C2\u00A0monotherapy\u00C2\u00A0 in\u00C2\u00A0patients\u00C2\u00A0 with\u00C2\u00A0advanced\u00C2\u00A0solid\u00C2\u00A0malignancies.\u00C2\u00A0Clin\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02008\u00C2\u00A0Jan\u00C2\u00A015;14(2):502\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 25.\u00C2\u00A0Hagenbeek\u00C2\u00A0A,\u00C2\u00A0Gadeberg\u00C2\u00A0O,\u00C2\u00A0 Johnson\u00C2\u00A0 P,\u00C2\u00A0M\u00C3\u00B8ller\u00C2\u00A0 Pedersen\u00C2\u00A0 L,\u00C2\u00A0Walewski\u00C2\u00A0 J,\u00C2\u00A0Hellmann\u00C2\u00A0A,\u00C2\u00A0 Link\u00C2\u00A0 BK,\u00C2\u00A0 Robak\u00C2\u00A0T,\u00C2\u00A0Wojtukiewicz\u00C2\u00A0M,\u00C2\u00A0Pfreundschuh\u00C2\u00A0M,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0First\u00C2\u00A0clinical\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0ofatumumab,\u00C2\u00A0a\u00C2\u00A0novel\u00C2\u00A0fully\u00C2\u00A0 human\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0in\u00C2\u00A0relapsed\u00C2\u00A0or\u00C2\u00A0refractory\u00C2\u00A0follicular\u00C2\u00A0lymphoma:\u00C2\u00A0Results\u00C2\u00A0of\u00C2\u00A0 a\u00C2\u00A0phase\u00C2\u00A01/2\u00C2\u00A0trial.\u00C2\u00A0Blood\u00C2\u00A02008\u00C2\u00A0June\u00C2\u00A015;111(12):5486\u00E2\u0080\u009095.\u00C2\u00A0 \u00C2\u00A0 26.\u00C2\u00A0Burtrum\u00C2\u00A0D,\u00C2\u00A0Zhu\u00C2\u00A0Z,\u00C2\u00A0Lu\u00C2\u00A0D,\u00C2\u00A0Anderson\u00C2\u00A0DM,\u00C2\u00A0Prewett\u00C2\u00A0M,\u00C2\u00A0Pereira\u00C2\u00A0DS,\u00C2\u00A0Bassi\u00C2\u00A0R,\u00C2\u00A0Abdullah\u00C2\u00A0R,\u00C2\u00A0Hooper\u00C2\u00A0AT,\u00C2\u00A0 Koo\u00C2\u00A0H,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0A\u00C2\u00A0 fully\u00C2\u00A0human\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0 to\u00C2\u00A0 the\u00C2\u00A0 insulin\u00E2\u0080\u0090like\u00C2\u00A0growth\u00C2\u00A0 factor\u00C2\u00A0 I\u00C2\u00A0 receptor\u00C2\u00A0 blocks\u00C2\u00A0 ligand\u00E2\u0080\u0090dependent\u00C2\u00A0signaling\u00C2\u00A0and\u00C2\u00A0 inhibits\u00C2\u00A0human\u00C2\u00A0tumor\u00C2\u00A0growth\u00C2\u00A0 in\u00C2\u00A0vivo.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02003\u00C2\u00A0 Dec\u00C2\u00A015;63(24):8912\u00E2\u0080\u009021.\u00C2\u00A0 \u00C2\u00A0 27.\u00C2\u00A0Coiffier\u00C2\u00A0B,\u00C2\u00A0 Lepage\u00C2\u00A0E,\u00C2\u00A0Briere\u00C2\u00A0 J,\u00C2\u00A0Herbrecht\u00C2\u00A0R,\u00C2\u00A0Tilly\u00C2\u00A0H,\u00C2\u00A0Bouabdallah\u00C2\u00A0R,\u00C2\u00A0Morel\u00C2\u00A0P,\u00C2\u00A0Van\u00C2\u00A0Den\u00C2\u00A0Neste\u00C2\u00A0E,\u00C2\u00A0 Salles\u00C2\u00A0G,\u00C2\u00A0Gaulard\u00C2\u00A0P,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0CHOP\u00C2\u00A0chemotherapy\u00C2\u00A0plus\u00C2\u00A0 rituximab\u00C2\u00A0compared\u00C2\u00A0with\u00C2\u00A0CHOP\u00C2\u00A0alone\u00C2\u00A0 in\u00C2\u00A0 elderly\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0diffuse\u00C2\u00A0large\u00E2\u0080\u0090B\u00E2\u0080\u0090cell\u00C2\u00A0lymphoma.\u00C2\u00A0N\u00C2\u00A0Engl\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A02002\u00C2\u00A0Jan\u00C2\u00A024;346(4):235\u00E2\u0080\u009042.\u00C2\u00A0 \u00C2\u00A0 28.\u00C2\u00A0McLaughlin\u00C2\u00A0P,\u00C2\u00A0Grillo\u00E2\u0080\u0090Lopez\u00C2\u00A0AJ,\u00C2\u00A0Link\u00C2\u00A0BK,\u00C2\u00A0Levy\u00C2\u00A0R,\u00C2\u00A0Czuczman\u00C2\u00A0MS,\u00C2\u00A0Williams\u00C2\u00A0ME,\u00C2\u00A0Heyman\u00C2\u00A0MR,\u00C2\u00A0Bence\u00E2\u0080\u0090 Bruckler\u00C2\u00A0 I,\u00C2\u00A0White\u00C2\u00A0 CA,\u00C2\u00A0 Cabanillas\u00C2\u00A0 F,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0Rituximab\u00C2\u00A0 chimeric\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0monoclonal\u00C2\u00A0 antibody\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0176\u00C2\u00A0\u00C2\u00A0 therapy\u00C2\u00A0 for\u00C2\u00A0 relapsed\u00C2\u00A0 indolent\u00C2\u00A0 lymphoma:\u00C2\u00A0Half\u00C2\u00A0of\u00C2\u00A0patients\u00C2\u00A0 respond\u00C2\u00A0 to\u00C2\u00A0 a\u00C2\u00A0 four\u00E2\u0080\u0090dose\u00C2\u00A0 treatment\u00C2\u00A0 program.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A01998\u00C2\u00A0Aug;16(8):2825\u00E2\u0080\u009033.\u00C2\u00A0 \u00C2\u00A0 29.\u00C2\u00A0 Leavy\u00C2\u00A0 O.\u00C2\u00A0 Therapeutic\u00C2\u00A0 antibodies:\u00C2\u00A0 Past,\u00C2\u00A0 present\u00C2\u00A0 and\u00C2\u00A0 future.\u00C2\u00A0 Nat\u00C2\u00A0 Rev\u00C2\u00A0 Immunol\u00C2\u00A0 2010\u00C2\u00A0 May;10(5):297.\u00C2\u00A0 \u00C2\u00A0 30.\u00C2\u00A0\u00C2\u00A0Aggarwal\u00C2\u00A0S.\u00C2\u00A0What's\u00C2\u00A0fueling\u00C2\u00A0the\u00C2\u00A0biotech\u00C2\u00A0engine\u00E2\u0080\u0090\u00E2\u0080\u00902008.\u00C2\u00A0Nat\u00C2\u00A0Biotechnol\u00C2\u00A02009\u00C2\u00A0Nov;27(11):987\u00E2\u0080\u009093.\u00C2\u00A0 \u00C2\u00A0 31.\u00C2\u00A0 Beck\u00C2\u00A0 A,\u00C2\u00A0Wurch\u00C2\u00A0 T,\u00C2\u00A0 Bailly\u00C2\u00A0 C,\u00C2\u00A0 Corvaia\u00C2\u00A0 N.\u00C2\u00A0 Strategies\u00C2\u00A0 and\u00C2\u00A0 challenges\u00C2\u00A0 for\u00C2\u00A0 the\u00C2\u00A0 next\u00C2\u00A0 generation\u00C2\u00A0 of\u00C2\u00A0 therapeutic\u00C2\u00A0antibodies.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Immunol\u00C2\u00A02010\u00C2\u00A0May;10(5):345\u00E2\u0080\u009052.\u00C2\u00A0 \u00C2\u00A0 32.\u00C2\u00A0Cobleigh\u00C2\u00A0MA,\u00C2\u00A0Vogel\u00C2\u00A0CL,\u00C2\u00A0Tripathy\u00C2\u00A0D,\u00C2\u00A0Robert\u00C2\u00A0NJ,\u00C2\u00A0Scholl\u00C2\u00A0S,\u00C2\u00A0Fehrenbacher\u00C2\u00A0L,\u00C2\u00A0Wolter\u00C2\u00A0JM,\u00C2\u00A0Paton\u00C2\u00A0V,\u00C2\u00A0Shak\u00C2\u00A0 S,\u00C2\u00A0Lieberman\u00C2\u00A0G,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Multinational\u00C2\u00A0 study\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0efficacy\u00C2\u00A0and\u00C2\u00A0 safety\u00C2\u00A0of\u00C2\u00A0humanized\u00C2\u00A0anti\u00E2\u0080\u0090HER2\u00C2\u00A0 monoclonal\u00C2\u00A0antibody\u00C2\u00A0 in\u00C2\u00A0women\u00C2\u00A0who\u00C2\u00A0have\u00C2\u00A0HER2\u00E2\u0080\u0090overexpressing\u00C2\u00A0metastatic\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0that\u00C2\u00A0 has\u00C2\u00A0progressed\u00C2\u00A0after\u00C2\u00A0chemotherapy\u00C2\u00A0 for\u00C2\u00A0metastatic\u00C2\u00A0disease.\u00C2\u00A0 J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A01999\u00C2\u00A0Sep;17(9):2639\u00E2\u0080\u0090 48.\u00C2\u00A0 \u00C2\u00A0 33.\u00C2\u00A0Slamon\u00C2\u00A0DJ,\u00C2\u00A0Leyland\u00E2\u0080\u0090Jones\u00C2\u00A0B,\u00C2\u00A0Shak\u00C2\u00A0S,\u00C2\u00A0Fuchs\u00C2\u00A0H,\u00C2\u00A0Paton\u00C2\u00A0V,\u00C2\u00A0Bajamonde\u00C2\u00A0A,\u00C2\u00A0Fleming\u00C2\u00A0T,\u00C2\u00A0Eiermann\u00C2\u00A0W,\u00C2\u00A0 Wolter\u00C2\u00A0J,\u00C2\u00A0Pegram\u00C2\u00A0M,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Use\u00C2\u00A0of\u00C2\u00A0chemotherapy\u00C2\u00A0plus\u00C2\u00A0a\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0against\u00C2\u00A0HER2\u00C2\u00A0for\u00C2\u00A0 metastatic\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0that\u00C2\u00A0overexpresses\u00C2\u00A0HER2.\u00C2\u00A0N\u00C2\u00A0Engl\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A02001\u00C2\u00A0Mar\u00C2\u00A015;344(11):783\u00E2\u0080\u009092.\u00C2\u00A0 \u00C2\u00A0 34.\u00C2\u00A0Lundin\u00C2\u00A0J,\u00C2\u00A0Kimby\u00C2\u00A0E,\u00C2\u00A0Bjorkholm\u00C2\u00A0M,\u00C2\u00A0Broliden\u00C2\u00A0PA,\u00C2\u00A0Celsing\u00C2\u00A0F,\u00C2\u00A0Hjalmar\u00C2\u00A0V,\u00C2\u00A0Mollgard\u00C2\u00A0L,\u00C2\u00A0Rebello\u00C2\u00A0P,\u00C2\u00A0Hale\u00C2\u00A0G,\u00C2\u00A0 Waldmann\u00C2\u00A0H,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Phase\u00C2\u00A0II\u00C2\u00A0trial\u00C2\u00A0of\u00C2\u00A0subcutaneous\u00C2\u00A0anti\u00E2\u0080\u0090CD52\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0alemtuzumab\u00C2\u00A0 (campath\u00E2\u0080\u00901H)\u00C2\u00A0as\u00C2\u00A0 first\u00E2\u0080\u0090line\u00C2\u00A0 treatment\u00C2\u00A0 for\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0chronic\u00C2\u00A0 lymphocytic\u00C2\u00A0 leukemia\u00C2\u00A0 (B\u00E2\u0080\u0090 CLL).\u00C2\u00A0Blood\u00C2\u00A02002\u00C2\u00A0Aug\u00C2\u00A01;100(3):768\u00E2\u0080\u009073.\u00C2\u00A0 \u00C2\u00A0 35.\u00C2\u00A0Van\u00C2\u00A0Cutsem\u00C2\u00A0E,\u00C2\u00A0Kohne\u00C2\u00A0CH,\u00C2\u00A0Hitre\u00C2\u00A0E,\u00C2\u00A0Zaluski\u00C2\u00A0J,\u00C2\u00A0Chang\u00C2\u00A0Chien\u00C2\u00A0CR,\u00C2\u00A0Makhson\u00C2\u00A0A,\u00C2\u00A0D'Haens\u00C2\u00A0G,\u00C2\u00A0Pinter\u00C2\u00A0T,\u00C2\u00A0Lim\u00C2\u00A0 R,\u00C2\u00A0Bodoky\u00C2\u00A0G,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Cetuximab\u00C2\u00A0and\u00C2\u00A0chemotherapy\u00C2\u00A0as\u00C2\u00A0 initial\u00C2\u00A0treatment\u00C2\u00A0for\u00C2\u00A0metastatic\u00C2\u00A0colorectal\u00C2\u00A0 cancer.\u00C2\u00A0N\u00C2\u00A0Engl\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A02009\u00C2\u00A0Apr\u00C2\u00A02;360(14):1408\u00E2\u0080\u009017.\u00C2\u00A0 \u00C2\u00A0 36.\u00C2\u00A0Hurwitz\u00C2\u00A0H,\u00C2\u00A0Fehrenbacher\u00C2\u00A0L,\u00C2\u00A0Novotny\u00C2\u00A0W,\u00C2\u00A0Cartwright\u00C2\u00A0T,\u00C2\u00A0Hainsworth\u00C2\u00A0J,\u00C2\u00A0Heim\u00C2\u00A0W,\u00C2\u00A0Berlin\u00C2\u00A0J,\u00C2\u00A0Baron\u00C2\u00A0A,\u00C2\u00A0 Griffing\u00C2\u00A0 S,\u00C2\u00A0 Holmgren\u00C2\u00A0 E,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Bevacizumab\u00C2\u00A0 plus\u00C2\u00A0 irinotecan,\u00C2\u00A0 fluorouracil,\u00C2\u00A0 and\u00C2\u00A0 leucovorin\u00C2\u00A0 for\u00C2\u00A0 metastatic\u00C2\u00A0colorectal\u00C2\u00A0cancer.\u00C2\u00A0N\u00C2\u00A0Engl\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A02004\u00C2\u00A0Jun\u00C2\u00A03;350(23):2335\u00E2\u0080\u009042.\u00C2\u00A0 \u00C2\u00A0 37.\u00C2\u00A0Wierda\u00C2\u00A0WG,\u00C2\u00A0Kipps\u00C2\u00A0TJ,\u00C2\u00A0Mayer\u00C2\u00A0 J,\u00C2\u00A0Stilgenbauer\u00C2\u00A0S,\u00C2\u00A0Williams\u00C2\u00A0CD,\u00C2\u00A0Hellmann\u00C2\u00A0A,\u00C2\u00A0Robak\u00C2\u00A0T,\u00C2\u00A0Furman\u00C2\u00A0RR,\u00C2\u00A0 Hillmen\u00C2\u00A0P,\u00C2\u00A0Trneny\u00C2\u00A0M,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Ofatumumab\u00C2\u00A0as\u00C2\u00A0single\u00E2\u0080\u0090agent\u00C2\u00A0CD20\u00C2\u00A0 immunotherapy\u00C2\u00A0 in\u00C2\u00A0fludarabine\u00E2\u0080\u0090 refractory\u00C2\u00A0chronic\u00C2\u00A0lymphocytic\u00C2\u00A0leukemia.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A02010\u00C2\u00A0Apr\u00C2\u00A01;28(10):1749\u00E2\u0080\u009055.\u00C2\u00A0 \u00C2\u00A0 38.\u00C2\u00A0Baselga\u00C2\u00A0 J,\u00C2\u00A0Gelmon\u00C2\u00A0KA,\u00C2\u00A0Verma\u00C2\u00A0S,\u00C2\u00A0Wardley\u00C2\u00A0A,\u00C2\u00A0Conte\u00C2\u00A0P,\u00C2\u00A0Miles\u00C2\u00A0D,\u00C2\u00A0Bianchi\u00C2\u00A0G,\u00C2\u00A0Cortes\u00C2\u00A0 J,\u00C2\u00A0McNally\u00C2\u00A0VA,\u00C2\u00A0 Ross\u00C2\u00A0GA,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Phase\u00C2\u00A0II\u00C2\u00A0trial\u00C2\u00A0of\u00C2\u00A0pertuzumab\u00C2\u00A0and\u00C2\u00A0trastuzumab\u00C2\u00A0in\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0human\u00C2\u00A0epidermal\u00C2\u00A0 growth\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 2\u00E2\u0080\u0093Positive\u00C2\u00A0 metastatic\u00C2\u00A0 breast\u00C2\u00A0 cancer\u00C2\u00A0 that\u00C2\u00A0 progressed\u00C2\u00A0 during\u00C2\u00A0 prior\u00C2\u00A0 trastuzumab\u00C2\u00A0therapy.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A02010\u00C2\u00A0Mar\u00C2\u00A01;28(7):1138\u00E2\u0080\u009044.\u00C2\u00A0 \u00C2\u00A0 39.\u00C2\u00A0Baselga\u00C2\u00A0 J,\u00C2\u00A0 Swain\u00C2\u00A0 SM.\u00C2\u00A0CLEOPATRA:\u00C2\u00A0A\u00C2\u00A0phase\u00C2\u00A0 III\u00C2\u00A0 evaluation\u00C2\u00A0of\u00C2\u00A0pertuzumab\u00C2\u00A0 and\u00C2\u00A0 trastuzumab\u00C2\u00A0 for\u00C2\u00A0 HER2\u00E2\u0080\u0090positive\u00C2\u00A0metastatic\u00C2\u00A0breast\u00C2\u00A0cancer.\u00C2\u00A0Clin\u00C2\u00A0Breast\u00C2\u00A0Cancer\u00C2\u00A02010\u00C2\u00A0Dec\u00C2\u00A01;10(6):489\u00E2\u0080\u009091.\u00C2\u00A0 \u00C2\u00A0 40.\u00C2\u00A0Goldenberg\u00C2\u00A0DM,\u00C2\u00A0Morschhauser\u00C2\u00A0F,\u00C2\u00A0Wegener\u00C2\u00A0WA.\u00C2\u00A0Veltuzumab\u00C2\u00A0(humanized\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0monoclonal\u00C2\u00A0 antibody):\u00C2\u00A0Characterization,\u00C2\u00A0current\u00C2\u00A0clinical\u00C2\u00A0results,\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0prospects.\u00C2\u00A0Leuk\u00C2\u00A0Lymphoma\u00C2\u00A02010\u00C2\u00A0 May;51(5):747\u00E2\u0080\u009055.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0177\u00C2\u00A0\u00C2\u00A0 41.\u00C2\u00A0Morschhauser\u00C2\u00A0F,\u00C2\u00A0Leonard\u00C2\u00A0JP,\u00C2\u00A0Fayad\u00C2\u00A0L,\u00C2\u00A0Coiffier\u00C2\u00A0B,\u00C2\u00A0Petillon\u00C2\u00A0MO,\u00C2\u00A0Coleman\u00C2\u00A0M,\u00C2\u00A0Schuster\u00C2\u00A0SJ,\u00C2\u00A0Dyer\u00C2\u00A0MJ,\u00C2\u00A0 Horne\u00C2\u00A0H,\u00C2\u00A0 Teoh\u00C2\u00A0N,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0Humanized\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 antibody,\u00C2\u00A0 veltuzumab,\u00C2\u00A0 in\u00C2\u00A0 refractory/recurrent\u00C2\u00A0 non\u00E2\u0080\u0090hodgkin's\u00C2\u00A0lymphoma:\u00C2\u00A0Phase\u00C2\u00A0I/II\u00C2\u00A0results.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A02009\u00C2\u00A0Jul\u00C2\u00A010;27(20):3346\u00E2\u0080\u009053.\u00C2\u00A0 \u00C2\u00A0 42.\u00C2\u00A0Clark\u00C2\u00A0MR.\u00C2\u00A0IgG\u00C2\u00A0effector\u00C2\u00A0mechanisms.\u00C2\u00A0Chem\u00C2\u00A0Immunol\u00C2\u00A01997;65:88\u00E2\u0080\u0090110.\u00C2\u00A0 \u00C2\u00A0 43.\u00C2\u00A0Natsume\u00C2\u00A0A,\u00C2\u00A0Niwa\u00C2\u00A0R,\u00C2\u00A0Satoh\u00C2\u00A0M.\u00C2\u00A0 Improving\u00C2\u00A0effector\u00C2\u00A0functions\u00C2\u00A0of\u00C2\u00A0antibodies\u00C2\u00A0for\u00C2\u00A0cancer\u00C2\u00A0treatment:\u00C2\u00A0 Enhancing\u00C2\u00A0ADCC\u00C2\u00A0and\u00C2\u00A0CDC.\u00C2\u00A0Drug\u00C2\u00A0Des\u00C2\u00A0Devel\u00C2\u00A0Ther\u00C2\u00A02009\u00C2\u00A0Sep\u00C2\u00A021;3:7\u00E2\u0080\u009016.\u00C2\u00A0 \u00C2\u00A0 44.\u00C2\u00A0 Lim\u00C2\u00A0 SH,\u00C2\u00A0 Beers\u00C2\u00A0 SA,\u00C2\u00A0 French\u00C2\u00A0 RR,\u00C2\u00A0 Johnson\u00C2\u00A0 PW,\u00C2\u00A0 Glennie\u00C2\u00A0 MJ,\u00C2\u00A0 Cragg\u00C2\u00A0 MS.\u00C2\u00A0 Anti\u00E2\u0080\u0090CD20\u00C2\u00A0 monoclonal\u00C2\u00A0 antibodies:\u00C2\u00A0Historical\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0perspectives.\u00C2\u00A0Haematologica\u00C2\u00A02010\u00C2\u00A0Jan;95(1):135\u00E2\u0080\u009043.\u00C2\u00A0 \u00C2\u00A0 45.\u00C2\u00A0Deans\u00C2\u00A0JP,\u00C2\u00A0Li\u00C2\u00A0H,\u00C2\u00A0Polyak\u00C2\u00A0MJ.\u00C2\u00A0CD20\u00E2\u0080\u0090mediated\u00C2\u00A0apoptosis:\u00C2\u00A0Signalling\u00C2\u00A0through\u00C2\u00A0 lipid\u00C2\u00A0rafts.\u00C2\u00A0Immunology\u00C2\u00A0 2002\u00C2\u00A0Oct;107(2):176\u00E2\u0080\u009082.\u00C2\u00A0 \u00C2\u00A0 46.\u00C2\u00A0Golay\u00C2\u00A0 J,\u00C2\u00A0 Zaffaroni\u00C2\u00A0 L,\u00C2\u00A0 Vaccari\u00C2\u00A0 T,\u00C2\u00A0 Lazzari\u00C2\u00A0M,\u00C2\u00A0 Borleri\u00C2\u00A0GM,\u00C2\u00A0 Bernasconi\u00C2\u00A0 S,\u00C2\u00A0 Tedesco\u00C2\u00A0 F,\u00C2\u00A0 Rambaldi\u00C2\u00A0 A,\u00C2\u00A0 Introna\u00C2\u00A0M.\u00C2\u00A0Biologic\u00C2\u00A0response\u00C2\u00A0of\u00C2\u00A0B\u00C2\u00A0lymphoma\u00C2\u00A0cells\u00C2\u00A0to\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0rituximab\u00C2\u00A0 in\u00C2\u00A0 vitro:\u00C2\u00A0 CD55\u00C2\u00A0 and\u00C2\u00A0 CD59\u00C2\u00A0 regulate\u00C2\u00A0 complement\u00E2\u0080\u0090mediated\u00C2\u00A0 cell\u00C2\u00A0 lysis.\u00C2\u00A0 Blood\u00C2\u00A0 2000\u00C2\u00A0 Jun\u00C2\u00A0 15;95(12):3900\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 47.\u00C2\u00A0Polyak\u00C2\u00A0MJ,\u00C2\u00A0Li\u00C2\u00A0H,\u00C2\u00A0Shariat\u00C2\u00A0N,\u00C2\u00A0Deans\u00C2\u00A0 JP.\u00C2\u00A0CD20\u00C2\u00A0homo\u00E2\u0080\u0090oligomers\u00C2\u00A0physically\u00C2\u00A0associate\u00C2\u00A0with\u00C2\u00A0the\u00C2\u00A0B\u00C2\u00A0cell\u00C2\u00A0 antigen\u00C2\u00A0receptor:\u00C2\u00A0Dissociation\u00C2\u00A0upon\u00C2\u00A0receptor\u00C2\u00A0engagement\u00C2\u00A0and\u00C2\u00A0recruitment\u00C2\u00A0of\u00C2\u00A0phosphoproteins\u00C2\u00A0 and\u00C2\u00A0calmodulin\u00E2\u0080\u0090binding\u00C2\u00A0proteins.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A02008\u00C2\u00A0Jul\u00C2\u00A04;283(27):18545\u00E2\u0080\u009052.\u00C2\u00A0 \u00C2\u00A0 48.\u00C2\u00A0Jefferis\u00C2\u00A0R,\u00C2\u00A0Lund\u00C2\u00A0J.\u00C2\u00A0Interaction\u00C2\u00A0sites\u00C2\u00A0on\u00C2\u00A0human\u00C2\u00A0IgG\u00E2\u0080\u0090fc\u00C2\u00A0for\u00C2\u00A0Fc\u00CE\u00B3R:\u00C2\u00A0Current\u00C2\u00A0models.\u00C2\u00A0Immunol\u00C2\u00A0Lett\u00C2\u00A02002\u00C2\u00A0 Jun\u00C2\u00A03;82(1\u00E2\u0080\u00902):57\u00E2\u0080\u009065.\u00C2\u00A0 \u00C2\u00A0 49.\u00C2\u00A0 Jazirehi\u00C2\u00A0 AR,\u00C2\u00A0 Bonavida\u00C2\u00A0 B.\u00C2\u00A0 Cellular\u00C2\u00A0 and\u00C2\u00A0molecular\u00C2\u00A0 signal\u00C2\u00A0 transduction\u00C2\u00A0 pathways\u00C2\u00A0modulated\u00C2\u00A0 by\u00C2\u00A0 rituximab\u00C2\u00A0 (rituxan,\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 mAb)\u00C2\u00A0 in\u00C2\u00A0 non\u00E2\u0080\u0090hodgkin's\u00C2\u00A0 lymphoma:\u00C2\u00A0 Implications\u00C2\u00A0 in\u00C2\u00A0 chemosensitization\u00C2\u00A0and\u00C2\u00A0therapeutic\u00C2\u00A0intervention.\u00C2\u00A0Oncogene\u00C2\u00A02005\u00C2\u00A0Mar\u00C2\u00A024;24(13):2121\u00E2\u0080\u009043.\u00C2\u00A0 \u00C2\u00A0 50.\u00C2\u00A0Cartron\u00C2\u00A0G,\u00C2\u00A0Watier\u00C2\u00A0H,\u00C2\u00A0Golay\u00C2\u00A0J,\u00C2\u00A0Solal\u00E2\u0080\u0090Celigny\u00C2\u00A0P.\u00C2\u00A0From\u00C2\u00A0the\u00C2\u00A0bench\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0bedside:\u00C2\u00A0Ways\u00C2\u00A0to\u00C2\u00A0 improve\u00C2\u00A0 rituximab\u00C2\u00A0efficacy.\u00C2\u00A0Blood\u00C2\u00A02004\u00C2\u00A0Nov\u00C2\u00A01;104(9):2635\u00E2\u0080\u009042.\u00C2\u00A0 \u00C2\u00A0 51.\u00C2\u00A0 Stolz\u00C2\u00A0 C,\u00C2\u00A0 Schuler\u00C2\u00A0 M.\u00C2\u00A0 Molecular\u00C2\u00A0 mechanisms\u00C2\u00A0 of\u00C2\u00A0 resistance\u00C2\u00A0 to\u00C2\u00A0 rituximab\u00C2\u00A0 and\u00C2\u00A0 pharmacologic\u00C2\u00A0 strategies\u00C2\u00A0for\u00C2\u00A0its\u00C2\u00A0circumvention.\u00C2\u00A0Leuk\u00C2\u00A0Lymphoma\u00C2\u00A02009\u00C2\u00A0Jun;50(6):873\u00E2\u0080\u009085.\u00C2\u00A0 \u00C2\u00A0 52.\u00C2\u00A0Walport\u00C2\u00A0MJ.\u00C2\u00A0Complement.\u00C2\u00A0N\u00C2\u00A0Engl\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A02001\u00C2\u00A0Apr\u00C2\u00A05;344(14):1058\u00E2\u0080\u009066.\u00C2\u00A0 \u00C2\u00A0 53.\u00C2\u00A0Zhou\u00C2\u00A0X,\u00C2\u00A0Hu\u00C2\u00A0W,\u00C2\u00A0Qin\u00C2\u00A0X.\u00C2\u00A0The\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0complement\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0rituximab\u00C2\u00A0for\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0 lymphoma:\u00C2\u00A0Implications\u00C2\u00A0for\u00C2\u00A0therapy.\u00C2\u00A0The\u00C2\u00A0Oncologist\u00C2\u00A02008\u00C2\u00A0September\u00C2\u00A001;13(9):954\u00E2\u0080\u009066.\u00C2\u00A0 \u00C2\u00A0 54.\u00C2\u00A0Wang\u00C2\u00A0SY,\u00C2\u00A0Weiner\u00C2\u00A0G.\u00C2\u00A0Complement\u00C2\u00A0and\u00C2\u00A0cellular\u00C2\u00A0cytotoxicity\u00C2\u00A0in\u00C2\u00A0antibody\u00C2\u00A0therapy\u00C2\u00A0of\u00C2\u00A0cancer.\u00C2\u00A0Expert\u00C2\u00A0 Opin\u00C2\u00A0Biol\u00C2\u00A0Ther\u00C2\u00A02008\u00C2\u00A0Jun;8(6):759\u00E2\u0080\u009068.\u00C2\u00A0 \u00C2\u00A0 55.\u00C2\u00A0 Dall'Acqua\u00C2\u00A0WF,\u00C2\u00A0 Cook\u00C2\u00A0 KE,\u00C2\u00A0 Damschroder\u00C2\u00A0MM,\u00C2\u00A0Woods\u00C2\u00A0 RM,\u00C2\u00A0Wu\u00C2\u00A0 H.\u00C2\u00A0Modulation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 effector\u00C2\u00A0 functions\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 human\u00C2\u00A0 IgG1\u00C2\u00A0 through\u00C2\u00A0 engineering\u00C2\u00A0 of\u00C2\u00A0 its\u00C2\u00A0 hinge\u00C2\u00A0 region.\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A0 2006\u00C2\u00A0 Jul\u00C2\u00A0 15;177(2):1129\u00E2\u0080\u009038.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0178\u00C2\u00A0\u00C2\u00A0 56.\u00C2\u00A0 Idusogie\u00C2\u00A0 EE,\u00C2\u00A0 Wong\u00C2\u00A0 PY,\u00C2\u00A0 Presta\u00C2\u00A0 LG,\u00C2\u00A0 Gazzano\u00E2\u0080\u0090Santoro\u00C2\u00A0 H,\u00C2\u00A0 Totpal\u00C2\u00A0 K,\u00C2\u00A0 Ultsch\u00C2\u00A0 M,\u00C2\u00A0 Mulkerrin\u00C2\u00A0 MG.\u00C2\u00A0 Engineered\u00C2\u00A0 antibodies\u00C2\u00A0 with\u00C2\u00A0 increased\u00C2\u00A0 activity\u00C2\u00A0 to\u00C2\u00A0 recruit\u00C2\u00A0 complement.\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A0 2001\u00C2\u00A0 Feb\u00C2\u00A0 15;166(4):2571\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 57.\u00C2\u00A0Natsume\u00C2\u00A0A,\u00C2\u00A0In\u00C2\u00A0M,\u00C2\u00A0Takamura\u00C2\u00A0H,\u00C2\u00A0Nakagawa\u00C2\u00A0T,\u00C2\u00A0Shimizu\u00C2\u00A0Y,\u00C2\u00A0Kitajima\u00C2\u00A0K,\u00C2\u00A0Wakitani\u00C2\u00A0M,\u00C2\u00A0Ohta\u00C2\u00A0S,\u00C2\u00A0Satoh\u00C2\u00A0M,\u00C2\u00A0 Shitara\u00C2\u00A0 K,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Engineered\u00C2\u00A0 antibodies\u00C2\u00A0 of\u00C2\u00A0 IgG1/IgG3\u00C2\u00A0mixed\u00C2\u00A0 isotype\u00C2\u00A0with\u00C2\u00A0 enhanced\u00C2\u00A0 cytotoxic\u00C2\u00A0 activities.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02008\u00C2\u00A0May\u00C2\u00A015;68(10):3863\u00E2\u0080\u009072.\u00C2\u00A0 \u00C2\u00A0 58.\u00C2\u00A0 Satoh\u00C2\u00A0 M,\u00C2\u00A0 Iida\u00C2\u00A0 S,\u00C2\u00A0 Shitara\u00C2\u00A0 K.\u00C2\u00A0 Non\u00E2\u0080\u0090fucosylated\u00C2\u00A0 therapeutic\u00C2\u00A0 antibodies\u00C2\u00A0 as\u00C2\u00A0 next\u00E2\u0080\u0090generation\u00C2\u00A0 therapeutic\u00C2\u00A0antibodies.\u00C2\u00A0Expert\u00C2\u00A0Opin\u00C2\u00A0Biol\u00C2\u00A0Ther\u00C2\u00A02006\u00C2\u00A0Nov;6(11):1161\u00E2\u0080\u009073.\u00C2\u00A0 \u00C2\u00A0 59.\u00C2\u00A0Weitzman\u00C2\u00A0J,\u00C2\u00A0Betancur\u00C2\u00A0M,\u00C2\u00A0Boissel\u00C2\u00A0L,\u00C2\u00A0Rabinowitz\u00C2\u00A0AP,\u00C2\u00A0Klein\u00C2\u00A0A,\u00C2\u00A0Klingemann\u00C2\u00A0H.\u00C2\u00A0Variable\u00C2\u00A0contribution\u00C2\u00A0 of\u00C2\u00A0 monoclonal\u00C2\u00A0 antibodies\u00C2\u00A0 to\u00C2\u00A0 ADCC\u00C2\u00A0 in\u00C2\u00A0 patients\u00C2\u00A0 with\u00C2\u00A0 chronic\u00C2\u00A0 lymphocytic\u00C2\u00A0 leukemia.\u00C2\u00A0 Leuk\u00C2\u00A0 Lymphoma\u00C2\u00A02009\u00C2\u00A0Aug;50(8):1361\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 60.\u00C2\u00A0Shields\u00C2\u00A0RL,\u00C2\u00A0Lai\u00C2\u00A0J,\u00C2\u00A0Keck\u00C2\u00A0R,\u00C2\u00A0O'Connell\u00C2\u00A0LY,\u00C2\u00A0Hong\u00C2\u00A0K,\u00C2\u00A0Meng\u00C2\u00A0YG,\u00C2\u00A0Weikert\u00C2\u00A0SHA,\u00C2\u00A0Presta\u00C2\u00A0LG.\u00C2\u00A0Lack\u00C2\u00A0of\u00C2\u00A0fucose\u00C2\u00A0 on\u00C2\u00A0 human\u00C2\u00A0 IgG1\u00C2\u00A0 N\u00E2\u0080\u0090linked\u00C2\u00A0 oligosaccharide\u00C2\u00A0 improves\u00C2\u00A0 binding\u00C2\u00A0 to\u00C2\u00A0 human\u00C2\u00A0 Fc\u00CE\u00B3RIII\u00C2\u00A0 and\u00C2\u00A0 antibody\u00E2\u0080\u0090 dependent\u00C2\u00A0cellular\u00C2\u00A0toxicity.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A02002\u00C2\u00A0July\u00C2\u00A026;277(30):26733\u00E2\u0080\u009040.\u00C2\u00A0 \u00C2\u00A0 61.\u00C2\u00A0Kanda\u00C2\u00A0Y,\u00C2\u00A0Yamada\u00C2\u00A0T,\u00C2\u00A0Mori\u00C2\u00A0K,\u00C2\u00A0Okazaki\u00C2\u00A0A,\u00C2\u00A0Inoue\u00C2\u00A0M,\u00C2\u00A0Kitajima\u00E2\u0080\u0090Miyama\u00C2\u00A0K,\u00C2\u00A0Kuni\u00E2\u0080\u0090Kamochi\u00C2\u00A0R,\u00C2\u00A0Nakano\u00C2\u00A0R,\u00C2\u00A0 Yano\u00C2\u00A0K,\u00C2\u00A0Kakita\u00C2\u00A0S,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Comparison\u00C2\u00A0of\u00C2\u00A0biological\u00C2\u00A0activity\u00C2\u00A0among\u00C2\u00A0nonfucosylated\u00C2\u00A0therapeutic\u00C2\u00A0IgG1\u00C2\u00A0 antibodies\u00C2\u00A0with\u00C2\u00A0 three\u00C2\u00A0 different\u00C2\u00A0N\u00E2\u0080\u0090linked\u00C2\u00A0 fc\u00C2\u00A0 oligosaccharides:\u00C2\u00A0 The\u00C2\u00A0 high\u00E2\u0080\u0090mannose,\u00C2\u00A0 hybrid,\u00C2\u00A0 and\u00C2\u00A0 complex\u00C2\u00A0types.\u00C2\u00A0Glycobiology\u00C2\u00A02007\u00C2\u00A0Jan\u00C2\u00A01;17(1):104\u00E2\u0080\u009018.\u00C2\u00A0 \u00C2\u00A0 62.\u00C2\u00A0Oganesyan\u00C2\u00A0V,\u00C2\u00A0Damschroder\u00C2\u00A0MM,\u00C2\u00A0Leach\u00C2\u00A0W,\u00C2\u00A0Wu\u00C2\u00A0H,\u00C2\u00A0Dall\u00E2\u0080\u0099Acqua\u00C2\u00A0WF.\u00C2\u00A0Structural\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0 mutated,\u00C2\u00A0ADCC\u00E2\u0080\u0090enhanced\u00C2\u00A0human\u00C2\u00A0fc\u00C2\u00A0fragment.\u00C2\u00A0Mol\u00C2\u00A0Immunol\u00C2\u00A02008\u00C2\u00A04;45(7):1872\u00E2\u0080\u009082.\u00C2\u00A0 \u00C2\u00A0 63.\u00C2\u00A0Shields\u00C2\u00A0RL,\u00C2\u00A0Namenuk\u00C2\u00A0AK,\u00C2\u00A0Hong\u00C2\u00A0K,\u00C2\u00A0Meng\u00C2\u00A0YG,\u00C2\u00A0Rae\u00C2\u00A0J,\u00C2\u00A0Briggs\u00C2\u00A0J,\u00C2\u00A0Xie\u00C2\u00A0D,\u00C2\u00A0Lai\u00C2\u00A0J,\u00C2\u00A0Stadlen\u00C2\u00A0A,\u00C2\u00A0Li\u00C2\u00A0B,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0High\u00C2\u00A0 resolution\u00C2\u00A0mapping\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0site\u00C2\u00A0on\u00C2\u00A0human\u00C2\u00A0 IgG1\u00C2\u00A0for\u00C2\u00A0Fc\u00CE\u00B3RI,\u00C2\u00A0Fc\u00CE\u00B3RII,\u00C2\u00A0Fc\u00CE\u00B3RIII,\u00C2\u00A0and\u00C2\u00A0FcRn\u00C2\u00A0and\u00C2\u00A0 design\u00C2\u00A0of\u00C2\u00A0IgG1\u00C2\u00A0variants\u00C2\u00A0with\u00C2\u00A0improved\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0Fc\u00CE\u00B3R.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A02001\u00C2\u00A0Mar\u00C2\u00A02;276(9):6591\u00E2\u0080\u0090 604.\u00C2\u00A0 \u00C2\u00A0 64.\u00C2\u00A0Lazar\u00C2\u00A0GA,\u00C2\u00A0Dang\u00C2\u00A0W,\u00C2\u00A0Karki\u00C2\u00A0S,\u00C2\u00A0Vafa\u00C2\u00A0O,\u00C2\u00A0Peng\u00C2\u00A0JS,\u00C2\u00A0Hyun\u00C2\u00A0L,\u00C2\u00A0Chan\u00C2\u00A0C,\u00C2\u00A0Chung\u00C2\u00A0HS,\u00C2\u00A0Eivazi\u00C2\u00A0A,\u00C2\u00A0Yoder\u00C2\u00A0SC,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 Engineered\u00C2\u00A0antibody\u00C2\u00A0fc\u00C2\u00A0variants\u00C2\u00A0with\u00C2\u00A0enhanced\u00C2\u00A0effector\u00C2\u00A0function.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A02006\u00C2\u00A0Mar\u00C2\u00A0 14;103(11):4005\u00E2\u0080\u009010.\u00C2\u00A0 \u00C2\u00A0 65.\u00C2\u00A0 Agus\u00C2\u00A0 DB,\u00C2\u00A0 Gordon\u00C2\u00A0MS,\u00C2\u00A0 Taylor\u00C2\u00A0 C,\u00C2\u00A0 Natale\u00C2\u00A0 RB,\u00C2\u00A0 Karlan\u00C2\u00A0 B,\u00C2\u00A0Mendelson\u00C2\u00A0 DS,\u00C2\u00A0 Press\u00C2\u00A0MF,\u00C2\u00A0 Allison\u00C2\u00A0 DE,\u00C2\u00A0 Sliwkowski\u00C2\u00A0 MX,\u00C2\u00A0 Lieberman\u00C2\u00A0 G,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Phase\u00C2\u00A0 I\u00C2\u00A0 clinical\u00C2\u00A0 study\u00C2\u00A0 of\u00C2\u00A0 pertuzumab,\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 HER\u00C2\u00A0 dimerization\u00C2\u00A0inhibitor,\u00C2\u00A0in\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0advanced\u00C2\u00A0cancer.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A02005\u00C2\u00A0Apr\u00C2\u00A010;23(11):2534\u00E2\u0080\u0090 43.\u00C2\u00A0 \u00C2\u00A0 66.\u00C2\u00A0Hall\u00C2\u00A0PS,\u00C2\u00A0Cameron\u00C2\u00A0DA.\u00C2\u00A0Current\u00C2\u00A0perspective\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0trastuzumab.\u00C2\u00A0Eur\u00C2\u00A0J\u00C2\u00A0Cancer\u00C2\u00A02009\u00C2\u00A01;45(1):12\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 67.\u00C2\u00A0Yarden\u00C2\u00A0Y,\u00C2\u00A0Sliwkowski\u00C2\u00A0MX.\u00C2\u00A0Untangling\u00C2\u00A0 the\u00C2\u00A0ErbB\u00C2\u00A0 signalling\u00C2\u00A0network.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Mol\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A02001\u00C2\u00A0 Feb;2(2):127\u00E2\u0080\u009037.\u00C2\u00A0 \u00C2\u00A0 68.\u00C2\u00A0 Sliwkowski\u00C2\u00A0 MX,\u00C2\u00A0 Lofgren\u00C2\u00A0 JA,\u00C2\u00A0 Lewis\u00C2\u00A0 GD,\u00C2\u00A0 Hotaling\u00C2\u00A0 TE,\u00C2\u00A0 Fendly\u00C2\u00A0 BM,\u00C2\u00A0 Fox\u00C2\u00A0 JA.\u00C2\u00A0 Nonclinical\u00C2\u00A0 studies\u00C2\u00A0 addressing\u00C2\u00A0 the\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0of\u00C2\u00A0 trastuzumab\u00C2\u00A0 (herceptin).\u00C2\u00A0Semin\u00C2\u00A0Oncol\u00C2\u00A01999\u00C2\u00A0Aug;26(4\u00C2\u00A0 Suppl\u00C2\u00A012):60\u00E2\u0080\u009070.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0179\u00C2\u00A0\u00C2\u00A0 69.\u00C2\u00A0 Valabrega\u00C2\u00A0G,\u00C2\u00A0Montemurro\u00C2\u00A0 F,\u00C2\u00A0 Aglietta\u00C2\u00A0M.\u00C2\u00A0 Trastuzumab:\u00C2\u00A0Mechanism\u00C2\u00A0 of\u00C2\u00A0 action,\u00C2\u00A0 resistance\u00C2\u00A0 and\u00C2\u00A0 future\u00C2\u00A0perspectives\u00C2\u00A0in\u00C2\u00A0HER2\u00E2\u0080\u0090overexpressing\u00C2\u00A0breast\u00C2\u00A0cancer.\u00C2\u00A0Ann\u00C2\u00A0Oncol\u00C2\u00A02007\u00C2\u00A0Jun\u00C2\u00A01;18(6):977\u00E2\u0080\u009084.\u00C2\u00A0 \u00C2\u00A0 70.\u00C2\u00A0Hudis\u00C2\u00A0CA.\u00C2\u00A0Trastuzumab\u00C2\u00A0\u00E2\u0080\u0094\u00C2\u00A0mechanism\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0and\u00C2\u00A0use\u00C2\u00A0in\u00C2\u00A0clinical\u00C2\u00A0practice.\u00C2\u00A0N\u00C2\u00A0Engl\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A02007\u00C2\u00A0Jul\u00C2\u00A0 5;357(1):39\u00E2\u0080\u009051.\u00C2\u00A0 \u00C2\u00A0 71.\u00C2\u00A0DeGrendele\u00C2\u00A0H,\u00C2\u00A0 Jain\u00C2\u00A0VK.\u00C2\u00A0 The\u00C2\u00A0 anti\u00E2\u0080\u0090HER2\u00C2\u00A0monoclonal\u00C2\u00A0 antibody\u00C2\u00A0 pertuzumab\u00C2\u00A0may\u00C2\u00A0 be\u00C2\u00A0 effective\u00C2\u00A0 in\u00C2\u00A0 androgen\u00E2\u0080\u0090independent\u00C2\u00A0prostate\u00C2\u00A0cancer.\u00C2\u00A0Clin\u00C2\u00A0Genitourin\u00C2\u00A0Canc\u00C2\u00A02003;2(3):143\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 72.\u00C2\u00A0 Nahta\u00C2\u00A0 R,\u00C2\u00A0 Hung\u00C2\u00A0 M,\u00C2\u00A0 Esteva\u00C2\u00A0 FJ.\u00C2\u00A0 The\u00C2\u00A0 HER\u00E2\u0080\u00902\u00E2\u0080\u0090targeting\u00C2\u00A0 antibodies\u00C2\u00A0 trastuzumab\u00C2\u00A0 and\u00C2\u00A0 pertuzumab\u00C2\u00A0 synergistically\u00C2\u00A0inhibit\u00C2\u00A0the\u00C2\u00A0survival\u00C2\u00A0of\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0cells.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02004\u00C2\u00A0Apr\u00C2\u00A01;64(7):2343\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0 73.\u00C2\u00A0 Scheuer\u00C2\u00A0 W,\u00C2\u00A0 Friess\u00C2\u00A0 T,\u00C2\u00A0 Burtscher\u00C2\u00A0 H,\u00C2\u00A0 Bossenmaier\u00C2\u00A0 B,\u00C2\u00A0 Endl\u00C2\u00A0 J,\u00C2\u00A0 Hasmann\u00C2\u00A0 M.\u00C2\u00A0 Strongly\u00C2\u00A0 enhanced\u00C2\u00A0 antitumor\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 trastuzumab\u00C2\u00A0 and\u00C2\u00A0 pertuzumab\u00C2\u00A0 combination\u00C2\u00A0 treatment\u00C2\u00A0 on\u00C2\u00A0HER2\u00E2\u0080\u0090positive\u00C2\u00A0 human\u00C2\u00A0xenograft\u00C2\u00A0tumor\u00C2\u00A0models.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02009\u00C2\u00A0Dec\u00C2\u00A015;69(24):9330\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0 74.\u00C2\u00A0 Janas\u00C2\u00A0 E,\u00C2\u00A0 Priest\u00C2\u00A0 R,\u00C2\u00A0 Wilde\u00C2\u00A0 JI,\u00C2\u00A0 White\u00C2\u00A0 JH,\u00C2\u00A0 Malhotra\u00C2\u00A0 R.\u00C2\u00A0 Rituxan\u00C2\u00A0 (anti\u00E2\u0080\u0090CD20\u00C2\u00A0 antibody)\u00E2\u0080\u0090induced\u00C2\u00A0 translocation\u00C2\u00A0 of\u00C2\u00A0 CD20\u00C2\u00A0 into\u00C2\u00A0 lipid\u00C2\u00A0 rafts\u00C2\u00A0 is\u00C2\u00A0 crucial\u00C2\u00A0 for\u00C2\u00A0 calcium\u00C2\u00A0 influx\u00C2\u00A0 and\u00C2\u00A0 apoptosis.\u00C2\u00A0 Clin\u00C2\u00A0 Exp\u00C2\u00A0 Immunol\u00C2\u00A02005\u00C2\u00A0Mar;139(3):439\u00E2\u0080\u009046.\u00C2\u00A0 \u00C2\u00A0 75.\u00C2\u00A0Zhang\u00C2\u00A0N,\u00C2\u00A0Khawli\u00C2\u00A0LA,\u00C2\u00A0Hu\u00C2\u00A0P,\u00C2\u00A0Epstein\u00C2\u00A0AL.\u00C2\u00A0Generation\u00C2\u00A0of\u00C2\u00A0rituximab\u00C2\u00A0polymer\u00C2\u00A0may\u00C2\u00A0cause\u00C2\u00A0hyper\u00E2\u0080\u0090cross\u00E2\u0080\u0090 linking\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 non\u00E2\u0080\u0090hodgkin's\u00C2\u00A0 lymphomas.\u00C2\u00A0 Clin\u00C2\u00A0 Cancer\u00C2\u00A0 Res\u00C2\u00A0 2005\u00C2\u00A0 Aug\u00C2\u00A0 15;11(16):5971\u00E2\u0080\u009080.\u00C2\u00A0 \u00C2\u00A0 76.\u00C2\u00A0 Pedersen\u00C2\u00A0 IM,\u00C2\u00A0 Buhl\u00C2\u00A0 AM,\u00C2\u00A0 Klausen\u00C2\u00A0 P,\u00C2\u00A0Geisler\u00C2\u00A0 CH,\u00C2\u00A0 Jurlander\u00C2\u00A0 J.\u00C2\u00A0 The\u00C2\u00A0 chimeric\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 antibody\u00C2\u00A0 rituximab\u00C2\u00A0induces\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0chronic\u00C2\u00A0lymphocytic\u00C2\u00A0leukemia\u00C2\u00A0cells\u00C2\u00A0through\u00C2\u00A0a\u00C2\u00A0p38\u00C2\u00A0mitogen\u00C2\u00A0 activated\u00C2\u00A0protein\u00E2\u0080\u0090kinase\u00E2\u0080\u0090dependent\u00C2\u00A0mechanism.\u00C2\u00A0Blood\u00C2\u00A02002\u00C2\u00A0Feb\u00C2\u00A015;99(4):1314\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 77.\u00C2\u00A0Shan\u00C2\u00A0D,\u00C2\u00A0Ledbetter\u00C2\u00A0JA,\u00C2\u00A0Press\u00C2\u00A0OW.\u00C2\u00A0Apoptosis\u00C2\u00A0of\u00C2\u00A0malignant\u00C2\u00A0human\u00C2\u00A0B\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0ligation\u00C2\u00A0of\u00C2\u00A0CD20\u00C2\u00A0with\u00C2\u00A0 monoclonal\u00C2\u00A0antibodies.\u00C2\u00A0Blood\u00C2\u00A01998\u00C2\u00A0Mar\u00C2\u00A01;91(5):1644\u00E2\u0080\u009052.\u00C2\u00A0 \u00C2\u00A0 78.\u00C2\u00A0Hanahan\u00C2\u00A0D,\u00C2\u00A0Weinberg\u00C2\u00A0RA.\u00C2\u00A0The\u00C2\u00A0hallmarks\u00C2\u00A0of\u00C2\u00A0cancer.\u00C2\u00A0Cell\u00C2\u00A02000\u00C2\u00A0Jan\u00C2\u00A07;100(1):57\u00E2\u0080\u009070.\u00C2\u00A0 \u00C2\u00A0 79.\u00C2\u00A0Pennarun\u00C2\u00A0B,\u00C2\u00A0Meijer\u00C2\u00A0A,\u00C2\u00A0de\u00C2\u00A0Vries\u00C2\u00A0EG,\u00C2\u00A0Kleibeuker\u00C2\u00A0JH,\u00C2\u00A0Kruyt\u00C2\u00A0F,\u00C2\u00A0de\u00C2\u00A0Jong\u00C2\u00A0S.\u00C2\u00A0Playing\u00C2\u00A0the\u00C2\u00A0DISC:\u00C2\u00A0Turning\u00C2\u00A0on\u00C2\u00A0 TRAIL\u00C2\u00A0 death\u00C2\u00A0 receptor\u00E2\u0080\u0090mediated\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 cancer.\u00C2\u00A0 Biochim\u00C2\u00A0 Biophys\u00C2\u00A0 Acta\u00C2\u00A0 2010\u00C2\u00A0 Apr;1805(2):123\u00E2\u0080\u009040.\u00C2\u00A0 \u00C2\u00A0 80.\u00C2\u00A0 Cotter\u00C2\u00A0 TG.\u00C2\u00A0 Apoptosis\u00C2\u00A0 and\u00C2\u00A0 cancer:\u00C2\u00A0 The\u00C2\u00A0 genesis\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 research\u00C2\u00A0 field.\u00C2\u00A0 Nat\u00C2\u00A0 Rev\u00C2\u00A0 Cancer\u00C2\u00A0 2009\u00C2\u00A0 Jul;9(7):501\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 81.\u00C2\u00A0Ghavami\u00C2\u00A0S,\u00C2\u00A0Hashemi\u00C2\u00A0M,\u00C2\u00A0Ande\u00C2\u00A0SR,\u00C2\u00A0Yeganeh\u00C2\u00A0B,\u00C2\u00A0Xiao\u00C2\u00A0W,\u00C2\u00A0Eshraghi\u00C2\u00A0M,\u00C2\u00A0Bus\u00C2\u00A0CJ,\u00C2\u00A0Kadkhoda\u00C2\u00A0K,\u00C2\u00A0Wiechec\u00C2\u00A0 E,\u00C2\u00A0Halayko\u00C2\u00A0AJ,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Apoptosis\u00C2\u00A0and\u00C2\u00A0cancer:\u00C2\u00A0Mutations\u00C2\u00A0within\u00C2\u00A0caspase\u00C2\u00A0genes.\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A0Genet\u00C2\u00A02009\u00C2\u00A0 Aug;46(8):497\u00E2\u0080\u0090510.\u00C2\u00A0 \u00C2\u00A0 82.\u00C2\u00A0Ashkenazi\u00C2\u00A0A.\u00C2\u00A0Targeting\u00C2\u00A0death\u00C2\u00A0and\u00C2\u00A0decoy\u00C2\u00A0 receptors\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 tumour\u00E2\u0080\u0090necrosis\u00C2\u00A0 factor\u00C2\u00A0 superfamily.\u00C2\u00A0 Nat\u00C2\u00A0Rev\u00C2\u00A0Cancer\u00C2\u00A02002\u00C2\u00A0Jun;2(6):420\u00E2\u0080\u009030.\u00C2\u00A0 \u00C2\u00A0 83.\u00C2\u00A0Danial\u00C2\u00A0NN,\u00C2\u00A0Korsmeyer\u00C2\u00A0SJ.\u00C2\u00A0Cell\u00C2\u00A0death:\u00C2\u00A0Critical\u00C2\u00A0control\u00C2\u00A0points.\u00C2\u00A0Cell\u00C2\u00A02004\u00C2\u00A0Jan\u00C2\u00A023;116(2):205\u00E2\u0080\u009019.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0180\u00C2\u00A0\u00C2\u00A0 84.\u00C2\u00A0Vermes\u00C2\u00A0 I,\u00C2\u00A0Haanen\u00C2\u00A0C,\u00C2\u00A0Steffens\u00E2\u0080\u0090Nakken\u00C2\u00A0H,\u00C2\u00A0Reutellingsperger\u00C2\u00A0C.\u00C2\u00A0A\u00C2\u00A0novel\u00C2\u00A0assay\u00C2\u00A0 for\u00C2\u00A0apoptosis\u00C2\u00A0 flow\u00C2\u00A0 cytometric\u00C2\u00A0detection\u00C2\u00A0of\u00C2\u00A0phosphatidylserine\u00C2\u00A0expression\u00C2\u00A0on\u00C2\u00A0early\u00C2\u00A0apoptotic\u00C2\u00A0cells\u00C2\u00A0using\u00C2\u00A0fluorescein\u00C2\u00A0 labelled\u00C2\u00A0annexin\u00C2\u00A0V.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0Methods\u00C2\u00A01995\u00C2\u00A0Jul\u00C2\u00A017;184(1):39\u00E2\u0080\u009051.\u00C2\u00A0 \u00C2\u00A0 85.\u00C2\u00A0Koopman\u00C2\u00A0G,\u00C2\u00A0Reutelingsperger\u00C2\u00A0C,\u00C2\u00A0Kuijten\u00C2\u00A0G,\u00C2\u00A0Keehnen\u00C2\u00A0R,\u00C2\u00A0Pals\u00C2\u00A0S,\u00C2\u00A0van\u00C2\u00A0Oers\u00C2\u00A0M.\u00C2\u00A0Annexin\u00C2\u00A0V\u00C2\u00A0for\u00C2\u00A0flow\u00C2\u00A0 cytometric\u00C2\u00A0detection\u00C2\u00A0of\u00C2\u00A0phosphatidylserine\u00C2\u00A0expression\u00C2\u00A0on\u00C2\u00A0B\u00C2\u00A0cells\u00C2\u00A0undergoing\u00C2\u00A0apoptosis.\u00C2\u00A0Blood\u00C2\u00A0 1994\u00C2\u00A0Sept\u00C2\u00A01;84(5):1415\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 86.\u00C2\u00A0 Strasser\u00C2\u00A0 A.\u00C2\u00A0 The\u00C2\u00A0 role\u00C2\u00A0 of\u00C2\u00A0 BH3\u00E2\u0080\u0090only\u00C2\u00A0 proteins\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 immune\u00C2\u00A0 system.\u00C2\u00A0 Nat\u00C2\u00A0 Rev\u00C2\u00A0 Immunol\u00C2\u00A0 2005\u00C2\u00A0 Mar;5(3):189\u00E2\u0080\u0090200.\u00C2\u00A0 \u00C2\u00A0 87.\u00C2\u00A0Kohlhaas\u00C2\u00A0SL,\u00C2\u00A0Craxton\u00C2\u00A0A,\u00C2\u00A0Sun\u00C2\u00A0XM,\u00C2\u00A0Pinkoski\u00C2\u00A0MJ,\u00C2\u00A0Cohen\u00C2\u00A0GM.\u00C2\u00A0Receptor\u00E2\u0080\u0090mediated\u00C2\u00A0endocytosis\u00C2\u00A0is\u00C2\u00A0not\u00C2\u00A0 required\u00C2\u00A0for\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00E2\u0080\u0090related\u00C2\u00A0apoptosis\u00E2\u0080\u0090inducing\u00C2\u00A0ligand\u00C2\u00A0(TRAIL)\u00E2\u0080\u0090induced\u00C2\u00A0apoptosis.\u00C2\u00A0 J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A02007\u00C2\u00A0Apr\u00C2\u00A027;282(17):12831\u00E2\u0080\u009041.\u00C2\u00A0 \u00C2\u00A0 88.\u00C2\u00A0Ghetie\u00C2\u00A0MA,\u00C2\u00A0Bright\u00C2\u00A0H,\u00C2\u00A0Vitetta\u00C2\u00A0ES.\u00C2\u00A0Homodimers\u00C2\u00A0but\u00C2\u00A0not\u00C2\u00A0monomers\u00C2\u00A0of\u00C2\u00A0rituxan\u00C2\u00A0(chimeric\u00C2\u00A0anti\u00E2\u0080\u0090CD20)\u00C2\u00A0 induce\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0human\u00C2\u00A0B\u00E2\u0080\u0090lymphoma\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0synergize\u00C2\u00A0with\u00C2\u00A0a\u00C2\u00A0chemotherapeutic\u00C2\u00A0agent\u00C2\u00A0and\u00C2\u00A0 an\u00C2\u00A0immunotoxin.\u00C2\u00A0Blood\u00C2\u00A02001\u00C2\u00A0Mar\u00C2\u00A01;97(5):1392\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 89.\u00C2\u00A0Miller\u00C2\u00A0K,\u00C2\u00A0Meng\u00C2\u00A0G,\u00C2\u00A0Liu\u00C2\u00A0J,\u00C2\u00A0Hurst\u00C2\u00A0A,\u00C2\u00A0Hsei\u00C2\u00A0V,\u00C2\u00A0Wong\u00C2\u00A0WL,\u00C2\u00A0Ekert\u00C2\u00A0R,\u00C2\u00A0Lawrence\u00C2\u00A0D,\u00C2\u00A0Sherwood\u00C2\u00A0S,\u00C2\u00A0DeForge\u00C2\u00A0L,\u00C2\u00A0 et\u00C2\u00A0al.\u00C2\u00A0Design,\u00C2\u00A0construction,\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0analyses\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0antibodies.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A02003\u00C2\u00A0May\u00C2\u00A0 1;170(9):4854\u00E2\u0080\u009061.\u00C2\u00A0 \u00C2\u00A0 90.\u00C2\u00A0Rossi\u00C2\u00A0EA,\u00C2\u00A0Goldenberg\u00C2\u00A0DM,\u00C2\u00A0Cardillo\u00C2\u00A0TM,\u00C2\u00A0Stein\u00C2\u00A0R,\u00C2\u00A0Wang\u00C2\u00A0Y,\u00C2\u00A0Chang\u00C2\u00A0CH.\u00C2\u00A0Novel\u00C2\u00A0designs\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0 humanized\u00C2\u00A0 antibodies\u00C2\u00A0 as\u00C2\u00A0 improved\u00C2\u00A0 lymphoma\u00C2\u00A0 therapeutics.\u00C2\u00A0 Cancer\u00C2\u00A0 Res\u00C2\u00A0 2008\u00C2\u00A0Oct\u00C2\u00A0 15;68(20):8384\u00E2\u0080\u009092.\u00C2\u00A0 \u00C2\u00A0 91.\u00C2\u00A0Deyev\u00C2\u00A0 SM,\u00C2\u00A0 Lebedenko\u00C2\u00A0 EN.\u00C2\u00A0Multivalency:\u00C2\u00A0 The\u00C2\u00A0 hallmark\u00C2\u00A0of\u00C2\u00A0 antibodies\u00C2\u00A0 used\u00C2\u00A0 for\u00C2\u00A0optimization\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0targeting\u00C2\u00A0by\u00C2\u00A0design.\u00C2\u00A0Bioessays\u00C2\u00A02008\u00C2\u00A0Sep;30(9):904\u00E2\u0080\u009018.\u00C2\u00A0 \u00C2\u00A0 92.\u00C2\u00A0Martos\u00C2\u00A0V,\u00C2\u00A0Castre\u00C3\u00B1o\u00C2\u00A0P,\u00C2\u00A0Valero\u00C2\u00A0 J,\u00C2\u00A0de\u00C2\u00A0Mendoza\u00C2\u00A0 J.\u00C2\u00A0Binding\u00C2\u00A0 to\u00C2\u00A0protein\u00C2\u00A0 surfaces\u00C2\u00A0by\u00C2\u00A0 supramolecular\u00C2\u00A0 multivalent\u00C2\u00A0scaffolds.\u00C2\u00A0Curr\u00C2\u00A0Opin\u00C2\u00A0Chem\u00C2\u00A0Biol\u00C2\u00A02008\u00C2\u00A012;12(6):698\u00E2\u0080\u0090706.\u00C2\u00A0 \u00C2\u00A0 93.\u00C2\u00A0Kitov\u00C2\u00A0PI,\u00C2\u00A0Mulvey\u00C2\u00A0GL,\u00C2\u00A0Griener\u00C2\u00A0TP,\u00C2\u00A0Lipinski\u00C2\u00A0T,\u00C2\u00A0Solomon\u00C2\u00A0D,\u00C2\u00A0Paszkiewicz\u00C2\u00A0E,\u00C2\u00A0Jacobson\u00C2\u00A0JM,\u00C2\u00A0Sadowska\u00C2\u00A0JM,\u00C2\u00A0 Suzuki\u00C2\u00A0 M,\u00C2\u00A0 Yamamura\u00C2\u00A0 K,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 In\u00C2\u00A0 vivo\u00C2\u00A0 supramolecular\u00C2\u00A0 templating\u00C2\u00A0 enhances\u00C2\u00A0 the\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 multivalent\u00C2\u00A0ligands:\u00C2\u00A0A\u00C2\u00A0potential\u00C2\u00A0therapeutic\u00C2\u00A0against\u00C2\u00A0the\u00C2\u00A0escherichia\u00C2\u00A0coli\u00C2\u00A0O157\u00C2\u00A0AB5\u00C2\u00A0toxins.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0 Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A02008\u00C2\u00A0Nov\u00C2\u00A04;105(44):16837\u00E2\u0080\u009042.\u00C2\u00A0 \u00C2\u00A0 94.\u00C2\u00A0Patri\u00C2\u00A0AK,\u00C2\u00A0Majoros\u00C2\u00A0IJ,\u00C2\u00A0Baker\u00C2\u00A0JR.\u00C2\u00A0Dendritic\u00C2\u00A0polymer\u00C2\u00A0macromolecular\u00C2\u00A0carriers\u00C2\u00A0for\u00C2\u00A0drug\u00C2\u00A0delivery.\u00C2\u00A0Curr\u00C2\u00A0 Opin\u00C2\u00A0Chem\u00C2\u00A0Biol\u00C2\u00A02002\u00C2\u00A0Aug;6(4):466\u00E2\u0080\u009071.\u00C2\u00A0 \u00C2\u00A0 95.\u00C2\u00A0Patri\u00C2\u00A0AK,\u00C2\u00A0Myc\u00C2\u00A0A,\u00C2\u00A0Beals\u00C2\u00A0J,\u00C2\u00A0Thomas\u00C2\u00A0TP,\u00C2\u00A0Bander\u00C2\u00A0NH,\u00C2\u00A0Baker\u00C2\u00A0JR.\u00C2\u00A0Synthesis\u00C2\u00A0and\u00C2\u00A0in\u00C2\u00A0vitro\u00C2\u00A0testing\u00C2\u00A0of\u00C2\u00A0J591\u00C2\u00A0 antibody\u00E2\u0080\u0090dendrimer\u00C2\u00A0 conjugates\u00C2\u00A0 for\u00C2\u00A0 targeted\u00C2\u00A0 prostate\u00C2\u00A0 cancer\u00C2\u00A0 therapy.\u00C2\u00A0 Bioconjug\u00C2\u00A0 Chem\u00C2\u00A0 2004\u00C2\u00A0 Nov;15(6):1174\u00E2\u0080\u009081.\u00C2\u00A0 \u00C2\u00A0 96.\u00C2\u00A0Weiss\u00C2\u00A0A,\u00C2\u00A0Preston\u00C2\u00A0TC,\u00C2\u00A0Popov\u00C2\u00A0 J,\u00C2\u00A0 Li\u00C2\u00A0Q,\u00C2\u00A0Wu\u00C2\u00A0S,\u00C2\u00A0Chou\u00C2\u00A0KC,\u00C2\u00A0Burt\u00C2\u00A0HM,\u00C2\u00A0Bally\u00C2\u00A0MB,\u00C2\u00A0Signorell\u00C2\u00A0R.\u00C2\u00A0Selective\u00C2\u00A0 recognition\u00C2\u00A0of\u00C2\u00A0rituximab\u00E2\u0080\u0090functionalized\u00C2\u00A0gold\u00C2\u00A0nanoparticles\u00C2\u00A0by\u00C2\u00A0 lymphoma\u00C2\u00A0cells\u00C2\u00A0studied\u00C2\u00A0with\u00C2\u00A03D\u00C2\u00A0 imaging.\u00C2\u00A0J\u00C2\u00A0Phys\u00C2\u00A0Chem\u00C2\u00A0C\u00C2\u00A02009\u00C2\u00A0Oct\u00C2\u00A021;113(47):20252\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0181\u00C2\u00A0\u00C2\u00A0 97.\u00C2\u00A0 Cuesta\u00C2\u00A0AM,\u00C2\u00A0 Sainz\u00E2\u0080\u0090Pastor\u00C2\u00A0N,\u00C2\u00A0 Bonet\u00C2\u00A0 J,\u00C2\u00A0Oliva\u00C2\u00A0 B,\u00C2\u00A0Alvarez\u00E2\u0080\u0090Vallina\u00C2\u00A0 L.\u00C2\u00A0Multivalent\u00C2\u00A0 antibodies:\u00C2\u00A0When\u00C2\u00A0 design\u00C2\u00A0surpasses\u00C2\u00A0evolution.\u00C2\u00A0Trends\u00C2\u00A0Biotechnol\u00C2\u00A02010\u00C2\u00A0Jul;28(7):355\u00E2\u0080\u009062.\u00C2\u00A0 \u00C2\u00A0 98.\u00C2\u00A0Coloma\u00C2\u00A0MJ,\u00C2\u00A0Clift\u00C2\u00A0A,\u00C2\u00A0Wims\u00C2\u00A0L,\u00C2\u00A0Morrison\u00C2\u00A0SL.\u00C2\u00A0The\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0carbohydrate\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0assembly\u00C2\u00A0and\u00C2\u00A0function\u00C2\u00A0 of\u00C2\u00A0polymeric\u00C2\u00A0IgG.\u00C2\u00A0Mol\u00C2\u00A0Immunol\u00C2\u00A02000\u00C2\u00A0Dec;37(18):1081\u00E2\u0080\u009090.\u00C2\u00A0 \u00C2\u00A0 99.\u00C2\u00A0Mammen\u00C2\u00A0M,\u00C2\u00A0Choi\u00C2\u00A0SK,\u00C2\u00A0Whitesides\u00C2\u00A0GM.\u00C2\u00A0Polyvalent\u00C2\u00A0interactions\u00C2\u00A0in\u00C2\u00A0biological\u00C2\u00A0systems:\u00C2\u00A0Implications\u00C2\u00A0 for\u00C2\u00A0design\u00C2\u00A0and\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0ligands\u00C2\u00A0and\u00C2\u00A0inhibitors.\u00C2\u00A0Angew\u00C2\u00A0Chem\u00C2\u00A0Int\u00C2\u00A0Edit\u00C2\u00A01998;37:2754\u00E2\u0080\u009094.\u00C2\u00A0 \u00C2\u00A0 100.\u00C2\u00A0Dam\u00C2\u00A0TK,\u00C2\u00A0Brewer\u00C2\u00A0CF.\u00C2\u00A0Effects\u00C2\u00A0of\u00C2\u00A0clustered\u00C2\u00A0epitopes\u00C2\u00A0 in\u00C2\u00A0multivalent\u00C2\u00A0 ligand\u00E2\u0080\u0090receptor\u00C2\u00A0 interactions.\u00C2\u00A0 Biochemistry\u00C2\u00A02008\u00C2\u00A0Aug\u00C2\u00A019;47(33):8470\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0 101.\u00C2\u00A0Martos\u00C2\u00A0V,\u00C2\u00A0Castreno\u00C2\u00A0P,\u00C2\u00A0Valero\u00C2\u00A0 J,\u00C2\u00A0de\u00C2\u00A0Mendoza\u00C2\u00A0 J.\u00C2\u00A0Binding\u00C2\u00A0 to\u00C2\u00A0protein\u00C2\u00A0surfaces\u00C2\u00A0by\u00C2\u00A0supramolecular\u00C2\u00A0 multivalent\u00C2\u00A0scaffolds.\u00C2\u00A0Curr\u00C2\u00A0Opin\u00C2\u00A0Chem\u00C2\u00A0Biol\u00C2\u00A02008\u00C2\u00A0Dec;12(6):698\u00E2\u0080\u0090706.\u00C2\u00A0 \u00C2\u00A0 102.\u00C2\u00A0Gestwicki\u00C2\u00A0JE,\u00C2\u00A0Cairo\u00C2\u00A0CW,\u00C2\u00A0Strong\u00C2\u00A0LE,\u00C2\u00A0Oetjen\u00C2\u00A0KA,\u00C2\u00A0Kiessling\u00C2\u00A0LL.\u00C2\u00A0 Influencing\u00C2\u00A0receptor\u00E2\u0080\u0090ligand\u00C2\u00A0binding\u00C2\u00A0 mechanisms\u00C2\u00A0with\u00C2\u00A0multivalent\u00C2\u00A0 ligand\u00C2\u00A0architecture.\u00C2\u00A0J\u00C2\u00A0Am\u00C2\u00A0Chem\u00C2\u00A0Soc\u00C2\u00A02002\u00C2\u00A0Dec\u00C2\u00A018;124(50):14922\u00E2\u0080\u0090 33.\u00C2\u00A0 \u00C2\u00A0 103.\u00C2\u00A0 Rinker\u00C2\u00A0 S,\u00C2\u00A0 Ke\u00C2\u00A0 Y,\u00C2\u00A0 Liu\u00C2\u00A0 Y,\u00C2\u00A0 Chhabra\u00C2\u00A0 R,\u00C2\u00A0 Yan\u00C2\u00A0 H.\u00C2\u00A0 Self\u00E2\u0080\u0090assembled\u00C2\u00A0 DNA\u00C2\u00A0 nanostructures\u00C2\u00A0 for\u00C2\u00A0 distance\u00E2\u0080\u0090 dependent\u00C2\u00A0multivalent\u00C2\u00A0ligand\u00E2\u0080\u0090protein\u00C2\u00A0binding.\u00C2\u00A0Nat\u00C2\u00A0Nanotechnol\u00C2\u00A02008\u00C2\u00A0Jul;3(7):418\u00E2\u0080\u009022.\u00C2\u00A0 \u00C2\u00A0 104.\u00C2\u00A0Diestler\u00C2\u00A0DJ,\u00C2\u00A0Knapp\u00C2\u00A0EW.\u00C2\u00A0Statistical\u00C2\u00A0thermodynamics\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0stability\u00C2\u00A0of\u00C2\u00A0multivalent\u00C2\u00A0ligand\u00E2\u0080\u0090receptor\u00C2\u00A0 complexes.\u00C2\u00A0Phys\u00C2\u00A0Rev\u00C2\u00A0Lett\u00C2\u00A02008\u00C2\u00A0May\u00C2\u00A02;100(17):178101.\u00C2\u00A0 \u00C2\u00A0 105.\u00C2\u00A0Feng\u00C2\u00A0 JQ,\u00C2\u00A0Mozdzanowska\u00C2\u00A0K,\u00C2\u00A0Gerhard\u00C2\u00A0W.\u00C2\u00A0Complement\u00C2\u00A0component\u00C2\u00A0C1q\u00C2\u00A0enhances\u00C2\u00A0the\u00C2\u00A0biological\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 influenza\u00C2\u00A0 virus\u00C2\u00A0 hemagglutinin\u00E2\u0080\u0090specific\u00C2\u00A0 antibodies\u00C2\u00A0 depending\u00C2\u00A0 on\u00C2\u00A0 their\u00C2\u00A0 fine\u00C2\u00A0 antigen\u00C2\u00A0 specificity\u00C2\u00A0and\u00C2\u00A0heavy\u00E2\u0080\u0090chain\u00C2\u00A0isotype.\u00C2\u00A0J\u00C2\u00A0Virol\u00C2\u00A02002\u00C2\u00A0Feb;76(3):1369\u00E2\u0080\u009078.\u00C2\u00A0 \u00C2\u00A0 106.\u00C2\u00A0Schumaker\u00C2\u00A0VN,\u00C2\u00A0Zavodszky\u00C2\u00A0P,\u00C2\u00A0Poon\u00C2\u00A0PH.\u00C2\u00A0Activation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0first\u00C2\u00A0component\u00C2\u00A0of\u00C2\u00A0complement.\u00C2\u00A0Annu\u00C2\u00A0 Rev\u00C2\u00A0Immunol\u00C2\u00A01987;5:21\u00E2\u0080\u009042.\u00C2\u00A0 \u00C2\u00A0 107.\u00C2\u00A0George\u00C2\u00A0SR,\u00C2\u00A0O'Dowd\u00C2\u00A0BF,\u00C2\u00A0Lee\u00C2\u00A0SP.\u00C2\u00A0G\u00E2\u0080\u0090protein\u00E2\u0080\u0090coupled\u00C2\u00A0receptor\u00C2\u00A0oligomerization\u00C2\u00A0and\u00C2\u00A0its\u00C2\u00A0potential\u00C2\u00A0for\u00C2\u00A0 drug\u00C2\u00A0discovery.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Drug\u00C2\u00A0Discov\u00C2\u00A02002\u00C2\u00A0Oct;1(10):808\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 108.\u00C2\u00A0Simons\u00C2\u00A0K,\u00C2\u00A0Toomre\u00C2\u00A0D.\u00C2\u00A0Lipid\u00C2\u00A0rafts\u00C2\u00A0and\u00C2\u00A0signal\u00C2\u00A0transduction.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Mol\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A02000\u00C2\u00A0Oct;1(1):31\u00E2\u0080\u0090 9.\u00C2\u00A0 \u00C2\u00A0 109.\u00C2\u00A0Hommelgaard\u00C2\u00A0AM,\u00C2\u00A0 Lerdrup\u00C2\u00A0M,\u00C2\u00A0 van\u00C2\u00A0Deurs\u00C2\u00A0B.\u00C2\u00A0Association\u00C2\u00A0with\u00C2\u00A0membrane\u00C2\u00A0protrusions\u00C2\u00A0makes\u00C2\u00A0 ErbB2\u00C2\u00A0an\u00C2\u00A0internalization\u00E2\u0080\u0090resistant\u00C2\u00A0receptor.\u00C2\u00A0Mol\u00C2\u00A0Biol\u00C2\u00A0Cell\u00C2\u00A02004\u00C2\u00A0Apr;15(4):1557\u00E2\u0080\u009067.\u00C2\u00A0 \u00C2\u00A0 110.\u00C2\u00A0Friedlander\u00C2\u00A0E,\u00C2\u00A0Arndt\u00E2\u0080\u0090Jovin\u00C2\u00A0DJ,\u00C2\u00A0Nagy\u00C2\u00A0P,\u00C2\u00A0Jovin\u00C2\u00A0TM,\u00C2\u00A0Szollosi\u00C2\u00A0J,\u00C2\u00A0Vereb\u00C2\u00A0G.\u00C2\u00A0Signal\u00C2\u00A0transduction\u00C2\u00A0of\u00C2\u00A0erbB\u00C2\u00A0 receptors\u00C2\u00A0 in\u00C2\u00A0 trastuzumab\u00C2\u00A0 (herceptin)\u00C2\u00A0sensitive\u00C2\u00A0and\u00C2\u00A0 resistant\u00C2\u00A0cell\u00C2\u00A0 lines:\u00C2\u00A0Local\u00C2\u00A0 stimulation\u00C2\u00A0using\u00C2\u00A0 magnetic\u00C2\u00A0microspheres\u00C2\u00A0 as\u00C2\u00A0 assessed\u00C2\u00A0 by\u00C2\u00A0 quantitative\u00C2\u00A0 digital\u00C2\u00A0microscopy.\u00C2\u00A0 Cytom\u00C2\u00A0 Part\u00C2\u00A0 A\u00C2\u00A0 2005\u00C2\u00A0 Oct;67(2):161\u00E2\u0080\u009071.\u00C2\u00A0 \u00C2\u00A0 111.\u00C2\u00A0Nagy\u00C2\u00A0P,\u00C2\u00A0Vereb\u00C2\u00A0G,\u00C2\u00A0Sebestyen\u00C2\u00A0Z,\u00C2\u00A0Horvath\u00C2\u00A0G,\u00C2\u00A0Lockett\u00C2\u00A0SJ,\u00C2\u00A0Damjanovich\u00C2\u00A0S,\u00C2\u00A0Park\u00C2\u00A0JW,\u00C2\u00A0Jovin\u00C2\u00A0TM,\u00C2\u00A0Szollosi\u00C2\u00A0 J.\u00C2\u00A0Lipid\u00C2\u00A0rafts\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0 local\u00C2\u00A0density\u00C2\u00A0of\u00C2\u00A0ErbB\u00C2\u00A0proteins\u00C2\u00A0 influence\u00C2\u00A0the\u00C2\u00A0biological\u00C2\u00A0role\u00C2\u00A0of\u00C2\u00A0homo\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0 heteroassociations\u00C2\u00A0of\u00C2\u00A0ErbB2.\u00C2\u00A0J\u00C2\u00A0Cell\u00C2\u00A0Sci\u00C2\u00A02002\u00C2\u00A0Nov\u00C2\u00A015;115(Pt\u00C2\u00A022):4251\u00E2\u0080\u009062.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0182\u00C2\u00A0\u00C2\u00A0 112.\u00C2\u00A0Ghatak\u00C2\u00A0 S,\u00C2\u00A0Misra\u00C2\u00A0S,\u00C2\u00A0Toole\u00C2\u00A0BP.\u00C2\u00A0Hyaluronan\u00C2\u00A0 constitutively\u00C2\u00A0 regulates\u00C2\u00A0ErbB2\u00C2\u00A0phosphorylation\u00C2\u00A0and\u00C2\u00A0 signaling\u00C2\u00A0complex\u00C2\u00A0formation\u00C2\u00A0in\u00C2\u00A0carcinoma\u00C2\u00A0cells.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A02005\u00C2\u00A0Mar\u00C2\u00A011;280(10):8875\u00E2\u0080\u009083.\u00C2\u00A0 \u00C2\u00A0 113.\u00C2\u00A0Misra\u00C2\u00A0S,\u00C2\u00A0Ghatak\u00C2\u00A0S,\u00C2\u00A0Toole\u00C2\u00A0BP.\u00C2\u00A0Regulation\u00C2\u00A0of\u00C2\u00A0MDR1\u00C2\u00A0expression\u00C2\u00A0and\u00C2\u00A0drug\u00C2\u00A0resistance\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0positive\u00C2\u00A0 feedback\u00C2\u00A0 loop\u00C2\u00A0 involving\u00C2\u00A0hyaluronan,\u00C2\u00A0phosphoinositide\u00C2\u00A03\u00E2\u0080\u0090kinase,\u00C2\u00A0and\u00C2\u00A0ErbB2.\u00C2\u00A0 J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A02005\u00C2\u00A0 May\u00C2\u00A027;280(21):20310\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 114.\u00C2\u00A0Oliveira\u00C2\u00A0S,\u00C2\u00A0Schiffelers\u00C2\u00A0RM,\u00C2\u00A0van\u00C2\u00A0der\u00C2\u00A0Veeken\u00C2\u00A0 J,\u00C2\u00A0van\u00C2\u00A0der\u00C2\u00A0Meel\u00C2\u00A0R,\u00C2\u00A0Vongpromek\u00C2\u00A0R,\u00C2\u00A0van\u00C2\u00A0Bergen\u00C2\u00A0En\u00C2\u00A0 Henegouwen\u00C2\u00A0 PM,\u00C2\u00A0 Storm\u00C2\u00A0 G,\u00C2\u00A0 Roovers\u00C2\u00A0 RC.\u00C2\u00A0 Downregulation\u00C2\u00A0 of\u00C2\u00A0 EGFR\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 novel\u00C2\u00A0 multivalent\u00C2\u00A0 nanobody\u00E2\u0080\u0090liposome\u00C2\u00A0platform.\u00C2\u00A0J\u00C2\u00A0Control\u00C2\u00A0Release\u00C2\u00A02010\u00C2\u00A0Jul\u00C2\u00A014;145(2):165\u00E2\u0080\u009075.\u00C2\u00A0 \u00C2\u00A0 115.\u00C2\u00A0 Reff\u00C2\u00A0 ME,\u00C2\u00A0 Carner\u00C2\u00A0 K,\u00C2\u00A0 Chambers\u00C2\u00A0 KS,\u00C2\u00A0 Chinn\u00C2\u00A0 PC,\u00C2\u00A0 Leonard\u00C2\u00A0 JE,\u00C2\u00A0 Raab\u00C2\u00A0 R,\u00C2\u00A0 Newman\u00C2\u00A0 RA,\u00C2\u00A0 Hanna\u00C2\u00A0 N,\u00C2\u00A0 Anderson\u00C2\u00A0DR.\u00C2\u00A0Depletion\u00C2\u00A0of\u00C2\u00A0B\u00C2\u00A0cells\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0by\u00C2\u00A0a\u00C2\u00A0chimeric\u00C2\u00A0mouse\u00C2\u00A0human\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0to\u00C2\u00A0 CD20.\u00C2\u00A0Blood\u00C2\u00A01994\u00C2\u00A0Jan\u00C2\u00A015;83(2):435\u00E2\u0080\u009045.\u00C2\u00A0 \u00C2\u00A0 116.\u00C2\u00A0Gancz\u00C2\u00A0D,\u00C2\u00A0Fishelson\u00C2\u00A0Z.\u00C2\u00A0Cancer\u00C2\u00A0resistance\u00C2\u00A0to\u00C2\u00A0complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity\u00C2\u00A0(CDC):\u00C2\u00A0Problem\u00E2\u0080\u0090 oriented\u00C2\u00A0research\u00C2\u00A0and\u00C2\u00A0development.\u00C2\u00A0Mol\u00C2\u00A0Immunol\u00C2\u00A02009\u00C2\u00A0Sep;46(14):2794\u00E2\u0080\u0090800.\u00C2\u00A0 \u00C2\u00A0 117.\u00C2\u00A0Flieger\u00C2\u00A0D,\u00C2\u00A0Renoth\u00C2\u00A0S,\u00C2\u00A0Beier\u00C2\u00A0 I,\u00C2\u00A0Sauerbruch\u00C2\u00A0T,\u00C2\u00A0Schmidt\u00E2\u0080\u0090Wolf\u00C2\u00A0 I.\u00C2\u00A0Mechanism\u00C2\u00A0of\u00C2\u00A0cytotoxicity\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0chimeric\u00C2\u00A0mouse\u00C2\u00A0human\u00C2\u00A0monoclonal\u00C2\u00A0antibody\u00C2\u00A0IDEC\u00E2\u0080\u0090C2B8\u00C2\u00A0 in\u00C2\u00A0CD20\u00E2\u0080\u0090expressing\u00C2\u00A0 lymphoma\u00C2\u00A0cell\u00C2\u00A0 lines.\u00C2\u00A0Cell\u00C2\u00A0Immunol\u00C2\u00A02000\u00C2\u00A0Aug\u00C2\u00A025;204(1):55\u00E2\u0080\u009063.\u00C2\u00A0 \u00C2\u00A0 118.\u00C2\u00A0Di\u00C2\u00A0Gaetano\u00C2\u00A0N,\u00C2\u00A0Cittera\u00C2\u00A0E,\u00C2\u00A0Nota\u00C2\u00A0R,\u00C2\u00A0Vecchi\u00C2\u00A0A,\u00C2\u00A0Grieco\u00C2\u00A0V,\u00C2\u00A0Scanziani\u00C2\u00A0E,\u00C2\u00A0Botto\u00C2\u00A0M,\u00C2\u00A0 Introna\u00C2\u00A0M,\u00C2\u00A0Golay\u00C2\u00A0J.\u00C2\u00A0 Complement\u00C2\u00A0 activation\u00C2\u00A0 determines\u00C2\u00A0 the\u00C2\u00A0 therapeutic\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 rituximab\u00C2\u00A0 in\u00C2\u00A0 vivo.\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A0 2003\u00C2\u00A0Aug\u00C2\u00A01;171(3):1581\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 119.\u00C2\u00A0Kennedy\u00C2\u00A0AD,\u00C2\u00A0Beum\u00C2\u00A0PV,\u00C2\u00A0Solga\u00C2\u00A0MD,\u00C2\u00A0DiLillo\u00C2\u00A0DJ,\u00C2\u00A0Lindorfer\u00C2\u00A0MA,\u00C2\u00A0Hess\u00C2\u00A0CE,\u00C2\u00A0Densmore\u00C2\u00A0JJ,\u00C2\u00A0Williams\u00C2\u00A0ME,\u00C2\u00A0 Taylor\u00C2\u00A0RP.\u00C2\u00A0Rituximab\u00C2\u00A0 infusion\u00C2\u00A0promotes\u00C2\u00A0 rapid\u00C2\u00A0 complement\u00C2\u00A0depletion\u00C2\u00A0and\u00C2\u00A0acute\u00C2\u00A0CD20\u00C2\u00A0 loss\u00C2\u00A0 in\u00C2\u00A0 chronic\u00C2\u00A0lymphocytic\u00C2\u00A0leukemia.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A02004\u00C2\u00A0Mar\u00C2\u00A01;172(5):3280\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 120.\u00C2\u00A0Guo\u00C2\u00A0B,\u00C2\u00A0Ma\u00C2\u00A0ZW,\u00C2\u00A0Li\u00C2\u00A0H,\u00C2\u00A0Xu\u00C2\u00A0GL,\u00C2\u00A0Zheng\u00C2\u00A0P,\u00C2\u00A0Zhu\u00C2\u00A0B,\u00C2\u00A0Wu\u00C2\u00A0YZ,\u00C2\u00A0Zou\u00C2\u00A0Q.\u00C2\u00A0Mapping\u00C2\u00A0of\u00C2\u00A0binding\u00C2\u00A0epitopes\u00C2\u00A0of\u00C2\u00A0a\u00C2\u00A0 human\u00C2\u00A0 decay\u00E2\u0080\u0090accelerating\u00C2\u00A0 factor\u00C2\u00A0 monoclonal\u00C2\u00A0 antibody\u00C2\u00A0 capable\u00C2\u00A0 of\u00C2\u00A0 enhancing\u00C2\u00A0 rituximab\u00E2\u0080\u0090 mediated\u00C2\u00A0complement\u00E2\u0080\u0090dependent\u00C2\u00A0cytotoxicity.\u00C2\u00A0Clin\u00C2\u00A0Immunol\u00C2\u00A02008\u00C2\u00A0Aug;128(2):155\u00E2\u0080\u009063.\u00C2\u00A0 \u00C2\u00A0 121.\u00C2\u00A0Clynes\u00C2\u00A0RA,\u00C2\u00A0Towers\u00C2\u00A0TL,\u00C2\u00A0Presta\u00C2\u00A0LG,\u00C2\u00A0Ravetch\u00C2\u00A0JV.\u00C2\u00A0Inhibitory\u00C2\u00A0fc\u00C2\u00A0receptors\u00C2\u00A0modulate\u00C2\u00A0in\u00C2\u00A0vivo\u00C2\u00A0cytoxicity\u00C2\u00A0 against\u00C2\u00A0tumor\u00C2\u00A0targets.\u00C2\u00A0Nat\u00C2\u00A0Med\u00C2\u00A02000\u00C2\u00A0Apr;6(4):443\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0 122.\u00C2\u00A0Koene\u00C2\u00A0HR,\u00C2\u00A0Kleijer\u00C2\u00A0M,\u00C2\u00A0Algra\u00C2\u00A0 J,\u00C2\u00A0Roos\u00C2\u00A0D,\u00C2\u00A0von\u00C2\u00A0dem\u00C2\u00A0Borne\u00C2\u00A0AE,\u00C2\u00A0de\u00C2\u00A0Haas\u00C2\u00A0M.\u00C2\u00A0Fc\u00C2\u00A0gammaRIIIa\u00E2\u0080\u0090158V/F\u00C2\u00A0 polymorphism\u00C2\u00A0influences\u00C2\u00A0the\u00C2\u00A0binding\u00C2\u00A0of\u00C2\u00A0IgG\u00C2\u00A0by\u00C2\u00A0natural\u00C2\u00A0killer\u00C2\u00A0cell\u00C2\u00A0fc\u00C2\u00A0gammaRIIIa,\u00C2\u00A0independently\u00C2\u00A0 of\u00C2\u00A0the\u00C2\u00A0fc\u00C2\u00A0gammaRIIIa\u00E2\u0080\u009048L/R/H\u00C2\u00A0phenotype.\u00C2\u00A0Blood\u00C2\u00A01997\u00C2\u00A0Aug\u00C2\u00A01;90(3):1109\u00E2\u0080\u009014.\u00C2\u00A0 \u00C2\u00A0 123.\u00C2\u00A0Cartron\u00C2\u00A0G,\u00C2\u00A0Dacheux\u00C2\u00A0L,\u00C2\u00A0Salles\u00C2\u00A0G,\u00C2\u00A0Solal\u00E2\u0080\u0090Celigny\u00C2\u00A0P,\u00C2\u00A0Bardos\u00C2\u00A0P,\u00C2\u00A0Colombat\u00C2\u00A0P,\u00C2\u00A0Watier\u00C2\u00A0H.\u00C2\u00A0Therapeutic\u00C2\u00A0 activity\u00C2\u00A0 of\u00C2\u00A0 humanized\u00C2\u00A0 anti\u00E2\u0080\u0090CD20\u00C2\u00A0monoclonal\u00C2\u00A0 antibody\u00C2\u00A0 and\u00C2\u00A0 polymorphism\u00C2\u00A0 in\u00C2\u00A0 IgG\u00C2\u00A0 fc\u00C2\u00A0 receptor\u00C2\u00A0 FcgammaRIIIa\u00C2\u00A0gene.\u00C2\u00A0Blood\u00C2\u00A02002\u00C2\u00A0Feb\u00C2\u00A01;99(3):754\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 124.\u00C2\u00A0 Inagaki\u00C2\u00A0A,\u00C2\u00A0 Ishida\u00C2\u00A0T,\u00C2\u00A0Yano\u00C2\u00A0H,\u00C2\u00A0 Ishii\u00C2\u00A0T,\u00C2\u00A0Kusumoto\u00C2\u00A0S,\u00C2\u00A0 Ito\u00C2\u00A0A,\u00C2\u00A0Ri\u00C2\u00A0M,\u00C2\u00A0Mori\u00C2\u00A0F,\u00C2\u00A0Ding\u00C2\u00A0J,\u00C2\u00A0Komatsu\u00C2\u00A0H,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0 Expression\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0ULBP\u00C2\u00A0ligands\u00C2\u00A0for\u00C2\u00A0NKG2D\u00C2\u00A0by\u00C2\u00A0B\u00E2\u0080\u0090NHL\u00C2\u00A0cells\u00C2\u00A0plays\u00C2\u00A0an\u00C2\u00A0important\u00C2\u00A0role\u00C2\u00A0in\u00C2\u00A0determining\u00C2\u00A0 their\u00C2\u00A0susceptibility\u00C2\u00A0to\u00C2\u00A0rituximab\u00E2\u0080\u0090induced\u00C2\u00A0ADCC.\u00C2\u00A0Int\u00C2\u00A0J\u00C2\u00A0Cancer\u00C2\u00A02009\u00C2\u00A0Jul\u00C2\u00A01;125(1):212\u00E2\u0080\u009021.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0183\u00C2\u00A0\u00C2\u00A0 125.\u00C2\u00A0 Carbone\u00C2\u00A0 E,\u00C2\u00A0 Ruggiero\u00C2\u00A0 G,\u00C2\u00A0 Terrazzano\u00C2\u00A0 G,\u00C2\u00A0 Palomba\u00C2\u00A0 C,\u00C2\u00A0 Manzo\u00C2\u00A0 C,\u00C2\u00A0 Fontana\u00C2\u00A0 S,\u00C2\u00A0 Spits\u00C2\u00A0 H,\u00C2\u00A0 Karre\u00C2\u00A0 K,\u00C2\u00A0 Zappacosta\u00C2\u00A0 S.\u00C2\u00A0 A\u00C2\u00A0 new\u00C2\u00A0 mechanism\u00C2\u00A0 of\u00C2\u00A0 NK\u00C2\u00A0 cell\u00C2\u00A0 cytotoxicity\u00C2\u00A0 activation:\u00C2\u00A0 The\u00C2\u00A0 CD40\u00E2\u0080\u0090CD40\u00C2\u00A0 ligand\u00C2\u00A0 interaction.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A01997\u00C2\u00A0Jun\u00C2\u00A016;185(12):2053\u00E2\u0080\u009060.\u00C2\u00A0 \u00C2\u00A0 126.\u00C2\u00A0Wilson\u00C2\u00A0 JL,\u00C2\u00A0 Charo\u00C2\u00A0 J,\u00C2\u00A0Martin\u00E2\u0080\u0090Fontecha\u00C2\u00A0 A,\u00C2\u00A0 Dellabona\u00C2\u00A0 P,\u00C2\u00A0 Casorati\u00C2\u00A0 G,\u00C2\u00A0 Chambers\u00C2\u00A0 BJ,\u00C2\u00A0 Kiessling\u00C2\u00A0 R,\u00C2\u00A0 Bejarano\u00C2\u00A0MT,\u00C2\u00A0Ljunggren\u00C2\u00A0HG.\u00C2\u00A0NK\u00C2\u00A0cell\u00C2\u00A0triggering\u00C2\u00A0by\u00C2\u00A0the\u00C2\u00A0human\u00C2\u00A0costimulatory\u00C2\u00A0molecules\u00C2\u00A0CD80\u00C2\u00A0and\u00C2\u00A0 CD86.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A01999\u00C2\u00A0Oct\u00C2\u00A015;163(8):4207\u00E2\u0080\u009012.\u00C2\u00A0 \u00C2\u00A0 127.\u00C2\u00A0Tucker\u00C2\u00A0CA,\u00C2\u00A0Bebb\u00C2\u00A0G,\u00C2\u00A0Klasa\u00C2\u00A0RJ,\u00C2\u00A0Chhanabhai\u00C2\u00A0M,\u00C2\u00A0Lestou\u00C2\u00A0V,\u00C2\u00A0Horsman\u00C2\u00A0DE,\u00C2\u00A0Gascoyne\u00C2\u00A0RD,\u00C2\u00A0Wiestner\u00C2\u00A0A,\u00C2\u00A0 Masin\u00C2\u00A0D,\u00C2\u00A0Bally\u00C2\u00A0M,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Four\u00C2\u00A0human\u00C2\u00A0 t(11;14)(q13;q32)\u00E2\u0080\u0090containing\u00C2\u00A0cell\u00C2\u00A0 lines\u00C2\u00A0having\u00C2\u00A0classic\u00C2\u00A0and\u00C2\u00A0 variant\u00C2\u00A0features\u00C2\u00A0of\u00C2\u00A0mantle\u00C2\u00A0cell\u00C2\u00A0lymphoma.\u00C2\u00A0Leuk\u00C2\u00A0Res\u00C2\u00A02006\u00C2\u00A0Apr;30(4):449\u00E2\u0080\u009057.\u00C2\u00A0 \u00C2\u00A0 128.\u00C2\u00A0 Hope\u00C2\u00A0MJ,\u00C2\u00A0 Bally\u00C2\u00A0MB,\u00C2\u00A0Webb\u00C2\u00A0 G,\u00C2\u00A0 Cullis\u00C2\u00A0 PR.\u00C2\u00A0 Production\u00C2\u00A0 of\u00C2\u00A0 large\u00C2\u00A0 unilamellar\u00C2\u00A0 vesicles\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 rapid\u00C2\u00A0 extrusion\u00C2\u00A0 procedure.\u00C2\u00A0 Characterization\u00C2\u00A0 of\u00C2\u00A0 size\u00C2\u00A0 distribution,\u00C2\u00A0 trapped\u00C2\u00A0 volume\u00C2\u00A0 and\u00C2\u00A0 ability\u00C2\u00A0 to\u00C2\u00A0 maintain\u00C2\u00A0a\u00C2\u00A0membrane\u00C2\u00A0potential.\u00C2\u00A0BBA\u00E2\u0080\u0090Biomembranes\u00C2\u00A01985;812(1):55\u00E2\u0080\u009065.\u00C2\u00A0 \u00C2\u00A0 129.\u00C2\u00A0 Mayer\u00C2\u00A0 LD,\u00C2\u00A0 Hope\u00C2\u00A0 MJ,\u00C2\u00A0 Cullis\u00C2\u00A0 PR.\u00C2\u00A0 Vesicles\u00C2\u00A0 of\u00C2\u00A0 variable\u00C2\u00A0 sizes\u00C2\u00A0 produced\u00C2\u00A0 by\u00C2\u00A0 a\u00C2\u00A0 rapid\u00C2\u00A0 extrusion\u00C2\u00A0 procedure.\u00C2\u00A0Biochim\u00C2\u00A0Biophys\u00C2\u00A0Acta\u00C2\u00A01986\u00C2\u00A0Jun\u00C2\u00A013;858(1):161\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 130.\u00C2\u00A0Chiu\u00C2\u00A0GN,\u00C2\u00A0Bally\u00C2\u00A0MB,\u00C2\u00A0Mayer\u00C2\u00A0LD.\u00C2\u00A0Targeting\u00C2\u00A0of\u00C2\u00A0antibody\u00C2\u00A0conjugated,\u00C2\u00A0phosphatidylserine\u00E2\u0080\u0090containing\u00C2\u00A0 liposomes\u00C2\u00A0to\u00C2\u00A0vascular\u00C2\u00A0cell\u00C2\u00A0adhesion\u00C2\u00A0molecule\u00C2\u00A01\u00C2\u00A0for\u00C2\u00A0controlled\u00C2\u00A0thrombogenesis.\u00C2\u00A0Biochim\u00C2\u00A0Biophys\u00C2\u00A0 Acta\u00C2\u00A02003\u00C2\u00A0Jun\u00C2\u00A027;1613(1\u00E2\u0080\u00902):115\u00E2\u0080\u009021.\u00C2\u00A0 \u00C2\u00A0 131.\u00C2\u00A0Gomez\u00E2\u0080\u0090Roman\u00C2\u00A0VR,\u00C2\u00A0Florese\u00C2\u00A0RH,\u00C2\u00A0Patterson\u00C2\u00A0LJ,\u00C2\u00A0Peng\u00C2\u00A0B,\u00C2\u00A0Venzon\u00C2\u00A0D,\u00C2\u00A0Aldrich\u00C2\u00A0K,\u00C2\u00A0Robert\u00E2\u0080\u0090Guroff\u00C2\u00A0M.\u00C2\u00A0A\u00C2\u00A0 simplified\u00C2\u00A0method\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0rapid\u00C2\u00A0fluorometric\u00C2\u00A0assessment\u00C2\u00A0of\u00C2\u00A0antibody\u00E2\u0080\u0090dependent\u00C2\u00A0cell\u00E2\u0080\u0090mediated\u00C2\u00A0 cytotoxicity.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0Methods\u00C2\u00A02006\u00C2\u00A0Jan\u00C2\u00A020;308(1\u00E2\u0080\u00902):53\u00E2\u0080\u009067.\u00C2\u00A0 \u00C2\u00A0 132.\u00C2\u00A0Estrov\u00C2\u00A0Z,\u00C2\u00A0Talpaz\u00C2\u00A0M,\u00C2\u00A0Ku\u00C2\u00A0S,\u00C2\u00A0Harris\u00C2\u00A0D,\u00C2\u00A0Van\u00C2\u00A0Q,\u00C2\u00A0Beran\u00C2\u00A0M,\u00C2\u00A0Hirsch\u00E2\u0080\u0090Ginsberg\u00C2\u00A0C,\u00C2\u00A0Huh\u00C2\u00A0Y,\u00C2\u00A0Yee\u00C2\u00A0G,\u00C2\u00A0Kurzrock\u00C2\u00A0R.\u00C2\u00A0 Z\u00E2\u0080\u0090138:\u00C2\u00A0 A\u00C2\u00A0 new\u00C2\u00A0 mature\u00C2\u00A0 B\u00E2\u0080\u0090cell\u00C2\u00A0 acute\u00C2\u00A0 lymphoblastic\u00C2\u00A0 leukemia\u00C2\u00A0 cell\u00C2\u00A0 line\u00C2\u00A0 from\u00C2\u00A0 a\u00C2\u00A0 patient\u00C2\u00A0 with\u00C2\u00A0 transformed\u00C2\u00A0chronic\u00C2\u00A0lymphocytic\u00C2\u00A0leukemia.\u00C2\u00A0Leuk\u00C2\u00A0Res\u00C2\u00A01998\u00C2\u00A0Apr;22(4):341\u00E2\u0080\u009053.\u00C2\u00A0 \u00C2\u00A0 133.\u00C2\u00A0Melo\u00C2\u00A0 JV,\u00C2\u00A0 Foroni\u00C2\u00A0 L,\u00C2\u00A0Brito\u00E2\u0080\u0090Babapulle\u00C2\u00A0V,\u00C2\u00A0 Luzzatto\u00C2\u00A0 L,\u00C2\u00A0Catovsky\u00C2\u00A0D.\u00C2\u00A0The\u00C2\u00A0establishment\u00C2\u00A0of\u00C2\u00A0 cell\u00C2\u00A0 lines\u00C2\u00A0 from\u00C2\u00A0chronic\u00C2\u00A0B\u00C2\u00A0cell\u00C2\u00A0leukaemias:\u00C2\u00A0Evidence\u00C2\u00A0of\u00C2\u00A0leukaemic\u00C2\u00A0origin\u00C2\u00A0by\u00C2\u00A0karyotypic\u00C2\u00A0abnormalities\u00C2\u00A0and\u00C2\u00A0ig\u00C2\u00A0 gene\u00C2\u00A0rearrangement.\u00C2\u00A0Clin\u00C2\u00A0Exp\u00C2\u00A0Immunol\u00C2\u00A01988\u00C2\u00A0Jul;73(1):23\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 134.\u00C2\u00A0 Bowles\u00C2\u00A0 JA,\u00C2\u00A0 Weiner\u00C2\u00A0 GJ.\u00C2\u00A0 CD16\u00C2\u00A0 polymorphisms\u00C2\u00A0 and\u00C2\u00A0 NK\u00C2\u00A0 activation\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0 monoclonal\u00C2\u00A0 antibody\u00E2\u0080\u0090coated\u00C2\u00A0target\u00C2\u00A0cells.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0Methods\u00C2\u00A02005\u00C2\u00A0Sep;304(1\u00E2\u0080\u00902):88\u00E2\u0080\u009099.\u00C2\u00A0 \u00C2\u00A0 135.\u00C2\u00A0Welsh\u00C2\u00A0RM,\u00C2\u00A0Brubaker\u00C2\u00A0 JO,\u00C2\u00A0Vargas\u00E2\u0080\u0090Cortes\u00C2\u00A0M,\u00C2\u00A0O'Donnell\u00C2\u00A0CL.\u00C2\u00A0Natural\u00C2\u00A0 killer\u00C2\u00A0 (NK)\u00C2\u00A0 cell\u00C2\u00A0 response\u00C2\u00A0 to\u00C2\u00A0 virus\u00C2\u00A0 infections\u00C2\u00A0 in\u00C2\u00A0mice\u00C2\u00A0with\u00C2\u00A0 severe\u00C2\u00A0 combined\u00C2\u00A0 immunodeficiency.\u00C2\u00A0 the\u00C2\u00A0 stimulation\u00C2\u00A0of\u00C2\u00A0NK\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 NK\u00C2\u00A0 cell\u00E2\u0080\u0090dependent\u00C2\u00A0 control\u00C2\u00A0 of\u00C2\u00A0 virus\u00C2\u00A0 infections\u00C2\u00A0 occur\u00C2\u00A0 independently\u00C2\u00A0 of\u00C2\u00A0 T\u00C2\u00A0 and\u00C2\u00A0 B\u00C2\u00A0 cell\u00C2\u00A0 function.\u00C2\u00A0J\u00C2\u00A0Exp\u00C2\u00A0Med\u00C2\u00A01991\u00C2\u00A0May\u00C2\u00A01;173(5):1053\u00E2\u0080\u009063.\u00C2\u00A0 \u00C2\u00A0 136.\u00C2\u00A0Dayde\u00C2\u00A0D,\u00C2\u00A0Ternant\u00C2\u00A0D,\u00C2\u00A0Ohresser\u00C2\u00A0M,\u00C2\u00A0Lerondel\u00C2\u00A0S,\u00C2\u00A0Pesnel\u00C2\u00A0S,\u00C2\u00A0Watier\u00C2\u00A0H,\u00C2\u00A0Le\u00C2\u00A0Pape\u00C2\u00A0A,\u00C2\u00A0Bardos\u00C2\u00A0P,\u00C2\u00A0Paintaud\u00C2\u00A0 G,\u00C2\u00A0Cartron\u00C2\u00A0G.\u00C2\u00A0Tumor\u00C2\u00A0burden\u00C2\u00A0influences\u00C2\u00A0exposure\u00C2\u00A0and\u00C2\u00A0response\u00C2\u00A0to\u00C2\u00A0rituximab:\u00C2\u00A0Pharmacokinetic\u00E2\u0080\u0090 pharmacodynamic\u00C2\u00A0modeling\u00C2\u00A0using\u00C2\u00A0a\u00C2\u00A0syngeneic\u00C2\u00A0bioluminescent\u00C2\u00A0murine\u00C2\u00A0model\u00C2\u00A0expressing\u00C2\u00A0human\u00C2\u00A0 CD20.\u00C2\u00A0Blood\u00C2\u00A02009\u00C2\u00A0Apr\u00C2\u00A016;113(16):3765\u00E2\u0080\u009072.\u00C2\u00A0 \u00C2\u00A0 137.\u00C2\u00A0 Sofou\u00C2\u00A0 S.\u00C2\u00A0 Surface\u00E2\u0080\u0090active\u00C2\u00A0 liposomes\u00C2\u00A0 for\u00C2\u00A0 targeted\u00C2\u00A0 cancer\u00C2\u00A0 therapy.\u00C2\u00A0 Nanomedicine\u00C2\u00A0 2007\u00C2\u00A0 Oct;2(5):711\u00E2\u0080\u009024.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0184\u00C2\u00A0\u00C2\u00A0 \u00C2\u00A0 138.\u00C2\u00A0Young\u00C2\u00A0LS,\u00C2\u00A0Rickinson\u00C2\u00A0AB.\u00C2\u00A0Epstein\u00E2\u0080\u0090barr\u00C2\u00A0virus:\u00C2\u00A040\u00C2\u00A0years\u00C2\u00A0on.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Cancer\u00C2\u00A02004\u00C2\u00A0Oct;4(10):757\u00E2\u0080\u009068.\u00C2\u00A0 \u00C2\u00A0 139.\u00C2\u00A0Howard\u00C2\u00A0OM,\u00C2\u00A0Gribben\u00C2\u00A0JG,\u00C2\u00A0Neuberg\u00C2\u00A0DS,\u00C2\u00A0Grossbard\u00C2\u00A0M,\u00C2\u00A0Poor\u00C2\u00A0C,\u00C2\u00A0Janicek\u00C2\u00A0MJ,\u00C2\u00A0Shipp\u00C2\u00A0MA.\u00C2\u00A0Rituximab\u00C2\u00A0 and\u00C2\u00A0CHOP\u00C2\u00A0 induction\u00C2\u00A0therapy\u00C2\u00A0 for\u00C2\u00A0newly\u00C2\u00A0diagnosed\u00C2\u00A0mantle\u00E2\u0080\u0090cell\u00C2\u00A0 lymphoma:\u00C2\u00A0Molecular\u00C2\u00A0complete\u00C2\u00A0 responses\u00C2\u00A0are\u00C2\u00A0not\u00C2\u00A0predictive\u00C2\u00A0of\u00C2\u00A0progression\u00E2\u0080\u0090free\u00C2\u00A0survival.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A02002\u00C2\u00A0Mar\u00C2\u00A01;20(5):1288\u00E2\u0080\u0090 94.\u00C2\u00A0 \u00C2\u00A0 140.\u00C2\u00A0Cheson\u00C2\u00A0BD,\u00C2\u00A0 Leonard\u00C2\u00A0 JP.\u00C2\u00A0Monoclonal\u00C2\u00A0antibody\u00C2\u00A0 therapy\u00C2\u00A0 for\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0non\u00E2\u0080\u0090hodgkin's\u00C2\u00A0 lymphoma.\u00C2\u00A0N\u00C2\u00A0 Engl\u00C2\u00A0J\u00C2\u00A0Med\u00C2\u00A02008\u00C2\u00A0Aug\u00C2\u00A07;359(6):613\u00E2\u0080\u009026.\u00C2\u00A0 \u00C2\u00A0 141.\u00C2\u00A0 Allen\u00C2\u00A0 TM,\u00C2\u00A0 Brandeis\u00C2\u00A0 E,\u00C2\u00A0 Hansen\u00C2\u00A0 CB,\u00C2\u00A0 Kao\u00C2\u00A0 GY,\u00C2\u00A0 Zalipsky\u00C2\u00A0 S.\u00C2\u00A0 A\u00C2\u00A0 new\u00C2\u00A0 strategy\u00C2\u00A0 for\u00C2\u00A0 attachment\u00C2\u00A0 of\u00C2\u00A0 antibodies\u00C2\u00A0 to\u00C2\u00A0 sterically\u00C2\u00A0 stabilized\u00C2\u00A0 liposomes\u00C2\u00A0 resulting\u00C2\u00A0 in\u00C2\u00A0 efficient\u00C2\u00A0 targeting\u00C2\u00A0 to\u00C2\u00A0 cancer\u00C2\u00A0 cells.\u00C2\u00A0 Biochim\u00C2\u00A0Biophys\u00C2\u00A0Acta\u00C2\u00A01995\u00C2\u00A0Jul\u00C2\u00A026;1237(2):99\u00E2\u0080\u0090108.\u00C2\u00A0 \u00C2\u00A0 142.\u00C2\u00A0Ishida\u00C2\u00A0T,\u00C2\u00A0Iden\u00C2\u00A0DL,\u00C2\u00A0Allen\u00C2\u00A0TM.\u00C2\u00A0A\u00C2\u00A0combinatorial\u00C2\u00A0approach\u00C2\u00A0to\u00C2\u00A0producing\u00C2\u00A0sterically\u00C2\u00A0stabilized\u00C2\u00A0(stealth)\u00C2\u00A0 immunoliposomal\u00C2\u00A0drugs.\u00C2\u00A0FEBS\u00C2\u00A0Lett\u00C2\u00A01999\u00C2\u00A0Oct\u00C2\u00A022;460(1):129\u00E2\u0080\u009033.\u00C2\u00A0 \u00C2\u00A0 143.\u00C2\u00A0Karve\u00C2\u00A0S,\u00C2\u00A0Alaouie\u00C2\u00A0A,\u00C2\u00A0Zhou\u00C2\u00A0Y,\u00C2\u00A0Rotolo\u00C2\u00A0J,\u00C2\u00A0Sofou\u00C2\u00A0S.\u00C2\u00A0The\u00C2\u00A0use\u00C2\u00A0of\u00C2\u00A0pH\u00E2\u0080\u0090triggered\u00C2\u00A0leaky\u00C2\u00A0heterogeneities\u00C2\u00A0on\u00C2\u00A0 rigid\u00C2\u00A0 lipid\u00C2\u00A0 bilayers\u00C2\u00A0 to\u00C2\u00A0 improve\u00C2\u00A0 intracellular\u00C2\u00A0 trafficking\u00C2\u00A0 and\u00C2\u00A0 therapeutic\u00C2\u00A0 potential\u00C2\u00A0 of\u00C2\u00A0 targeted\u00C2\u00A0 liposomal\u00C2\u00A0immunochemotherapy.\u00C2\u00A0Biomaterials\u00C2\u00A02009\u00C2\u00A010;30(30):6055\u00E2\u0080\u009064.\u00C2\u00A0 \u00C2\u00A0 144.\u00C2\u00A0Hansen\u00C2\u00A0CB,\u00C2\u00A0 Kao\u00C2\u00A0GY,\u00C2\u00A0Moase\u00C2\u00A0 EH,\u00C2\u00A0 Zalipsky\u00C2\u00A0 S,\u00C2\u00A0Allen\u00C2\u00A0 TM.\u00C2\u00A0Attachment\u00C2\u00A0of\u00C2\u00A0 antibodies\u00C2\u00A0 to\u00C2\u00A0 sterically\u00C2\u00A0 stabilized\u00C2\u00A0 liposomes:\u00C2\u00A0 Evaluation,\u00C2\u00A0 comparison\u00C2\u00A0 and\u00C2\u00A0 optimization\u00C2\u00A0 of\u00C2\u00A0 coupling\u00C2\u00A0 procedures.\u00C2\u00A0 BBA\u00E2\u0080\u0090 Biomembranes\u00C2\u00A01995;1239(2):133\u00E2\u0080\u009044.\u00C2\u00A0 \u00C2\u00A0 145.\u00C2\u00A0Moreira\u00C2\u00A0JN,\u00C2\u00A0Ishida\u00C2\u00A0T,\u00C2\u00A0Gaspar\u00C2\u00A0R,\u00C2\u00A0Allen\u00C2\u00A0TM.\u00C2\u00A0Use\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0post\u00E2\u0080\u0090insertion\u00C2\u00A0technique\u00C2\u00A0to\u00C2\u00A0insert\u00C2\u00A0peptide\u00C2\u00A0 ligands\u00C2\u00A0 into\u00C2\u00A0pre\u00E2\u0080\u0090formed\u00C2\u00A0 stealth\u00C2\u00A0 liposomes\u00C2\u00A0with\u00C2\u00A0 retention\u00C2\u00A0of\u00C2\u00A0binding\u00C2\u00A0activity\u00C2\u00A0and\u00C2\u00A0 cytotoxicity.\u00C2\u00A0 Pharm\u00C2\u00A0Res\u00C2\u00A02002\u00C2\u00A0Mar;19(3):265\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 146.\u00C2\u00A0Loughrey\u00C2\u00A0HC,\u00C2\u00A0Choi\u00C2\u00A0LS,\u00C2\u00A0Wong\u00C2\u00A0KF,\u00C2\u00A0Cullis\u00C2\u00A0PR,\u00C2\u00A0Bally\u00C2\u00A0MB.\u00C2\u00A0Preparation\u00C2\u00A0of\u00C2\u00A0streptavidin\u00E2\u0080\u0090liposomes\u00C2\u00A0for\u00C2\u00A0 use\u00C2\u00A0 in\u00C2\u00A0 ligand\u00E2\u0080\u0090specific\u00C2\u00A0 targeting\u00C2\u00A0 applications.\u00C2\u00A0 In:\u00C2\u00A0 Gregory\u00C2\u00A0 Gregoriadis,\u00C2\u00A0 editor.\u00C2\u00A0 Liposome\u00C2\u00A0 technology:\u00C2\u00A0Vol.\u00C2\u00A03:\u00C2\u00A0 Interactions\u00C2\u00A0of\u00C2\u00A0 liposomes\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0biological\u00C2\u00A0milieu.\u00C2\u00A02nd\u00C2\u00A0ed.\u00C2\u00A0Boca\u00C2\u00A0Raton,\u00C2\u00A0 Fla.:\u00C2\u00A0CRC\u00C2\u00A0Press;\u00C2\u00A01993.\u00C2\u00A0 \u00C2\u00A0 147.\u00C2\u00A0 Wen\u00C2\u00A0 H,\u00C2\u00A0 DeCory\u00C2\u00A0 TR,\u00C2\u00A0 Borejsza\u00E2\u0080\u0090Wysocki\u00C2\u00A0 W,\u00C2\u00A0 Durst\u00C2\u00A0 RA.\u00C2\u00A0 Investigation\u00C2\u00A0 of\u00C2\u00A0 NeutrAvidin\u00E2\u0080\u0090tagged\u00C2\u00A0 liposomal\u00C2\u00A0nanovesicles\u00C2\u00A0as\u00C2\u00A0universal\u00C2\u00A0detection\u00C2\u00A0 reagents\u00C2\u00A0 for\u00C2\u00A0bioanalytical\u00C2\u00A0assays.\u00C2\u00A0Talanta,\u00C2\u00A02006\u00C2\u00A0 Feb\u00C2\u00A015;68(4):1264\u00E2\u0080\u009072.\u00C2\u00A0 \u00C2\u00A0 148.\u00C2\u00A0Ke\u00C2\u00A0S,\u00C2\u00A0Wright\u00C2\u00A0JC,\u00C2\u00A0Kwon\u00C2\u00A0GS.\u00C2\u00A0Intermolecular\u00C2\u00A0interaction\u00C2\u00A0of\u00C2\u00A0avidin\u00C2\u00A0and\u00C2\u00A0PEGylated\u00C2\u00A0biotin.\u00C2\u00A0Bioconjug\u00C2\u00A0 Chem\u00C2\u00A02007;18(6):2109\u00E2\u0080\u009014.\u00C2\u00A0 \u00C2\u00A0 149.\u00C2\u00A0Strachan\u00C2\u00A0E,\u00C2\u00A0Mallia\u00C2\u00A0AK,\u00C2\u00A0Cox\u00C2\u00A0 JM,\u00C2\u00A0Antharavally\u00C2\u00A0B,\u00C2\u00A0Desai\u00C2\u00A0S,\u00C2\u00A0Sykaluk\u00C2\u00A0L,\u00C2\u00A0O'Sullivan\u00C2\u00A0V,\u00C2\u00A0Bell\u00C2\u00A0PA.\u00C2\u00A0Solid\u00E2\u0080\u0090 phase\u00C2\u00A0biotinylation\u00C2\u00A0of\u00C2\u00A0antibodies.\u00C2\u00A0J\u00C2\u00A0Mol\u00C2\u00A0Recognit\u00C2\u00A02004\u00C2\u00A0May\u00E2\u0080\u0090Jun;17(3):268\u00E2\u0080\u009076.\u00C2\u00A0 \u00C2\u00A0 150.\u00C2\u00A0 You\u00C2\u00A0 WW,\u00C2\u00A0 Haugland\u00C2\u00A0 RP,\u00C2\u00A0 Ryan\u00C2\u00A0 DK,\u00C2\u00A0 Haugland\u00C2\u00A0 RP.\u00C2\u00A0 3\u00E2\u0080\u0090(4\u00E2\u0080\u0090carboxybenzoyl)quinoline\u00E2\u0080\u00902\u00E2\u0080\u0090 carboxaldehyde,\u00C2\u00A0a\u00C2\u00A0reagent\u00C2\u00A0with\u00C2\u00A0broad\u00C2\u00A0dynamic\u00C2\u00A0range\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0assay\u00C2\u00A0of\u00C2\u00A0proteins\u00C2\u00A0and\u00C2\u00A0lipoproteins\u00C2\u00A0 in\u00C2\u00A0solution.\u00C2\u00A0Anal\u00C2\u00A0Biochem\u00C2\u00A01997\u00C2\u00A0Jan\u00C2\u00A015;244(2):277\u00E2\u0080\u009082.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0185\u00C2\u00A0\u00C2\u00A0 151.\u00C2\u00A0 Nag\u00C2\u00A0 A,\u00C2\u00A0Mitra\u00C2\u00A0 G,\u00C2\u00A0 Ghosh\u00C2\u00A0 PC.\u00C2\u00A0 A\u00C2\u00A0 colorimetric\u00C2\u00A0 assay\u00C2\u00A0 for\u00C2\u00A0 estimation\u00C2\u00A0 of\u00C2\u00A0 polyethylene\u00C2\u00A0 glycol\u00C2\u00A0 and\u00C2\u00A0 polyethylene\u00C2\u00A0 glycolated\u00C2\u00A0 protein\u00C2\u00A0 using\u00C2\u00A0 ammonium\u00C2\u00A0 ferrothiocyanate.\u00C2\u00A0 Anal\u00C2\u00A0 Biochem\u00C2\u00A0 1996\u00C2\u00A0 Jun\u00C2\u00A0 1;237(2):224\u00E2\u0080\u009031.\u00C2\u00A0 \u00C2\u00A0 152.\u00C2\u00A0Lawrence\u00C2\u00A0MJ,\u00C2\u00A0Barlow\u00C2\u00A0DJ,\u00C2\u00A0Harvey\u00C2\u00A0RD,\u00C2\u00A0Heenan\u00C2\u00A0RK.\u00C2\u00A0Development\u00C2\u00A0of\u00C2\u00A0non\u00E2\u0080\u0090ionic\u00C2\u00A0surfactant\u00C2\u00A0vesicles\u00C2\u00A0 as\u00C2\u00A0drug\u00C2\u00A0delivery\u00C2\u00A0vehicles.\u00C2\u00A0In:\u00C2\u00A0B.\u00C2\u00A0H.\u00C2\u00A0Robinson,\u00C2\u00A0editor.\u00C2\u00A0Self\u00E2\u0080\u0090assembly.\u00C2\u00A0Amsterdam:\u00C2\u00A0IOS\u00C2\u00A0Press;\u00C2\u00A02003.\u00C2\u00A0 \u00C2\u00A0 153.\u00C2\u00A0Moghimi\u00C2\u00A0SM,\u00C2\u00A0Hunter\u00C2\u00A0AC,\u00C2\u00A0Murray\u00C2\u00A0JC.\u00C2\u00A0Long\u00E2\u0080\u0090circulating\u00C2\u00A0and\u00C2\u00A0target\u00E2\u0080\u0090specific\u00C2\u00A0nanoparticles:\u00C2\u00A0Theory\u00C2\u00A0 to\u00C2\u00A0practice.\u00C2\u00A0Pharmacol\u00C2\u00A0Rev\u00C2\u00A02001\u00C2\u00A0Jun\u00C2\u00A01;53(2):283\u00E2\u0080\u0090318.\u00C2\u00A0 \u00C2\u00A0 154.\u00C2\u00A0Ying\u00C2\u00A0X,\u00C2\u00A0Wen\u00C2\u00A0H,\u00C2\u00A0Lu\u00C2\u00A0W,\u00C2\u00A0Du\u00C2\u00A0J,\u00C2\u00A0Guo\u00C2\u00A0J,\u00C2\u00A0Tian\u00C2\u00A0W,\u00C2\u00A0Men\u00C2\u00A0Y,\u00C2\u00A0Zhang\u00C2\u00A0Y,\u00C2\u00A0Li\u00C2\u00A0R,\u00C2\u00A0Yang\u00C2\u00A0T,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Dual\u00E2\u0080\u0090targeting\u00C2\u00A0 daunorubicin\u00C2\u00A0 liposomes\u00C2\u00A0 improve\u00C2\u00A0the\u00C2\u00A0therapeutic\u00C2\u00A0efficacy\u00C2\u00A0of\u00C2\u00A0brain\u00C2\u00A0glioma\u00C2\u00A0 in\u00C2\u00A0animals.\u00C2\u00A0J\u00C2\u00A0Control\u00C2\u00A0 Release\u00C2\u00A02010\u00C2\u00A0Jan\u00C2\u00A025;141(2):183\u00E2\u0080\u009092.\u00C2\u00A0 \u00C2\u00A0 155.\u00C2\u00A0Yang\u00C2\u00A0T,\u00C2\u00A0Choi\u00C2\u00A0MK,\u00C2\u00A0Cui\u00C2\u00A0FD,\u00C2\u00A0Kim\u00C2\u00A0 JS,\u00C2\u00A0Chung\u00C2\u00A0SJ,\u00C2\u00A0Shim\u00C2\u00A0CK,\u00C2\u00A0Kim\u00C2\u00A0DD.\u00C2\u00A0Preparation\u00C2\u00A0and\u00C2\u00A0evaluation\u00C2\u00A0of\u00C2\u00A0 paclitaxel\u00E2\u0080\u0090loaded\u00C2\u00A0PEGylated\u00C2\u00A0immunoliposome.\u00C2\u00A0J\u00C2\u00A0Control\u00C2\u00A0Release\u00C2\u00A02007\u00C2\u00A0Jul\u00C2\u00A031;120(3):169\u00E2\u0080\u009077.\u00C2\u00A0 \u00C2\u00A0 156.\u00C2\u00A0Epstein\u00C2\u00A0H,\u00C2\u00A0Gutman\u00C2\u00A0D,\u00C2\u00A0Cohen\u00E2\u0080\u0090Sela\u00C2\u00A0E,\u00C2\u00A0Haber\u00C2\u00A0E,\u00C2\u00A0Elmalak\u00C2\u00A0O,\u00C2\u00A0Koroukhov\u00C2\u00A0N,\u00C2\u00A0Danenberg\u00C2\u00A0HD,\u00C2\u00A0Golomb\u00C2\u00A0 G.\u00C2\u00A0Preparation\u00C2\u00A0of\u00C2\u00A0alendronate\u00C2\u00A0 liposomes\u00C2\u00A0for\u00C2\u00A0enhanced\u00C2\u00A0stability\u00C2\u00A0and\u00C2\u00A0bioactivity:\u00C2\u00A0 In\u00C2\u00A0vitro\u00C2\u00A0and\u00C2\u00A0 in\u00C2\u00A0 vivo\u00C2\u00A0characterization.\u00C2\u00A0AAPS\u00C2\u00A0J\u00C2\u00A02008\u00C2\u00A0Dec;10(4):505\u00E2\u0080\u009015.\u00C2\u00A0 \u00C2\u00A0 157.\u00C2\u00A0Morton\u00C2\u00A0RE,\u00C2\u00A0Evans\u00C2\u00A0TA.\u00C2\u00A0Modification\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0bicinchoninic\u00C2\u00A0acid\u00C2\u00A0protein\u00C2\u00A0assay\u00C2\u00A0 to\u00C2\u00A0eliminate\u00C2\u00A0 lipid\u00C2\u00A0 interference\u00C2\u00A0in\u00C2\u00A0determining\u00C2\u00A0lipoprotein\u00C2\u00A0protein\u00C2\u00A0content.\u00C2\u00A0Anal\u00C2\u00A0Biochem\u00C2\u00A01992;204(2):332\u00E2\u0080\u00904.\u00C2\u00A0 \u00C2\u00A0 158.\u00C2\u00A0Hale\u00C2\u00A0JE,\u00C2\u00A0Beidler\u00C2\u00A0DE.\u00C2\u00A0Purification\u00C2\u00A0of\u00C2\u00A0humanized\u00C2\u00A0murine\u00C2\u00A0and\u00C2\u00A0murine\u00C2\u00A0monoclonal\u00C2\u00A0antibodies\u00C2\u00A0using\u00C2\u00A0 immobilized\u00C2\u00A0metal\u00E2\u0080\u0090affinity\u00C2\u00A0chromatography.\u00C2\u00A0Anal\u00C2\u00A0Biochem\u00C2\u00A01994\u00C2\u00A010;222(1):29\u00E2\u0080\u009033.\u00C2\u00A0 \u00C2\u00A0 159.\u00C2\u00A0 Gaberc\u00E2\u0080\u0090Porekar\u00C2\u00A0 V,\u00C2\u00A0Menart\u00C2\u00A0 V.\u00C2\u00A0 Perspectives\u00C2\u00A0 of\u00C2\u00A0 immobilized\u00E2\u0080\u0090metal\u00C2\u00A0 affinity\u00C2\u00A0 chromatography.\u00C2\u00A0 J\u00C2\u00A0 Biochem\u00C2\u00A0Biophys\u00C2\u00A0Methods\u00C2\u00A02001\u00C2\u00A0Oct\u00C2\u00A030;49(1\u00E2\u0080\u00903):335\u00E2\u0080\u009060.\u00C2\u00A0 \u00C2\u00A0 160.\u00C2\u00A0Denkhaus\u00C2\u00A0E,\u00C2\u00A0Salnikow\u00C2\u00A0K.\u00C2\u00A0Nickel\u00C2\u00A0essentiality,\u00C2\u00A0 toxicity,\u00C2\u00A0and\u00C2\u00A0carcinogenicity.\u00C2\u00A0Crit\u00C2\u00A0Rev\u00C2\u00A0Oncol\u00C2\u00A02002\u00C2\u00A0 4;42(1):35\u00E2\u0080\u009056.\u00C2\u00A0 \u00C2\u00A0 161.\u00C2\u00A0Hiller\u00C2\u00A0Y,\u00C2\u00A0Gershoni\u00C2\u00A0JM,\u00C2\u00A0Bayer\u00C2\u00A0EA,\u00C2\u00A0Wilchek\u00C2\u00A0M.\u00C2\u00A0Biotin\u00C2\u00A0binding\u00C2\u00A0to\u00C2\u00A0avidin.\u00C2\u00A0Oligosaccharide\u00C2\u00A0side\u00C2\u00A0chain\u00C2\u00A0 not\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0ligand\u00C2\u00A0association.\u00C2\u00A0Biochem\u00C2\u00A0J\u00C2\u00A01987\u00C2\u00A0Nov\u00C2\u00A015;248(1):167\u00E2\u0080\u009071.\u00C2\u00A0 \u00C2\u00A0 162.\u00C2\u00A0Loughrey\u00C2\u00A0H,\u00C2\u00A0Bally\u00C2\u00A0MB,\u00C2\u00A0Cullis\u00C2\u00A0PR.\u00C2\u00A0A\u00C2\u00A0non\u00E2\u0080\u0090covalent\u00C2\u00A0method\u00C2\u00A0of\u00C2\u00A0attaching\u00C2\u00A0antibodies\u00C2\u00A0to\u00C2\u00A0 liposomes.\u00C2\u00A0 Biochim\u00C2\u00A0Biophys\u00C2\u00A0Acta\u00C2\u00A01987\u00C2\u00A0Jul\u00C2\u00A010;901(1):157\u00E2\u0080\u009060.\u00C2\u00A0 \u00C2\u00A0 163.\u00C2\u00A0Loughrey\u00C2\u00A0HC,\u00C2\u00A0Choi\u00C2\u00A0LS,\u00C2\u00A0Cullis\u00C2\u00A0PR,\u00C2\u00A0Bally\u00C2\u00A0MB.\u00C2\u00A0Optimized\u00C2\u00A0procedures\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0coupling\u00C2\u00A0of\u00C2\u00A0proteins\u00C2\u00A0to\u00C2\u00A0 liposomes.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A0Methods\u00C2\u00A01990\u00C2\u00A0Aug\u00C2\u00A028;132(1):25\u00E2\u0080\u009035.\u00C2\u00A0 \u00C2\u00A0 164.\u00C2\u00A0 Jue\u00C2\u00A0 R,\u00C2\u00A0 Lambert\u00C2\u00A0 JM,\u00C2\u00A0 Pierce\u00C2\u00A0 LR,\u00C2\u00A0 Traut\u00C2\u00A0 RR.\u00C2\u00A0 Addition\u00C2\u00A0 of\u00C2\u00A0 sulfhydryl\u00C2\u00A0 groups\u00C2\u00A0 of\u00C2\u00A0 escherichia\u00C2\u00A0 coli\u00C2\u00A0 ribosomes\u00C2\u00A0 by\u00C2\u00A0 protein\u00C2\u00A0 modification\u00C2\u00A0 with\u00C2\u00A0 2\u00E2\u0080\u0090iminothiolane\u00C2\u00A0 (methyl\u00C2\u00A0 4\u00E2\u0080\u0090mercaptobutyrimidate).\u00C2\u00A0 Biochemistry\u00C2\u00A01978;17(25):5399\u00E2\u0080\u0090406.\u00C2\u00A0 \u00C2\u00A0 165.\u00C2\u00A0Wong\u00C2\u00A0SS.\u00C2\u00A0Chemistry\u00C2\u00A0of\u00C2\u00A0protein\u00C2\u00A0conjugation\u00C2\u00A0and\u00C2\u00A0cross\u00E2\u0080\u0090linking.\u00C2\u00A0Boca\u00C2\u00A0Raton,\u00C2\u00A0Fla.:\u00C2\u00A0CRC\u00C2\u00A0Press;\u00C2\u00A01991.\u00C2\u00A0 \u00C2\u00A0 166.\u00C2\u00A0 Wang\u00C2\u00A0 X,\u00C2\u00A0 Yang\u00C2\u00A0 L,\u00C2\u00A0 Chen\u00C2\u00A0 Z,\u00C2\u00A0 Shin\u00C2\u00A0 DM.\u00C2\u00A0 Application\u00C2\u00A0 of\u00C2\u00A0 nanotechnology\u00C2\u00A0 in\u00C2\u00A0 cancer\u00C2\u00A0 therapy\u00C2\u00A0 and\u00C2\u00A0 imaging.\u00C2\u00A0CA\u00E2\u0080\u0090Cancer\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A02008\u00C2\u00A0Mar\u00C2\u00A01;58(2):97\u00E2\u0080\u0090110.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0186\u00C2\u00A0\u00C2\u00A0 167.\u00C2\u00A0Sanvicens\u00C2\u00A0N,\u00C2\u00A0Marco\u00C2\u00A0MP.\u00C2\u00A0Multifunctional\u00C2\u00A0nanoparticles\u00C2\u00A0\u00E2\u0080\u0093\u00C2\u00A0properties\u00C2\u00A0and\u00C2\u00A0prospects\u00C2\u00A0for\u00C2\u00A0their\u00C2\u00A0use\u00C2\u00A0 in\u00C2\u00A0human\u00C2\u00A0medicine.\u00C2\u00A0Trends\u00C2\u00A0Biotechnol\u00C2\u00A02008\u00C2\u00A08;26(8):425\u00E2\u0080\u009033.\u00C2\u00A0 \u00C2\u00A0 168.\u00C2\u00A0Bezombes\u00C2\u00A0C,\u00C2\u00A0Grazide\u00C2\u00A0S,\u00C2\u00A0Garret\u00C2\u00A0C,\u00C2\u00A0Fabre\u00C2\u00A0C,\u00C2\u00A0Quillet\u00E2\u0080\u0090Mary\u00C2\u00A0A,\u00C2\u00A0Muller\u00C2\u00A0S,\u00C2\u00A0 Jaffrezou\u00C2\u00A0 JP,\u00C2\u00A0 Laurent\u00C2\u00A0G.\u00C2\u00A0 Rituximab\u00C2\u00A0antiproliferative\u00C2\u00A0effect\u00C2\u00A0in\u00C2\u00A0B\u00E2\u0080\u0090lymphoma\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0associated\u00C2\u00A0with\u00C2\u00A0acid\u00E2\u0080\u0090sphingomyelinase\u00C2\u00A0 activation\u00C2\u00A0in\u00C2\u00A0raft\u00C2\u00A0microdomains.\u00C2\u00A0Blood\u00C2\u00A02004\u00C2\u00A0Aug\u00C2\u00A015;104(4):1166\u00E2\u0080\u009073.\u00C2\u00A0 \u00C2\u00A0 169.\u00C2\u00A0Semac\u00C2\u00A0I,\u00C2\u00A0Palomba\u00C2\u00A0C,\u00C2\u00A0Kulangara\u00C2\u00A0K,\u00C2\u00A0Klages\u00C2\u00A0N,\u00C2\u00A0van\u00C2\u00A0Echten\u00E2\u0080\u0090Deckert\u00C2\u00A0G,\u00C2\u00A0Borisch\u00C2\u00A0B,\u00C2\u00A0Hoessli\u00C2\u00A0DC.\u00C2\u00A0Anti\u00E2\u0080\u0090 CD20\u00C2\u00A0 therapeutic\u00C2\u00A0 antibody\u00C2\u00A0 rituximab\u00C2\u00A0 modifies\u00C2\u00A0 the\u00C2\u00A0 functional\u00C2\u00A0 organization\u00C2\u00A0 of\u00C2\u00A0 rafts/microdomains\u00C2\u00A0of\u00C2\u00A0B\u00C2\u00A0lymphoma\u00C2\u00A0cells.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02003\u00C2\u00A0Jan\u00C2\u00A015;63(2):534\u00E2\u0080\u009040.\u00C2\u00A0 \u00C2\u00A0 170.\u00C2\u00A0Cillessen\u00C2\u00A0SA,\u00C2\u00A0Hess\u00C2\u00A0CJ,\u00C2\u00A0Hooijberg\u00C2\u00A0E,\u00C2\u00A0Castricum\u00C2\u00A0KC,\u00C2\u00A0Kortman\u00C2\u00A0P,\u00C2\u00A0Denkers\u00C2\u00A0F,\u00C2\u00A0Vos\u00C2\u00A0W,\u00C2\u00A0van\u00C2\u00A0de\u00C2\u00A0Wiel\u00C2\u00A0MA,\u00C2\u00A0 Schuurhuis\u00C2\u00A0 GJ,\u00C2\u00A0 Ossenkoppele\u00C2\u00A0 GJ,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Inhibition\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 intrinsic\u00C2\u00A0 apoptosis\u00C2\u00A0 pathway\u00C2\u00A0 downstream\u00C2\u00A0 of\u00C2\u00A0 caspase\u00E2\u0080\u00909\u00C2\u00A0 activation\u00C2\u00A0 causes\u00C2\u00A0 chemotherapy\u00C2\u00A0 resistance\u00C2\u00A0 in\u00C2\u00A0 diffuse\u00C2\u00A0 large\u00C2\u00A0 B\u00E2\u0080\u0090cell\u00C2\u00A0 lymphoma.\u00C2\u00A0Clin\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02007\u00C2\u00A0Dec\u00C2\u00A01;13(23):7012\u00E2\u0080\u009021.\u00C2\u00A0 \u00C2\u00A0 171.\u00C2\u00A0 Peter\u00C2\u00A0 ME,\u00C2\u00A0 Krammer\u00C2\u00A0 PH.\u00C2\u00A0 The\u00C2\u00A0 CD95(APO\u00E2\u0080\u00901/Fas)\u00C2\u00A0 DISC\u00C2\u00A0 and\u00C2\u00A0 beyond.\u00C2\u00A0 Cell\u00C2\u00A0 Death\u00C2\u00A0 Differ\u00C2\u00A0 2003\u00C2\u00A0 Jan;10(1):26\u00E2\u0080\u009035.\u00C2\u00A0 \u00C2\u00A0 172.\u00C2\u00A0Hehlgans\u00C2\u00A0 T,\u00C2\u00A0 Pfeffer\u00C2\u00A0 K.\u00C2\u00A0 The\u00C2\u00A0 intriguing\u00C2\u00A0biology\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 tumour\u00C2\u00A0 necrosis\u00C2\u00A0 factor/tumour\u00C2\u00A0necrosis\u00C2\u00A0 factor\u00C2\u00A0receptor\u00C2\u00A0superfamily:\u00C2\u00A0Players,\u00C2\u00A0rules\u00C2\u00A0and\u00C2\u00A0the\u00C2\u00A0games.\u00C2\u00A0Immunology\u00C2\u00A02005\u00C2\u00A0May;115(1):1\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 173.\u00C2\u00A0Carswell\u00C2\u00A0EA,\u00C2\u00A0Old\u00C2\u00A0 LJ,\u00C2\u00A0Kassel\u00C2\u00A0RL,\u00C2\u00A0Green\u00C2\u00A0S,\u00C2\u00A0 Fiore\u00C2\u00A0N,\u00C2\u00A0Williamson\u00C2\u00A0B.\u00C2\u00A0An\u00C2\u00A0endotoxin\u00E2\u0080\u0090induced\u00C2\u00A0 serum\u00C2\u00A0 factor\u00C2\u00A0that\u00C2\u00A0causes\u00C2\u00A0necrosis\u00C2\u00A0of\u00C2\u00A0tumors.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A01975\u00C2\u00A0Sep;72(9):3666\u00E2\u0080\u009070.\u00C2\u00A0 \u00C2\u00A0 174.\u00C2\u00A0Guicciardi\u00C2\u00A0ME,\u00C2\u00A0Gores\u00C2\u00A0GJ.\u00C2\u00A0Life\u00C2\u00A0and\u00C2\u00A0death\u00C2\u00A0by\u00C2\u00A0death\u00C2\u00A0receptors.\u00C2\u00A0FASEB\u00C2\u00A0J\u00C2\u00A02009\u00C2\u00A0Jun;23(6):1625\u00E2\u0080\u009037.\u00C2\u00A0 \u00C2\u00A0 175.\u00C2\u00A0 Schutze\u00C2\u00A0 S,\u00C2\u00A0 Tchikov\u00C2\u00A0 V,\u00C2\u00A0 Schneider\u00E2\u0080\u0090Brachert\u00C2\u00A0 W.\u00C2\u00A0 Regulation\u00C2\u00A0 of\u00C2\u00A0 TNFR1\u00C2\u00A0 and\u00C2\u00A0 CD95\u00C2\u00A0 signalling\u00C2\u00A0 by\u00C2\u00A0 receptor\u00C2\u00A0compartmentalization.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Mol\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A02008\u00C2\u00A0Aug;9(8):655\u00E2\u0080\u009062.\u00C2\u00A0 \u00C2\u00A0 176.\u00C2\u00A0Gonzalvez\u00C2\u00A0 F,\u00C2\u00A0Ashkenazi\u00C2\u00A0A.\u00C2\u00A0New\u00C2\u00A0 insights\u00C2\u00A0 into\u00C2\u00A0 apoptosis\u00C2\u00A0 signaling\u00C2\u00A0 by\u00C2\u00A0Apo2L/TRAIL.\u00C2\u00A0Oncogene\u00C2\u00A0 2010\u00C2\u00A0Aug\u00C2\u00A026;29(34):4752\u00E2\u0080\u009065.\u00C2\u00A0 \u00C2\u00A0 177.\u00C2\u00A0 Aggarwal\u00C2\u00A0 BB.\u00C2\u00A0 Signalling\u00C2\u00A0 pathways\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 TNF\u00C2\u00A0 superfamily:\u00C2\u00A0 A\u00C2\u00A0 double\u00E2\u0080\u0090edged\u00C2\u00A0 sword.\u00C2\u00A0 Nat\u00C2\u00A0 Rev\u00C2\u00A0 Immunol\u00C2\u00A02003\u00C2\u00A0Sep;3(9):745\u00E2\u0080\u009056.\u00C2\u00A0 \u00C2\u00A0 178.\u00C2\u00A0Wajant\u00C2\u00A0H.\u00C2\u00A0Death\u00C2\u00A0receptors.\u00C2\u00A0Essays\u00C2\u00A0Biochem\u00C2\u00A02003;39:53\u00E2\u0080\u009071.\u00C2\u00A0 \u00C2\u00A0 179.\u00C2\u00A0 Chan\u00C2\u00A0 KF,\u00C2\u00A0 Siegel\u00C2\u00A0 MR,\u00C2\u00A0 Lenardo\u00C2\u00A0 JM.\u00C2\u00A0 Signaling\u00C2\u00A0 by\u00C2\u00A0 the\u00C2\u00A0 TNF\u00C2\u00A0 receptor\u00C2\u00A0 superfamily\u00C2\u00A0 and\u00C2\u00A0 T\u00C2\u00A0 cell\u00C2\u00A0 homeostasis.\u00C2\u00A0Immunity\u00C2\u00A02000\u00C2\u00A0Oct;13(4):419\u00E2\u0080\u009022.\u00C2\u00A0 \u00C2\u00A0 180.\u00C2\u00A0Chan\u00C2\u00A0FK,\u00C2\u00A0Chun\u00C2\u00A0HJ,\u00C2\u00A0Zheng\u00C2\u00A0 L,\u00C2\u00A0 Siegel\u00C2\u00A0RM,\u00C2\u00A0Bui\u00C2\u00A0KL,\u00C2\u00A0 Lenardo\u00C2\u00A0MJ.\u00C2\u00A0A\u00C2\u00A0domain\u00C2\u00A0 in\u00C2\u00A0TNF\u00C2\u00A0 receptors\u00C2\u00A0 that\u00C2\u00A0 mediates\u00C2\u00A0 ligand\u00E2\u0080\u0090independent\u00C2\u00A0 receptor\u00C2\u00A0 assembly\u00C2\u00A0 and\u00C2\u00A0 signaling.\u00C2\u00A0 Science\u00C2\u00A0 2000\u00C2\u00A0 Jun\u00C2\u00A0 30;288(5475):2351\u00E2\u0080\u00904.\u00C2\u00A0 \u00C2\u00A0 181.\u00C2\u00A0Beltinger\u00C2\u00A0C,\u00C2\u00A0Fulda\u00C2\u00A0S,\u00C2\u00A0Kammertoens\u00C2\u00A0T,\u00C2\u00A0Meyer\u00C2\u00A0E,\u00C2\u00A0Uckert\u00C2\u00A0W,\u00C2\u00A0Debatin\u00C2\u00A0KM.\u00C2\u00A0Herpes\u00C2\u00A0 simplex\u00C2\u00A0virus\u00C2\u00A0 thymidine\u00C2\u00A0 kinase/ganciclovir\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 involves\u00C2\u00A0 ligand\u00E2\u0080\u0090independent\u00C2\u00A0 death\u00C2\u00A0 receptor\u00C2\u00A0 aggregation\u00C2\u00A0and\u00C2\u00A0activation\u00C2\u00A0of\u00C2\u00A0caspases.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A01999\u00C2\u00A0Jul\u00C2\u00A020;96(15):8699\u00E2\u0080\u0090704.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0187\u00C2\u00A0\u00C2\u00A0 182.\u00C2\u00A0Ozsoy\u00C2\u00A0HZ,\u00C2\u00A0 Sivasubramanian\u00C2\u00A0N,\u00C2\u00A0Wieder\u00C2\u00A0 ED,\u00C2\u00A0Pedersen\u00C2\u00A0 S,\u00C2\u00A0Mann\u00C2\u00A0DL.\u00C2\u00A0Oxidative\u00C2\u00A0 stress\u00C2\u00A0promotes\u00C2\u00A0 ligand\u00E2\u0080\u0090independent\u00C2\u00A0and\u00C2\u00A0enhanced\u00C2\u00A0ligand\u00E2\u0080\u0090dependent\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0receptor\u00C2\u00A0signaling.\u00C2\u00A0J\u00C2\u00A0 Biol\u00C2\u00A0Chem\u00C2\u00A02008\u00C2\u00A0Aug\u00C2\u00A022;283(34):23419\u00E2\u0080\u009028.\u00C2\u00A0 \u00C2\u00A0 183.\u00C2\u00A0Yi\u00C2\u00A0 JS,\u00C2\u00A0Choo\u00C2\u00A0HJ,\u00C2\u00A0Cho\u00C2\u00A0BR,\u00C2\u00A0Kim\u00C2\u00A0HM,\u00C2\u00A0Kim\u00C2\u00A0YN,\u00C2\u00A0Ham\u00C2\u00A0YM,\u00C2\u00A0Ko\u00C2\u00A0YG.\u00C2\u00A0Ginsenoside\u00C2\u00A0Rh2\u00C2\u00A0 induces\u00C2\u00A0 ligand\u00E2\u0080\u0090 independent\u00C2\u00A0 fas\u00C2\u00A0 activation\u00C2\u00A0 via\u00C2\u00A0 lipid\u00C2\u00A0 raft\u00C2\u00A0 disruption.\u00C2\u00A0 Biochem\u00C2\u00A0 Biophys\u00C2\u00A0 Res\u00C2\u00A0 Commun\u00C2\u00A0 2009\u00C2\u00A0 Jul\u00C2\u00A0 24;385(2):154\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 184.\u00C2\u00A0 Day\u00C2\u00A0 TW,\u00C2\u00A0 Huang\u00C2\u00A0 S,\u00C2\u00A0 Safa\u00C2\u00A0 AR.\u00C2\u00A0 c\u00E2\u0080\u0090FLIP\u00C2\u00A0 knockdown\u00C2\u00A0 induces\u00C2\u00A0 ligand\u00E2\u0080\u0090independent\u00C2\u00A0 DR5\u00E2\u0080\u0090,\u00C2\u00A0 FADD\u00E2\u0080\u0090,\u00C2\u00A0 caspase\u00E2\u0080\u00908\u00E2\u0080\u0090,\u00C2\u00A0 and\u00C2\u00A0 caspase\u00E2\u0080\u00909\u00E2\u0080\u0090dependent\u00C2\u00A0 apoptosis\u00C2\u00A0 in\u00C2\u00A0 breast\u00C2\u00A0 cancer\u00C2\u00A0 cells.\u00C2\u00A0 Biochem\u00C2\u00A0 Pharmacol\u00C2\u00A0 2008\u00C2\u00A0Dec\u00C2\u00A015;76(12):1694\u00E2\u0080\u0090704.\u00C2\u00A0 \u00C2\u00A0 185.\u00C2\u00A0 Sheikh\u00C2\u00A0MS,\u00C2\u00A0Antinore\u00C2\u00A0MJ,\u00C2\u00A0Huang\u00C2\u00A0 Y,\u00C2\u00A0 Fornace\u00C2\u00A0AJ,Jr.\u00C2\u00A0Ultraviolet\u00E2\u0080\u0090irradiation\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 is\u00C2\u00A0 mediated\u00C2\u00A0 via\u00C2\u00A0 ligand\u00C2\u00A0 independent\u00C2\u00A0 activation\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 1.\u00C2\u00A0 Oncogene\u00C2\u00A0 1998\u00C2\u00A0Nov\u00C2\u00A019;17(20):2555\u00E2\u0080\u009063.\u00C2\u00A0 \u00C2\u00A0 186.\u00C2\u00A0Muppidi\u00C2\u00A0JR,\u00C2\u00A0Siegel\u00C2\u00A0RM.\u00C2\u00A0Ligand\u00E2\u0080\u0090independent\u00C2\u00A0redistribution\u00C2\u00A0of\u00C2\u00A0fas\u00C2\u00A0(CD95)\u00C2\u00A0into\u00C2\u00A0lipid\u00C2\u00A0rafts\u00C2\u00A0mediates\u00C2\u00A0 clonotypic\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0death.\u00C2\u00A0Nat\u00C2\u00A0Immunol\u00C2\u00A02004\u00C2\u00A0Feb;5(2):182\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 187.\u00C2\u00A0Hsiang\u00C2\u00A0Y,\u00C2\u00A0Lihou\u00C2\u00A0MG,\u00C2\u00A0Liu\u00C2\u00A0LF.\u00C2\u00A0Arrest\u00C2\u00A0of\u00C2\u00A0replication\u00C2\u00A0forks\u00C2\u00A0by\u00C2\u00A0drug\u00E2\u0080\u0090stabilized\u00C2\u00A0topoisomerase\u00C2\u00A0 I\u00E2\u0080\u0090DNA\u00C2\u00A0 cleavable\u00C2\u00A0 complexes\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0mechanism\u00C2\u00A0 of\u00C2\u00A0 cell\u00C2\u00A0 killing\u00C2\u00A0 by\u00C2\u00A0 camptothecin.\u00C2\u00A0 Cancer\u00C2\u00A0 Res\u00C2\u00A0 1989\u00C2\u00A0 Sep\u00C2\u00A0 15;49(18):5077\u00E2\u0080\u009082.\u00C2\u00A0 \u00C2\u00A0 188.\u00C2\u00A0Gao\u00C2\u00A0X,\u00C2\u00A0Picchi\u00C2\u00A0A,\u00C2\u00A0Zhang\u00C2\u00A0C.\u00C2\u00A0Upregulation\u00C2\u00A0of\u00C2\u00A0TNF\u00E2\u0080\u0090alpha\u00C2\u00A0and\u00C2\u00A0 receptors\u00C2\u00A0contribute\u00C2\u00A0 to\u00C2\u00A0endothelial\u00C2\u00A0 dysfunction\u00C2\u00A0in\u00C2\u00A0zucker\u00C2\u00A0diabetic\u00C2\u00A0rats.\u00C2\u00A0Am\u00C2\u00A0J\u00C2\u00A0Biomed\u00C2\u00A0Sci\u00C2\u00A02010;2(1):1\u00E2\u0080\u009012.\u00C2\u00A0 \u00C2\u00A0 189.\u00C2\u00A0Cook\u00C2\u00A0EB,\u00C2\u00A0Stahl\u00C2\u00A0JL,\u00C2\u00A0Graziano\u00C2\u00A0FM,\u00C2\u00A0Barney\u00C2\u00A0NP.\u00C2\u00A0Regulation\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0receptor\u00C2\u00A0for\u00C2\u00A0TNFalpha,\u00C2\u00A0TNFR1,\u00C2\u00A0in\u00C2\u00A0 human\u00C2\u00A0conjunctival\u00C2\u00A0epithelial\u00C2\u00A0cells.\u00C2\u00A0Invest\u00C2\u00A0Ophthalmol\u00C2\u00A0Vis\u00C2\u00A0Sci\u00C2\u00A02008\u00C2\u00A0Sep;49(9):3992\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 190.\u00C2\u00A0 Cubillas\u00C2\u00A0 R,\u00C2\u00A0 Kintner\u00C2\u00A0 K,\u00C2\u00A0 Phillips\u00C2\u00A0 F,\u00C2\u00A0 Karandikar\u00C2\u00A0NJ,\u00C2\u00A0 Thiele\u00C2\u00A0DL,\u00C2\u00A0 Brown\u00C2\u00A0GR.\u00C2\u00A0 Tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00C2\u00A0 receptor\u00C2\u00A0 1\u00C2\u00A0 expression\u00C2\u00A0 is\u00C2\u00A0 upregulated\u00C2\u00A0 in\u00C2\u00A0 dendritic\u00C2\u00A0 cells\u00C2\u00A0 in\u00C2\u00A0 patients\u00C2\u00A0 with\u00C2\u00A0 chronic\u00C2\u00A0 HCV\u00C2\u00A0 who\u00C2\u00A0 respond\u00C2\u00A0to\u00C2\u00A0therapy.\u00C2\u00A0Hepat\u00C2\u00A0Res\u00C2\u00A0Treat\u00C2\u00A02010;2010:429243.\u00C2\u00A0 \u00C2\u00A0 191.\u00C2\u00A0Rahman\u00C2\u00A0MM,\u00C2\u00A0McFadden\u00C2\u00A0G.\u00C2\u00A0Modulation\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0by\u00C2\u00A0microbial\u00C2\u00A0pathogens.\u00C2\u00A0PLoS\u00C2\u00A0 Pathog\u00C2\u00A02006\u00C2\u00A0Feb;2(2):e4.\u00C2\u00A0 \u00C2\u00A0 192.\u00C2\u00A0Deb\u00C2\u00A0S,\u00C2\u00A0Amin\u00C2\u00A0S,\u00C2\u00A0Imir\u00C2\u00A0AG,\u00C2\u00A0Yilmaz\u00C2\u00A0MB,\u00C2\u00A0Suzuki\u00C2\u00A0T,\u00C2\u00A0Sasano\u00C2\u00A0H,\u00C2\u00A0Bulun\u00C2\u00A0SE.\u00C2\u00A0Estrogen\u00C2\u00A0regulates\u00C2\u00A0expression\u00C2\u00A0 of\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0receptors\u00C2\u00A0 in\u00C2\u00A0breast\u00C2\u00A0adipose\u00C2\u00A0fibroblasts.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Endocrinol\u00C2\u00A0Metab\u00C2\u00A02004\u00C2\u00A0 Aug;89(8):4018\u00E2\u0080\u009024.\u00C2\u00A0 \u00C2\u00A0 193.\u00C2\u00A0Berger\u00C2\u00A0AC,\u00C2\u00A0Alexander\u00C2\u00A0HR,\u00C2\u00A0Wu\u00C2\u00A0PC,\u00C2\u00A0Tang\u00C2\u00A0G,\u00C2\u00A0Gnant\u00C2\u00A0MF,\u00C2\u00A0Mixon\u00C2\u00A0A,\u00C2\u00A0Turner\u00C2\u00A0ES,\u00C2\u00A0Libutti\u00C2\u00A0SK.\u00C2\u00A0Tumour\u00C2\u00A0 necrosis\u00C2\u00A0factor\u00C2\u00A0receptor\u00C2\u00A0I\u00C2\u00A0(p55)\u00C2\u00A0is\u00C2\u00A0upregulated\u00C2\u00A0on\u00C2\u00A0endothelial\u00C2\u00A0cells\u00C2\u00A0by\u00C2\u00A0exposure\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0tumour\u00E2\u0080\u0090 derived\u00C2\u00A0 cytokine\u00C2\u00A0 endothelial\u00C2\u00A0 monocyte\u00E2\u0080\u0090\u00C2\u00A0 activating\u00C2\u00A0 polypeptide\u00C2\u00A0 II\u00C2\u00A0 (EMAP\u00E2\u0080\u0090II).\u00C2\u00A0 Cytokine\u00C2\u00A0 2000\u00C2\u00A0 Jul;12(7):992\u00E2\u0080\u00901000.\u00C2\u00A0 \u00C2\u00A0 194.\u00C2\u00A0de\u00C2\u00A0Totero\u00C2\u00A0D,\u00C2\u00A0Tazzari\u00C2\u00A0PL,\u00C2\u00A0Capaia\u00C2\u00A0M,\u00C2\u00A0Montera\u00C2\u00A0MP,\u00C2\u00A0Clavio\u00C2\u00A0M,\u00C2\u00A0Balleari\u00C2\u00A0E,\u00C2\u00A0Foa\u00C2\u00A0R,\u00C2\u00A0Gobbi\u00C2\u00A0M.\u00C2\u00A0CD40\u00C2\u00A0 triggering\u00C2\u00A0 enhances\u00C2\u00A0 fludarabine\u00E2\u0080\u0090induced\u00C2\u00A0 apoptosis\u00C2\u00A0 of\u00C2\u00A0 chronic\u00C2\u00A0 lymphocytic\u00C2\u00A0 leukemia\u00C2\u00A0 B\u00E2\u0080\u0090cells\u00C2\u00A0 through\u00C2\u00A0 autocrine\u00C2\u00A0 release\u00C2\u00A0 of\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0 factor\u00E2\u0080\u0090alpha\u00C2\u00A0 and\u00C2\u00A0 interferon\u00E2\u0080\u0090gama\u00C2\u00A0 and\u00C2\u00A0 tumor\u00C2\u00A0 necrosis\u00C2\u00A0factor\u00C2\u00A0receptor\u00E2\u0080\u0090I\u00E2\u0080\u0090II\u00C2\u00A0upregulation.\u00C2\u00A0Haematologica\u00C2\u00A02003\u00C2\u00A0Feb;88(2):148\u00E2\u0080\u009058.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0188\u00C2\u00A0\u00C2\u00A0 195.\u00C2\u00A0Grell\u00C2\u00A0M,\u00C2\u00A0Douni\u00C2\u00A0E,\u00C2\u00A0Wajant\u00C2\u00A0H,\u00C2\u00A0L\u00C3\u00B6hden\u00C2\u00A0M,\u00C2\u00A0Clauss\u00C2\u00A0M,\u00C2\u00A0Maxeiner\u00C2\u00A0B,\u00C2\u00A0Georgopoulos\u00C2\u00A0S,\u00C2\u00A0Lesslauer\u00C2\u00A0W,\u00C2\u00A0 Kollias\u00C2\u00A0G,\u00C2\u00A0Pfizenmaier\u00C2\u00A0K,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0The\u00C2\u00A0transmembrane\u00C2\u00A0form\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0 is\u00C2\u00A0the\u00C2\u00A0prime\u00C2\u00A0 activating\u00C2\u00A0ligand\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A080\u00C2\u00A0kDa\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0receptor.\u00C2\u00A0Cell\u00C2\u00A01995;83(5):793\u00E2\u0080\u0090802.\u00C2\u00A0 \u00C2\u00A0 196.\u00C2\u00A0Muppidi\u00C2\u00A0JR,\u00C2\u00A0Tschopp\u00C2\u00A0J,\u00C2\u00A0Siegel\u00C2\u00A0RM.\u00C2\u00A0Life\u00C2\u00A0and\u00C2\u00A0death\u00C2\u00A0decisions:\u00C2\u00A0Secondary\u00C2\u00A0complexes\u00C2\u00A0and\u00C2\u00A0lipid\u00C2\u00A0rafts\u00C2\u00A0 in\u00C2\u00A0TNF\u00C2\u00A0receptor\u00C2\u00A0family\u00C2\u00A0signal\u00C2\u00A0transduction.\u00C2\u00A0Immunity\u00C2\u00A02004\u00C2\u00A0Oct;21(4):461\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 197.\u00C2\u00A0Micheau\u00C2\u00A0 O,\u00C2\u00A0 Tschopp\u00C2\u00A0 J.\u00C2\u00A0 Induction\u00C2\u00A0 of\u00C2\u00A0 TNF\u00C2\u00A0 receptor\u00C2\u00A0 I\u00E2\u0080\u0090mediated\u00C2\u00A0 apoptosis\u00C2\u00A0 via\u00C2\u00A0 two\u00C2\u00A0 sequential\u00C2\u00A0 signaling\u00C2\u00A0complexes.\u00C2\u00A0Cell\u00C2\u00A02003\u00C2\u00A0Jul\u00C2\u00A025;114(2):181\u00E2\u0080\u009090.\u00C2\u00A0 \u00C2\u00A0 198.\u00C2\u00A0Legler\u00C2\u00A0DF,\u00C2\u00A0Micheau\u00C2\u00A0O,\u00C2\u00A0Doucey\u00C2\u00A0MA,\u00C2\u00A0Tschopp\u00C2\u00A0J,\u00C2\u00A0Bron\u00C2\u00A0C.\u00C2\u00A0Recruitment\u00C2\u00A0of\u00C2\u00A0TNF\u00C2\u00A0receptor\u00C2\u00A01\u00C2\u00A0to\u00C2\u00A0 lipid\u00C2\u00A0 rafts\u00C2\u00A0 is\u00C2\u00A0essential\u00C2\u00A0for\u00C2\u00A0TNFalpha\u00E2\u0080\u0090mediated\u00C2\u00A0NF\u00E2\u0080\u0090kappaB\u00C2\u00A0activation.\u00C2\u00A0 Immunity\u00C2\u00A02003\u00C2\u00A0May;18(5):655\u00E2\u0080\u0090 64.\u00C2\u00A0 \u00C2\u00A0 199.\u00C2\u00A0Singer\u00C2\u00A0SJ,\u00C2\u00A0Nicolson\u00C2\u00A0GL.\u00C2\u00A0The\u00C2\u00A0fluid\u00C2\u00A0mosaic\u00C2\u00A0model\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0structure\u00C2\u00A0of\u00C2\u00A0cell\u00C2\u00A0membranes.\u00C2\u00A0Science\u00C2\u00A01972\u00C2\u00A0 Feb\u00C2\u00A018;175(23):720\u00E2\u0080\u009031.\u00C2\u00A0 \u00C2\u00A0 200.\u00C2\u00A0Vereb\u00C2\u00A0G,\u00C2\u00A0Szollosi\u00C2\u00A0J,\u00C2\u00A0Matko\u00C2\u00A0J,\u00C2\u00A0Nagy\u00C2\u00A0P,\u00C2\u00A0Farkas\u00C2\u00A0T,\u00C2\u00A0Vigh\u00C2\u00A0L,\u00C2\u00A0Matyus\u00C2\u00A0L,\u00C2\u00A0Waldmann\u00C2\u00A0TA,\u00C2\u00A0Damjanovich\u00C2\u00A0S.\u00C2\u00A0 Dynamic,\u00C2\u00A0yet\u00C2\u00A0structured:\u00C2\u00A0The\u00C2\u00A0cell\u00C2\u00A0membrane\u00C2\u00A0three\u00C2\u00A0decades\u00C2\u00A0after\u00C2\u00A0the\u00C2\u00A0singer\u00E2\u0080\u0090nicolson\u00C2\u00A0model.\u00C2\u00A0P\u00C2\u00A0 Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A02003\u00C2\u00A0Jul\u00C2\u00A08;100(14):8053\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 201.\u00C2\u00A0Simons\u00C2\u00A0K,\u00C2\u00A0Ehehalt\u00C2\u00A0R.\u00C2\u00A0Cholesterol,\u00C2\u00A0lipid\u00C2\u00A0rafts,\u00C2\u00A0and\u00C2\u00A0disease.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Invest\u00C2\u00A02002\u00C2\u00A0Sep;110(5):597\u00E2\u0080\u0090603.\u00C2\u00A0 \u00C2\u00A0 202.\u00C2\u00A0 Carver\u00C2\u00A0 LA,\u00C2\u00A0 Schnitzer\u00C2\u00A0 JE,\u00C2\u00A0 Anderson\u00C2\u00A0 RG,\u00C2\u00A0Mohla\u00C2\u00A0 S.\u00C2\u00A0 Role\u00C2\u00A0 of\u00C2\u00A0 caveolae\u00C2\u00A0 and\u00C2\u00A0 lipid\u00C2\u00A0 rafts\u00C2\u00A0 in\u00C2\u00A0 cancer:\u00C2\u00A0 Workshop\u00C2\u00A0summary\u00C2\u00A0and\u00C2\u00A0future\u00C2\u00A0needs.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02003\u00C2\u00A0Oct\u00C2\u00A015;63(20):6571\u00E2\u0080\u00904.\u00C2\u00A0 \u00C2\u00A0 203.\u00C2\u00A0Li\u00C2\u00A0YC,\u00C2\u00A0Park\u00C2\u00A0MJ,\u00C2\u00A0Ye\u00C2\u00A0SK,\u00C2\u00A0Kim\u00C2\u00A0CW,\u00C2\u00A0Kim\u00C2\u00A0YN.\u00C2\u00A0Elevated\u00C2\u00A0 levels\u00C2\u00A0of\u00C2\u00A0cholesterol\u00E2\u0080\u0090rich\u00C2\u00A0 lipid\u00C2\u00A0rafts\u00C2\u00A0 in\u00C2\u00A0cancer\u00C2\u00A0 cells\u00C2\u00A0 are\u00C2\u00A0 correlated\u00C2\u00A0with\u00C2\u00A0 apoptosis\u00C2\u00A0 sensitivity\u00C2\u00A0 induced\u00C2\u00A0 by\u00C2\u00A0 cholesterol\u00E2\u0080\u0090depleting\u00C2\u00A0 agents.\u00C2\u00A0Am\u00C2\u00A0 J\u00C2\u00A0 Pathol\u00C2\u00A02006\u00C2\u00A0Apr;168(4):1107\u00E2\u0080\u009018.\u00C2\u00A0 \u00C2\u00A0 204.\u00C2\u00A0 Zhuang\u00C2\u00A0 L,\u00C2\u00A0 Lin\u00C2\u00A0 J,\u00C2\u00A0 Lu\u00C2\u00A0ML,\u00C2\u00A0 Solomon\u00C2\u00A0 KR,\u00C2\u00A0 Freeman\u00C2\u00A0MR.\u00C2\u00A0 Cholesterol\u00E2\u0080\u0090rich\u00C2\u00A0 lipid\u00C2\u00A0 rafts\u00C2\u00A0mediate\u00C2\u00A0 akt\u00E2\u0080\u0090 regulated\u00C2\u00A0survival\u00C2\u00A0in\u00C2\u00A0prostate\u00C2\u00A0cancer\u00C2\u00A0cells.\u00C2\u00A0Cancer\u00C2\u00A0Res\u00C2\u00A02002\u00C2\u00A0Apr\u00C2\u00A015;62(8):2227\u00E2\u0080\u009031.\u00C2\u00A0 \u00C2\u00A0 205.\u00C2\u00A0 Lajoie\u00C2\u00A0 P,\u00C2\u00A0 Partridge\u00C2\u00A0 EA,\u00C2\u00A0Guay\u00C2\u00A0G,\u00C2\u00A0Goetz\u00C2\u00A0 JG,\u00C2\u00A0 Pawling\u00C2\u00A0 J,\u00C2\u00A0 Lagana\u00C2\u00A0A,\u00C2\u00A0 Joshi\u00C2\u00A0B,\u00C2\u00A0Dennis\u00C2\u00A0 JW,\u00C2\u00A0Nabi\u00C2\u00A0 IR.\u00C2\u00A0 Plasma\u00C2\u00A0membrane\u00C2\u00A0domain\u00C2\u00A0organization\u00C2\u00A0regulates\u00C2\u00A0EGFR\u00C2\u00A0signaling\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0cells.\u00C2\u00A0J\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A02007\u00C2\u00A0 Oct\u00C2\u00A022;179(2):341\u00E2\u0080\u009056.\u00C2\u00A0 \u00C2\u00A0 206.\u00C2\u00A0Pike\u00C2\u00A0LJ.\u00C2\u00A0Rafts\u00C2\u00A0defined:\u00C2\u00A0A\u00C2\u00A0report\u00C2\u00A0on\u00C2\u00A0 the\u00C2\u00A0keystone\u00C2\u00A0symposium\u00C2\u00A0on\u00C2\u00A0 lipid\u00C2\u00A0rafts\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0 function.\u00C2\u00A0 J\u00C2\u00A0 Lipid\u00C2\u00A0Res\u00C2\u00A02006\u00C2\u00A0Jul;47(7):1597\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 207.\u00C2\u00A0Hancock\u00C2\u00A0JF.\u00C2\u00A0Lipid\u00C2\u00A0rafts:\u00C2\u00A0Contentious\u00C2\u00A0only\u00C2\u00A0from\u00C2\u00A0simplistic\u00C2\u00A0standpoints.\u00C2\u00A0Nat\u00C2\u00A0Rev\u00C2\u00A0Mol\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A02006\u00C2\u00A0 Jun;7(6):456\u00E2\u0080\u009062.\u00C2\u00A0 \u00C2\u00A0 208.\u00C2\u00A0Simons\u00C2\u00A0K,\u00C2\u00A0Ikonen\u00C2\u00A0E.\u00C2\u00A0Functional\u00C2\u00A0rafts\u00C2\u00A0in\u00C2\u00A0cell\u00C2\u00A0membranes.\u00C2\u00A0Nature\u00C2\u00A01997\u00C2\u00A0Jun\u00C2\u00A05;387(6633):569\u00E2\u0080\u009072.\u00C2\u00A0 \u00C2\u00A0 209.\u00C2\u00A0Brown\u00C2\u00A0DA,\u00C2\u00A0London\u00C2\u00A0E.\u00C2\u00A0Structure\u00C2\u00A0and\u00C2\u00A0 function\u00C2\u00A0of\u00C2\u00A0sphingolipid\u00E2\u0080\u0090\u00C2\u00A0and\u00C2\u00A0cholesterol\u00E2\u0080\u0090rich\u00C2\u00A0membrane\u00C2\u00A0 rafts.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A02000\u00C2\u00A0Jun\u00C2\u00A09;275(23):17221\u00E2\u0080\u00904.\u00C2\u00A0 \u00C2\u00A0 210.\u00C2\u00A0Edidin\u00C2\u00A0M.\u00C2\u00A0The\u00C2\u00A0state\u00C2\u00A0of\u00C2\u00A0 lipid\u00C2\u00A0rafts:\u00C2\u00A0From\u00C2\u00A0model\u00C2\u00A0membranes\u00C2\u00A0to\u00C2\u00A0cells.\u00C2\u00A0Annu\u00C2\u00A0Rev\u00C2\u00A0Biophys\u00C2\u00A0Biomol\u00C2\u00A0 Struct\u00C2\u00A02003;32:257\u00E2\u0080\u009083.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0189\u00C2\u00A0\u00C2\u00A0 211.\u00C2\u00A0Brown\u00C2\u00A0DA,\u00C2\u00A0London\u00C2\u00A0E.\u00C2\u00A0Functions\u00C2\u00A0of\u00C2\u00A0 lipid\u00C2\u00A0rafts\u00C2\u00A0 in\u00C2\u00A0biological\u00C2\u00A0membranes.\u00C2\u00A0Annu\u00C2\u00A0Rev\u00C2\u00A0Cell\u00C2\u00A0Dev\u00C2\u00A0Biol\u00C2\u00A0 1998;14:111\u00E2\u0080\u009036.\u00C2\u00A0 \u00C2\u00A0 212.\u00C2\u00A0Stauffer\u00C2\u00A0TP,\u00C2\u00A0Meyer\u00C2\u00A0T.\u00C2\u00A0Compartmentalized\u00C2\u00A0 IgE\u00C2\u00A0 receptor\u00E2\u0080\u0090mediated\u00C2\u00A0 signal\u00C2\u00A0 transduction\u00C2\u00A0 in\u00C2\u00A0 living\u00C2\u00A0 cells.\u00C2\u00A0J\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A01997\u00C2\u00A0Dec\u00C2\u00A015;139(6):1447\u00E2\u0080\u009054.\u00C2\u00A0 \u00C2\u00A0 213.\u00C2\u00A0Matko\u00C2\u00A0J,\u00C2\u00A0Szollosi\u00C2\u00A0J.\u00C2\u00A0Landing\u00C2\u00A0of\u00C2\u00A0immune\u00C2\u00A0receptors\u00C2\u00A0and\u00C2\u00A0signal\u00C2\u00A0proteins\u00C2\u00A0on\u00C2\u00A0lipid\u00C2\u00A0rafts:\u00C2\u00A0A\u00C2\u00A0safe\u00C2\u00A0way\u00C2\u00A0to\u00C2\u00A0 be\u00C2\u00A0spatio\u00E2\u0080\u0090temporally\u00C2\u00A0coordinated?\u00C2\u00A0Immunol\u00C2\u00A0Lett\u00C2\u00A02002\u00C2\u00A0Jun\u00C2\u00A03;82(1\u00E2\u0080\u00902):3\u00E2\u0080\u009015.\u00C2\u00A0 \u00C2\u00A0 214.\u00C2\u00A0 Kenworthy\u00C2\u00A0 AK,\u00C2\u00A0 Nichols\u00C2\u00A0 BJ,\u00C2\u00A0 Remmert\u00C2\u00A0 CL,\u00C2\u00A0 Hendrix\u00C2\u00A0 GM,\u00C2\u00A0 Kumar\u00C2\u00A0M,\u00C2\u00A0 Zimmerberg\u00C2\u00A0 J,\u00C2\u00A0 Lippincott\u00E2\u0080\u0090 Schwartz\u00C2\u00A0J.\u00C2\u00A0Dynamics\u00C2\u00A0of\u00C2\u00A0putative\u00C2\u00A0raft\u00E2\u0080\u0090associated\u00C2\u00A0proteins\u00C2\u00A0at\u00C2\u00A0the\u00C2\u00A0cell\u00C2\u00A0surface.\u00C2\u00A0J\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A02004\u00C2\u00A0Jun\u00C2\u00A0 7;165(5):735\u00E2\u0080\u009046.\u00C2\u00A0 \u00C2\u00A0 215.\u00C2\u00A0Foster\u00C2\u00A0LJ,\u00C2\u00A0De\u00C2\u00A0Hoog\u00C2\u00A0CL,\u00C2\u00A0Mann\u00C2\u00A0M.\u00C2\u00A0Unbiased\u00C2\u00A0quantitative\u00C2\u00A0proteomics\u00C2\u00A0of\u00C2\u00A0 lipid\u00C2\u00A0 rafts\u00C2\u00A0 reveals\u00C2\u00A0high\u00C2\u00A0 specificity\u00C2\u00A0for\u00C2\u00A0signaling\u00C2\u00A0factors.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A02003\u00C2\u00A0May\u00C2\u00A013;100(10):5813\u00E2\u0080\u00908.\u00C2\u00A0 \u00C2\u00A0 216.\u00C2\u00A0Magee\u00C2\u00A0 AI,\u00C2\u00A0 Parmryd\u00C2\u00A0 I.\u00C2\u00A0 Detergent\u00E2\u0080\u0090resistant\u00C2\u00A0membranes\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 protein\u00C2\u00A0 composition\u00C2\u00A0 of\u00C2\u00A0 lipid\u00C2\u00A0 rafts.\u00C2\u00A0Genome\u00C2\u00A0Biol\u00C2\u00A02003;4(11):234.\u00C2\u00A0 \u00C2\u00A0 217.\u00C2\u00A0 Rajendran\u00C2\u00A0 L,\u00C2\u00A0 Simons\u00C2\u00A0 K.\u00C2\u00A0 Lipid\u00C2\u00A0 rafts\u00C2\u00A0 and\u00C2\u00A0membrane\u00C2\u00A0 dynamics.\u00C2\u00A0 J\u00C2\u00A0 Cell\u00C2\u00A0 Sci\u00C2\u00A0 2005\u00C2\u00A0Mar\u00C2\u00A0 15;118(Pt\u00C2\u00A0 6):1099\u00E2\u0080\u0090102.\u00C2\u00A0 \u00C2\u00A0 218.\u00C2\u00A0 Silvius\u00C2\u00A0 JR.\u00C2\u00A0Partitioning\u00C2\u00A0of\u00C2\u00A0membrane\u00C2\u00A0molecules\u00C2\u00A0between\u00C2\u00A0 raft\u00C2\u00A0 and\u00C2\u00A0non\u00E2\u0080\u0090raft\u00C2\u00A0domains:\u00C2\u00A0 Insights\u00C2\u00A0 from\u00C2\u00A0model\u00E2\u0080\u0090membrane\u00C2\u00A0studies.\u00C2\u00A0Biochim\u00C2\u00A0Biophys\u00C2\u00A0Acta\u00C2\u00A02005\u00C2\u00A0Dec\u00C2\u00A030;1746(3):193\u00E2\u0080\u0090202.\u00C2\u00A0 \u00C2\u00A0 219.\u00C2\u00A0Simons\u00C2\u00A0K,\u00C2\u00A0Vaz\u00C2\u00A0WL.\u00C2\u00A0Model\u00C2\u00A0systems,\u00C2\u00A0 lipid\u00C2\u00A0rafts,\u00C2\u00A0and\u00C2\u00A0cell\u00C2\u00A0membranes.\u00C2\u00A0Annu\u00C2\u00A0Rev\u00C2\u00A0Biophys\u00C2\u00A0Biomol\u00C2\u00A0 Struct\u00C2\u00A02004;33:269\u00E2\u0080\u009095.\u00C2\u00A0 \u00C2\u00A0 220.\u00C2\u00A0London\u00C2\u00A0E.\u00C2\u00A0How\u00C2\u00A0principles\u00C2\u00A0of\u00C2\u00A0domain\u00C2\u00A0formation\u00C2\u00A0 in\u00C2\u00A0model\u00C2\u00A0membranes\u00C2\u00A0may\u00C2\u00A0explain\u00C2\u00A0ambiguities\u00C2\u00A0 concerning\u00C2\u00A0lipid\u00C2\u00A0raft\u00C2\u00A0formation\u00C2\u00A0in\u00C2\u00A0cells.\u00C2\u00A0Biochim\u00C2\u00A0Biophys\u00C2\u00A0Acta\u00C2\u00A02005\u00C2\u00A0Dec\u00C2\u00A030;1746(3):203\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 221.\u00C2\u00A0Dietrich\u00C2\u00A0C,\u00C2\u00A0Volovyk\u00C2\u00A0ZN,\u00C2\u00A0Levi\u00C2\u00A0M,\u00C2\u00A0Thompson\u00C2\u00A0NL,\u00C2\u00A0Jacobson\u00C2\u00A0K.\u00C2\u00A0Partitioning\u00C2\u00A0of\u00C2\u00A0thy\u00E2\u0080\u00901,\u00C2\u00A0GM1,\u00C2\u00A0and\u00C2\u00A0cross\u00E2\u0080\u0090 linked\u00C2\u00A0 phospholipid\u00C2\u00A0 analogs\u00C2\u00A0 into\u00C2\u00A0 lipid\u00C2\u00A0 rafts\u00C2\u00A0 reconstituted\u00C2\u00A0 in\u00C2\u00A0 supported\u00C2\u00A0 model\u00C2\u00A0 membrane\u00C2\u00A0 monolayers.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A02001\u00C2\u00A0Sep\u00C2\u00A011;98(19):10642\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 222.\u00C2\u00A0Kahya\u00C2\u00A0N,\u00C2\u00A0Brown\u00C2\u00A0DA,\u00C2\u00A0 Schwille\u00C2\u00A0P.\u00C2\u00A0Raft\u00C2\u00A0partitioning\u00C2\u00A0 and\u00C2\u00A0dynamic\u00C2\u00A0behavior\u00C2\u00A0of\u00C2\u00A0human\u00C2\u00A0placental\u00C2\u00A0 alkaline\u00C2\u00A0phosphatase\u00C2\u00A0in\u00C2\u00A0giant\u00C2\u00A0unilamellar\u00C2\u00A0vesicles.\u00C2\u00A0Biochemistry\u00C2\u00A02005\u00C2\u00A0May\u00C2\u00A024;44(20):7479\u00E2\u0080\u009089.\u00C2\u00A0 \u00C2\u00A0 223.\u00C2\u00A0 Harder\u00C2\u00A0 T,\u00C2\u00A0 Engelhardt\u00C2\u00A0 KR.\u00C2\u00A0Membrane\u00C2\u00A0 domains\u00C2\u00A0 in\u00C2\u00A0 lymphocytes\u00C2\u00A0 \u00E2\u0080\u0090\u00C2\u00A0 from\u00C2\u00A0 lipid\u00C2\u00A0 rafts\u00C2\u00A0 to\u00C2\u00A0 protein\u00C2\u00A0 scaffolds.\u00C2\u00A0Traffic\u00C2\u00A02004\u00C2\u00A0Apr;5(4):265\u00E2\u0080\u009075.\u00C2\u00A0 \u00C2\u00A0 224.\u00C2\u00A0Harder\u00C2\u00A0T,\u00C2\u00A0Scheiffele\u00C2\u00A0P,\u00C2\u00A0Verkade\u00C2\u00A0P,\u00C2\u00A0Simons\u00C2\u00A0K.\u00C2\u00A0Lipid\u00C2\u00A0domain\u00C2\u00A0structure\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0plasma\u00C2\u00A0membrane\u00C2\u00A0 revealed\u00C2\u00A0by\u00C2\u00A0patching\u00C2\u00A0of\u00C2\u00A0membrane\u00C2\u00A0components.\u00C2\u00A0J\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A01998\u00C2\u00A0May\u00C2\u00A018;141(4):929\u00E2\u0080\u009042.\u00C2\u00A0 \u00C2\u00A0 225.\u00C2\u00A0 Brown\u00C2\u00A0 DA,\u00C2\u00A0 Rose\u00C2\u00A0 JK.\u00C2\u00A0 Sorting\u00C2\u00A0 of\u00C2\u00A0 GPI\u00E2\u0080\u0090anchored\u00C2\u00A0 proteins\u00C2\u00A0 to\u00C2\u00A0 glycolipid\u00E2\u0080\u0090enriched\u00C2\u00A0 membrane\u00C2\u00A0 subdomains\u00C2\u00A0during\u00C2\u00A0transport\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0apical\u00C2\u00A0cell\u00C2\u00A0surface.\u00C2\u00A0Cell\u00C2\u00A01992\u00C2\u00A0Feb\u00C2\u00A07;68(3):533\u00E2\u0080\u009044.\u00C2\u00A0 \u00C2\u00A0 226.\u00C2\u00A0Schroeder\u00C2\u00A0R,\u00C2\u00A0London\u00C2\u00A0E,\u00C2\u00A0Brown\u00C2\u00A0D.\u00C2\u00A0Interactions\u00C2\u00A0between\u00C2\u00A0saturated\u00C2\u00A0acyl\u00C2\u00A0chains\u00C2\u00A0confer\u00C2\u00A0detergent\u00C2\u00A0 resistance\u00C2\u00A0 on\u00C2\u00A0 lipids\u00C2\u00A0 and\u00C2\u00A0 glycosylphosphatidylinositol\u00C2\u00A0 (GPI)\u00E2\u0080\u0090anchored\u00C2\u00A0 proteins:\u00C2\u00A0 GPI\u00E2\u0080\u0090anchored\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0190\u00C2\u00A0\u00C2\u00A0 proteins\u00C2\u00A0 in\u00C2\u00A0 liposomes\u00C2\u00A0 and\u00C2\u00A0 cells\u00C2\u00A0 show\u00C2\u00A0 similar\u00C2\u00A0 behavior.\u00C2\u00A0 P\u00C2\u00A0 Natl\u00C2\u00A0 Acad\u00C2\u00A0 Sci\u00C2\u00A0 USA\u00C2\u00A0 1994\u00C2\u00A0 Dec\u00C2\u00A0 6;91(25):12130\u00E2\u0080\u00904.\u00C2\u00A0 \u00C2\u00A0 227.\u00C2\u00A0 Kurzchalia\u00C2\u00A0 TV,\u00C2\u00A0 Hartmann\u00C2\u00A0 E,\u00C2\u00A0 Dupree\u00C2\u00A0 P.\u00C2\u00A0 Guilty\u00C2\u00A0 by\u00C2\u00A0 insolubility\u00E2\u0080\u0090\u00E2\u0080\u0090does\u00C2\u00A0 a\u00C2\u00A0 protein's\u00C2\u00A0 detergent\u00C2\u00A0 insolubility\u00C2\u00A0reflect\u00C2\u00A0a\u00C2\u00A0caveolar\u00C2\u00A0location?\u00C2\u00A0Trends\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A01995\u00C2\u00A0May;5(5):187\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 228.\u00C2\u00A0Lichtenberg\u00C2\u00A0D,\u00C2\u00A0Goni\u00C2\u00A0FM,\u00C2\u00A0Heerklotz\u00C2\u00A0H.\u00C2\u00A0Detergent\u00E2\u0080\u0090resistant\u00C2\u00A0membranes\u00C2\u00A0should\u00C2\u00A0not\u00C2\u00A0be\u00C2\u00A0identified\u00C2\u00A0 with\u00C2\u00A0membrane\u00C2\u00A0rafts.\u00C2\u00A0Trends\u00C2\u00A0Biochem\u00C2\u00A0Sci\u00C2\u00A02005\u00C2\u00A0Aug;30(8):430\u00E2\u0080\u00906.\u00C2\u00A0 \u00C2\u00A0 229.\u00C2\u00A0Brown\u00C2\u00A0DA.\u00C2\u00A0 Lipid\u00C2\u00A0 rafts,\u00C2\u00A0detergent\u00E2\u0080\u0090resistant\u00C2\u00A0membranes,\u00C2\u00A0and\u00C2\u00A0 raft\u00C2\u00A0 targeting\u00C2\u00A0 signals.\u00C2\u00A0Physiology\u00C2\u00A0 2006\u00C2\u00A0Dec;21:430\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 230.\u00C2\u00A0 Ahmed\u00C2\u00A0 SN,\u00C2\u00A0 Brown\u00C2\u00A0 DA,\u00C2\u00A0 London\u00C2\u00A0 E.\u00C2\u00A0 On\u00C2\u00A0 the\u00C2\u00A0 origin\u00C2\u00A0 of\u00C2\u00A0 sphingolipid/cholesterol\u00E2\u0080\u0090rich\u00C2\u00A0 detergent\u00E2\u0080\u0090 insoluble\u00C2\u00A0cell\u00C2\u00A0membranes:\u00C2\u00A0Physiological\u00C2\u00A0concentrations\u00C2\u00A0of\u00C2\u00A0cholesterol\u00C2\u00A0and\u00C2\u00A0sphingolipid\u00C2\u00A0 induce\u00C2\u00A0 formation\u00C2\u00A0 of\u00C2\u00A0 a\u00C2\u00A0 detergent\u00E2\u0080\u0090insoluble,\u00C2\u00A0 liquid\u00E2\u0080\u0090ordered\u00C2\u00A0 lipid\u00C2\u00A0 phase\u00C2\u00A0 in\u00C2\u00A0 model\u00C2\u00A0 membranes.\u00C2\u00A0 Biochemistry\u00C2\u00A01997\u00C2\u00A0Sep\u00C2\u00A09;36(36):10944\u00E2\u0080\u009053.\u00C2\u00A0 \u00C2\u00A0 231.\u00C2\u00A0Dietrich\u00C2\u00A0C,\u00C2\u00A0Bagatolli\u00C2\u00A0LA,\u00C2\u00A0Volovyk\u00C2\u00A0ZN,\u00C2\u00A0Thompson\u00C2\u00A0NL,\u00C2\u00A0Levi\u00C2\u00A0M,\u00C2\u00A0 Jacobson\u00C2\u00A0K,\u00C2\u00A0Gratton\u00C2\u00A0E.\u00C2\u00A0Lipid\u00C2\u00A0rafts\u00C2\u00A0 reconstituted\u00C2\u00A0in\u00C2\u00A0model\u00C2\u00A0membranes.\u00C2\u00A0Biophys\u00C2\u00A0J\u00C2\u00A02001\u00C2\u00A03;80(3):1417\u00E2\u0080\u009028.\u00C2\u00A0 \u00C2\u00A0 232.\u00C2\u00A0H.\u00C2\u00A0H.\u00C2\u00A0Triton\u00C2\u00A0promotes\u00C2\u00A0domain\u00C2\u00A0 formation\u00C2\u00A0 in\u00C2\u00A0 lipid\u00C2\u00A0raft\u00C2\u00A0mixtures.\u00C2\u00A0Biophys\u00C2\u00A0 J\u00C2\u00A02002\u00C2\u00A011;83(5):2693\u00E2\u0080\u0090 701.\u00C2\u00A0 \u00C2\u00A0 233.\u00C2\u00A0 Schuck\u00C2\u00A0 S,\u00C2\u00A0 Honsho\u00C2\u00A0M,\u00C2\u00A0 Ekroos\u00C2\u00A0 K,\u00C2\u00A0 Shevchenko\u00C2\u00A0 A,\u00C2\u00A0 Simons\u00C2\u00A0 K.\u00C2\u00A0 Resistance\u00C2\u00A0 of\u00C2\u00A0 cell\u00C2\u00A0membranes\u00C2\u00A0 to\u00C2\u00A0 different\u00C2\u00A0detergents.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0Sci\u00C2\u00A0USA\u00C2\u00A02003\u00C2\u00A0May\u00C2\u00A013;100(10):5795\u00E2\u0080\u0090800.\u00C2\u00A0 \u00C2\u00A0 234.\u00C2\u00A0 Lingwood\u00C2\u00A0 D,\u00C2\u00A0 Simons\u00C2\u00A0 K.\u00C2\u00A0 Detergent\u00C2\u00A0 resistance\u00C2\u00A0 as\u00C2\u00A0 a\u00C2\u00A0 tool\u00C2\u00A0 in\u00C2\u00A0membrane\u00C2\u00A0 research.\u00C2\u00A0 Nat\u00C2\u00A0 Protoc\u00C2\u00A0 2007;2(9):2159\u00E2\u0080\u009065.\u00C2\u00A0 \u00C2\u00A0 235.\u00C2\u00A0Field\u00C2\u00A0KA,\u00C2\u00A0Holowka\u00C2\u00A0D,\u00C2\u00A0Baird\u00C2\u00A0B.\u00C2\u00A0Fc\u00C2\u00A0epsilon\u00C2\u00A0RI\u00E2\u0080\u0090mediated\u00C2\u00A0 recruitment\u00C2\u00A0of\u00C2\u00A0p53/56lyn\u00C2\u00A0 to\u00C2\u00A0detergent\u00E2\u0080\u0090 resistant\u00C2\u00A0membrane\u00C2\u00A0 domains\u00C2\u00A0 accompanies\u00C2\u00A0 cellular\u00C2\u00A0 signaling.\u00C2\u00A0 P\u00C2\u00A0Natl\u00C2\u00A0 Acad\u00C2\u00A0 Sci\u00C2\u00A0USA\u00C2\u00A0 1995\u00C2\u00A0 Sep\u00C2\u00A0 26;92(20):9201\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 236.\u00C2\u00A0Gombos\u00C2\u00A0 I,\u00C2\u00A0Bacso\u00C2\u00A0Z,\u00C2\u00A0Detre\u00C2\u00A0C,\u00C2\u00A0Nagy\u00C2\u00A0H,\u00C2\u00A0Goda\u00C2\u00A0K,\u00C2\u00A0Andrasfalvy\u00C2\u00A0M,\u00C2\u00A0 Szabo\u00C2\u00A0G,\u00C2\u00A0Matko\u00C2\u00A0 J.\u00C2\u00A0Cholesterol\u00C2\u00A0 sensitivity\u00C2\u00A0of\u00C2\u00A0detergent\u00C2\u00A0 resistance:\u00C2\u00A0A\u00C2\u00A0 rapid\u00C2\u00A0 flow\u00C2\u00A0 cytometric\u00C2\u00A0 test\u00C2\u00A0 for\u00C2\u00A0detecting\u00C2\u00A0 constitutive\u00C2\u00A0or\u00C2\u00A0 induced\u00C2\u00A0raft\u00C2\u00A0association\u00C2\u00A0of\u00C2\u00A0membrane\u00C2\u00A0proteins.\u00C2\u00A0Cytom\u00C2\u00A0Part\u00C2\u00A0A\u00C2\u00A02004\u00C2\u00A0Oct;61(2):117\u00E2\u0080\u009026.\u00C2\u00A0 \u00C2\u00A0 237.\u00C2\u00A0Macdonald\u00C2\u00A0 JL,\u00C2\u00A0Pike\u00C2\u00A0LJ.\u00C2\u00A0A\u00C2\u00A0simplified\u00C2\u00A0method\u00C2\u00A0 for\u00C2\u00A0the\u00C2\u00A0preparation\u00C2\u00A0of\u00C2\u00A0detergent\u00E2\u0080\u0090free\u00C2\u00A0 lipid\u00C2\u00A0rafts.\u00C2\u00A0 J\u00C2\u00A0 Lipid\u00C2\u00A0Res\u00C2\u00A02005\u00C2\u00A0May\u00C2\u00A001;46(5):1061\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 238.\u00C2\u00A0Simons\u00C2\u00A0K,\u00C2\u00A0Ikonen\u00C2\u00A0E.\u00C2\u00A0How\u00C2\u00A0cells\u00C2\u00A0handle\u00C2\u00A0cholesterol.\u00C2\u00A0Science\u00C2\u00A02000\u00C2\u00A0December\u00C2\u00A001;290(5497):1721\u00E2\u0080\u0090 6.\u00C2\u00A0 \u00C2\u00A0 239.\u00C2\u00A0 Christian\u00C2\u00A0 AE,\u00C2\u00A0 Haynes\u00C2\u00A0MP,\u00C2\u00A0 Phillips\u00C2\u00A0MC,\u00C2\u00A0 Rothblat\u00C2\u00A0 GH.\u00C2\u00A0 Use\u00C2\u00A0 of\u00C2\u00A0 cyclodextrins\u00C2\u00A0 for\u00C2\u00A0manipulating\u00C2\u00A0 cellular\u00C2\u00A0cholesterol\u00C2\u00A0content.\u00C2\u00A0J\u00C2\u00A0Lipid\u00C2\u00A0Res\u00C2\u00A01997\u00C2\u00A0Nov;38(11):2264\u00E2\u0080\u009072.\u00C2\u00A0 \u00C2\u00A0 240.\u00C2\u00A0Drake\u00C2\u00A0DR,\u00C2\u00A0Braciale\u00C2\u00A0 TJ.\u00C2\u00A0Cutting\u00C2\u00A0 edge:\u00C2\u00A0 Lipid\u00C2\u00A0 raft\u00C2\u00A0 integrity\u00C2\u00A0 affects\u00C2\u00A0 the\u00C2\u00A0 efficiency\u00C2\u00A0of\u00C2\u00A0MHC\u00C2\u00A0 class\u00C2\u00A0 I\u00C2\u00A0 tetramer\u00C2\u00A0 binding\u00C2\u00A0 and\u00C2\u00A0 cell\u00C2\u00A0 surface\u00C2\u00A0 TCR\u00C2\u00A0 arrangement\u00C2\u00A0 on\u00C2\u00A0 CD8+\u00C2\u00A0 T\u00C2\u00A0 cells.\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A0 2001\u00C2\u00A0 Jun\u00C2\u00A0 15;166(12):7009\u00E2\u0080\u009013.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0191\u00C2\u00A0\u00C2\u00A0 241.\u00C2\u00A0Py\u00C3\u00B6r\u00C3\u00A4l\u00C3\u00A4\u00C2\u00A0K,\u00C2\u00A0Pedersen\u00C2\u00A0TR,\u00C2\u00A0Kjekshus\u00C2\u00A0J,\u00C2\u00A0Faergeman\u00C2\u00A0O,\u00C2\u00A0Olsson\u00C2\u00A0AG,\u00C2\u00A0Thorgeirsson\u00C2\u00A0G,\u00C2\u00A0The\u00C2\u00A0Scandinavian\u00C2\u00A0 Simvastatin\u00C2\u00A0 Survival\u00C2\u00A0 Study\u00C2\u00A0 (4S)\u00C2\u00A0 Group.\u00C2\u00A0 Cholesterol\u00C2\u00A0 lowering\u00C2\u00A0 with\u00C2\u00A0 simvastatin\u00C2\u00A0 improves\u00C2\u00A0 prognosis\u00C2\u00A0 of\u00C2\u00A0 diabetic\u00C2\u00A0 patients\u00C2\u00A0 with\u00C2\u00A0 coronary\u00C2\u00A0 heart\u00C2\u00A0 disease:\u00C2\u00A0 A\u00C2\u00A0 subgroup\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 scandinavian\u00C2\u00A0simvastatin\u00C2\u00A0survival\u00C2\u00A0study\u00C2\u00A0(4S).\u00C2\u00A0Diabetes\u00C2\u00A0Care\u00C2\u00A01997\u00C2\u00A0April\u00C2\u00A001;20(4):614\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 242.\u00C2\u00A0 Ponce\u00C2\u00A0 J,\u00C2\u00A0 de\u00C2\u00A0 la\u00C2\u00A0Ossa\u00C2\u00A0NP,\u00C2\u00A0Hurtado\u00C2\u00A0O,\u00C2\u00A0Millan\u00C2\u00A0M,\u00C2\u00A0Arenillas\u00C2\u00A0 JF,\u00C2\u00A0D\u00C3\u00A1valos\u00C2\u00A0A,\u00C2\u00A0Gasull\u00C2\u00A0 T.\u00C2\u00A0 Simvastatin\u00C2\u00A0 reduces\u00C2\u00A0the\u00C2\u00A0association\u00C2\u00A0of\u00C2\u00A0NMDA\u00C2\u00A0receptors\u00C2\u00A0to\u00C2\u00A0lipid\u00C2\u00A0rafts.\u00C2\u00A0Stroke\u00C2\u00A02008\u00C2\u00A0April\u00C2\u00A001;39(4):1269\u00E2\u0080\u009075.\u00C2\u00A0 \u00C2\u00A0 243.\u00C2\u00A0Zhuang\u00C2\u00A0 L,\u00C2\u00A0Kim\u00C2\u00A0 J,\u00C2\u00A0Adam\u00C2\u00A0RM,\u00C2\u00A0Solomon\u00C2\u00A0KR,\u00C2\u00A0Freeman\u00C2\u00A0MR.\u00C2\u00A0Cholesterol\u00C2\u00A0 targeting\u00C2\u00A0alters\u00C2\u00A0 lipid\u00C2\u00A0 raft\u00C2\u00A0 composition\u00C2\u00A0 and\u00C2\u00A0 cell\u00C2\u00A0 survival\u00C2\u00A0 in\u00C2\u00A0 prostate\u00C2\u00A0 cancer\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 xenografts.\u00C2\u00A0 J\u00C2\u00A0 Clin\u00C2\u00A0 Invest\u00C2\u00A0 2005\u00C2\u00A0 Apr;115(4):959\u00E2\u0080\u009068.\u00C2\u00A0 \u00C2\u00A0 244.\u00C2\u00A0Algeciras\u00E2\u0080\u0090Schimnich\u00C2\u00A0A,\u00C2\u00A0Shen\u00C2\u00A0L,\u00C2\u00A0Barnhart\u00C2\u00A0BC,\u00C2\u00A0Murmann\u00C2\u00A0AE,\u00C2\u00A0Burkhardt\u00C2\u00A0 JK,\u00C2\u00A0Peter\u00C2\u00A0ME.\u00C2\u00A0Molecular\u00C2\u00A0 ordering\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0initial\u00C2\u00A0signaling\u00C2\u00A0events\u00C2\u00A0of\u00C2\u00A0CD95.\u00C2\u00A0Mol\u00C2\u00A0Cell\u00C2\u00A0Biol\u00C2\u00A02002\u00C2\u00A0Jan;22(1):207\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 245.\u00C2\u00A0Brown\u00C2\u00A0BK,\u00C2\u00A0Song\u00C2\u00A0W.\u00C2\u00A0The\u00C2\u00A0actin\u00C2\u00A0cytoskeleton\u00C2\u00A0 is\u00C2\u00A0required\u00C2\u00A0for\u00C2\u00A0the\u00C2\u00A0trafficking\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0B\u00C2\u00A0cell\u00C2\u00A0antigen\u00C2\u00A0 receptor\u00C2\u00A0to\u00C2\u00A0the\u00C2\u00A0late\u00C2\u00A0endosomes.\u00C2\u00A0Traffic\u00C2\u00A02001\u00C2\u00A0Jun;2(6):414\u00E2\u0080\u009027.\u00C2\u00A0 \u00C2\u00A0 246.\u00C2\u00A0 Beum\u00C2\u00A0 PV,\u00C2\u00A0 Lindorfer\u00C2\u00A0 MA,\u00C2\u00A0 Taylor\u00C2\u00A0 RP.\u00C2\u00A0 Within\u00C2\u00A0 peripheral\u00C2\u00A0 blood\u00C2\u00A0 mononuclear\u00C2\u00A0 cells,\u00C2\u00A0 antibody\u00E2\u0080\u0090 dependent\u00C2\u00A0cellular\u00C2\u00A0cytotoxicity\u00C2\u00A0of\u00C2\u00A0rituximab\u00E2\u0080\u0090opsonized\u00C2\u00A0daudi\u00C2\u00A0cells\u00C2\u00A0is\u00C2\u00A0promoted\u00C2\u00A0by\u00C2\u00A0NK\u00C2\u00A0cells\u00C2\u00A0and\u00C2\u00A0 inhibited\u00C2\u00A0by\u00C2\u00A0monocytes\u00C2\u00A0due\u00C2\u00A0to\u00C2\u00A0shaving.\u00C2\u00A0J\u00C2\u00A0Immunol\u00C2\u00A02008\u00C2\u00A0Aug\u00C2\u00A015;181(4):2916\u00E2\u0080\u009024.\u00C2\u00A0 \u00C2\u00A0 247.\u00C2\u00A0Radeva\u00C2\u00A0G,\u00C2\u00A0Sharom\u00C2\u00A0FJ.\u00C2\u00A0Isolation\u00C2\u00A0and\u00C2\u00A0characterization\u00C2\u00A0of\u00C2\u00A0lipid\u00C2\u00A0rafts\u00C2\u00A0with\u00C2\u00A0different\u00C2\u00A0properties\u00C2\u00A0from\u00C2\u00A0 RBL\u00E2\u0080\u00902H3\u00C2\u00A0(rat\u00C2\u00A0basophilic\u00C2\u00A0leukaemia)\u00C2\u00A0cells.\u00C2\u00A0Biochem\u00C2\u00A0J\u00C2\u00A02004\u00C2\u00A0May\u00C2\u00A015;380(Pt\u00C2\u00A01):219\u00E2\u0080\u009030.\u00C2\u00A0 \u00C2\u00A0 248.\u00C2\u00A0Mitchell\u00C2\u00A0 JS,\u00C2\u00A0Kanca\u00C2\u00A0O,\u00C2\u00A0McIntyre\u00C2\u00A0BW.\u00C2\u00A0 Lipid\u00C2\u00A0microdomain\u00C2\u00A0 clustering\u00C2\u00A0 induces\u00C2\u00A0a\u00C2\u00A0 redistribution\u00C2\u00A0of\u00C2\u00A0 antigen\u00C2\u00A0 recognition\u00C2\u00A0and\u00C2\u00A0 adhesion\u00C2\u00A0molecules\u00C2\u00A0on\u00C2\u00A0human\u00C2\u00A0T\u00C2\u00A0 lymphocytes.\u00C2\u00A0 J\u00C2\u00A0 Immunol\u00C2\u00A02002\u00C2\u00A0Mar\u00C2\u00A0 15;168(6):2737\u00E2\u0080\u009044.\u00C2\u00A0 \u00C2\u00A0 249.\u00C2\u00A0Im\u00C2\u00A0JS,\u00C2\u00A0Arora\u00C2\u00A0P,\u00C2\u00A0Bricard\u00C2\u00A0G,\u00C2\u00A0Molano\u00C2\u00A0A,\u00C2\u00A0Venkataswamy\u00C2\u00A0MM,\u00C2\u00A0Baine\u00C2\u00A0I,\u00C2\u00A0Jerud\u00C2\u00A0ES,\u00C2\u00A0Goldberg\u00C2\u00A0MF,\u00C2\u00A0Baena\u00C2\u00A0 A,\u00C2\u00A0Yu\u00C2\u00A0KO,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Kinetics\u00C2\u00A0and\u00C2\u00A0cellular\u00C2\u00A0site\u00C2\u00A0of\u00C2\u00A0glycolipid\u00C2\u00A0 loading\u00C2\u00A0control\u00C2\u00A0 the\u00C2\u00A0outcome\u00C2\u00A0of\u00C2\u00A0natural\u00C2\u00A0 killer\u00C2\u00A0T\u00C2\u00A0cell\u00C2\u00A0activation.\u00C2\u00A0Immunity\u00C2\u00A02009\u00C2\u00A0Jun\u00C2\u00A019;30(6):888\u00E2\u0080\u009098.\u00C2\u00A0 \u00C2\u00A0 250.\u00C2\u00A0 Wolf\u00C2\u00A0 Z,\u00C2\u00A0 Orso\u00C2\u00A0 E,\u00C2\u00A0 Werner\u00C2\u00A0 T,\u00C2\u00A0 Klunemann\u00C2\u00A0 HH,\u00C2\u00A0 Schmitz\u00C2\u00A0 G.\u00C2\u00A0 Monocyte\u00C2\u00A0 cholesterol\u00C2\u00A0 homeostasis\u00C2\u00A0 correlates\u00C2\u00A0with\u00C2\u00A0 the\u00C2\u00A0presence\u00C2\u00A0of\u00C2\u00A0detergent\u00C2\u00A0 resistant\u00C2\u00A0membrane\u00C2\u00A0microdomains.\u00C2\u00A0Cytom\u00C2\u00A0 Part\u00C2\u00A0A\u00C2\u00A0 2007\u00C2\u00A0Jul;71(7):486\u00E2\u0080\u009094.\u00C2\u00A0 \u00C2\u00A0 251.\u00C2\u00A0Calzolari\u00C2\u00A0A,\u00C2\u00A0Raggi\u00C2\u00A0C,\u00C2\u00A0Deaglio\u00C2\u00A0S,\u00C2\u00A0Sposi\u00C2\u00A0NM,\u00C2\u00A0Stafsnes\u00C2\u00A0M,\u00C2\u00A0Fecchi\u00C2\u00A0K,\u00C2\u00A0Parolini\u00C2\u00A0I,\u00C2\u00A0Malavasi\u00C2\u00A0F,\u00C2\u00A0Peschle\u00C2\u00A0C,\u00C2\u00A0 Sargiacomo\u00C2\u00A0M,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0TfR2\u00C2\u00A0localizes\u00C2\u00A0in\u00C2\u00A0lipid\u00C2\u00A0raft\u00C2\u00A0domains\u00C2\u00A0and\u00C2\u00A0is\u00C2\u00A0released\u00C2\u00A0in\u00C2\u00A0exosomes\u00C2\u00A0to\u00C2\u00A0activate\u00C2\u00A0 signal\u00C2\u00A0transduction\u00C2\u00A0along\u00C2\u00A0the\u00C2\u00A0MAPK\u00C2\u00A0pathway.\u00C2\u00A0J\u00C2\u00A0Cell\u00C2\u00A0Sci\u00C2\u00A02006\u00C2\u00A0Nov\u00C2\u00A01;119(Pt\u00C2\u00A021):4486\u00E2\u0080\u009098.\u00C2\u00A0 \u00C2\u00A0 252.\u00C2\u00A0Nellis\u00C2\u00A0DF,\u00C2\u00A0 Ekstrom\u00C2\u00A0DL,\u00C2\u00A0Kirpotin\u00C2\u00A0DB,\u00C2\u00A0 Zhu\u00C2\u00A0 J,\u00C2\u00A0Andersson\u00C2\u00A0R,\u00C2\u00A0Broadt\u00C2\u00A0 TL,\u00C2\u00A0Ouellette\u00C2\u00A0 TF,\u00C2\u00A0Perkins\u00C2\u00A0 SC,\u00C2\u00A0 Roach\u00C2\u00A0 JM,\u00C2\u00A0 Drummond\u00C2\u00A0 DC,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Preclinical\u00C2\u00A0 manufacture\u00C2\u00A0 of\u00C2\u00A0 an\u00C2\u00A0 anti\u00E2\u0080\u0090HER2\u00C2\u00A0 scFv\u00E2\u0080\u0090PEG\u00E2\u0080\u0090DSPE,\u00C2\u00A0 liposome\u00E2\u0080\u0090inserting\u00C2\u00A0conjugate.\u00C2\u00A01.\u00C2\u00A0Gram\u00E2\u0080\u0090scale\u00C2\u00A0production\u00C2\u00A0and\u00C2\u00A0purification.\u00C2\u00A0Biotechnol\u00C2\u00A0Prog\u00C2\u00A02005\u00C2\u00A0 Jan\u00E2\u0080\u0090Feb;21(1):205\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 253.\u00C2\u00A0Nellis\u00C2\u00A0DF,\u00C2\u00A0Giardina\u00C2\u00A0SL,\u00C2\u00A0Janini\u00C2\u00A0GM,\u00C2\u00A0Shenoy\u00C2\u00A0SR,\u00C2\u00A0Marks\u00C2\u00A0JD,\u00C2\u00A0Tsai\u00C2\u00A0R,\u00C2\u00A0Drummond\u00C2\u00A0DC,\u00C2\u00A0Hong\u00C2\u00A0K,\u00C2\u00A0Park\u00C2\u00A0JW,\u00C2\u00A0 Ouellette\u00C2\u00A0 TF,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Preclinical\u00C2\u00A0 manufacture\u00C2\u00A0 of\u00C2\u00A0 anti\u00E2\u0080\u0090HER2\u00C2\u00A0 liposome\u00E2\u0080\u0090inserting,\u00C2\u00A0 scFv\u00E2\u0080\u0090PEG\u00E2\u0080\u0090lipid\u00C2\u00A0 conjugate.\u00C2\u00A02.\u00C2\u00A0Conjugate\u00C2\u00A0micelle\u00C2\u00A0identity,\u00C2\u00A0purity,\u00C2\u00A0stability,\u00C2\u00A0and\u00C2\u00A0potency\u00C2\u00A0analysis.\u00C2\u00A0Biotechnol\u00C2\u00A0Prog\u00C2\u00A0 2005\u00C2\u00A0Jan\u00E2\u0080\u0090Feb;21(1):221\u00E2\u0080\u009032.\u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0192\u00C2\u00A0\u00C2\u00A0 254.\u00C2\u00A0 Smith\u00C2\u00A0 LM,\u00C2\u00A0Parce\u00C2\u00A0 JW,\u00C2\u00A0 Smith\u00C2\u00A0BA,\u00C2\u00A0McConnell\u00C2\u00A0HM.\u00C2\u00A0Antibodies\u00C2\u00A0bound\u00C2\u00A0 to\u00C2\u00A0 lipid\u00C2\u00A0haptens\u00C2\u00A0 in\u00C2\u00A0model\u00C2\u00A0 membranes\u00C2\u00A0 diffuse\u00C2\u00A0 as\u00C2\u00A0 rapidly\u00C2\u00A0 as\u00C2\u00A0 the\u00C2\u00A0 lipids\u00C2\u00A0 themselves.\u00C2\u00A0 P\u00C2\u00A0 Natl\u00C2\u00A0 Acad\u00C2\u00A0 Sci\u00C2\u00A0 USA\u00C2\u00A0 1979\u00C2\u00A0 Sep;76(9):4177\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 255.\u00C2\u00A0Smith\u00C2\u00A0MR.\u00C2\u00A0Rituximab\u00C2\u00A0(monoclonal\u00C2\u00A0anti\u00E2\u0080\u0090CD20\u00C2\u00A0antibody):\u00C2\u00A0Mechanisms\u00C2\u00A0of\u00C2\u00A0action\u00C2\u00A0and\u00C2\u00A0resistance.\u00C2\u00A0 Oncogene\u00C2\u00A02003\u00C2\u00A0Oct\u00C2\u00A020;22(47):7359\u00E2\u0080\u009068.\u00C2\u00A0 \u00C2\u00A0 256.\u00C2\u00A0Kolch\u00C2\u00A0W,\u00C2\u00A0Calder\u00C2\u00A0M,\u00C2\u00A0Gilbert\u00C2\u00A0D.\u00C2\u00A0When\u00C2\u00A0kinases\u00C2\u00A0meet\u00C2\u00A0mathematics:\u00C2\u00A0The\u00C2\u00A0systems\u00C2\u00A0biology\u00C2\u00A0of\u00C2\u00A0MAPK\u00C2\u00A0 signalling.\u00C2\u00A0FEBS\u00C2\u00A0Lett\u00C2\u00A02005\u00C2\u00A0Mar\u00C2\u00A021;579(8):1891\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 257.\u00C2\u00A0 Kholodenko\u00C2\u00A0 BN.\u00C2\u00A0 Cell\u00E2\u0080\u0090signalling\u00C2\u00A0 dynamics\u00C2\u00A0 in\u00C2\u00A0 time\u00C2\u00A0 and\u00C2\u00A0 space.\u00C2\u00A0 Nat\u00C2\u00A0 Rev\u00C2\u00A0 Mol\u00C2\u00A0 Cell\u00C2\u00A0 Biol\u00C2\u00A0 2006\u00C2\u00A0 Mar;7(3):165\u00E2\u0080\u009076.\u00C2\u00A0 \u00C2\u00A0 258.\u00C2\u00A0Kontermann\u00C2\u00A0RE,\u00C2\u00A0Munkel\u00C2\u00A0S,\u00C2\u00A0Neumeyer\u00C2\u00A0J,\u00C2\u00A0Muller\u00C2\u00A0D,\u00C2\u00A0Branschadel\u00C2\u00A0M,\u00C2\u00A0Scheurich\u00C2\u00A0P,\u00C2\u00A0Pfizenmaier\u00C2\u00A0K.\u00C2\u00A0A\u00C2\u00A0 humanized\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0receptor\u00C2\u00A01\u00C2\u00A0(TNFR1)\u00E2\u0080\u0090specific\u00C2\u00A0antagonistic\u00C2\u00A0antibody\u00C2\u00A0for\u00C2\u00A0selective\u00C2\u00A0 inhibition\u00C2\u00A0of\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0(TNF)\u00C2\u00A0action.\u00C2\u00A0J\u00C2\u00A0Immunother\u00C2\u00A02008\u00C2\u00A0Apr;31(3):225\u00E2\u0080\u009034.\u00C2\u00A0 \u00C2\u00A0 259.\u00C2\u00A0 McDermott\u00C2\u00A0 MF,\u00C2\u00A0 Aksentijevich\u00C2\u00A0 I,\u00C2\u00A0 Galon\u00C2\u00A0 J,\u00C2\u00A0 McDermott\u00C2\u00A0 EM,\u00C2\u00A0 Ogunkolade\u00C2\u00A0 BW,\u00C2\u00A0 Centola\u00C2\u00A0 M,\u00C2\u00A0 Mansfield\u00C2\u00A0E,\u00C2\u00A0Gadina\u00C2\u00A0M,\u00C2\u00A0Karenko\u00C2\u00A0L,\u00C2\u00A0Pettersson\u00C2\u00A0T,\u00C2\u00A0et\u00C2\u00A0al.\u00C2\u00A0Germline\u00C2\u00A0mutations\u00C2\u00A0 in\u00C2\u00A0the\u00C2\u00A0extracellular\u00C2\u00A0 domains\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 55\u00C2\u00A0 kDa\u00C2\u00A0 TNF\u00C2\u00A0 receptor,\u00C2\u00A0 TNFR1,\u00C2\u00A0 define\u00C2\u00A0 a\u00C2\u00A0 family\u00C2\u00A0 of\u00C2\u00A0 dominantly\u00C2\u00A0 inherited\u00C2\u00A0 autoinflammatory\u00C2\u00A0syndromes.\u00C2\u00A0Cell\u00C2\u00A01999\u00C2\u00A0Apr\u00C2\u00A02;97(1):133\u00E2\u0080\u009044.\u00C2\u00A0 \u00C2\u00A0 260.\u00C2\u00A0Ryan\u00C2\u00A0JG,\u00C2\u00A0Aksentijevich\u00C2\u00A0I.\u00C2\u00A0Tumor\u00C2\u00A0necrosis\u00C2\u00A0factor\u00C2\u00A0receptor\u00E2\u0080\u0090associated\u00C2\u00A0periodic\u00C2\u00A0syndrome:\u00C2\u00A0Toward\u00C2\u00A0 a\u00C2\u00A0molecular\u00C2\u00A0understanding\u00C2\u00A0of\u00C2\u00A0 the\u00C2\u00A0 systemic\u00C2\u00A0autoinflammatory\u00C2\u00A0diseases.\u00C2\u00A0Arthritis\u00C2\u00A0Rheum\u00C2\u00A02009\u00C2\u00A0 Jan;60(1):8\u00E2\u0080\u009011.\u00C2\u00A0 \u00C2\u00A0 261.\u00C2\u00A0Kimberley\u00C2\u00A0FC,\u00C2\u00A0Lobito\u00C2\u00A0AA,\u00C2\u00A0Siegel\u00C2\u00A0RM,\u00C2\u00A0Screaton\u00C2\u00A0GR.\u00C2\u00A0Falling\u00C2\u00A0into\u00C2\u00A0TRAPS\u00E2\u0080\u0090\u00E2\u0080\u0090receptor\u00C2\u00A0misfolding\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0 TNF\u00C2\u00A0receptor\u00C2\u00A01\u00E2\u0080\u0090associated\u00C2\u00A0periodic\u00C2\u00A0fever\u00C2\u00A0syndrome.\u00C2\u00A0Arthritis\u00C2\u00A0Res\u00C2\u00A0Ther\u00C2\u00A02007;9(4):217.\u00C2\u00A0 \u00C2\u00A0 262.\u00C2\u00A0 Lobito\u00C2\u00A0AA,\u00C2\u00A0Gabriel\u00C2\u00A0TL,\u00C2\u00A0Medema\u00C2\u00A0 JP,\u00C2\u00A0Kimberley\u00C2\u00A0 FC.\u00C2\u00A0Disease\u00C2\u00A0 causing\u00C2\u00A0mutations\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0TNF\u00C2\u00A0and\u00C2\u00A0 TNFR\u00C2\u00A0 superfamilies:\u00C2\u00A0 Focus\u00C2\u00A0 on\u00C2\u00A0molecular\u00C2\u00A0mechanisms\u00C2\u00A0 driving\u00C2\u00A0 disease.\u00C2\u00A0 Trends\u00C2\u00A0Mol\u00C2\u00A0Med\u00C2\u00A0 2011\u00C2\u00A0 Sep;17(9):494\u00E2\u0080\u0090505.\u00C2\u00A0 \u00C2\u00A0 263.\u00C2\u00A0 Vogelstein\u00C2\u00A0 B,\u00C2\u00A0 Kinzler\u00C2\u00A0 KW.\u00C2\u00A0 Cancer\u00C2\u00A0 genes\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 pathways\u00C2\u00A0 they\u00C2\u00A0 control.\u00C2\u00A0 Nat\u00C2\u00A0 Med\u00C2\u00A0 2004\u00C2\u00A0 Aug;10(8):789\u00E2\u0080\u009099.\u00C2\u00A0 \u00C2\u00A0 264.\u00C2\u00A0Dechant\u00C2\u00A0MJ,\u00C2\u00A0Fellenberg\u00C2\u00A0J,\u00C2\u00A0Scheuerpflug\u00C2\u00A0CG,\u00C2\u00A0Ewerbeck\u00C2\u00A0V,\u00C2\u00A0Debatin\u00C2\u00A0KM.\u00C2\u00A0Mutation\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0 apoptotic\u00C2\u00A0 \"death\u00E2\u0080\u0090receptors\"\u00C2\u00A0 and\u00C2\u00A0 the\u00C2\u00A0 adaptors\u00C2\u00A0 TRADD\u00C2\u00A0 and\u00C2\u00A0 FADD/MORT\u00E2\u0080\u00901\u00C2\u00A0 in\u00C2\u00A0 osteosarcoma\u00C2\u00A0 tumor\u00C2\u00A0samples\u00C2\u00A0and\u00C2\u00A0osteosarcoma\u00C2\u00A0cell\u00C2\u00A0lines.\u00C2\u00A0Int\u00C2\u00A0J\u00C2\u00A0Cancer\u00C2\u00A02004\u00C2\u00A0May\u00C2\u00A01;109(5):661\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 265.\u00C2\u00A0Park\u00C2\u00A0A,\u00C2\u00A0Baichwal\u00C2\u00A0VR.\u00C2\u00A0Systematic\u00C2\u00A0mutational\u00C2\u00A0analysis\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0death\u00C2\u00A0domain\u00C2\u00A0of\u00C2\u00A0the\u00C2\u00A0tumor\u00C2\u00A0necrosis\u00C2\u00A0 factor\u00C2\u00A0receptor\u00C2\u00A01\u00E2\u0080\u0090associated\u00C2\u00A0protein\u00C2\u00A0TRADD.\u00C2\u00A0J\u00C2\u00A0Biol\u00C2\u00A0Chem\u00C2\u00A01996\u00C2\u00A0Apr\u00C2\u00A019;271(16):9858\u00E2\u0080\u009062.\u00C2\u00A0 \u00C2\u00A0 266.\u00C2\u00A0Dechant\u00C2\u00A0MJ,\u00C2\u00A0Scheuerpflug\u00C2\u00A0CG,\u00C2\u00A0Pauly\u00C2\u00A0E,\u00C2\u00A0van\u00C2\u00A0der\u00C2\u00A0Werff\u00C2\u00A0Ten\u00C2\u00A0Bosch,J.,\u00C2\u00A0Debatin\u00C2\u00A0KM,\u00C2\u00A0Fellenberg\u00C2\u00A0 J.\u00C2\u00A0 Screening,\u00C2\u00A0 identification,\u00C2\u00A0 and\u00C2\u00A0 functional\u00C2\u00A0 analysis\u00C2\u00A0 of\u00C2\u00A0 three\u00C2\u00A0 novel\u00C2\u00A0missense\u00C2\u00A0 mutations\u00C2\u00A0 in\u00C2\u00A0 the\u00C2\u00A0 TRADD\u00C2\u00A0gene\u00C2\u00A0in\u00C2\u00A0children\u00C2\u00A0with\u00C2\u00A0ALL\u00C2\u00A0and\u00C2\u00A0ALPS.\u00C2\u00A0Pediatr\u00C2\u00A0Blood\u00C2\u00A0Cancer\u00C2\u00A02008\u00C2\u00A0Nov;51(5):616\u00E2\u0080\u009020.\u00C2\u00A0 \u00C2\u00A0 267.\u00C2\u00A0Sassano\u00C2\u00A0A,\u00C2\u00A0Platanias\u00C2\u00A0LC.\u00C2\u00A0Statins\u00C2\u00A0in\u00C2\u00A0tumor\u00C2\u00A0suppression.\u00C2\u00A0Cancer\u00C2\u00A0Lett\u00C2\u00A02008\u00C2\u00A0Feb\u00C2\u00A018;260(1\u00E2\u0080\u00902):11\u00E2\u0080\u00909.\u00C2\u00A0 \u00C2\u00A0 \u00E2\u0080\u00A2\u00C2\u00A0193\u00C2\u00A0\u00C2\u00A0 268.\u00C2\u00A0 Katano\u00C2\u00A0 H,\u00C2\u00A0 Pesnicak\u00C2\u00A0 L,\u00C2\u00A0 Cohen\u00C2\u00A0 JI.\u00C2\u00A0 Simvastatin\u00C2\u00A0 induces\u00C2\u00A0 apoptosis\u00C2\u00A0 of\u00C2\u00A0 epstein\u00E2\u0080\u0090barr\u00C2\u00A0 virus\u00C2\u00A0 (EBV)\u00E2\u0080\u0090 transformed\u00C2\u00A0lymphoblastoid\u00C2\u00A0cell\u00C2\u00A0lines\u00C2\u00A0and\u00C2\u00A0delays\u00C2\u00A0development\u00C2\u00A0of\u00C2\u00A0EBV\u00C2\u00A0lymphomas.\u00C2\u00A0P\u00C2\u00A0Natl\u00C2\u00A0Acad\u00C2\u00A0 Sci\u00C2\u00A0USA\u00C2\u00A02004\u00C2\u00A0Apr\u00C2\u00A06;101(14):4960\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 269.\u00C2\u00A0 Ahn\u00C2\u00A0 KS,\u00C2\u00A0 Sethi\u00C2\u00A0 G,\u00C2\u00A0 Chaturvedi\u00C2\u00A0 MM,\u00C2\u00A0 Aggarwal\u00C2\u00A0 BB.\u00C2\u00A0 Simvastatin,\u00C2\u00A0 3\u00E2\u0080\u0090hydroxy\u00E2\u0080\u00903\u00E2\u0080\u0090methylglutaryl\u00C2\u00A0 coenzyme\u00C2\u00A0A\u00C2\u00A0 reductase\u00C2\u00A0 inhibitor,\u00C2\u00A0suppresses\u00C2\u00A0osteoclastogenesis\u00C2\u00A0 induced\u00C2\u00A0by\u00C2\u00A0 receptor\u00C2\u00A0activator\u00C2\u00A0 of\u00C2\u00A0nuclear\u00C2\u00A0factor\u00E2\u0080\u0090kappaB\u00C2\u00A0 ligand\u00C2\u00A0through\u00C2\u00A0modulation\u00C2\u00A0of\u00C2\u00A0NF\u00E2\u0080\u0090kappaB\u00C2\u00A0pathway.\u00C2\u00A0 Int\u00C2\u00A0J\u00C2\u00A0Cancer\u00C2\u00A02008\u00C2\u00A0 Oct\u00C2\u00A015;123(8):1733\u00E2\u0080\u009040.\u00C2\u00A0 \u00C2\u00A0 270.\u00C2\u00A0Ahn\u00C2\u00A0KS,\u00C2\u00A0Sethi\u00C2\u00A0G,\u00C2\u00A0Aggarwal\u00C2\u00A0BB.\u00C2\u00A0Reversal\u00C2\u00A0of\u00C2\u00A0chemoresistance\u00C2\u00A0and\u00C2\u00A0enhancement\u00C2\u00A0of\u00C2\u00A0apoptosis\u00C2\u00A0by\u00C2\u00A0 statins\u00C2\u00A0 through\u00C2\u00A0 down\u00E2\u0080\u0090regulation\u00C2\u00A0 of\u00C2\u00A0 the\u00C2\u00A0 NF\u00E2\u0080\u0090kappaB\u00C2\u00A0 pathway.\u00C2\u00A0 Biochem\u00C2\u00A0 Pharmacol\u00C2\u00A0 2008\u00C2\u00A0 Feb\u00C2\u00A0 15;75(4):907\u00E2\u0080\u009013.\u00C2\u00A0 \u00C2\u00A0 271.\u00C2\u00A0Aberg\u00C2\u00A0M,\u00C2\u00A0Wickstrom\u00C2\u00A0M,\u00C2\u00A0Siegbahn\u00C2\u00A0A.\u00C2\u00A0Simvastatin\u00C2\u00A0induces\u00C2\u00A0apoptosis\u00C2\u00A0in\u00C2\u00A0human\u00C2\u00A0breast\u00C2\u00A0cancer\u00C2\u00A0cells\u00C2\u00A0 in\u00C2\u00A0a\u00C2\u00A0NFkappaB\u00E2\u0080\u0090dependent\u00C2\u00A0manner\u00C2\u00A0and\u00C2\u00A0abolishes\u00C2\u00A0 the\u00C2\u00A0anti\u00E2\u0080\u0090apoptotic\u00C2\u00A0 signaling\u00C2\u00A0of\u00C2\u00A0TF/FVIIa\u00C2\u00A0and\u00C2\u00A0 TF/FVIIa/FXa.\u00C2\u00A0Thromb\u00C2\u00A0Res\u00C2\u00A02008;122(2):191\u00E2\u0080\u0090202.\u00C2\u00A0 \u00C2\u00A0 272.\u00C2\u00A0Martirosyan\u00C2\u00A0A,\u00C2\u00A0Clendening\u00C2\u00A0J,\u00C2\u00A0Goard\u00C2\u00A0C,\u00C2\u00A0Penn\u00C2\u00A0L.\u00C2\u00A0Lovastatin\u00C2\u00A0 induces\u00C2\u00A0apoptosis\u00C2\u00A0of\u00C2\u00A0ovarian\u00C2\u00A0cancer\u00C2\u00A0 cells\u00C2\u00A0 and\u00C2\u00A0 synergizes\u00C2\u00A0 with\u00C2\u00A0 doxorubicin:\u00C2\u00A0 Potential\u00C2\u00A0 therapeutic\u00C2\u00A0 relevance.\u00C2\u00A0 BMC\u00C2\u00A0 Cancer\u00C2\u00A0 2010;10(1):103.\u00C2\u00A0 \u00C2\u00A0 273.\u00C2\u00A0Glynn\u00C2\u00A0SA,\u00C2\u00A0O'Sullivan\u00C2\u00A0D,\u00C2\u00A0Eustace\u00C2\u00A0AJ,\u00C2\u00A0Clynes\u00C2\u00A0M,\u00C2\u00A0O'Donovan\u00C2\u00A0N.\u00C2\u00A0The\u00C2\u00A03\u00E2\u0080\u0090hydroxy\u00E2\u0080\u00903\u00E2\u0080\u0090methylglutaryl\u00E2\u0080\u0090 coenzyme\u00C2\u00A0A\u00C2\u00A0 reductase\u00C2\u00A0 inhibitors,\u00C2\u00A0 simvastatin,\u00C2\u00A0 lovastatin\u00C2\u00A0 and\u00C2\u00A0mevastatin\u00C2\u00A0 inhibit\u00C2\u00A0 proliferation\u00C2\u00A0 and\u00C2\u00A0invasion\u00C2\u00A0of\u00C2\u00A0melanoma\u00C2\u00A0cells.\u00C2\u00A0BMC\u00C2\u00A0Cancer\u00C2\u00A02008\u00C2\u00A0Jan\u00C2\u00A016;8:9.\u00C2\u00A0 \u00C2\u00A0 274.\u00C2\u00A0 Schmidmaier\u00C2\u00A0 R,\u00C2\u00A0 Baumann\u00C2\u00A0 P,\u00C2\u00A0 Bumeder\u00C2\u00A0 I,\u00C2\u00A0Meinhardt\u00C2\u00A0 G,\u00C2\u00A0 Straka\u00C2\u00A0 C,\u00C2\u00A0 Emmerich\u00C2\u00A0 B.\u00C2\u00A0 First\u00C2\u00A0 clinical\u00C2\u00A0 experience\u00C2\u00A0with\u00C2\u00A0simvastatin\u00C2\u00A0to\u00C2\u00A0overcome\u00C2\u00A0drug\u00C2\u00A0resistance\u00C2\u00A0in\u00C2\u00A0refractory\u00C2\u00A0multiple\u00C2\u00A0myeloma.\u00C2\u00A0Eur\u00C2\u00A0J\u00C2\u00A0 Haematol\u00C2\u00A02007\u00C2\u00A0Sep;79(3):240\u00E2\u0080\u00903.\u00C2\u00A0 \u00C2\u00A0 275.\u00C2\u00A0Fortuny\u00C2\u00A0J,\u00C2\u00A0de\u00C2\u00A0Sanjose\u00C2\u00A0S,\u00C2\u00A0Becker\u00C2\u00A0N,\u00C2\u00A0Maynadie\u00C2\u00A0M,\u00C2\u00A0Cocco\u00C2\u00A0PL,\u00C2\u00A0Staines\u00C2\u00A0A,\u00C2\u00A0Foretova\u00C2\u00A0L,\u00C2\u00A0Vornanen\u00C2\u00A0M,\u00C2\u00A0 Brennan\u00C2\u00A0 P,\u00C2\u00A0 Nieters\u00C2\u00A0 A,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Statin\u00C2\u00A0 use\u00C2\u00A0 and\u00C2\u00A0 risk\u00C2\u00A0 of\u00C2\u00A0 lymphoid\u00C2\u00A0 neoplasms:\u00C2\u00A0 Results\u00C2\u00A0 from\u00C2\u00A0 the\u00C2\u00A0 european\u00C2\u00A0 case\u00E2\u0080\u0090control\u00C2\u00A0 study\u00C2\u00A0 EPILYMPH.\u00C2\u00A0 Cancer\u00C2\u00A0 Epidemiol\u00C2\u00A0 Biomarkers\u00C2\u00A0 Prev\u00C2\u00A0 2006\u00C2\u00A0 May;15(5):921\u00E2\u0080\u00905.\u00C2\u00A0 \u00C2\u00A0 276.\u00C2\u00A0Winiarska\u00C2\u00A0M,\u00C2\u00A0Bil\u00C2\u00A0 J,\u00C2\u00A0Wilczek\u00C2\u00A0E,\u00C2\u00A0Wilczynski\u00C2\u00A0GM,\u00C2\u00A0 Lekka\u00C2\u00A0M,\u00C2\u00A0Engelberts\u00C2\u00A0PJ,\u00C2\u00A0Mackus\u00C2\u00A0WJ,\u00C2\u00A0Gorska\u00C2\u00A0E,\u00C2\u00A0 Bojarski\u00C2\u00A0 L,\u00C2\u00A0 Stoklosa\u00C2\u00A0 T,\u00C2\u00A0 et\u00C2\u00A0 al.\u00C2\u00A0 Statins\u00C2\u00A0 impair\u00C2\u00A0 antitumor\u00C2\u00A0 effects\u00C2\u00A0 of\u00C2\u00A0 rituximab\u00C2\u00A0 by\u00C2\u00A0 inducing\u00C2\u00A0 conformational\u00C2\u00A0changes\u00C2\u00A0of\u00C2\u00A0CD20.\u00C2\u00A0PLoS\u00C2\u00A0Med\u00C2\u00A02008\u00C2\u00A0Mar\u00C2\u00A025;5(3):e64.\u00C2\u00A0 \u00C2\u00A0 277.\u00C2\u00A0Nowakowski\u00C2\u00A0GS,\u00C2\u00A0Maurer\u00C2\u00A0MJ,\u00C2\u00A0Habermann\u00C2\u00A0 TM,\u00C2\u00A0Ansell\u00C2\u00A0 SM,\u00C2\u00A0Macon\u00C2\u00A0WR,\u00C2\u00A0 Ristow\u00C2\u00A0 KM,\u00C2\u00A0Allmer\u00C2\u00A0 C,\u00C2\u00A0 Slager\u00C2\u00A0SL,\u00C2\u00A0Witzig\u00C2\u00A0TE,\u00C2\u00A0Cerhan\u00C2\u00A0 JR.\u00C2\u00A0Statin\u00C2\u00A0use\u00C2\u00A0and\u00C2\u00A0prognosis\u00C2\u00A0 in\u00C2\u00A0patients\u00C2\u00A0with\u00C2\u00A0diffuse\u00C2\u00A0 large\u00C2\u00A0B\u00E2\u0080\u0090cell\u00C2\u00A0 lymphoma\u00C2\u00A0and\u00C2\u00A0follicular\u00C2\u00A0lymphoma\u00C2\u00A0in\u00C2\u00A0the\u00C2\u00A0rituximab\u00C2\u00A0era.\u00C2\u00A0J\u00C2\u00A0Clin\u00C2\u00A0Oncol\u00C2\u00A02010\u00C2\u00A0Jan\u00C2\u00A020;28(3):412\u00E2\u0080\u00907.\u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0 \u00C2\u00A0"@en . "Thesis/Dissertation"@en . "2012-05"@en . "10.14288/1.0072637"@en . "eng"@en . "Interdisciplinary Oncology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "Attribution-NonCommercial-NoDerivatives 4.0 International"@en . "http://creativecommons.org/licenses/by-nc-nd/4.0/"@en . "Graduate"@en . "Multivalent monoclonal antibodies as improved cancer therapies : proof-of-concept and mechanistic studies using rituximab-lipid nanoparticles"@en . "Text"@en . "http://hdl.handle.net/2429/41804"@en .