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Immunostimulatory properties and mechanisms of action of encapsulated methylated cpg oligodeoxynucleotides de Jong, Susan Rachel Dean 2007

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IMMUNOSTIMULATORY PROPERTIES AND MECHANISMS OF ACTION OF ENCAPSULATED METHYLATED CpG OLIGODEOXYNUCLEOTIDES  by SUSAN RACHEL DEAN DE JONG B.Sc., Carleton University, 2002 B.A., Carleton University, 1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Biochemistry and Molecular Biology)  THE UNIVERISITY OF BRITISH COLUMBIA December, 2007 © Susan Rachel Dean de Jong, 2007  ABSTRACT  Immunostimulatory oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through the triggering of the endosomally localized TLR9 by a poorly understood mechanism. The immunopotency and broad range of activity of CpG ODN makes it a promising immunotherapeutic for the treatment and prevention of cancer and other diseases. However, rapid degradation of ODN by serum nucleases, low levels of accumulation in target tissue and lack of specificity for and poor uptake into target cells following systemic administration pose significant hurdles for the clinical application of CpG ODN. This thesis describes the immunostimulatory properties of CpG ODN encapsulated in liposomal nanoparticles (LN), a delivery system that overcomes many of the problems impeding the clinical development of "free" ODN. In particular, it is shown that LN delivery of CpG ODN specifically targets the ODN for uptake by immune cells in vivo, providing a basis for significantly enhanced immunostimulatory activity, including more potent innate and adaptive immune responses, that ultimately improve anti-tumour efficacy. A particular focus of this thesis concerns previous observations that methylated sequences in ODN (mCpG ODN) are immunologically inert. It is shown that encapsulation of mCpG ODN in LN results in immunostimulatory activity that is equal to or greater than that observed for LN formulations of the equivalent unmethylated form as judged by various immune parameters and anti-tumour efficacy. Further, it is shown that both LN-mCpG ODN and LNCpG ODN exert their immunostimulatory effects via TLR9 based on preliminary in vitro results and confirmed by studies performed in TLR9 knockout animals. The mechanisms responsible for the differentiation between both CpG ODN and mCpG ODN and how encapsulation endows immunostimulatory potential are explored. It is shown that 11  discrimination occurs upstream of TLR9 and that the lack of immunological activity of free mCpG ODN is not due to differences in uptake, trafficking to endosomal compartments or ability to bind to TLR9, when compared with CpG ODN, but rather due to its ability to colocalize with TLR9 in the endosomal compartment. It is proposed that whereas the uptake of free CpG ODN results in the induction of the Src Family Kinase signalling cascade which mediates the migration of TLR9 from the ER to the late endosome, the uptake of free mCpG ODN does not. However, it is suggested that encapsulation bypasses the methylation specific recognition of CpG ODN, allowing for the activation of SFK signalling resulting in subsequent co-localization of TLR9 and mCpG ODN in the endosome thus initiating immunostimulatory activity.  TABLE OF CONTENTS  ABSTRACT^  ii  TABLE OF CONTENTS ^  iv  LIST OF TABLES ^  viii  LIST OF FIGURES ^  ix  ABBREVIATIONS ^  xi  ACKNOWLEDGEMENTS ^  xv  DEDICATION ^ CO-AUTHORSHIP STATEMENT ^ CHAPTER 1: Introduction ^ 1.1 Background ^  xvii xviii 1  1  1.1.1 Inception of immunotherapy ^  1  1.1.2 Danger signals and PRRs ^  3  1.1.3 Stimulatory DNA sequences ^  6  1.1.4 TLR9 structure and expression ^  8  1.1.5 Signal transduction via TLR9 ^  9  1.2 Mechanism of action of CpG ODN — delivery of stimulatory DNA ^ 12 1.2.1 Internalization of CpG ODN ^  12  1.2.2 Trafficking of CpG ODN ^  15  1.2.3 Binding of CpG ODN to TLR9 ^  16  1.2.4 Molecular and cellular effects of CpG ODN ^  17  1.2.5 Immunostimulatory activity of pathogenic and eukaryotic DNA ^ 20 1.3 Therapeutic potential of CpG ODN ^  22  1.3.1 Immunotherapeutic applications of CpG ODN ^  22  1.3.2 Adjuvant activity of CpG ODN ^  23 iv  1.3.3 Anti-tumour efficacy of CpG ODN ^ 1.4 Liposomal carriers for CpG ODN ^  24 28  1.4.1 Shortcomings of free CpG ODN ^  28  1.4.2 Complexes of CpG ODN with cationic lipid ^  29  1.4.3 Stabilized antisense lipid particles ^  31  1.4.4 Therapeutic potential of LN-CpG ODN ^  34  1.5 Thesis Objectives ^  36  CHAPTER 2: Encapsulation in Liposomal Nanoparticles Enhances the Immunostimulatory, Adjuvant and Anti-tumour Activity of Subcutaneously Administered CpG ODN ^ 38 2.1 Introduction ^  38  2.2 Materials and methods ^  42  2.2.1 Animals and cell lines ^  42  2.2.2 ODN and preparation of liposomal nanoparticles ^  42  2.2.3 Cell uptake analysis ^  44  2.2.4 Ex-vivo analysis of immune parameters ^  44  2.2.4.1 Plasma cytokine analysis ^ 2.2.4.2 Cell activation analysis ^ 2.2.4.3 Assessment of antigen-specific CD8 T lymphocytes ^ 2.2.4.3.1 MHC tetramer assay ^ 2.2.4.3.2 Cytotoxicity assay ^ 2.2.4.3.3 Cytokine secretion assay ^  44 45 45 46 46 47  2.2.5 Tumour challenge efficacy studies ^  47  2.2.6 Statistical analyses ^  48  2.3 Results ^  49  2.3.1 Encapsulation in LN enhances uptake of CpG ODN by immune effector cells in the lymph nodes and spleen after subcutaneous administration ^ 49 2.3.2 Encapsulation of CpG ODN in LN enhances immune cell activation ^ 51 2.3.3 Encapsulation of CpG ODN in LN enhances the generation of antigen-specific immune responses ^ 54 2.3.4 Anti-tumour efficacy of encapsulated ODN as a vaccine adjuvant in xenogeneic and syngeneic tumour models ^ 58 2.4 Discussion ^ CHAPTER 3: Synthetic Methylated CpG ODNs are Potent in vivo Adjuvants When Delivered in Liposomal Nanoparticles ^  62  67  V  3.1 Introduction ^  67  3.2 Materials and methods ^  70  3.2.1 Animals and cell lines ^  70  3.2.2 Preparation of liposomal ODN ^  70  3.2.3 Analysis of innate and adaptive immunopotency ^  71  3.2.3.1 Immune cell activation ^ 3.2.3.2 Plasma cytokine levels ^ 3.2.3.3 MHC tetramer assay ^ 3.2.3.4 Cytotoxicity assay ^ 3.2.3.5 Cytokine secretion assay ^ 3.2.3.6 Antigen-specific anti-tumour activity ^  71 72 72 72 73 73  3.2.4 Role of TLR9 in immunostimulatory activity of mCpG ODN ^ 74 3.2.4.1 Upregulation of TLR9 ^ 3.2.4.2 Nitric oxide production ^ 3.2.4.3 Inhibition by chloroquine ^ 3.2.4.4 TLR9-K0 studies ^ 3.2.5 Statistical analyses ^ 3.3 Results ^  74 74 74 75 75 76  3.3.1 Methylated CpG ODN induces potent innate and adaptive immune responses when delivered in liposomal nanoparticles ^ 76 3.3.2 Adaptive immune responses mediated by immunization with LN-mCpG ODN mediates effective anti-tumour activity ^ 82 3.3.3 LN-mCpG ODN induces its immunostimulatory activity through TLR9 ^ 85 3.4 Discussion ^  90  CHAPTER 4: Lipid Encapsulation Promotes Subcellular co-localization of Methylated CpG ODN and TLR9: a new model for the immunostimulatory activity of CpG DNA ^ 95 4.1 Introduction ^  95  4.2 Materials and methods ^  98  4.2.1 Reagents ^  98  4.2.2 Mice and cell lines ^  98  4.2.3 Preparation of liposomal nanoparticles ^  99  4.2.4 Cell uptake and immune response ^  99  4.2.5 Flow cytometry ^  100  4.2.6 Endosomal trafficking and localization ^  100  4.2.7 Immunofluorescence ^  101  4.2.8 Confocal microscopy ^  102  vi  4.2.9 Statistical analyses ^ 4.3 Results ^  102 104  4.3.1 The methylation status of CpG ODN does not affect ODN uptake and intracellular trafficking by immune cells whether presented in free or LNencapsulated form  104  4.3.2 Free CpG, LN-CpG and LN-mCpG ODN co-localize with TLR9 in the late endosomal compartment but free mCpG ODN does not ^  108  4.3.3 Co-localization with TLR9 and immunostimulatory activity is mediated via a SFK signalling cascade ^ 112 4.3.4 Free CpG ODN and encapsulated CpG and mCpG ODN enable co-localization by inducing TLR9 mobilization to LAMP1+ endosomes ^ 117 4.4 Discussion ^ CHAPTER 5: Concluding Remarks ^  121 125  5.1 Summary and significance of research ^  125  5.2 Future work ^  127  REFERENCES^  130  APPENDIX ^  150  vii  LIST OF TABLES Table 3.1 Encapsulation in Liposomal Nanoparticles Confers Immunostimulatory Activity on a Variety of Methylated CpG Oligodeoxytnucleotides ^ 84  viii  LIST OF FIGURES Fig 1.1 TLR9-mediated signalling pathway ^  10  Fig 1.2 Innate and adaptive immune responses of CpG ODN ^  18  Fig 1.3 Cryo-electron microscopy of SALP ^  33  Fig 2.1 Uptake of free and liposome nanoparticulate formulations of CpG ODN by immune cells in spleen and lymph nodes following s.c. administration ^  50  Fig 2.2 Comparison of immune cell activation following s.c. treatment with free and encapsulated CpG ODN ^  51  Fig 2.3 Comparison of plasma cytokine induction following s.c. treatment with free and encapsulated CpG ODN ^  53  Fig 2.4 Frequency of antigen-specific OVA-MHC tetramer+ CD8+ cells following s.c. immunization with OVA adjuvanated with free or encapsulated CpG ODN ^ 55 Fig 2.5 Frequency of antigen-specific, IFNy secreting CD8+ cells following s.c. immunization with OVA and free or encapsulated CpG ODN ^ 56 Fig 2.6 Antigen-specific cytolytic activity of splenocytes against E.G7-OVA cells following s ^ c. immunization with OVA adjuvanated with free or encapsulated CpG ODN ^ 57 Fig 2.7 Antigen-specific anti-tumour activity following prophylactic s.c. immunization with OVA adjuvanated with free or encapsulated CpG ODN in a E.G7-OVA xenogeneic tumour model 59 Fig 2.8 Antigen-specific anti-tumour activity following prophylactic s.c. immunization with TRP-2 adjuvanated with free or encapsulated CpG ODN in a E.G7-OVA syngeneic tumour model 60 Fig 3.1 Methylated CpG ODN induces potent immune cell activation when encapsulated in liposomal nanoparticles ^  77  Fig 3.2 Encapsulated methylated CpG ODN induces elevated plasma cytokine levels ^ 78 Fig 3.3 Liposomal nanoparticulate methylated CpG ODN adjuvanates potent, antigen-specific adaptive cellular immune responses after s.c. immunization with OVA ^ 81 Fig 3.4 Liposomal nanoparticulate methylated CpG ODN induces potent, antigen-specific antitumour activity following prophylactic immunization with OVA adjuvanated with free or encapsulated CpG ODN in a E.G7-OVA syngeneic tumour model ^ 83 Fig 3.5 Liposomal nanoparticulate, unmethylated and methylated CpG ODN induce similar responses ^  86  Fig 3.6 Liposomal nanoparticulate, methylated CpG ODN mediates immune cell activation through TLR9 ^  89  Fig 4.1 The uptake of free and LN-encapsulated CpG ODN by RAW264.7 cells and BMDC is not influenced by the methylation status of the ODN ^ 105 Fig 4.2 The uptake of free and LN-encapsulated CpG ODN by immune cells in spleen and lymph nodes following s.c. administration is not influenced by the methylation status of the ODN 106 ix  Fig 4.3 The trafficking of free and LN-encapsulated CpG ODN following uptake into RAW264.7 cells is not influenced by the methylation status of the ODN ^ 107 Fig 4.4 The trafficking of free and LN-encapsulated CpG ODN following uptake into BMDC is not influenced by the methylation status of the ODN ^ 108 Fig 4.5 Free CpG ODN, LN-mCpG-ODN and LN-CpG ODN co-localize with TLR9 in LAMP1+ compartments in vivo but free mCpG ODN does not ^ 109 Fig 4.6 Empty LN co-localize with TLR9 but PP2 inhibits the localization TLR9 to endosomes containing empty LN and LAMP1 ^ 110 Fig 4.7 Co-localization of CpG ODN with TLR9 in LAMP1+ endosomes of splenic DCs ^ 111 Fig 4.8 Src family kinase inhibitor PP2 inhibits the localization of TLR9 to LAMP1 containing endosomes ^ 113 Fig 4.9 Inhibition of CpG ODN and TLR9 co-localization in LAMP1+ endosomes of splenic DCs by PP2 ^ 114 Fig 4.10 CpG-mediated immune activation is a SFK dependent process ^ 114 Fig 4.11 Plasma cytokine induction is a SFK dependent process ^  116  Fig 4.12 Co-localization is mediated by TLR9 mobilization and trafficking to the LAMP1+ compartment ^  118  Fig 4.13 Mobilization of TLR9 to LAMP1+ endosomes of splenic DCs in control and PP2 treated mice ^ 119 Fig 4.14 Trafficking of CpG ODN to the LAMP1+ endosomes of splenic DCs in control and PP2 treated mice ^ 119  ABBREVIATIONS Ab^antibody Ag^antigen ALT^alanine aminotransferase ANOVA^analysis of variance AP-1^activating protein-1 APC^allophycocyanin (FACS fluorophore) APC^antigen presenting cell AST^aspartate aminotransferase BCG^M bovis Bacillus Calmette-Guerin BCR^B cell receptor bDNA^bacterial DNA CD^clusters of differentiation CFA^complete Freund's adjuvant COX^cyclooxygenase CpG^cytosine-guanine CTL^cytotoxic T lymphocyte DC^dendritic cells DD^death domain DEAE^diethylaminoethyl DODMA^1,2-dioleyloxy-N,N-dimethy1-3-amino propane DMEM^Dulbecco's modified Eagle medium DSPC^1,2-distearoyl-sn-glycero-3-phosphocholine EDTA^ethylenediaminetetraacetic acid EEA 1^early endosomal antigen-1 eGFR^epidermal growth factor receptor ELISA^enzyme-linked immunosorbent assay ERK^extracellular signal-regulated kinase FACS^fluorescence-activated cell sorter FBS^foetal bovine serum FITC^fluorescein isothiocyanate HBS^HEPES buffered saline xi  HEL^hen egg lysozyme IFA^incomplete Freund's adjuvant IFN^interferon IKK^inhibitor of kappa kinase IL^interleukin i.p.^intraperitoneal IRAK^IL-1R-associated kinase IRF^interferon regulatory factor i.v.^intravenous INK^Jun n-terminal kinase KO^knock-out LAMP1^lysosome associated membrane protein 1 LE^late endosome LN^liposomal nanoparticle LPS^lipopolysaccharide LRR^leucine rich repeat MAGE^melanoma antigen-encoding gene MAP^mitogen activated kinase MARCO^macrophage receptor with a collagenous structure MART^melanoma-associated antigen recognized by T cells MCP^macrophage chemo-attractant protein mDC^myeloid dendritic cell MEK^MAP-ERK kinase MHC^major histocompatability complex MKK^mitogen-activated protein kinase kinase MPL^monophosphoryl lipid MyD88^myeloid differentiation primary response protein 88 NFK13^nuclear factor KB NK^natural killer NOS^nitric oxide synthase NSCLC^non-small cell lung cancer ODN^oligodeoxynucleotide xii  OVA^ovalbumin PAMP^pathogen associated molecular pattern PBMC^peripheral blood mononuclear cell PBS^phosphate buffered saline PCR^polymerase chain reaction pDC^plasmacytoid dendritic cell pDNA^plasmid DNA PEG-DMG^3-0-[2'-(w-monomethoxypolyethylene glycob000)succinoyl] -1,2-dimyrsitoyl-sn-glycerol PFV^pre-formed vesicle PG^prostaglandin PI3K^phosphatidylinosito1-3-0H kinase PM^plasma membrane PO^phosphodiester POPC^1-palmitoy1-2-oleoyl-sn-glycero-3-phosphocholine PRR^pathogen recognition receptor PS^phosphorothioate RES^reticuloendothelial system ROS^reactive oxygen species SALP^stabilized antisense lipid particle s.c.^subcutaneous SCID^severe combined immune deficiency SFK^src family kinase SPLP^stabilized plasmid lipid particle SRA^class A scavenger receptor TAA^tumour-associated antigen TAB^TAK1 binding protein TAK^transforming growth factor-13-activated protein kinase TH^helper T cell TIR^TLR/IL-1R homology domain TLR^toll-like receptor TNF^tumour necrosis factor  TRAF^tumour necrosis factor receptor-associated factor TRP^tyrosinase-related protein Ubc^ubiquitin conjugating enzyme Uev^ubiquitin conjugating enzyme variant WT^wild type  xiv  ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor Dr. Pieter Cullis for his commitment to helping see this research through to its completion and for his guidance during its development. This research would not have been possible without the guidance and support of my co-supervisor Dr. Ying Tam. I would like to thank Dr. Tam for his enthusiasm, patience, focus, ability to keep me on track and those many 'passive aggressive' phone conversations. Many thanks are given to my committee members, Dr. Alice Mui (Jack Bell Research Centre) and Dr. Ross MacGillivray (Centre for Blood Research), who were supportive throughout and listened to my trials and tribulations between stacks of books and papers, or over pints of British beer. A warm thank-you also to Jeff Hewitt (Centre for Blood Research) who guided me through PCR, helped me genotype my mice and was always available for consultation and moral support. Special thanks to Kaley Wilson for the many hours spent proof-reading my papers, discussing experiments and helping me with many sleepless all-night(s) experiments. Thanks also to May Kazem who performed many tail-vein injections in the early hours of the morning and often stayed late to help me. I thank both Kaley and May for their support and encouragement, for making those very long experiments something to look forward to and giving meaning to the words cooperation and friendship. Particular thanks to Kim Wong for providing an island of sanity and fruitful bits of advice over a cup of tea. I am also indebted to Elaine Taylor at the Jack Bell vivarium who persistently and successfully bred the TLR9 KO mice by threatening to show them 'how to' videos, and to Miguel Pacheco, Julian Kaye and the rest of the gang at the Animal Resource Unit (UBC) for their kindness and support to me and my little four legged friends.  XV  I am eternally grateful for the support of my family. Firstly, to my mother Kathleen Dean, who always had an encouraging word and unwavering faith in me. Secondly, I would like to thank my husband and best friend Mike who has been my source of encouragement and inspiration, and without whose love and support I would never had gone to Graduate school Bzzzzz Finally, I wish to recognize William, Oscar, Marigold and Brindle and all the other animals with whom I have shared this journey; their unconditional love for their pet human brought me back to earth on many occasions.  xvi  To all creatures great and small  xvii  CO-AUTHORSHIP STATEMENT In Chapter 2, I was assisted in the determination of the generation of Ag-specific immune responses by G. Chikh and L. Sekirov at Inex Pharmaceuticals. The anti-tumour efficacy of encapsulated CpG ODN in xenogeneic and syngeneic tumour models was also assessed in collaboration with Inex staff. In Chapter 3, the examination of anti-tumour activity in animal tumour models was conducted in collaboration with G. Chikh and L. Sekirov. Mating pairs of TLR9 KO mice were kindly provided by Dr Jan Dutz and animal studies were conducted at the Animal Resource Units (ARU) at Jack Bell and the UBC hospital by S. de Jong. In Chapter 4, confocal images were obtained in collaboration with Dr G. Basha.  xviii  CHAPTER 1: Introduction  1.1 Background 1.1.1 Inception of immunotherapy  The origins of employing pathogens to confer immunological protection against more virulent strains of a disease, and against infectious and malignant diseases in general, can be found in the history of smallpox: A very serious and highly contagious disease with a 20-60% mortality rate. The observation that, upon recovery, a person would appear to be resistant to reinfection, led to the practice in China, India, and later the Ottoman Empire, of exposing uninfected individuals to dried smallpox lesions from individuals with a mild case of the disease, (a process termed variolation from latin varius, meaning `spotted'), in essence inducing a mild case of the disease to confer immunity to more virulent strains. Lady Mary Wortley Montagu (1689-1762; who was personally affected by smallpox), as the wife of the British ambassador to Turkey, introduced the Turkish practice of variolation into England, bringing it to the attention of the London College of Physicians and Surgeons, who successfully carried out experimental inoculations on condemned prisoners in 1723. The results of these trials as well as those of Zabdial Bolyston, an American physician who inoculated many individuals during the 1721 smallpox epidemic in Boston, led the Royal Society to conclude in 1727, that this practice reduced the risk of acquiring smallpox by 90%. However, despite its success as prophylaxis against smallpox, inoculation was accompanied by significant drawbacks including the risks of mortality from the introduced disease and the inoculated individuals actually infecting others and initiating an epidemic (Radetsky 1999). The next major development in immunization came in 1770 when a country doctor, Edward Jenner (1749-1823), observed that milkmaids did not become infected with smallpox in 1  spite of repeated exposure leading Jenner to postulate that infection of these milkmaids with cowpox, somehow protected them against the much more virulent smallpox. To test this hypothesis, he first infected a young boy with cowpox, allowed him to recover and then exposed him to smallpox. As anticipated, the boy did not contract smallpox, a result which Jenner reproduced in a number of other individuals. Jenner's process, called vaccination, coined from the Latin term for cow vacca, had two main advantages over variolation: It eliminated the risk of developing smallpox from the vaccination and prevented potential infection of others with smallpox (Radetsky 1999). Further developments in immunization were made by Louis Pasteur (1822-1895) and colleagues who first introduced the concept of using laboratory attenuated infectious agents for vaccination against infectious disease, specifically chicken cholera bacterium for the treatment of chicken cholera and followed shortly thereafter by weakened strains of both anthrax and rabies for the treatment of sheep and humans, respectively. A parallel strategy was also used for tuberculosis after immunization trials revealed that the bovine form of Mycobacterium bovis was as virulent in humans as Mycobacterium tuberculosis. This led French bacteriologist Albert Calmette (1863-1933) and veterinarian Jean-Marie Camille Guerin (1872-1961) to develop less virulent strains of M tuberculosis in the 1920's which were used in massive population inoculations in Europe. In one of the first reported uses of an immunostimulatory therapy for a nonbacterial disease, William Coley, a New York surgeon, in the 1890s performed a series of studies evaluating the anti-tumour activity of infectious agents (Weiner 2000) based on observations that sarcoma patients who developed superficial erysipelas streptococcal skin infection after surgery had better outcomes. In his initial studies, Coley found that live Streptococci injected directly into the tumours of patients was able to induce tumour regression. However, the high risk of infection led Coley to use heat-killed Streptococcus and Serratia bacteria, a preparation known as 2  "Coley's Toxin" which resulted in tumour regression in some patients, although at a rate less than with live organisms. Decades later, much of the anti-tumour activity of Coley's toxin has been attributed to endotoxin, although his original success was with Streptococcus, a grampositive organism that does not produce endotoxin (Weiner 2000). Further evidence for a potential role of the immune system in the control and treatment of cancer came in the early 1960's when it was observed that children vaccinated with M bovis Bacillus Calmette-Guerin (BCG) as infants had a lower incidence of childhood leukemia. This led to an investigation of the activity of BCG as a cancer immunotherapeutic, with efforts to identify the specific component(s) responsible for its anti-tumour activity. While trials in lung and skin cancer in the 1970's failed to demonstrate any efficacy, Alvar Morales at the National Cancer Institute reported that BCG markedly reduced bladder tumour recurrence in patients (Morales, Eidinger et al. 1976) and BCG currently represents the most commonly prescribed adjuvant immunotherapy for use in bladder cancer.  1.1.2 Danger signals and PRRs Although the pathogen-based vaccines of Jenner, Bolyston, Pasteur, Coley, Calmette and Guerin and others were effective, the basis of their activity was not known for many years. However, relatively recent discoveries have shed light onto the mechanisms by which these and similar immunotherapeutic strategies exert their therapeutic activity. The innate immune system, which was originally considered to be primitive and 'dumb' (Kaisho and Akira 2002) has more recently stimulated much interest with the discovery that it not only destroys pathogens through complement components but can also detect infectious agents through pattern recognition receptors (PRRs). It is now appreciated that the innate immune system represents a primary line of defence as well as playing a seminal role in the 3  initiation of adaptive host defences (Medzhitov, Preston-Hurlburt et al. 1997). PRRs specifically recognize and bind highly conserved molecular patterns present in many types of microbes, termed pathogen associated molecular patterns (PAMPs). They include such structures as lipopolysaccharide (LPS), peptidoglycan, lipopetides and bacterial DNA (Krieg and Davis 2001). PAMPs have three characteristics that make them targets for innate immune recognition: Firstly they are only produced by pathogens and not by the host cell, thus distinguishing between self and non-self; secondly, they are conserved between organisms of a given class, allowing for a limited set of PRRs to detect a wide range of pathogens; lastly, PAMPs are essential for pathogen survival and their mutation or loss are typically lethal (Ashkar and Rosenthal 2002). The best described PRRs are a family of proteins known as toll-like receptors (TLRs) (Krieg and Davis 2001; O'Neill 2004), the protein Toll being first identified in 1988 in the fruit fly Drosophila as a developmentally critical protein responsible for regulating a fly transcription factor equivalent to NFK13 (Dimarcq, Keppi et al. 1988). IL-1-receptor I and Toll share homology, both possessing a Toll-IL-1 receptor homology (TIR) domain (O'Neill 2004). A role for Toll in host defence was first postulated based on the observation that adult flies lacking Toll succumbed to fungal infections, leading to efforts to identify the roles for Toll in humans and other vertebrates (Lemaitre, Nicolas et al. 1996). This led to the discovery of the first human toll (hToll), which was shown to possess the ability to induce inflammatory cytokines and expression of co-stimulatory molecules (Medzhitov, Preston-Hurlburt et al. 1997) and, a year later, the identification of LPS as a ligand for a murine homologue of hToll (Poltorak, Smirnova et al. 1998; Hoshino, Takeuchi et al. 1999). Currently there are 13 TLRs identified, of which 10 are expressed in humans (TLRs 110) and 12 in mice (TLRs 1-9 and 11-13), each of which respond to a specific lipid, protein or nucleic acid PAMP (Kaisho and Akira 2006). TLR4 recognizes LPS, a cell wall constituent of 4  gram-negative bacteria, while peptidoglycans and triacyl and diacyl lipoproteins characteristic of gram-positive bacterial cell walls are recognized by TLR2 and TLR1/TLR2 and TLR2/TLR6, respectively (Kaisho and Akira 2006). The protein flagellin, which is a component of bacterial flagella is recognized by TLR5 and it has recently been determined that mouse TLR11 recognizes a protozoan-derived profilin-like protein found in uropathogenic bacteria (Kaisho and Akira 2006). TLR7 and TLR8 are involved in the recognition of virus-derived single-stranded (ss) RNAs as well as being stimulated by synthetic antiviral compounds, such as imiquimod, while TLR3 recognizes double-stranded (ds) RNA characteristic of virally infected cells (Kaisho and Akira 2006). In 2000, Hemmi and colleagues identified a receptor responsible for mediating the immunostimulatory effects of DNA (Hemmi, Takeuchi et al. 2000). By generating gene knockout mice, it was definitively demonstrated that TLR9 controls the cellular immune response to bacterial DNA (Kaisho and Akira 2006). TLRs have been found to posses a spatially distinct cellular distribution. Lipid or protein ligands are recognized on the plasma membrane by TLRs 1, 2, 4, 6 or TLRs 5, 11, respectively, whereas nucleic acid ligands are recognized by TLRs 3, 7, 9, localized to intracellular endosomal compartments (Kaisho and Akira 2006). TLR8 appears to be primarily intracellular with a small portion expressed on the cell surface (Miggin and O'Neill 2006). The basis for this distribution is likely related to the nature of their respective ligands. TLRs localized on the plasma membrane recognize those PAMPs which are present and recognizable on the surface of pathogens. Conversely, intracellular TLRs sense those PAMPs not immediately accessible when encountering a pathogen. It has been hypothesized that the intracellular environment may facilitate ligand recognition by the intracellular TLRs possibly by ligand modification as a consequence of lysosomal acidification (Miggin and O'Neill 2006). Additionally, the endosomal  5  localization of TLRs allows efficient detection of invading non-endogenous nucleic acids, while preventing 'accidental' stimulation by self DNA (Krieg 2006).  1.1.3 Stimulatory DNA sequences  Until the latter half of the 20th century, DNA was considered to be immunologically inert and irrelevant in the interaction between bacteria and its host. However, evidence accumulated over the last several decades has identified DNA as an important immunogenic constituent within pathogenic organisms. The first observation that bacterial DNA itself can stimulate the immune system came from studies by Tokunaga and Shimada with fractionated extracts of BCG (Tokunaga, Yamamoto et al. 1984). These researchers identified a purified nucleic acid fraction designated MY-1 (70% DNA and 28% RNA), which induced anti-tumour activity against nine different syngeneic mouse tumours. Interestingly, digestion with RNase enhanced MY-1 anti-tumour activity while DNase abolished it, implicating BCG DNA as the strong biological response modifier responsible for anti-tumour activity. In 1986 Shimada and colleagues showed BCGmediated tumour resistance was due to enhanced natural killer (NK) cell activity and the concomitant production of interferons (IFN) cc/13/y in vitro (Shimada, Yam et al. 1986; Mashiba, Matsunaga et al. 1988; Yamamoto, Yamamoto et al. 1992). In later studies, these researchers found that DNA from a variety of bacteria resulted in IFNy secretion and tumour regression whereas DNA from vertebrates did not (Yamamoto, Yamamoto et al. 1992). The importance of the nucleotide sequence of DNA and its relationship to immunostimulatory activity such as IFN induction was established by assessing the in vitro NK activity of randomly selected DNA sequences from the cDNA of M bovis BCG. These experiments led to the important discovery that ODNs containing a hexameric motif with an 6  unmethylated 5'- purine-purine-CpG-pyrimidine-pyrimidine — 3' sequence resulted in the most potent induction of NK cell activity (Kataoka, Yamamoto et al. 1992; Tokunaga, Yano et al. 1992; Yamamoto, Yamamoto et al. 1992) in both human as well as murine systems, suggesting a generalized mammalian system for cytokine induction (Pisetsky 1996). Kreig and colleagues further determined that the immune stimulatory effects of ds bacterial DNA can be mimicked by short synthetic ss ODNs containing one or more CpG motifs (Krieg, Yi et al. 1995; Scheule 2000). More recent research measuring apoptosis protection has further defined the sequence requirements for optimal immune activity as having a sequence length of greater than 14 bases (typically 14 to 24 bases), a 5'T, a central CpG, and the CpG must be flanked 3' by TT but not preceded by a C (Lenert, Goeken et al. 2006). Interestingly, the most active sequence GTCGTT appears to be conserved across several species, including domestic animals and humans, while inbred rabbits and mice respond maximally to the sequence GACGTT (Mutwiri, Pontarollo et al. 2003). In addition to the hexameric CpG motif, it appears that the specific flanking purine and pyrimidine residues, the number of motifs (usually two to four are optimal), the spacing between motifs, the presence of poly guanosine (poly-G) sequences and the ODN backbone have an effect on the level and type of immune response (Mutwiri, Pontarollo et al. 2003; Krieg 2006). Furthermore, it has been found that certain cells (both different immune cells in the same compartment and the same cell type in different compartments) may respond optimally to specific CpG sequences. All of these observations have led to the functional classification of three categories of synthetic immunostimulatory DNA based on their ability to elicit distinctive cellular responses (Wilson, Dar et al. 2006): Although somewhat vague in their specific criteria, briefly, A-class CpG (CpG-A) ODN, alternatively referred to as CpG-D, consist mainly of a phosphodiester (PO) backbone containing one or more CpG motifs in a self-complementary palindrome with poly-G on a phosphorothioate (PS) backbone at the 5' and/or 3' ends of the 7  sequence capable of forming complex higher-ordered structures known as G-tetrads. It should be noted that the majority of the work with CpG ODN has employed ODN with a PS backbone modification where one of the non-bridging oxygen atoms has been substituted for a sulphur in order to produce a more nuclease resistant sequence, as natural PO backbone linkages are extremely sensitive to nuclease degradation. While both B- and C-class CpG (CpG-B, also referred to as CpG-K, and CpG-C) both possess PS backbones, they are differentiated by the fact that CpG-B do not possess poly-G sequences and CpG-C have one or more 5' CpG motifs and a 3' palindrome that allows for duplex formation (Krieg 2006; Wilson, Dar et al. 2006). A fourth category, CpG-S, are suppressive ODNs typically characterized by the sequence CCT positioned 5' to a GGG sequence (Lenert 2005).  1.1.4 TLR9 structure and expression As previously noted, CpG DNA activity is mediated by TLR9. Like other TLRs, TLR9 is a type I transmembrane receptor characterized by a highly variable extracellular region containing a leucine-rich repeat (LRR) domain involved in ligand binding and an intracellular tail containing the highly conserved TIR (TLR/IL-1R) homology domain which mediates homotypic protein-protein interactions between TLRs and downstream signalling molecules (Miggin and O'Neill 2006). Although the structure of TLR9 has not been solved, the ectodomain or luminal portion of the protein is thought to contain 18 tandemly repeated LRRs flanked by a 31 amino acid long signal sequence region at the N-terminal end, and a cysteine rich domain of approximately 60 amino acids at the C-terminus (Medzhitov, Preston-Hurlburt et al. 1997). Based on the recently elucidated crystal structure of the TLR3 ectodomain, it is hypothesized that the TLR9 DNA binding site is comprised of 3 LRRs with extended inserts, one of which contains two CXXC motifs. This is similar to a motif in the ligand binding portion of a molecule 8  termed CpG binding protein, that coordinates a Zn2+ ion and binds directly to unmethylated CpG dinucleotide sequences (Bell, Mullen et al. 2003; Miggin and O'Neill 2006). TLR9 mRNA is expressed predominantly in DC, macrophage, B cells and NK cells, although the highest protein expression of TLR9 is found in B cells and DC (Ashkar and Rosenthal 2002). While in mice all splenic DC subsets (CD8+, CD4+, pDC and mDC) express TLR9, in humans, expression is only found in pDCs and type I IFN- producing cells. In tissues, TLR9 mRNA is detected in mouse spleen, lymph, lung and genital tract but not in liver, thymus, kidney or heart (Ashkar and Rosenthal 2002). With regard to intracellular distribution, TLR9 resides in the endoplasmic reticulum (ER) as it can not be stained directly on the cell surface and, as determined by experiments using fluorescent fusion proteins, is recruited to late endosomes/lysosomes after cells are stimulated with CpG ODN (Dalpke and Heeg 2004; Latz, Schoenemeyer et al. 2004; Leifer, Kennedy et al. 2004). The possible significance of the endosomal location of CpG ODN recognition is underlined by a study showing that chimeric TLR9 engineered to localize to the cell surface can respond to self-DNA but fails to respond to viral DNA (Barton, Kagan et al. 2006).  1.1.5 Signal transduction via TLR9  Despite their recognition of divergent PAMP ligands, most TLRs, including TLR9, share a common signalling pathway via the adaptor molecule, myeloid differentiation primary response protein 88 (MyD88), which has a TIR domain in its C-terminal region and a death domain (DD) in its N-terminal regions (Kaisho and Akira 2006; Tsan 2006). Downstream of MyD88 the signalling pathway bifurcates into NFicB and IRF-7 activating pathways (Kaisho and Akira 2006). NFKB is required for inflammatory gene expression, whereas IRF-7 is essential for type I IFN expression (Kaisho and Akira 2006) (Fig 1). 9  //,  Late Endosome/ Lysosome TLR9  (0) IRAK4 4RF 7 TRAF6 A.-^4-----Tab a Tab 3J  T7IMIC : .., K1 ,  _ .-----■____ jab1 all' limb Ub TAK't  CF1)  (13)  I® KK4 (12 I  410 I®  githirl/2  Activation Proliferation ^ Ig Secretion ^  Nucleus  Fig 1.1 TLR9-mediated signalling pathway DNA released from pathogens or host cells is taken up and delivered into endocytic vesicles where TLR9 is expressed. TLR9 interaction with DNA and subsequent stimulation recruit MyD88, followed by complex formation with IRAKs 1,4 and TRAF6 resulting in the activation of MAP kinases, IKK complex and IRF7, culminating in the upregulation of transcription factors including NFKB, AP-1 and IRF7.  As previously described, TLR9 is expressed in the ER and is recruited to late endosomal/lysosomal compartments after stimulation with CpG DNAs (Latz, Schoenemeyer et al. 2004). TLR9 recruits MyD88 through interaction of their respective TIR domains resulting in the formation of a signalling complex consisting of two members of the IL-1 receptor associated 10  kinase family (IRAK1 and IRAK4) and TNF receptor-associated factor 6 (TRAF6) (Kawai and Akira 2006; Tsan 2006). IRAK4 and IRAK1 are sequentially phosphorylated causing the complex to dissociate from MyD88, resulting in the activation of TNF receptor-associated factor 6 (TRAF6). TRAF6 forms a complex with Ubc13 and Uev1A, serving as the ubiquitin E3 ligase to promote synthesis of lysine 63-linked polyubiquitin chains (Kawai and Akira 2006). TRAF6 disengages from IRAKs 1 and 4 and interacts with a second complex consisting of transforming growth factor-13-activated protein kinase 1 (TAK1), a member of the mitogen-activated protein (MAP)3 kinase family and TABs 1, 2, 3. TRAF6 activates TAK1 in a ubiquitin-dependent manner (TAB2 and 3 bind the lysine 63-linked polyubiquitin chains via zinc finger domains, interactions which are required for TAK1 activation) which activates the MK (inhibitor of kappa kinase) complex, consisting of IKKa, IKKI3 and IKKy/Nemo. The IKK complex phosphorylates hcBs at its serine residues and targets it for ubiquitination and degradation by the proteasome pathway, allowing for the activation and translocation of NFKB to the nucleus and its binding to the KB site (Kawai and Akira 2006). TAK1 simultaneously phosphorylates MAP kinase kinases (MKK) 4 and 3/6, which subsequently activate c-Jun N-terminal kinase (JNK) and p38, respectively, resulting in the phosphorylation and activation of activating protein-1(AP-1). Both the NFKB and AP-1 proteins enter the nucleus where they activate target genes involved in cell activation, proliferation and Ig production. Extracellular signal-regulated kinase (ERK) is also activated in response to TLR ligands through the activation of MEK1 (MAP-ERK kinase) and MEK2, although the upstream kinase responsible for activating MEK1 and MEK2 in TLR signalling remains unknown (Kawai and Akira 2006). Although not shown in Figure 1.1, it has also been determined that TLR9 triggers type I IFN induction by pDCs which, unlike other DC, constitutively expresses high levels of IRF7 11  which interacts with MyD88, IRAK1 and TRAF6 to form a signalling complex but is specifically phosphorylated by IRAK1 downstream of MyD88 and IRAK4. Although TRAF6 E3 ubiquitin ligase activity is required for IRF7 activation, its function remains unclear. IRF5 and IRF8 are also implicated in TLR9 mediated responses although their roles are not known (Kawai and Akira 2006).  1.2 Mechanism of action of CpG ODN — delivery of stimulatory DNA 1.2.1 Internalization of CpG ODN  Receptor mediated endocytosis is a process whereby receptors on the plasma membrane capture substances or particles by binding to their specific ligands which can be divided morphologically and mechanistically into clathrin-dependent and -independent mechanisms. Clathrin mediated endocytosis is a well characterized major endocytic pathway in mammalian cells. Internalization of ligands (typically <150 nm) occurs through morphologically visible domains of the PM (termed coated pits) composed of the protein complex clathrin. Ligands, such as transferrin and Ag-Ab complexes, bind to receptors that either reside in or cluster to clathrin domains after binding, resulting in invagination of the coated pit to form a vesicle which is eventually pinched off and uncoated (Medina-Kauwe, Xie et al. 2005). In contrast, phagocytosis defines a receptor mediated, clathrin-independent process whereby large particles (>0.5 1.tm) such as bacteria, are internalized following ligand binding to surface receptors. This involves exocytosis at the site of attachment(s) and subsequent pseudopod extensions, supported by an extensive actin matrix, which wrap around and engulf the particle in a phagosome. Phagocytosis is usually restricted to macrophage and other phagocytic cells that specialize in uptake and digestion. Other mechanisms of internalization include caveolin-dependent endocytosis, marked by small flask-shaped invaginations of the PM coated with the coat-protein 12  caveolin-1, and fluid phase endocytosis/macropinocytosis, a non-receptor, non-clathrin mechanism of endocytosis transiently induced in response to mitogenic factors. Macropinocytosis can be readily detect morphologically as it is typically accompanied by actindriven ruffling of the plasma membrane followed by folding and pinching off of irregular sized vesicles (Medina-Kauwe, Xie et al. 2005). While both macrophage and endothelial cells are capable of macropinocytosis upon stimulation, immature DC are the only immune cells known that constitutively carry out macropinocytosis (West, Prescott et al. 2000; Bruewer, Utech et al. 2005). The uptake of ODN is an active process that is energy-, temperature- and time-dependent and generally sequence-independent but its mechanism has not been fully elucidated. Based primarily on competition analyses it has been argued that the uptake of CpG ODN is receptor mediated since inhibitory ODN block effects of stimulatory CpG ODN in B cells (Krieg 2002). A number of receptors and a variety of uncharacterized binding proteins have been investigated as potential mediators of uptake of CpG ODN and bacterial DNA (Loke, Stein et al. 1989; Yao, Corrias et al. 1996; Laktionov, Dazard et al. 1999; de Diesbach, Berens et al. 2000). These include Class A scavenger receptors (SRA) and macrophage receptor with a collagenous structure (MARCO) which are expressed predominately on macrophage and DCs and have been implicated in the scavenging and immune function of these cells (Jozefowski, Sulahian et al. 2006). However, PS CpG ODN in SRA KO mice show no differences in uptake, cellular distribution in tissues or immune activity compared to wild-type (WT) mice (Butler, Crooke et al. 2000; Zhu, Reich et al. 2001). Interestingly, CpG ODN have been reported to fail to stimulate IL-12 and NO production in MARCO deficient peritoneal macrophage, although this research has yet to be confirmed by others (Jozefowski, Sulahian et al. 2006). The integrin CD1 1 b/CD18 (Mac-1) has also been proposed to be involved in the uptake of CpG ODN 13  (Benimetskaya, Loike et al. 1997) although Mac-1 deficiency does not alter CpG-ODN signalling during ODN stimulation (Stacey, Sester et al. 2000; Dalpke and Heeg 2004). Although the cell surface receptor responsible for binding and translocation of CpG ODN to endosomes has yet to be identified, it is generally accepted that cellular uptake is required for immune stimulation (Wagner 2002; Lamphier, Sirois et al. 2006). In vitro studies show that CpG ODN immobilized on a solid support do not activate lymphocytes and similarly, CpG-ODN linked to latex, magnetic or gold beads cannot be taken up and thus, lose their stimulatory activity (Krieg 2002). Stimulatory and non-stimulatory ODN bind equally well to cell membranes and uptake of CpG ODN appears to have little or no sequence specificity, although in theory, a surface protein could bind DNA in a sequence-independent fashion but transduce a signal in a CpG-dependent fashion (Krieg, Yi et al. 1995). Most cell types are capable of taking up DNA, although certain cell types have higher rates of uptake than others (Krieg, Gmelig-Meyling et al. 1991; Zhao, Song et al. 1996; Hartmann, Krug et al. 1998). In vitro, monocytic and pro and pre B cells have the highest rates of ODN uptake, while T cells and neutrophils have low rates (Krieg 2002). In vivo, cells that are active phagocytically, such as macrophage, Kupffer cells and proximal tubular cells have all been shown to internalize ODNs although non-phagocytic cell types such as hepatocytes, vascular endothelial cells, bone marrow cells, non-phagocytic immune cell populations and other cell types in the skin have also been found to take up ODNs (Levin 1999). Uptake has been determined to be highly saturable and therefore rate limiting at higher concentrations, possibly as a result of the loss of cell surface DNA receptors due to ligand-driven endocytosis (Yasuda, Yu et al. 2005).  14  1.2.2 Trafficking of CpG ODN The mechanism of trafficking and endosomal maturation is also an imprecisely defined process. Ligands that are taken up by receptor mediated endocytosis are delivered rapidly to early (or sorting) endosomes that become acidified by ATP-dependent proton pumps to a pH of approximately 6.0. If the cargo is destined for degradation, the endosome becomes more acidified as proton pumps transport hydrogen ions into the vesicle lumen, and matures to a late endosome (pH 5.0-6.0). Eventually the late endosome merges with a lysosome (pH 5.0-5.5) transferring its contents for degradation (Medina-Kauwe, Xie et al. 2005). It has been extensively demonstrated that endosomal maturation is essential for TLR9 signalling, as compounds that block endosomal maturation, such as chloroquine, a diprotic weak base which can partition into acidic vesicles, and bafilomycin A, a specific v-ATPase inhibitor, prevent cellular activation by CpG-containing DNA but not by other PAMPs such as LPS (Hacker, Mischak et al. 1998; Yi and Krieg 1998; Manzel, Strekowski et al. 1999). Specifically, in vitro, chloroquine decreases cytokine release from macrophage, B cells and unfractionated human peripheral blood monocytes (PBMC) and ROS generation in J774 cells (Macfarlane and Manzel 1998; Yi, Tuetken et al. 1998; Manzel, Strekowski et al. 1999). In vivo, chloroquine significantly reduces serum levels of TNFa and IL-6 and protects mice and rats from lethal doses of CpG ODN (Hong, Jiang et al. 2004). High concentrations of chloroquine increase the pH of vesicles, reducing the activity of the enzymes involved in internalized ligand degradation thus prolonging their residence within these compartments (Asokan and Cho 2002). Research with flow cytometry and confocal microscopy has also revealed that chloroquine does not influence uptake nor the vesicular localization of CpG ODN but rather that delayed degradation of CpG ODN is the rate limiting step in the internalization of additional CpG ODN (Lamphier, Sirois et al. 2006). However, the observation that chloroquine can block the activity of CpG 15  ODN at concentrations well below those required for pH interference (Macfarlane and Manzel 1998) has prompted the hypothesis that chloroquine may act to block CpG activity by inhibiting the actual binding to TLR9 (Rutz, Metzger et al. 2004).  1.2.3 Binding of CpG ODN to TLR9 Several studies have indicated that CpG-DNA but not non-CpG ODN binds directly to TLR9, to initiate an immune response. Experiments using surface plasmon resonance biosensor technology show that free CpG ODN competed with immobilized CpG ODN for binding with TLR9 whereas non-CpG ODN failed to successfully compete (Rutz, Metzger et al. 2004). Similarly, it has been shown that TLR9 binds to unmethylated CpG-containing plasmid in a sequence-specific manner and is negatively influenced by the methylation status of the plasmid (Comelie, Hoebeke et al. 2004). DNA binding to TLR9 has also been found to preferentially occur at the acidic pH (6.5-5.0) found in endosomes and lysosomes, but poorly at physiological pH (7.4) suggesting the presence of regulatory mechanisms which only allow TLR9 activation in certain pH-defined subsets of endosomes thus corroborating the necessity for endosomal maturation and acidification for immune activation (Rutz, Metzger et al. 2004). Conversely, immunoprecipitation experiments show that both CpG ODN and non-stimulatory ODN are capable of capturing and binding TLR9. These findings are supported by the fact that nonstimulatory ODN can competitively inhibit activation by CpG ODN and compete for binding for TLR9 (Latz, Schoenemeyer et al. 2004). However, the binding of both CpG ODN and nonstimulatory ODN sequences to TLR9 does not explain the differences in their apparent functional response. Therefore, it has been suggested that TLR9 may actually bind to an assortment of nucleic acids but only respond to a subset of these, suggesting a signalling event that is subsequent to and distinct from DNA binding (Latz, Schoenemeyer et al. 2004; Yasuda, 16  Rutz et al. 2006). The understanding of the molecular events and the concept of transduction on the biochemical level (uptake, trafficking, binding, conformational change) in the activation of TLR9 by CpG ODN remains fragmentary and hypothetical. It is, therefore, of considerable importance to define the exact molecular mechanisms of TLR9 activation, as this will facilitate the development of TLR9-modulating therapies for a broad range of immune pathologies.  1.2.4 Molecular and cellular effects of CpG ODN  In many animal models, sequences bearing the active CpG motif have been shown to directly activate cells of the innate immune system such as monocytes, macrophage, and dendritic cells (DCs). CpG ODN directly stimulates DC and macrophage maturation, upregulating co-stimulatory cell surface molecules such as CD40 and CD86, intracellular adhesion molecule-1 (ICAM-1) and MHC Classes I and II molecules (Lipford, Heeg et al. 1998; Chu, Askew et al. 1999; Hartmann and Krieg 1999; Krieg 1999), as well as enhancing their Ag presentation capability via the MHCs. These effects render APCs capable of T cell activation (Lipford, Heeg et al. 1998; Hartmann, Weiner et al. 1999). In addition, the activation of these cells initiates various innate effects such as the production of pro- and anti- inflammatory cytokines, such as TNF a, IL-6, 10, 12 and 18 and IL-10 and IFNs, respectively as well as chemokines such as MIP1 (Stacey, Sweet et al. 1996; Chu, Askew et al. 1999). Activated DC up-regulate CCR7 and down-regulate CCR5 chemokine receptors which drives their migration and clustering in the marginal zone and outer T cell regions of the lymph nodes where they are better able to stimulate adaptive immune responses (Asselin-Paturel, Brizard et al. 2005; van Duin, Medzhitov et al. 2006). Furthermore, CpG ODN enhances the capability of DCs to crosspresent exogenous antigens to CD8 T cells. While Ag presentation on MHC I to CD8 and II to CD4 T cells classically involves cytosolic (endogenous) and acquired (exogenous) Ags, 17  respectively, DCs possess a mechanism that allows for exogenous Ags to be presented on MHC I (termed cross-presentation) and prime CD8 T cells (termed cross-priming). Therefore, CpG ODN enhances the importance of the exogenous pathway to the development of CTL responses  CpG ODN ^Innate responses:  CpG-ODN stimulate the immune system following uptake into dendritic cells and interaction with TLR 9^ 1FNT  I^  NK  •  IL-12 * Cytolytic APO^I TNFa IFNa113^I activity ( antigen display (in presence of Production of IgM  antigen) MHCs, activation markers Migrate to lymphoid tissue — increase APC function  t Proliferation Polyclonal activation IL-6, IL-12, chemokines  *Lk  Naive CD8+ T cell  THO  -7Tr  .4) Plasma B cell Secretion of IgG2a  TH1 Secretion of IFNT  CTL Killing I activity  Adaptive responses: Develop Ag-specific CTLs, Trcells Ag-specific Abs  Fig 1.2 Innate and adaptive immune responses of CpG ODN CpG ODN directly stimulates APCs up-regulating co-stimulatory cell surface, MHC molecules and the production of pro-and anti- inflammatory cytokines. Activated APCs induce the cytolytic ability of NK cells and concomitant IFNy secretion which further activates APCs in an amplification loop. Activated APCs are capable of T cell activation promoting a TH I /CTL response. CpG ODN synergizes with signals through the BCR resulting in B cell proliferation and inducing their differentiation into plasma cells.  18  against tumour and pathogens that do not directly infect APCs, promoting a TH1/CTL response to a much wider range of antigens. Early in the immune response, the chemokines secreted by activated APC attract NK cells where IL-12/IL-18 stimulation induces NK cell activation and concomitant IFNy secretion. In a self-amplification loop, IFNy further activates APCs to secrete IL-12 and is responsible for inducing various cellular effects, such as synergizing with secreted TNFoc to increase the bactericidal action of macrophage through induction of ROS such as NO and oxygen radicals (02') and up-regulating the expression of the anti-microbial inducible NO synthase (iNOS) (Chu, Askew et al. 1999; Scheule 2000; Chen, Zhang et al. 2001; Ghosh, Misukonis et al. 2001). The secretion of IL-12 by APCs has a pivotal role in bridging the innate and adaptive immune responses initiating a cascade of cellular effects stemming from its ability to induce TH1, rather than TH2, polarization. Briefly, a TH 1 response is characterised by the secretion of cytokines such as IL-12 that promote CTL responses and the production of complement-fixing and opsonizing antibody isotypes, such as IgG2., essential for the control of intracellular pathogens (Huang, Krieg et al. 1999). Conversely, a TH2 response is characterised by the secretion of IL-4, IL-5 and IL-10 and the generation of IgGi and IgE type antibodies and is optimal for mediating humoural responses against extracellular parasites and allergens (Wilson, Pitt et al. 2002). CpG ODN drives the differentiation of nave CD4 T cells to TH1 cells enabling activated macrophage to destroy intracellular pathogens and indirectly promotes the production of memory CD8+ T cells and CTL responses (Wilson et al., 2006). Furthermore, CpG ODN is capable of inducing CTL responses in the absence of TH 1 cell help, reportedly substituting for the CD4O-CD40 ligand interaction between APCs and TH1 cells required before an APC can activate CD8+ T cells (Sparwasser, Vabulas et al. 2000).  19  Although B cells are considered to be a major component of adaptive immune responses, they are also strongly stimulated by CpG ODN (Messina, Gilkeson et al. 1991; Liang and Lipsky 2000). CpG ODN synergizes with signals through the B cell receptor (BCR), leading to significant increases in B cell proliferation and inducing their differentiation into plasma cells (Krieg, Yi et al. 1995; Ashkar and Rosenthal 2002; Li, Bally et al. 2002; Poeck, Wagner et al. 2004). Interestingly, CpG ODN also drives isotype switching of B cells, inhibition of IgG1 and IgE switching, and immunoglobulin production independent of T cell help (Krieg, Yi et al. 1999; Van Uden and Raz 2000; Liu, Ohnishi et al. 2003). CpG ODN also increases B cell survival by preventing apoptosis and has been reported to protect against Fas mediated apoptosis by FAS down-regulation (Ashkar and Rosenthal 2002; Li, Bally et al. 2002). CpG ODN also enhance B cell APC function by upregulating co-stimulatory and MHC molecules and inducing inflammatory cytokines and chemokines such as IL-6, IL-10 and MIP1 (Davis, Weeratna et al. 1998; Van Uden and Raz 2000; Wilson, Pitt et al. 2002).  1.2.5 Immunostimulatory activity of pathogenic and eukaryotic DNA  Although CpG and other motifs have been shown to mediate the immune stimulatory effects of bacterial and synthetic DNA, it is clear that the mammalian immune system must be able to distinguish immunostimulatory pathogenic DNA from immunologically inactive self DNA and identify them as a "danger signal" (Ashkar and Rosenthal 2002), particularly in view of the fact that we are continually exposed to self DNA from apoptotic and necrotic cells during normal development, growth and maintenance. Certainly, early research by Yamamoto (1992) determined that DNA from a variety of bacterial, but not mammalian, sources could stimulate the NK response, suggesting that specific sequences or base modifications are critical for activity (Yamamoto, Yamamoto et al. 1992; Pisetsky 1996). Based on observations made by Bird and 20  others (1987), the ability to distinguish pathogenic and mammalian DNA may be due to three critical structural differences between bacterial and mammalian DNA: Firstly, while CpG dinucleotides are present at the expected frequency in bacterial DNA (1:16), they are suppressed in mammals, being found at approximately one-fourth of the expected frequency; secondly, the cytosine residue is commonly methylated in mammalian CpG DNA (approx 70-80%) whereas in bacterial DNA it remains unmethylated (Bird, Taggart et al. 1987; Hergersberg 1991); lastly, in mammals CpG motifs occur in the context of inhibitory flanking sequences. DNA methylation is important in a variety of regulatory events. It has been widely shown that methylation within promoters and enhancers halts transcriptional activity and is found in gene down-regulation, X-chromosome inactivation and the suppression of invading and endogenous viruses and transposons (Van Uden and Raz 2000). In fact, it has been theorized that methylation is responsible for the suppression of CpG in the vertebrate genome as methylated CpG is a locus for mutation to TpG during replication and organisms with fewer CpG tend to have more TpG and its complement CpA than expected (Van Uden and Raz 2000). It has also been extensively shown that immunostimulatory CpG motifs lose their stimulatory effect upon methylation of the 5' cytosine of the CpG motif (Krieg, Yi et al. 1995). However, since vertebrate genomic DNA still contains many unmethylated CpG motifs, it is evident that other mechanisms, in addition to differences in CpG frequency and methylation, must contribute to the relative immunostimulatory activity of pathogenic and eukaryotic DNA. Vertebrate DNA that has been completely unmethylated, however, still has no immune stimulatory effects providing evidence for the presence of immunosuppressive flanking sequences (Krieg, Yi et al. 1999). In vertebrates, inhibitory flanking regions typically include C and G nucleotides immediately 5' and 3' of the CpG motif, respectively (Tasheva and Roufa 1994) and interestingly, several adenoviruses have evolved genomes in which their CpG motifs are 21  associated with neutralizing sequences to inducing host immune responses (Krieg and Davis 2001).  1.3 Therapeutic potential of CpG ODN 1.3.1 Immunotherapeutic applications of CpG ODN  Interest in CpG ODN as an immunotherapeutic agent has been focused on four main areas. Firstly, ODNs have been evaluated in murine and primate models of allergy and asthma where it has been shown that delivering allergens together with CpG ODNs inhibits TH2 allergic responses by inducing T H 1-biased responses (Kline, Waldschmidt et al. 1998; Sur, Wild et al. 1999; Shirota, Sano et al. 2000). Secondly, ODNs can be used as a stand-alone therapy to act by non-specific, innate immune activation due to its strong T H 1 biasing effect (Chu, Targoni et al. 1997; Zimmermann, Egeter et al. 1998). Studies in mice have demonstrated that the innate immune defences activated by CpG ODN can protect and in some cases, direct a TH 1 response against a wide range of viral, bacterial and even some parasitic pathogens including lethal challenge with agents such as Leishmania major, Bacillus anthracis, Listeria monocytgenes, Francisella tularensis, and Ebola virus (Krieg 2006). Thirdly, CpG ODN can also improve function of APC and create a cytokine/chemokine milieu that is conducive to the development of an enhanced TH 1, Ag-specific immune response to co-administered vaccines as demonstrated in studies using model antigens such as hen egg lysozyme (HEL) (Chu, Targoni et al. 1997), ovalbumin (OVA) (Lipford, Sparwasser et al. 1997) and infectious disease vaccines such as hepatitis B (Davis, Weeratna et al. 1998) and influenza (Moldoveanu, Love-Homan et al. 1998; Cooper, Davis et al. 2004). Adjuvant activity of CpG ODN has been demonstrated in numerous animal species (Chu, Targoni et al. 1997; Davis, Weeratna et al. 1998; Ioannou, Gomis et al. 2002; Ioannou, Griebel et al. 2002). Finally, the ability of CpG ODN to induce Ag-specific 22  immune responses has been evaluated in tumour models using model tumour antigens (Weiner, Liu et al. 1997; Heckelsmiller, Beck et al. 2002). Clinical studies are currently underway in humans to evaluate CpG ODN therapy against asthma and allergy, (Bohle 2003; Leifer, Verthelyi et al. 2003; Tulic, Fiset et al. 2004) infectious disease, such as HBV (Cooper, Davis et al. 2004), influenza (Cooper, Davis et al. 2004) and cancer (Krieg 2007).  1.3.2 Adjuvant activity of CpG ODN  As previously mentioned, a TH1 response is essential for the control of intracellular pathogens (Huang, Krieg et al. 1999); conversely, a TH2 response is optimal for mediating humoural responses against extracellular parasites and allergens (Wilson, Pitt et al. 2002). Cytokines that promote a TH1 response typically tend to inhibit the TH2 response and vice versa (Scheule 2000). Currently, the only adjuvants licensed for use in humans (aluminium hydroxide, salts and MF59) stimulate primarily humoural responses and induce little or no CTL responses and augment these responses largely through enhanced delivery (Zaks, Jordan et al. 2006). In contrast, other common experimental immune adjuvants, such as complete Freund's adjuvants (CFA), monophosphoryl lipid (MPL) and CpG ODN function primarily as activators of innate immunity; CFA and MPL activate via TLR2 or TLR4 (Zaks, Jordan et al. 2006). CpG ODN promotes a strong TH 1 immune response and interestingly when combined with Alum shifts the TH2 response to a strongly TH1 biased response (Weeratna, McCluskie et al. 2000). In a mouse study comparing 19 different vaccine adjuvants with two tumour antigens (MUC1 peptide and GD3 ganglioside), CpG ODN induced the most TH 1 biased immune responses with the highest levels of cytokine secretion (Kim, Ragupathi et al. 2000). The attractiveness of CpG ODN as a potential adjuvant results from several mechanisms. Firstly, CpG ODN directly stimulates maturation of DC and macrophage thereby enhancing their 23  ability to express co-stimulatory molecules, to present antigen and cross prime, enhancing T-cell activation and the generation of antigen specific TH1 cells and CTLs through the production of IFNs and IL 12, even in the absence of CD4 T cell help (Ayash-Rashkovsky, Bentwich et al. 2005). Secondly, B cells are synergistically activated when stimulated by CpG ODN in the presence of an antigen, indicating cross-talk between the B cell receptor and CpG signalling pathways, and the possession of enhanced IgG class switching capabilities (Krieg, Yi et al. 1995). Lastly, CpG ODNs activate B cells to rapidly enter the G1 phase of the cell cycle and become resistant to spontaneous or induced apoptosis, contributing to a more sustained immune response (Yi, Hornbeck et al. 1996; Yi and Krieg 1998).  1.3.3 Anti-tumour efficacy of CpG ODN Significant efforts have been made towards developing CpG-based therapeutics for the treatment of malignant disease. Experimental and pre-clinical studies have explored the immunostimulatory capabilities of CpG ODN as a stand-alone monotherapy, a vaccine adjuvant in combination with specific antigens, an adjunct therapy with monoclonal Abs, or in combination with chemo- or radiation therapy for the treatment of cancer (Weigel, Rodeberg et al. 2003; Balsari, Tortoreto et al. 2004; Mason, Ariga et al. 2005; Taieb, Chaput et al. 2006; Wang, Rayburn et al. 2006). These last two applications are beyond the scope of this thesis, but are reviewed by A.M. Krieg (Krieg 2007). As a stand-alone therapy, CpG ODN has demonstrated cellular and humoural responses associated with immune stimulation, namely induction of cytokines, such as TNFcc, IL-12 and IFNs, enhanced antibody-dependent cellular cytotoxicity (ADCC) and up-regulation of MHC I and II and other co-stimulatory molecules on a variety of primary malignant B cells, including various lymphomas and chronic lymphocytic leukemia cells (Ballas, Rasmussen et al. 1996; 24  Cowdery, Chace et al. 1996; Lipford, Sparwasser et al. 1997; Decker, Schneller et al. 2000; Jahrsdorfer, Hartmann et al. 2001). In vivo, systemic administration of CpG ODN as a single agent demonstrates significant anti-tumour effects in a number of murine models such as protection of 80% of animals from a lethal challenge of B16 melanoma and rejection of established CNS-1 glioma tumours (Carpentier, Xie et al. 2000; Ballas, Krieg et al. 2001). In many tumour models, efficacy appears to be mediated predominantly by NK-dependent mechanisms related to enhanced cytolytic activity. For example, while CpG ODN effectively inhibited B16 melanoma and EL4 lymphoma in both immunocompetent and SCID mice deficient in B and T cells (but retaining NK function), removal of NK cells eliminated the antitumour response (Weiner 2000). However, efficacy in other models is dependent on a CD8+ CTL mechanism (Carpentier, Chen et al. 1999; Ballas, Krieg et al. 2001; Blazar, Krieg et al. 2001; Lonsdorf, Kuekrek et al. 2003). In murine models of both cervical cancer and glioma, tumour regression and extended survival were shown to be dependent upon CD8+ but not CD4+ T cells (Baines and Celis 2003; El Andaloussi, Sonabend et al. 2006). Likewise, peritumoural injection of CpG ODN significantly reduced melanoma skin tumours and metastatic potential (development of pulmonary colonies) based on CD8+ T cell activity. Interestingly, in this study, adoptive transfer of splenocytes obtained from CpG ODN treated mice reduced the number of previously established pulmonary colonies in recipient mice (Kunikata, Sano et al. 2004). The most clinically advanced CpG therapeutic is CpG 7909, a PS sequence optimized for immunostimulatory activity in humans under development by Coley Pharmaceuticals. It has been evaluated as a standalone agent and its anti-tumour activity has been recently demonstrated in clinical trials of non-Hodgkins lymphoma, cutaneous T cell lymphoma, melanoma and in renal cell carcinoma. CpG 7909 administered to 23 non-Hodgkins lymphoma patients induced an increase in NK cell concentrations and resulted in two late clinical responses (Link, Ballas et 25  al. 2006). Intratumoural and s.c. treatments for melanoma resulted in an activation of pDCs and indirectly mDCs (human mDCs do not express TLR9), one out of the five patients receiving intratumoural CpG 7909 showed complete regression of the injected lesion, partial responses occurred in two out of 20 melanoma patients receiving s.c. treatments (Pashenkov, Goess et al. 2006) and two of 35 patients with advanced renal cell carcinoma had partial responses. The best response was obtained in a trial of 28 patients with advanced cutaneous T cell lymphoma (who had failed an average of six prior therapies) where there were three complete and six partial responses (Krieg 2007). With regard to adaptive cellular anti-tumour immune responses, CTLs recognize Ag on tumour cells in the form of peptide fragments complexed with MHC I molecules derived from TAAs which correspond to proteins synthesized and subsequently processed by the tumour cell. However, tumour cells in general lack co-stimulatory molecules that are capable of delivering the second signal necessary for T-cell activation, resulting in T-cell tolerance. Therefore, a significant challenge in the development of cancer vaccines is to break immune tolerance against one or more TAA(s) and induce a tumour specific response strong enough to induce tumour cell destruction in established tumours (Weiner 2000). CpG ODN have been shown to be sufficient to overcome central tolerance (Ashkar and Rosenthal 2002; Krieg 2006). In relatively small tumours, stand-alone immunotherapy is sufficient for rejection. However, to induce the rejection of larger or poorly immunogenic tumours, CpG ODN generally need to be combined with either a tumour vaccine or with other effective anti-tumour strategies such as surgery or radio- and chemo- therapy. The utility of CpG ODN as a vaccine adjuvant for inducing Ag-specific humoural and cellular responses has been confirmed in animal studies using a wide variety of Ags. Compared to other adjuvants, CpG ODN have been reported to induce increased numbers of Ag-specific CD8+ and CD4+ T cells (Kim, Ragupathi et al. 1999; Davila and Celis 2000; 26  Warren, Dahle et al. 2000; Kim, Myoung et al. 2002; Miconnet, Koenig et al. 2002; Stern, Boehm et al. 2002; Zwaveling, Ferreira Mota et al. 2002). A peptide analogue of MART1/Melan-A (melanoma-associated antigen recognized by T cells), a melanocyte protein expressed in most melanomas, when combined with CpG ODN, induced T cells with greater specificity for the TAA, as reflected in a tetramer assay and increased cytotoxic activity compared with peptide emulsified in IFA (Miconnet, Koenig et al. 2002). Furthermore, mice injected with CpG ODN produced a higher titre of antigen-specific IgG2a than those treated with CFA (Weiner, Liu et al. 1997; Uhlmann, Brinckmann et al. 2002). CpG ODNs are the only adjuvants reported to induce anti-tumour responses strong enough to eliminate established tumours (Heckelsmiller, Rall et al. 2002; Zwaveling, Ferreira Mota et al. 2002). Repeated immunizations with OVA protein or peptide together with CpG ODN of mice bearing B16 OVA expressing melanomas resulted in a 1-100 fold increase in CTL responses and substantial therapeutic activity even when the vaccination was delayed until day 7 after tumour seeding (Davila and Celis 2000). Although tumour vaccines employing CpG ODN have shown superior results in mouse models, objective human clinical responses are rarely achieved (Rosenberg and Dudley 2004; Klinman 2006). A trial vaccination of the MARTI peptide in IFA combined with CpG ODN stimulated approximately half of the patients to generate increased frequencies of MARTI specific CD8+ T cells in the blood although anti-tumour efficacy was not observed (Speiser, Lienard et al. 2005; Appay, Speiser et al. 2006). A clinical trial of five patients using MAGE-3 (melanoma Ag-encoding gene) protein combined with CpG 7909 to treat NSCLC resulted in two partial responses that became apparent only after at least seven vaccinations (Krieg 2007). Coley granted Pfizer worldwide marketing rights to CpG 7909 (referred to as PF3512676 by Pfizer) for the treatment, control and prevention of multiple cancer indications. 27  Clinical trials of PF-3512676 (phase I) and of IMO-2055 (immune modulatory ODN- developed by Idera Pharmaceuticals) (phase II) as single agents are currently underway for chronic lymphocytic leukemia and renal cell carcinoma, respectively. Phase I trials have been initiated for CpG 7909 in combination with MART 1, tyrosinase peptide and IFA for the treatment of melanoma, and phase I/II and phase II trials have commenced for the treatment of breast cancer utilizing a combination of MAGE-3 or Her2 recombinant protein, respectively, combined with the adjuvant AS15 (comprised of CpG 7909, MPL, and QS-21 [saponin]).  1.4 Liposomal carriers for CpG ODN 1.4.1 Shortcomings of free CpG ODN  As summarized in the preceding sections, CpG-based therapeutics have shown very encouraging results in a variety of disease indications in animal models and, to a lesser extent, in some human pre-clinical and clinical trials. However, a number of challenges associated with the clinical use of free CpG-ODN remain, particularly the need for ODN stabilization. As previously noted, natural PO ODN are sensitive to nuclease degradation, rendering them inactive in their free form; therefore the majority of work has employed CpG ODN with nucleaseresistant, typically PS, backbone modifications (Krieg, Yi et al. 1995; Krieg 2004). Although PS modification is fairly effective in stabilizing ODN, it has several inherent disadvantages. These include non-sequence specific, chemical class-related toxic effects due to non-specific binding to proteins leading to acute toxicity due to complement activation and other haemodynamic changes and can result in occasional cardiovascular collapse and death. High doses of PS CpG ODN in mice also produce morphological changes in bone marrow and a reduction in the number of megakaryocytes concomitant with a severe decrease in the numbers of circulating platelets and an inhibition of clotting (Levin 1999). Secondly, in addition to these 28  shortcomings, free CpG ODN is characterized by unfavourable pharmacokinetics, lack of specificity for target cells and rapid and broad tissue disposition after systemic administration. Further, despite backbone modifications, the metabolism by exonucleases of PS ODN is rapid; almost immediately after i.v. administration, metabolites of the parent PS CpG ODN can be detected (Levin 1999; Krieg 2002). Finally, irrespective of rapid clearance and nuclease degradation, intracellular delivery is highly inefficient due to the inability of charged macromolecules to cross cell membranes. Although PS-modified CpG ODN exhibit increased cell membrane binding, when compared with unmodified forms, the actual amount of CpG ODN internalized compared with the amount absorbed to the cell surface is very small since transbilayer transport is minimal (Shi and Hoekstra 2004).  1.4.2 Complexes of CpG ODN with cationic lipid Due to the challenges associated with the administration of free CpG ODN, there has been much interest in developing alternative delivery methods. Lipid-based nanoparticulate (LN) systems have been used to enhance the potency of small molecule drugs due to the ability of small, long circulating LN systems to preferentially accumulate at disease sites such as tumour sites (Maurer, Fenske et al. 2001). The application of LN systems for the delivery of immunostimulatory ODN is suggested by the extensive accumulation of LN by macrophage and DC following administration (Gursel, Gursel et al. 2001; Mui, Raney et al. 2001), as well as the ability of LN to protect encapsulated ODN from serum nucleases, removing the requirement for chemically stabilizing the ODN. The high solubility of most ODN in aqueous solutions enables their encapsulation within lipid particles by a diverse range of strategies including thin film and reverse-phase hydration as well as detergent dialysis (Semple, Klimuk et al. 2000) to produce anionic and neutral liposomes 29  as well as modified liposomes altered to promote targeting and uptake. However, the ODN encapsulation efficiency of these "passive" encapsulation techniques is typically in the range of 3-10% limiting their practical application. In order to achieve higher encapsulation efficiencies, liposomal systems containing cationic lipids must be employed, for which high encapsulation efficiencies can be achieved through association of the positively charged lipids and the negatively charged phosphate molecules on the DNA backbone (Bally, Harvie et al. 1999). The resulting cationic lipid-ODN "complexes" are usually formed by the addition of ODN to the preformed cationic liposomes or cationic lipid, resulting in lipid-nucleic acid particles with heterogeneous morphology and size (Dass 2004). Complexes have been used extensively for the transfer of nucleic acids to cultured cells facilitating enhanced interaction with cell membranes resulting in increased endocytosismediated, intracellular nucleic acid delivery, when compared with free ODN (Feigner, Gadek et al. 1987). However, while complexes are very effective in vitro, they are not suitable for systemic delivery in vivo as they are rapidly eliminated from the blood, provide only partial nuclease protection to DNA and often exhibit toxic and sometimes lethal side effects particularly upon i.v. administration (Chiou, Tangco et al. 1994; Dash, Read et al. 1999; Hwang and Davis 2001; Faneca, Simoes et al. 2002). In complexes, the charged lipid is usually in excess resulting in a strong net positive charge on the surface of the complex which causes extensive non-specific interactions with cells, plasma proteins and other macromolecules in the circulation. In addition, complexes are thermodynamically unstable and display a tendency to grow into larger aggregates resulting in broad size distributions and structural heterogeneity (Maurer, Wong et al. 2001). Due to their initial targeting to the lungs following i.v. administration, extensive interactions with cells, proteins and other macromolecules in the circulation, and their strong tendency to aggregate over time, complexes can induce pulmonary embolisms (Mahato, Anwer 30  et al. 1998; Chesnoy and Huang 2000; Verbaan, Oussoren et al. 2001). Intravenous administration of complexes is also found to result in the depletion of several blood components and cell types, such as platelets and lymphocytes, an increase in clotting times, complement activation and significant hepatocellular toxicity, as shown by elevated serum levels of the transaminases alanine animotransferase (ALT) and aspartate animotransferase (AST) (Semple, Klimuk et al. 2001) (Tousignant, Zhao et al. 2003). Furthermore, additional adverse sideeffects have been reported including pulmonary toxicity (Dokka, Toledo et al. 2000; Tan, Liu et al. 2001) as well as erythrocyte agglutination (Eliyahu, Servel et al. 2002) and aggregationmediated microinfarction (Wright, Rosenthal et al. 1998).  1.4.3 Stabilized antisense lipid particles Stabilized antisense-lipid particles (SALP) have been developed as an alternative formulation to complexes that are more suited to in vivo applications. First, in order to achieve high levels of ODN encapsulation but avoid the problems associated with cationic lipids, ionisable cationic lipids with pK values below 7 are employed allowing for efficient ODN encapsulation at lower pH values where the cationic lipid is fully protonated, but results in particles that have little surface charge at physiological pH values. This low surface charge also reduces the toxicity of the SALP system and allows the long circulation lifetimes required for accumulation at disease site, such as tumour sites. Second, in order to achieve small stable LN systems, a polyethylene glycol (PEG) containing lipid is required during the formulation stage. However, the presence of a PEG coating inhibits association with cells and intracellular delivery; therefore, a PEG-lipid with relatively short acyl chains is employed, which allows the PEG-lipid to rapidly dissociate from the SALP following administration. The SALP system produces small homogeneous populations of discrete particles, exhibits high encapsulation efficiencies 31  approaching 90%, results in high ODN:lipid ratio (up to 0.20 w/w; corresponding to over 2000 ODN per LN) and long circulation lifetimes of 5 h or longer (Maurer, Wong et al. 2001; Semple, Klimuk et al. 2001). SALP were originally developed in response to a need to improve the delivery of antisense DNA to down-regulate genes involved in tumourigenesis. The antisense sequences tested originally were single stranded non-methylated ODNs complementary to the initiation codon region of the human and mouse proto-oncogene c-myc (INX-6295), human eGFR, and human and mouse ICAM (intercellular adhesion molecule-1). However, the discovery that SALP possessed ODN dependent immunostimulatory properties beyond its original role as a novel antisense formulation has led to the investigation of its clinical potential as an adjuvant primarily in the treatment of cancer (Mui, Raney et al. 2001). SALP LN systems can be used to encapsulate short single strands (16-24 bases) of DNA within a vesicle typically 80-120 nm in diameter (Semple, Klimuk et al. 2001). The liposomal formulation used in this work is composed of palmitoyloleoylphosphatidylcholine (POPC), cholesterol, 1,2-dioleyloxy-/V,N-dimethy1-3-amino propane (DODMA) and PEG-dimyristoyl glycerol (PEG-DMG) (25:45:20:10 mol%) and the ODN is typically encapsulated at an ODN:lipid ratio of 0.1 w/w. DODMA is the ionisable aminolipid required to achieve association and entrapment of the CpG ODN into the LN; this step is performed at pH 4 where DODMA is fully protonated. The other components include neutral structural lipids (POPC, cholesterol) and an exchangeable poly (ethylene glycol) (PEG)-conjugated lipid which as previously mentioned provides a surface steric barrier that inhibits vesicle aggregation and fusion during LN-CpG ODN formation but leaves the delivery system rapidly after administration (Semple, Klimuk et al. 2001). The formulation of LN-CpG ODN requires the introduction of the POPC/Cholesterol/DODMA/PEG-DMG mixture dissolved in ethanol, to an aqueous buffer at pH 4 (final ethanol content is 40% by volume) followed by extrusion to form pre-formed 32  vesicles (PFV). The PFV are incubated with aqueous buffer containing the dissolved CpG ODN, maintaining the ethanol content at 40% by volume. The negatively charged ODN then interact with the positively charged PFV which causes structural re-organizations facilitated by the presence of high levels of ethanol allowing for the formation of multi-lamellar vesicles with internalized ODN (Maurer, Wong et al. 2001).  Fig 1.3 Cryo-electron microscopy of SALP Structure of SALP at an ODN:lipid ratio of 0.25 (Semple, Klimuk et al. 2001).  The number of lamellae is dependent on the initial ODN to lipid ratio; higher concentrations of ODN can result in numerous lamellae (Semple, Klimuk et al. 2001). The subsequent removal of the ethanol by dialysis against a citrate buffer (pH 4) induces the spontaneous entrapment of the ODN and the stabilization of the particle. Upon neutralization of the encapsulated particles through dialysis against buffer at near physiological pH (7.6), externally bound CpG ODN is released. Non-encapsulated ODN is further removed by anion exchange chromatography. Encapsulation efficiency is typically 85-90% and is independent of  33  ODN sequence (Maurer, Wong et al. 2001). Cryo-electron microscopy indicates that the final particles consist of a mixed population of unilamellar and small multilamellar vesicles (80120nm diameter) (Semple, Klimuk et al. 2001).  1.4.4 Therapeutic potential of LN-CpG ODN The improved pharmacokinetics of SALP, the reduction in cationic lipid content and surface charge, the resistance to degradation and the homogeneous, stable nature of the SALP formulations have made these particles an attractive in vivo delivery system for nucleic acid based therapeutics. In vitro analysis confirms that ODN are completely protected from degradation by serum nucleases when contained within SALP (Semple, Klimuk et al. 2000; Gursel, Gursel et al. 2001) with metabolites being virtually undetectable following in vivo administration. This efficient protection enables the use of unmodified PO CpG-ODN, completely eliminating PS-associated complement and coagulation toxicities. Furthermore, PO ODN exhibit greater potency and induce enhanced immune responses (Mui, Raney et al. 2001), likely due to more efficient interactions with TLR-9 and possibly through TLR-9 independent pathways (Yasuda, Yu et al. 2005; Yasuda, Rutz et al. 2006). It is well known that liposomes are naturally taken up by APCs allowing for targeted delivery of CpG ODN to cells of the immune system compared to the non-specific distribution exhibited by free CpG-ODN, thereby enhancing the activity of CpG ODN (Rao and Alving 2000; Gursel, Gursel et al. 2001). The ability of SALP to be used as an immune adjuvant was first demonstrated by Mui and colleagues (Mui, Raney et al. 2001) who investigated the plasma cytokine induction based on the reported ability of SALP to target tumour vasculature and macrophage in vivo in a manner similar to other liposome-like particulate delivery systems (Rao and Alving 2000; Leonetti, Biroccio et al. 2001; Mui, Raney et al. 2001). It was found that 34  SALP (termed LN-CpG ODN in this thesis) dramatically increased the cytokines production compared with the same dose of free ODN, demonstrating the immune potency of these particulate delivery systems (Mui, Raney et al. 2001). Depending on the lipid composition, LN encapsulation can also prolong ODN circulation(Yu, Geary et al. 1999). Although the benefits of long circulating LN and subsequent passive drug accumulation at sites of disease have been well demonstrated for LN containing conventional drugs, the benefits for immunostimulatory agents are less clear since cells of the mononuclear phagocyte system (MPS), which clear LN from the circulation, are often the desired target. However, the localized delivery of CpG ODNs would be preferable in immunotherapeutic strategies where it is desirable to achieve recruitment and activation of local immune cells such as in solid tumours and to dampen localized autoimmune responses, etc. The benefits associated with improved pharmacokinetics and biodistribution have a direct impact on the potential for dose escalation. Preclinical studies in non-human primates have demonstrated that the systemic administration of PS CpG ODNs can results in severe and acute haemodynamic and cardiovascular effects which are generally observed at peak PS CpG ODNs plasma concentrations of >90 1.tg/m1 — representing an approximate dose of 10-15 mg/kg. In contrast SALP INX-6295 has been administered in these animals at doses of up to 40 mg/kg without any changes in cardiovascular or haematological parameters (Leonetti, Biroccio et al. 2001). In summary, the combination of the TH1-biased immunogenicity of CpG ODN coupled with the ability of LN-CpG ODN to protect the ODN payload from nuclease digestion and target the CpG ODN to disease sites offers exciting possibilities for improving the therapeutic potential of CpG-based immunotherapeutics, which has led to the studies conducted in this thesis. 35  1.5 Thesis Objectives  The immunopotency and broad range of activity of CpG ODN suggests promise as an immunomodulatory agent for the treatment and prevention of cancer and other diseases. However, despite encouraging preclinical and clinical data, rapid nuclease degradation, poor cellular uptake, unfavourable pharmacokinetics, lack of specificity for target cells and broad tissue disposition render them unsuitable for clinical use. Further, while complexes consisting of cationic lipids and CpG ODN have been found to preferentially target APCs, they do not fully protect the ODN from degradation, lead to unstable formulations and suffer from toxicity issues. These difficulties are reduced using the LN-CpG ODN delivery system which has low surface charge, fully protects encapsulated ODN and exhibits enhanced immunostimulatory effects compared with free CpG ODN following administration. In Chapter 2 of this thesis, the uptake of free and encapsulated, mCpG ODN and CpG ODN by cells of the spleen and lymph nodes are characterized and these results are correlated with the effect of encapsulation on the development of immune activation and adaptive responses. Furthermore, the ability of free and encapsulated CpG ODN to induce adaptive cellular responses against co-administered Ags and effective anti-tumour efficacy in both xenogeneic and syngeneic models of cancer are assessed. In Chapter 3, the generally accepted immunological dogma that mCpG ODN sequences are immunologically inert and that the unmethylated nature of bacterial and pathogenic viral CpG DNA are essential characteristics for immunostimulatory activity are investigated. The effect of encapsulation on the adjuvanticity of adaptive, cell-mediated immune responses, both quantitatively and functionally, are determined and its therapeutic relevance is assessed by evaluating the anti-tumour efficacy of encapsulated mCpG ODN in a tumour model and its ability to establish an immunological memory. To assess whether LN-mCpG ODN mediates its 36  effects through the TLR9 signalling pathway, a number of in vitro parameters known to be implicated in TLR9 mediated responses are investigated which are further examined in TLR9 KO mice. The results of Chapters 2 and 3 show that liposomal encapsulation of CpG ODN not only enhances the immune activity of CpG ODN but also endows immunostimulatory activity upon mCpG ODN. Thus in Chapter 4 the mechanisms responsible for the differentiation between both CpG ODN and mCpG ODN and the endowment of immunostimulatory potential by encapsulation are discussed. The question as to whether differential uptake and or trafficking to endosomal compartments could account for the disparity in observed immune responses between mCpG ODN and CpG ODN is considered and studies which examine possible preferential trafficking to late endosomal compartments and co-localization with TLR9 are conducted.  37  CHAPTER 2: Encapsulation in Liposomal Nanoparticles Enhances the Immunostimulatory, Adjuvant and Anti-tumour Activity of Subcutaneously Administered CpG ODN *  2.1 Introduction It is well established that bacterial DNA and synthetic ODN containing unmethylated CpG motifs are capable of inducing potent immune responses. CpG-containing DNA directly activates APCs such as DCs and B lymphocytes, resulting in enhanced Ag processing and presentation, upregulation of co-stimulatory molecules and secretion of immunomodulatory cytokines, including interleukin- (IL-) 6, 10, 12, 18 and interferon- (IFN-) cc/13. This, in turn, activates innate immune effector cells such as natural killer (NK) cells, results in the production of additional immunoactive cytokines (including IFNy) and chemokines and promotes the development of adaptive responses, with induction of Ag-specific effector cells such as cytotoxic and helper T lymphocytes (CTLs and TH cells respectively) (Krieg, Yi et al. 1995; Klinman, Yi et al. 1996; Bohle, Jahn-Schmid et al. 1999; Lipford, Bendigs et al. 2000; Hafner, Zawatzky et al. 2001; Van Uden, Tran et al. 2001). A hallmark of CpG ODN activity is its capacity to induce strong TH1-biased responses (Chu, Targoni et al. 1997; Brazolot Milian, Weeratna et al. 1998; Zimmermann, Egeter et al. 1998; Whitmore, Li et al. 1999; Weeratna, Brazolot Milian et al. 2001). Binding, internalization and trafficking of CpG ODN to the endosomal compartment by APCs are requisites for immunostimulatory activity (Krieg, Yi et al. 1995; Hacker, Mischak et  *A version of this chapter has been published. de Jong, S., Chikh, G., Sekirov, L., Raney, S., Semple, S., Klimuk, S., Ning, Y., Hope, M., Cullis, P., and Y. Tam (2007) Encapsulation in Liposomal Nanoparticles Enhances the Immunostimulatory, Adjuvant and Anti-tumor Activity of Subcutaneously Administered CpG ODN. Cancer Immunol Immunother 56(8):1251-1264  38  al. 1998; Takeshita, Leifer et al. 2001), allowing for the recognition and engagement by TLR9, a pattern recognition receptor specific for CpG DNA (Hemmi, Takeuchi et al. 2000; Bauer, Kirschning et al. 2001; Takeshita, Leifer et al. 2001; Ulevitch, Mathison et al. 2004). Its immunopotency and broad range of activity suggests that CpG ODN may be an effective immune modulator for the treatment and prophylaxis of a wide variety of diseases (Davis 1999; Hussain and Kline 2003; Ulevitch, Mathison et al. 2004; Becker 2005). In fact, CpG ODN are currently undergoing extensive clinical evaluation of their safety and tolerability, as well as their therapeutic potential as a single agent, adjunct therapy and vaccine adjuvant for treatment and prevention of malignant, infectious, allergic, inflammatory and autoimmune diseases (Cooper, Davis et al. 2004; Cooper, Davis et al. 2004; Krieg, Efler et al. 2004; Friedberg, Kim et al. 2005; Speiser, Lienard et al. 2005). Preliminary results from these studies are promising, showing that these compounds are well tolerated and mediate significant immune modulation (Cooper, Davis et al. 2004; Klinman, Currie et al. 2004; Krieg, Efler et al. 2004; Tulic, Fiset et al. 2004). Despite encouraging preclinical and clinical data, the use of free CpG ODN still has several disadvantages. First, free ODN have unfavourable pharmacokinetics and lack specificity for target cells, with rapid and broad tissue disposition after systemic administration. Furthermore, free ODN exhibit poor cellular uptake characteristics. Finally, ODN with natural phosphodiester (PO) backbone linkages are extremely sensitive to nuclease degradation, rendering them inactive in their free form (Zhao, Matson et al. 1993; Sands, Gorey-Feret et al. 1994; Agrawal, Temsamani et al. 1995). Therefore, the majority of the work with CpG ODN has employed ODN with nuclease-resistant phosphorothioate (PS) backbone modifications (Krieg, Yi et al. 1995; Cooper, Davis et al. 2004; Krieg 2004). While effectively enhancing stability, use of free PS-modified ODN also has inherent disadvantages, foremost being non39  sequence specific complement- and coagulation- related toxicities associated with PS-containing ODN (Levin 1999). To address these issues, we have previously reported on the development of a lipid-based nanoparticulate delivery system for ODN (Semple, Klimuk et al. 2000; Klimuk, Najar et al. 2004) that allows efficient encapsulation within liposomes possessing optimal characteristics (i.e., neutral surface charge, small particle size) for effective i.v. delivery and targeting of ODN to immune effector cells such as macrophages and DCs. Furthermore, liposomal encapsulation enhances intracellular delivery compared to equivalent doses of free CpG ODN through endocytosis-mediated cellular uptake (Semple, Klimuk et al. 2000; Li and Ma 2001). Finally, encapsulation completely protects ODN from nuclease degradation thus allowing for the use of the more immunopotent PO ODN (Mui, Raney et al. 2001). As a result, the use of lipid-based nanoparticulate delivery systems enhances targeting to and uptake by immune cells as well as providing an alternative to chemical modification to increase the stability of CpG ODNs in the circulation (Gokhale, Soldatenkov et al. 1997; Hacker, Mischak et al. 1998; Li and Ma 2001; Whitmore, Li et al. 2001). A recent study demonstrates that these factors result in improved immunostimulatory activity following i.v. administration as demonstrated by plasma cytokine levels and immune cell activation (Mui, Raney et al. 2001). While the benefits of encapsulation in nanoparticles are clear for i.v. administered CpG ODN, encapsulation may also improve the activity of locally delivered CpG ODN. This may be achieved through enhancing ODN uptake by phagocytic APCs, facilitating intra- and extracellular ODN trafficking, serving a source of drug (i.e. depot effect) and enabling the use of more immunopotent forms (i.e. PO) of CpG ODN. The ability of encapsulation to enhance activity of both locally and systemically administered CpG ODN drug is relevant for the development of LN-CpG ODN as a platform technology with broad immunostimulatory 40  applications. In particular, as a vaccine adjuvant, s.c. administration is the route of choice for many prophylactic and therapeutic vaccines aimed at inducing adaptive immune responses, allowing access to a large number of APCs. Therefore, it is of interest to define and evaluate the capacity of liposome encapsulation to enhance the activity of CpG ODN following local delivery. In this work, previous i.v. data is expanded upon, focusing primarily on the capacity of LN-CpG ODN to serve as an effective vaccine adjuvant to mediate Ag-specific immune responses. Specifically, the immunopotency of s.c. administered LN-CpG ODN and its ability to induce adaptive cellular responses against co-administered Ags and effective anti-tumour activity in animal models of cancer are investigated. As hypothesized, LN-CpG ODN demonstrated enhanced delivery and uptake by APCs following s.c. administration and, consistent with previous observations following i.v. administration, enhanced immunopotency, mediating more vigorous innate immune responses compared to free ODN. These enhanced responses were also reflected by a more potent induction of adaptive cellular responses by LNCpG ODN and ultimately, enhanced anti-tumour activity as a cancer vaccine adjuvant in combination with TAAs. These results confirm that encapsulation within LN profoundly enhances the immunopotency of CpG ODN and demonstrates the effectiveness of LN-CpG ODN in promoting the induction of Ag-specific cellular immune responses, thus dramatically increasing anti-tumour activity in various animal models. These results strongly suggest that liposomal encapsulation is an effective strategy to optimize the activity of CpG ODN and that LN-CpG ODN may be a suitable adjuvant for the development of effective vaccines for the treatment and prevention of cancer and other diseases.  41  2.2 Materials and methods 2.2.1 Animals and cell lines  Six- to 8-week-old female C57BL/6 and ICR mice were obtained from Charles River Laboratories (Saint-Constant, PQ, Canada) or Harlan (Indianapolis, IN). Mice were held in a pathogen-free environment and all procedures involving animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care. Murine B 1 6-F 1 0 melanoma and EL4 and E.G7-OVA (EL4 transfected to express OVA [43]) thymoma cells were obtained from the American Type Culture Collection (Manassas, VA). Parental EL4 and transfected E.G7-OVA thymoma cells were cultured in complete medium (CM) consisting of RPMI 1640 medium (Invitrogen, Burlington, ON, Canada) supplemented with penicillin G (100 U/ml), streptomycin sulphate (100 lag/m1), 5x10-5 M 13-mercaptoethanol and 10% foetal bovine serum. B16-F10 cells were cultured in CM supplemented with 0.1 mM non-essential amino acids and 1 mM sodium pyruvate, 0.3% glutamine and 50 m.g/m1 gentamicin.  2.2.2 ODN and preparation of liposomal nanoparticles  INX-6295 (5'-TAACGTTGAGGGGCAT-3'), a PS 16-mer ODN, was synthesized by Trilink BioTechnologies (San Diego, CA) for use in these studies. This ODN does not conform to any of the classes of CpG ODN previously described based on backbone chemistry, nucleotide sequence and immunostimulatory characteristics (Krieg 2002; Marshall, Fearon et al. 2003). INX-0167 contains a single CpG and a poly-G motif within a fully phosphorothioated ODN and when encapsulated, induces activation of wide variety of immune cells (unpublished data). INX-6295 was encapsulated in lipid nanoparticles containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Semple, Klimuk et al. 2001). Briefly, lipid combinations consisting of the bilayer forming lipids 42  distearoylphosphotidylcholine, or palmitoyloleoylphosphatidylcholine and cholesterol (Avanti Polar Lipids, Alabaster, AL), the ionisable lipid 1,2-dioleyloxy-3-N,N-dimethylaminopropane (for efficient ODN encapsulation) and the steric barrier lipid polyethylene glycol (PEG)dimyristol glycerol or PEG-ceramide  C14  (to prevent vesicle aggregation during formation) at a  molar ratio of 25/45/20/10 were solubilized in ethanol and added to 50 mM citrate buffer pH 4.0. The lipid mixture was passed twice through stacked 200 nm + 100 nm polycarbonate membranes (Whatman Nuclepore, Clifton, NJ) using a thermobarrel extruder (Lipex Biomembranes, Vancouver, B.C., Canada) to produce a homogeneous population of pre-formed vesicles approximately 100 nm in diameter. Oligodeoxynucleotides (3.33 mg/ml) were solubilized in citrate buffer followed by ethanol to a final ethanol concentration of 40%. The ODN and pre-formed vesicles were combined and incubated at room temperature for 2 h. The resulting vesicles were dialyzed first against citrate buffer followed by HEPES-buffered saline (HBS) at pH 7.58. Unencapsulated ODN was subsequently removed by anion exchange chromatography on DEAE-Sepharose CL-6B columns equilibrated in HBS. As previously described, this process results in discrete vesicles completely encapsulating the ODN within an aqueous interior, which are distinctly different from lipid complexes (Maurer, Wong et al. 2001; Semple, Klimuk et al. 2001). Oligonucleotide concentrations were determined by UV spectroscopy (260 nm) on a Beckman DU 640 spectrophotometer (Beckman Coulter, San Diego, CA) following solubilization of the samples in chloroform/methanol at a volume ratio of 1:2.1:1 chloroform/methanol/aqueous phase (sample/HBS). Lipid concentrations were determined using an inorganic phosphorous assay after separation of the lipids from the oligonucleotides by a Bligh and Dyer extraction (Bligh and Dyer 1959). The ODN-to-lipid ratio was typically 0.100.13 (w/w). Particle size, as determined by quasi-elastic light scattering using a NICOMP submicron particle sizer (Model 370, Santa Barbara, CA), was approximately 100 ± 25 nm. 43  2.2.3 Cell uptake analysis The effect of encapsulation on the uptake of CpG ODN by murine immune cell populations following s.c. administration was assessed in ICR mice injected with 5 mg/kg free or encapsulated 5' -carboxyfluorescein (FAM)-labelled INX-6295 PS ODN (Trilink Biotechnologies). For uptake analysis, mice were anaesthetized with ketamine/xylazine (3.2%/0.8%, v/v) 1, 4, 7 and 24 h post administration, and spleens and lymph nodes were collected and processed to single cell suspensions by passage through a sterile 100 pm nylon mesh (Becton Dickenson, Franklin Lakes, NJ). Splenocytes were depleted of red blood cells by ammonium chloride lysis. Cells were analyzed for ODN uptake (as judged by intensity of the fluorescently labelled ODN on a per cell basis) by specific immune cell populations (as determined by phenotype analysis; cell suspensions were stained with phycoerythrin [PE]conjugated or allophycocyanin [APC]-conjugated anti-CD11b, anti-CD1 lc, anti-CD8 and antiDX5 phenotype antibodies) using a 4-colour FACSort flow cytometer and CellQuest Pro software (BD Biosciences, San Jose, CA). All fluorescently labelled antibodies were obtained from BD Biosciences. Propidium iodide was used to exclude dead cells and 150,000 and 20,000 events were collected to analyze DCs or NK cells, macrophages, B cells, CD4 and CD8 Tlymphocytes, respectively.  2.2.4 Ex-vivo analysis of immune parameters 2.2.4.1 Plasma cytokine analysis The effect of encapsulation on the potency of ODN-mediated activation of murine immune cell populations was evaluated after s.c. administration of free and encapsulated ODN to ICR mice. For plasma cytokine levels, mice were anaesthetized as previously described and blood was collected via cardiac puncture into Vacutainer tubes containing EDTA (Becton 44  Dickinson). Plasma was isolated by centrifugation and frozen at —20°C until assayed. Plasma concentrations of IL-6, IL-10, MCP-1 and IF1\17 were determined using commercially available ELISA or cytometric bead array kits (both from BD Biosciences), as per the manufacturer's instructions.  2.2.4.2 Cell activation analysis  For cellular assays, peripheral blood, spleens and lymph nodes were collected after treatment and single cell suspensions were generated from the organs as described above. Blood and splenocytes were depleted of red blood cells by ammonium chloride lysis and analyzed for immune stimulation as judged by activation marker expression. Cell suspensions were stained with fluorescein isothyocianate (FITC)- and APC- labelled phenotype antibodies (anti-CD 1 lb and anti-CD 11c, anti-CD8, anti-DX5 respectively) in combination with PE-conjugated antibodies directed against the activation markers CD69 or CD86. Cell activation analyses were performed by flow cytometry as described above.  2.2.4.3 Assessment of antigen-specific CD8 T lymphocytes  The ability of free and encapsulated ODN to induce Ag-specific CD8 T lymphocytes was assessed in C57BL/6 mice immunized s.c. with hen egg albumin (ovalbumin or OVA) mixed with saline, free ODN or LN-CpG ODN using MHC tetramer, chromium release cytotoxicity, and IFN7 cytokine secretion assays.  45  2.2.4.3.1 MHC tetramer assay After immunization, the frequency of OVA-specific CD8 T-lymphocytes was determined using an MHC-tetramer assay. Briefly 5x106 spleen cells were incubated with PE-coupled H2Kb MHC tetramers containing the immunodominant peptide of OVA (SIINFEKL; BeckmanCoulter, Immunomics, San Diego, CA) and FITC-labelled anti-mouse-CD8 and PE-cyaninlabelled anti-TC113 phenotype antibodies (BD Biosciences) prior to analysis on a flow cytometer as previously described. One hundred and fifty thousand events were collected to analyze the frequency of OVA-specific CD8 T-lymphocytes in immunized animals.  2.2.4.3.2 Cytotoxicity assay The ability of cells from immunized animals to lyse target cells in an Ag-specific manner was assessed in splenocytes, either immediately following completion of the immunization regimen with OVA or after in vitro Ag-restimulation. For the latter, E.G7-OVA cells were treated with mitomycin C (50 lg/m1) and combined with splenocytes from immunized animals for 5 days with the addition of human recombinant IL-2 (100 IU/ml; BD Biosciences). Ovalbumin-specific cytotoxicity was assessed using a standard 4 h 51chromium (51Cr) release assay. Briefly, splenocytes were mixed in various effector:target ratios with 51Cr-loaded parental EL4 or OVA-transfected E.G7 cells and the percentage of cellular cytotoxicity was calculated on the basis of 51Cr released to the supernatant using the formula: % lysis =[(experimental cpm — spontaneous cpm) / (maximal cpm-spontaneous cpm)] x 100, where maximal cpm was achieved by complete lysis of 51Cr-labeled targets in 10% Triton X 100, spontaneous CPM was determined by incubating labelled targets in CM and Ag-specific killing was determined by comparison of cytotoxicity of 51Cr-labeled OVA-expressing and nonexpressing E.G7 and EL4 cells, respectively. 46  2.2.4.3.3 Cytokine secretion assay  Interferon--y secreting CD8 T-lymphocytes were detected using the IFNy secretion assay (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's instructions. This assay is designed to quantify Ag-specific CD8 T-lymphocytes by enumerating the number of CD8 Tlymphocytes that secrete IFNy in response to Ag stimulation. Briefly, splenocytes were restimulated with OVA-expressing APCs prior to incubation with a bispecific antibody designed to bind to activated T-lymphocytes via the CD25 activation marker and capture secreted IFNy. The frequency and phenotype of cells that responded to OVA-stimulation by actively secreting cytokines were determined by flow cytometry as described previously using a fluorescently labelled anti-IFNy antibody in combination with previously described fluorescently labelled phenotype antibodies.  2.2.5 Tumour challenge efficacy studies  The xenogeneic E.G7-OVA and syngeneic B16 C57BL/6 tumour models were used to determine the efficacy of encapsulated ODN as a vaccine adjuvant to induce Ag-specific anticancer immune responses. For the EG7-OVA model, mice were immunized prophylactically with OVA mixed with 100 jig free or encapsulated INX-6295 ODN adjuvant weekly for two or three weeks. One week following the last vaccination, mice were injected s.c. with 2.5x106 E.G7-OVA tumour cells into the left flank and monitored for tumour growth. Tumour size was assessed every second day using digital callipers (Mitutoyo, Mississauga, ON, Canada). Tumour volumes were calculated using the standard formula for tumour volumes, (length x width2)/2. For the syngeneic B16-tyrosinase related protein (TRP)-2 model, mice were immunized prophylactically with TRP-2 mixed with 100 lig free or encapsulated INX-6295 ODN adjuvant weekly for two or three weeks. Two days following the final vaccination, mice were injected i.v. 47  with 1.0 x 105 B16 cells. Mice were euthanized 18 days later, lungs were excised and metastases were enumerated using a stereomicroscope.  2.2.6 Statistical analyses  All statistical analyses were performed using SPSS Ver 14.0. Initially, a one-way analysis of variance (ANOVA) was used to statistically evaluate the differences between treatment groups. In the case of statistically significant results, the differences between treatment groups were assessed through the use of Bonferroni adjusted t-tests, a post-hoc test which controls for error rate. Probability (p) values less than .05 were considered significant.  48  2.3 Results 2.3.1 Encapsulation in LN enhances uptake of CpG ODN by immune effector cells in the lymph nodes and spleen after subcutaneous administration  Preliminary pharmacokinetic and biodistribution studies following s.c. administration using radiolabelled lipids and CpG ODN to determine the fate of administered LN-CpG ODN, indicates preferential accumulation in local draining lymph nodes on a per gram basis, as well as secondary accumulation patterns consistent with i.v. delivery, particularly in the spleen and liver, (manuscript in preparation) following s.c. administration. Based on these results, studies were undertaken to characterize the cellular uptake of s.c. administered free and LN-CpG ODN in the draining lymph nodes and spleen using fluorescently-labelled ODN. Results from these studies demonstrate a 2-9-fold enhancement of uptake for encapsulated ODN by phagocytic APCs including macrophages and DCs (as judged by expression of CD1 1 b and CD1 lc ± CD1 lb respectively; Fig 2.1) compared to free ODN in the lymph nodes over a 24 h period. Similarly, enhanced uptake for LN-CpG ODN is also observed in spleen following s.c. administration albeit at a much lower relative level than that observed in lymph nodes, with splenic uptake of free ODN being similar to control levels.  49  A  70 60 50 40 30 20 10  4  8  12  16  20  24  Time (h)  250  200  g- 150  100 z  0  50  -  0 0^4^8^12^16^20^24 Time (h)  160 140 — 120 U.  100 80 60 40 20 0 0  4^8^12  16  20  24  Time (h)  Fig 2.1 Uptake of free and liposome nanoparticulate formulations of CpG ODN by immune cells in spleen and lymph nodes following s.c. administration  Five mg/kg of free (denoted by closed symbols) or encapsulated (denoted by open symbols) fluorescently-labelled (5'-FAM) INX-6295 CpG ODN was administered s.c to mice (4 animals/group). After 1, 4, 7 and 24 h, animals were euthanized and spleens (denoted by diamonds) and lymph nodes (denoted by triangles) were harvested and processed to single cells. Samples were analyzed for uptake of the ODN (as judged by mean fluorescence intensity or MFI ± SD) by specific cell types (as judged by expression of the phenotype markers CD11 b [Panel A], CD1Ic [Panel B] and CD11b/CD11 c [Panel C]) by flow cytometry as outlined in the Materials and Methods. Background fluorescence levels of 17.5-18.3 were subtracted from the data. Data presented here is representative of 2 separate experiments.  50  2.3.2 Encapsulation of CpG ODN in LN enhances immune cell activation  To assess the effect of encapsulation on the immunopotency of CpG ODN, the expression of CD69, an early activation marker of T and B lymphocytes NK cells, macrophage and neutrophils (Sancho, Gomez et al. 2005) and CD86, a co-stimulatory molecule expressed primarily by activated APCs such as monocytes/macrophage and DCs were monitored COI 1 aCC69-Ive  ^  CD1 1 b/CDS9+ve  35 3.0 2.5 2.0  1.  1.5 1.0 0.5 0.0 CpG-CCN  ^  Encap CpG-CCN  ^  cosicces+ve  CpG-CCN  ^  Encap, CpG-CCN  12115/CDEt94ve  35 3.0 2.5 2.0 / 1.5 0.8 I 1.0 0.5 0.0  1  1-13S^CpG-CCN^Encap. CpG-OCN  COI leJCC*16-we 25 ^  2.0  I 1 5  1.0  05-  1-f3S  ^  CpG-OCN  ^  00 Encap. CpG-CON  F-ES^CpG-CON^Emap CpG-CCN  Fig 2.2 Comparison of immune cell activation following s.c. treatment with free and encapsulated CpG ODN Five mg/kg of free or encapsulated CpG ODN was administered sc. to ICR mice (4 animals/group). After 24 h, animals were euthanized and spleens harvested. Splenocytes were analyzed for expression of the CD69 and CD86 cell surface activation markers (% of total cell population ± SD) in conjunction with phenotype markers by flow cytometry as outlined in the Materials and Methods. Data presented here is representative of 3 separate experiments.  51  (Lenschow, Walunas et al. 1996; McAdam, Schweitzer et al. 1998; McAdam, Farkash et al. 2000). Similarly, the production of TH1 and TH2 cytokines (IFNy and IL-6, IL-10, respectively) as well as MCP-1 (a macrophage chemokine) was also assessed. These data were collected over a 72 h time period following administration of either free or encapsulated CpG ODN to ICR mice. Data presented here are representative of at least three independent experiments. Immune cell activation is observed in response to s.c. treatment with both free and encapsulated CpG ODN, with upregulation of CD69 and CD86 expression on spleen and lymph node (data not shown) cell populations that peaks at 24 h post injection. Based on CD69 expression, free CpG ODN induces a 44% increase in the number of activated CD1 1 b+ cells, a 4-fold increase in activated CD1 1 c+ cells and a 2-fold increase in activated DX5+ cells above control levels. No appreciable effect is observed in CD8+ cells (Fig 2.2). In contrast, the administration of encapsulated CpG ODN leads to a >5-fold, 12-fold, 4-fold and a 12 fold increase in activated CD1 lb, CD1 1 c, DX5 and CD8+ cells, respectively, compared to cells from HBS-treated control animals. This corresponds to a 4-fold increase in activated CD1 lb+ cells, a 3-fold increase in activated CD! 1c cells, a 2-fold increase in activated DX5+ cells, and a 12-fold increase in activated CD8+ cells above levels seen after administration of free CpG ODN. Similarly, the expression of the co-stimulatory molecule CD86 is upregulated approximately 2-fold in both CD1 lb+ and CD1 1 c+ cells in animals treated with free CpG ODN but at least 6-fold in those treated with LN-CpG ODN. An ANOVA was performed on each of the 6 groups of data shown in Fig 2.2. The results from each analysis showed that there is a statistically significant difference between the treatment groups at the p<.001 level. Further analysis using a Bonferroni (  test showed that the upregulation of both CD69 and CD86 was significantly increased (p<.005)  in mice treated with LN-CpG ODN compared with both the control and mice treated with free CpG-ODN. 52  •  The relative immunopotency of encapsulated CpG ODN compared to free is also reflected in plasma cytokine levels. Mice injected with free CpG ODN show modest increases in cytokine levels over 72 h (Fig 2.3) while LN-CpG ODN is able to exert a dramatic effect. 450 -  900  400 -  800  350  700 I  .1i- 300  ▪  250 a. — 200  -, E 500  -  o •  150  600 400 300  100  200  50  100  0 12^24^36^48  ^  so  ^  -  0 -1ao  72  Time (hrs)  Time (hrs) 3000  350 -  2500  300 250  ri 2000  200  a. 1500 — a.  12^24^36^48^60 ^ 72  0. 150  1000  z u-  2  50 -1  500  0 0^12^24^36^48  60  72  -50  Time (hrs)  12^24^36  48  ^  60  ^  72  Time (hrs)  Fig 2.3 Comparison of plasma cytokine induction following s.c. treatment with free and encapsulated CpG ODN Five mg/kg of free CpG ODN (denoted by closed squares) or encapsulated CpG ODN (denoted by open squares) was administered s.c. to ICR mice (4 animals/group). Blood was collected from animals by cardiac puncture, processed to collect plasma and cytokine levels (pg/ml + SE) were determined by ELISA or cytometric bead array. Data presented here is representative of 3 separate experiments and each data point represents an average of 4 animals.  The plasma concentration of IL-6 at the 7 h peak concentration is 240-fold above baseline while both IL-10 and MCP-1 expression is enhanced 14-fold at the 24 h peak time point. The concentration of IFNy is also greatly enhanced by encapsulation, showing a 250-fold enhancement in plasma levels above that of free CpG ODN. As expected, these data are similar to those results previously reported after i.v. administration of LN-CpG ODN demonstrating dramatic immunostimulation as judged by 53  plasma cytokines and cell activation marker levels compared to control animals and those treated with equivalent doses of free CpG ODN (Mui, Raney et al. 2001). Furthermore, results with other cytokines (eg IL-12) and activation markers (CD 16 and IL-12 receptor) show similar trends (data not shown) In summary, encapsulation dramatically enhances the immunogenicity of CpG ODN following s.c. administration compared to free CpG ODN, resulting in enhanced expression of cell activation markers CD69 and CD86 and elevated plasma cytokine/chemokine levels. No appreciable immunostimulatory activity, based on immune cell activation or plasma cytokines were detected in animals treated with empty liposomes (unpublished data).  2.3.3 Encapsulation of CpG ODN in LN enhances the generation of antigen-specific immune responses To assess the effect of encapsulation on the development of adaptive responses, studies with OVA were undertaken. Ovalbumin is widely studied as a model Ag and thus its antigenic determinants have been mapped and reagents and models are available to monitor both humoural and cell-mediated immune responses. In our studies, OVA was employed as an artificial TAA to evaluate the ability of LN-CpG ODN to act as an adjuvant in the generation of tumour-specific cell-mediated immune responses. In vitro immune parameters included quantitative and functional assessments of OVA-specific cytotoxic T-lymphocytes (CD81) using an MHC tetramer assay and cytotoxicity and cytokine secretion assays respectively. The ability of encapsulated CpG ODN to generate OVA-specific CD8 T-lymphocytes was assessed using a standard MHC tetramer assay on splenocytes from C57BL/6 mice immunized with OVA adjuvanated with free or encapsulated CpG ODN. The use of tetramers is a quantitative method of determining the frequency of Ag-specific CTL that is not dependent upon limiting dilution or in vitro culture methods. Using this assay, the percentage of 54  CD8/TCR-f3 OVA-tetramer positive cells is enhanced 4- and 20- fold following treatment with free and encapsulated CpG ODN, respectively, compared with control animals (Fig 2.4). 3000 +61 0 2500 0 gia Z.- 2000 co 0 co 1500  E 1000 s 500 0 Co  100 1.01■11 HBS  CpG ODN + OVA LN-CpG ODN + OVA  Fig 2.4 Frequency of antigen-specific OVA-MHC tetramer+ CD8+ cells following s.c. immunization with OVA adjuvanated with free or encapsulated CpG ODN C57BL/6 mice (4 animals/group) were immunized s.c. with OVA adjuvanated with 100 pig free or encapsulated CpG ODN. Splenocytes were isolated , incubated with an OVA-specific PE-labelled H-2Kb-SIINFEKL MHC tetramer in conjunction with fiuorescently labelled anti- CD8 and TCR(3 antibodies and analyzed by flow cytometry to quantitate the frequency of OVA-specific CD8 T-lymphocytes in animals following immunization. These data are derived from 5 separate studies and expressed as a percentage of control levels ± SD.  Further analysis revealed statistically significantly greater frequency of antigen-specific OVAMHC tetramer positive CD8 cells following immunization with OVA adjuvanated with LN-CpG OVA compared with free CpG ODN plus ova [t(7)=-2.157, p<.051 In addition to a quantitative assessment, the relative ability of liposome-encapsulated vs free CpG ODN to generate Ag-specific CTLs was also assessed functionally. One such functional assay used the secretion of IFNy (as an indicator of TH 1 response) by splenocytes from 55  C57BL/6 mice to monitor the number of T-lymphocytes in immunized animals capable of responding to Ag-specific stimulation. While a 2.5-fold increase in the percentage of CD8+ IFNy producing cells is observed in mice treated with free CpG ODN compared to control mice (Fig 2.5), a further significant increase in the percentage of IFNy-secreting CD8+ cells is obtained when mice are immunized with OVA adjuvanated with LN-CpG ODN [t(7)=-4.584, p<.05]. 1200  + 400 uc--0- 200 0 HBS  ^  CpG ODN + OVA LN-CpG ODN + OVA  Fig 2.5 Frequency of antigen-specific, IFNy secreting CD8+ cells following s.c. immunization with OVA and free or encapsulated CpG ODN C57BL/6 mice (4 animals/group) were immunized s.c. with OVA adjuvanated with 100 jig free or encapsulated CpG ODN. Splenocytes were isolated and activated as described in the Materials and Methods. Briefly, IFNy secreting ability of CD8+ cells was assessed after 8 h in vitro restimulation with OVA expressing E.G7 cells as determined by cytokine secretion assay. These data are derived from 5 separate studies and expressed as a percentage of control levels + SD.  As an additional functional assessment, the induction of Ag-specific CTL responses was assessed in a standard chromium release cytotoxicity assay. The relative ability of splenocytes, 56  isolated from animals immunized with OVA and encapsulated or free CpG ODN, to lyse OVAexpressing E.G7 target cells in an Ag-specific manner was assessed immediately after isolation or following 5 days of in vitro restimulation (IVR) with OVA-expressing APCs. In both primary and IVR evaluation of CTL activity, effector cells from control animals exhibit only minimal levels of target cell lysis while immunization with OVA and free CpG ODN results in a 2-fold and a 4-fold increase respectively (Fig 2.6). 60.0 50.0 0 0 40.0 $65 30.0 -  to  20.0  CpG LN-CpG ODN ODN  Primary Assay  HBS^CpG LN-CpG ODN ODN  IVR Assay  Fig 2.6 Antigen-specific cytolytic activity of splenocytes against E.G7-OVA cells following s.c. immunization with OVA adjuvanated with free or encapsulated CpG ODN C57BL/6 mice (4 animals/group) were immunized s.c. with OVA adjuvanated with 100 pg free or encapsulated CpG ODN. Splenocytes were isolated as described in the Materials and Methods and either used immediately (primary) or after 5 days in vitro restimulation (IVR) as effector cells in a standard 51Cr release assay. The percentage of chromium released from radiolabelled E.G7 (for specific lysis) and EL-4 (for non-specific lysis) targets after 4 h co-incubation with isolated splenocytes was used to calculate specific cytolytic activity ± SD. Effector cells and target cells were plated at a variety of ratios; the 100:1 effector-target ratio is shown.  However, significantly higher CTL responses in both the primary and the IVR assays are observed in mice that received OVA and LN-CpG ODN compared to mice vaccinated with free CpG ODN (p<.005 — Bonferroni adjusted t tests) and control animals. No Ag-specific 57  responses, as judged by either the quantitative or functional assays were detected following immunization with empty liposomes (unpublished data).  2.3.4 Anti-tumour efficacy of encapsulated ODN as a vaccine adjuvant in xenogeneic and syngeneic tumour models  Using the parental EL4 and OVA-expressing E.G7 tumour cell lines, the relative ability of free and encapsulated CpG ODN to induce anti-OVA immunity and effective anti-tumour efficacy was assessed. Specifically, C57BL/6 mice were immunized prophylactically with OVA combined with free or LN-CpG ODN, challenged s.c. with E.G7 tumour cells and monitored for effect on tumour growth. As a standardized control, these data were compared to animals immunized with OVA adjuvanated with complete Freund's adjuvant (CFA), a widely used research adjuvant against which the majority of new adjuvants are measured. Immunization with OVA alone or in combination with free CpG ODN results in only minor anti-tumour activity, with animals exhibiting vigorous tumour growth similar to that observed in control animals and those immunized with CFA alone (Fig 2.7). Alternatively, OVA adjuvanated with CFA induces a moderate anti-tumour response while mice immunized prophylactically with OVA and LNCpG ODN show pronounced responses, exhibiting complete tumour regression that persists prior to eventual tumour re-growth. Interestingly, the use of encapsulated CpG ODN as an adjuvant also results in less variability in response compared with CFA, most likely due to difficulties in obtaining a homogeneous Ag-adjuvant emulsion with CFA for immunizations. Furthermore, animals treated with CFA also develop local inflammatory responses and granuloma formation unlike those treated with LN-CpG ODN. However, while LN-CpG ODN elicits potent anti-tumour activity in the E.G7-OVA tumour model, it has been noted that OVA, being a xenogeneic Ag, is highly immunogenic. Therefore induction of effective anti-OVA immune responses would be expected to be relatively 58  • easy compared to true, syngeneic TAAs that are characterized by low immunogenicity and host tolerance. To address this concern, the ability of free and encapsulated CpG ODN to induce immune responses against TRP-2 was assessed in a B16 pulmonary metastasis model to establish whether free or encapsulated CpG ODN provides sufficient immunological stimulus to generate immune responses and therapeutic activity against self Ags.  4000 • 3500  HBS  —0—OVA —21r— CFA  3000  —A— CFA+OVA —N— CpG ODN+OVA  E 2500  —0-- LN-CpG ODN + OVA 2000 o 1500 I– 1000  500  0  ^^ 5  10^15^20  ^  25  Time Post-Tumour Cell Injection (Days)  Fig 2.7 Antigen-specific anti-tumour activity following prophylactic s.c. immunization with OVA adjuvanated with free or encapsulated CpG ODN in a E.G7-OVA xenogeneic tumour model C57BL/6 mice were immunized prophylactically s.c. with OVA adjuvanated with 100 fag free or encapsulated CpG ODN. One week following the last vaccination, mice were challenged s.c. with 2.5 x 106 E.G7-OVA cells and tumour growth was monitored. Tumour volume was calculated using the formula V–(LxW2)/2. Each data point represents the mean and standard deviation of a group of 5 animals.  TRP-2 is a tissue specific syngeneic Ag, expressed on melanoma, melanocytes and the retina and anti-B16 CTLs have been shown to recognize the immunodominant MHC class I (H-2Kb) epitope of TRP-2 (TRP181_188;) (Bloom, Perry-Lalley et al. 1997). Since TRP-2 has been identified as a potential TAA against which to target CTL responses for cancer therapy in 59  humans (van Elsas, Sutmuller et al. 2001), the murine TRP-2-B16 model provides a system that mimics human melanoma without the introduction of a xenogeneic Ag. C57BL/6 mice were immunized prophylactically with the immunodominant peptide TRP 181-188 co-administered with free or encapsulated CpG ODN, challenged i.v. with B16 cells and after 18 days, euthanized, lungs collected and lung metastases enumerated. Animals immunized with the TRP-2 peptide alone show no anti-tumour effect, with similar numbers of lung metastases (mean = 82) compared to untreated animals (data not shown). Compared with untreated animals, there is a non-significant but suggestive decrease in the total number of lung metastases in mice treated with TRP-2 peptide adjuvanated with CFA (mean = 46) and free CpG ODN (mean = 35) (Fig 2.8).  TRP2  ••^•  CFA+TRP2  CpG ODN+TRP2  ••  LN-CpG ODN + TRP2  40e• r 0^20^40^60^80^100^120 Total B16 Lung Metastases  Fig 2.8 Antigen-specific anti-tumour activity following prophylactic s.c. immunization with TRP-2 adjuvanated with free or encapsulated CpG ODN in a E.G7-OVA syngeneic tumour model C57BL/6 mice were immunized prophylactically s.c. with TRP-2 adjuvanated with 100 u.g free or encapsulated CpG ODN or CFA. Two days following final immunization, mice were injected i.v. with 1 x 105 B16 cells. Animals were euthanized on day 18, lungs were excised and metastases were enumerated using a stereomicroscope. The bar represents the mean number of lung metastases per group (n=4); circles represent individual animals.  60  However, animals immunized with TRP181_188 adjuvanated with encapsulated CpG ODN have a significant reduction in lung metastases (mean = 8) compared to control or CFA-adjuvanated animals (Bonferroni adjusted t tests; vs HBS: t(7) = 4.244, p<.05; and vs CFA: t(7) = 4.965, p<.01). A suggestive but non significant reduction in lung metastases was observed also between animals immunized with TRP-2 peptide adjuvanated with free vs encapsulated CpG ODN. No anti-tumour activity was observed for empty liposomes in either of these models (unpublished data).  61  2.4 Discussion  Broadly active biological response modifiers that target the innate as well as the adaptive arms of the immune system and enhance cell-mediated immunity are continuously being explored for their clinical potential in the treatment of cancer (Wysocka, Benoit et al. 2004). It has been clearly established that bacterial DNA or synthetic ODN containing CpG motifs have potent immunostimulatory properties through their interaction with TLR9 (Krieg 2003; Sfondrini, Balsari et al. 2003; Klinman 2004). CpG ODN-mediated stimulation of APCs induce the secretion of immunomodulatory cytokines and chemokines and promote the upregulation of costimulatory molecules which, in turn, activate NK and other innate immune cells. This results in elaboration of secondary cytokines such as IFNy and facilitates the priming and expansion of T-lymphocytes, ultimately giving rise to Ag-specific effector T-lymphocyte populations. This ability to elicit potent innate responses and directly and indirectly induce adaptive, cellular immunity supports the concept of CpG ODN as an effective cancer therapeutic with a variety of potential applications, including as a bioresponse modifier and vaccine adjuvant (Dalpke and Heeg 2004). While free CpG ODN mediate potent immunostimulatory effects, its limited delivery to target cells or tissues, poor cellular uptake, rapid degradation (for the native PO form) and toxic side-effects represent limitations for its clinical use (Mutwiri, Nichani et al. 2004). As an alternative strategy, it has been well demonstrated that encapsulation in liposomal nanoparticles has the capacity to dramatically alter the pharmacokinetic and biodistribution characteristics of a drug. In immunotherapeutic applications, encapsulation of immunomodulatory compounds results in targeted delivery to immune cells (Rao and Alving 2000). For antisense ODNs, encapsulation in liposomal nanoparticles has been shown to significantly enhance the plasma and tissue levels and improve anti-tumour effects in human melanoma and laryngeal squamous 62  carcinoma in vivo (Gokhale, Soldatenkov et al. 1997; Gokhale, McRae et al. 1999; Leonetti, Biroccio et al. 2001). Although all micro and nanoparticulate carrier systems preferentially accumulate in macrophages and professional APCs following systemic administration, traditional complexes consisting of cationic lipids and CpG ODN are not suitable for systemic delivery in humans as they are rapidly eliminated from the blood due to their large (micron) size and positive charge. The charge associated with complexes can also give rise to non-specific immunological activity as a result of complement activation via the alternative pathway, resulting in damage to the liver and other tissues (Levin 1999). However, the nanoparticulate lipid-based delivery system utilized in this study is characterized by low surface charge and small particle size, resulting in reduced clearance rates, relatively long circulation lifetimes and minimal toxicity (Semple, Klimuk et al. 2000; Klimuk, Najar et al. 2004). These nanoparticulate systems protect the ODNs from nuclease degradation and promotes uptake of relatively large amounts of intact ODN by target immune cells. In these studies it is shown that s.c. delivered LN-CpG ODN is preferentially delivered to and taken up by target APCs, providing, at least in part, the basis for the enhanced immunopotency of encapsulated CpG ODN. Using this formulation, the enhanced immunopotency of LN-CpG ODN compared to free ODN following i.v. administration has been previously demonstrated (Mui, Raney et al. 2001), consistent with other observations of immune stimulation and anti-tumour activity following systemic administration of lipid-DNA complexes for gene transfer applications (Dow, Fradkin et al. 1999; Whitmore, Li et al. 1999) and report here that a parallel response is induced following s.c. administration. Expression of activation markers such as CD69, which is low on resting immune cells such as NK and T-lymphocytes, and minimal in mice injected with free CpG ODN is dramatically upregulated on immune cell populations following s.c. (and i.v., as previously 63  reported (Mui, Raney et al. 2001)) administration of LN-CpG ODN. These results are supported by similar observations of plasma cytokine levels in which s.c. (and i.v. (Mui, Raney et al. 2001)) administered LN-CpG ODN dramatically enhances levels of a number of cytokines and chemokines compared to equivalent doses of free ODN, both in terms of the magnitude and duration of cytokine expression (Mui, Raney et al. 2001). Furthermore, this enhanced immunopotency is also reflected in the capacity of LN-CpG ODN to induce more vigorous adaptive immune responses. It has been well documented that CpG ODN have properties that make them ideal immune adjuvants for cancer vaccines (Miconnet, Koenig et al. 2002; Lonsdorf, Kuekrek et al. 2003; Uhlmann and Vollmer 2003; Wysocka, Benoit et al. 2004), promoting the generation of TH1-biased, Ag-specific immune responses against co-administered peptide and protein tumour Ags including vigorous cellmediated responses. Since priming and expansion of tumour-specific T-lymphocytes is considered to be an essential component of an effective anti-tumour immune response (Tulic, Fiset et al. 2004), it was of particular interest to assess the capacity of liposome encapsulation to enhance CpG ODN induced Ag-specific adaptive cellular responses. In these studies, the generation of high frequencies of functional Ag-specific CTLs, as assessed using MHC tetramer, IFN7 secretion and cytotoxicity assays, directly supports the characterization of LN-CpG ODN as a highly effective adjuvant that is able to promote generation of TAA-specific immune responses. While very low levels of Ag-specific T-lymphocytes are detected in splenocytes of control mice and animals immunized with TAAs adjuvanated with free CpG ODN, a concomitant increase in MHC-tetramer positive cells, IFN7 secretion and cytolytic activity is detected in the splenocytes of mice immunized with TAAs co-injected with LN-CpG ODN, indicating an induction of functional Ag-specific CD8+ T-lymphocytes. Thus, both quantitative and functional assays demonstrate that encapsulation results in a dramatic increase in the ability 64  of CpG ODN to support development of Ag-specific cytotoxic T-lymphocytes compared with free CpG ODN. Furthermore, although not described in detail here, LN-CpG ODN was also found to be effective in inducing humoural immune responses, resulting in elevated plasma levels of Ag-specific immunoglobulins, analysis of IgGI and IgG2a isotypes indicating either a slight TH1-biased or a balanced TH 1 /TH2 immune response (unpublished results). While evaluation of ex vivo immune parameters provides valuable insight into mechanisms of action and allows quantitative and functional comparisons of the immunostimulatory capacity of free and encapsulated CpG ODN, evaluation of anti-tumour efficacy provides a more relevant assessment of the ultimate potential of liposomal CpG ODN as a cancer therapeutic. Towards this end, the relative ability of LN-CpG ODN to provide antitumour activity was evaluated in a number of animal models. As a vaccine adjuvant, LN-CpG ODN is able to support the generation of protective immunity against tumour challenge, promoting significantly more effective tumour immunoprophylaxis than free CpG ODN as evidenced by the enhanced inhibition of tumour growth in animals compared to free CpG ODN or "gold standard" adjuvants such as CFA. Importantly, this therapeutic strategy is able to not only provide effective protection against model Ags, but is capable of breaking tolerance to self Ags, an aspect that is vital for the development of clinically relevant therapies. Encapsulated CpG ODN is sufficiently potent to induce immune responses against poorly immunogenic syngeneic Ags, providing effective anti-tumour activity against even aggressive tumours expressing only very low levels of MHC class I Ag (Lollini, De Giovanni et al. 1990). In summary, the immunological potency and therapeutic efficacy of immunostimulatory CpG ODN is greatly enhanced by encapsulation in liposomal nanoparticles. In the work described here, the previous observations of enhanced immunopotency of LN-CpG ODN compared to free CpG ODN are confirmed and further defined. In view of the interrelated 65  nature of immune regulation, it should perhaps not be surprising that this enhanced capacity of LN-CpG ODN to induce innate responses following i.v. administration would also be reflected in enhanced innate and adaptive immune responses following s.c. administration. Importantly, these observations are also extended, clearly demonstrating the potential of encapsulated CpG ODN to induce potent and effective in vivo anti-tumour activity. As an immune adjuvant to support vigorous adaptive cellular immune responses, encapsulated CpG ODN mediates effective anti-cancer activity, acting to reduce tumour burden and enhance survival. Thus, encapsulation of CpG ODN within liposomal nanoparticles offers an attractive strategy for significantly enhancing the activity of free CpG ODN and improving its therapeutic activity in the treatment of cancer.  66  CHAPTER 3: Synthetic Methylated CpG ODNs are Potent in vivo Adjuvants When Delivered in Liposomal Nanoparticles *  3.1 Introduction  Pathogenic DNA containing unmethylated CpG motifs is a well-recognized immunomodulator that can induce potent immune responses capable of providing significant protective and therapeutic immunity against a number of malignant and infectious diseases (Krieg, Love-Homan et al. 1998; Zimmermann, Egeter et al. 1998), properties that are shared with short, CpG-containing synthetic ODN (Wagner 2001). CpG-containing DNA is a member of a group of molecules known as pathogen-associated molecular patterns (PAMPs), highly conserved molecular motifs associated with a wide range of pathogens, other members of which include lipopolysaccharides and peptidylglycans/lipopeptides/lipoproteins from Gram negative and positive bacterial cell walls respectively, flagellin, and single- and double-stranded viral RNA. Eukaryotic organisms have evolved "pattern recognition receptors" or PRRs expressed in APCs that function to specifically recognize these PAMPS. The best described of these are the TLRs, of which 13 have been identified in mammals and 10 are expressed in humans (Kawai and Akira 2006). PAMP recognition acts as a "danger signal" indicative of bacterial and viral invasion that triggers rapid and potent innate and adaptive immune responses (Krieg 2002) characterized by activation of cytolytic cells, secretion of cytokines, chemokines and bactericidal effectors such as nitric oxide (NO) and induction of antigen (Ag) -specific cellular and humoural immunity (Krieg, Yi et al. 1995; Klinman, Yi et al. 1996; Utaisincharoen, Anuntagool et al. 2002).  * A version of this chapter has been submitted for publication to the Journal of Immunology Chikh, G., de Jong, S., Sekirov, L., Raney, S., Cullis, P., Dutz, J and Y. Tam (2007) Synthetic Methylated CpG ODNs are Potent in vivo Adjuvants When Delivered in Liposomal Nanoparticles  67  While most TLRs are localized on the surface of APCs, those specific for pathogenic nucleic acids (TLR3, 7-9) are largely localized to the endosomal compartment. Therefore, prerequisites for CpG DNA activity include internalization and endosomal trafficking (Hemmi, Takeuchi et al. 2000; Takeshita, Leifer et al. 2001; Wagner 2001). Since eukaryotic cells are continually exposed to self DNA during normal development, growth and maintenance, the ability to distinguish immunostimulatory from eukaryotic DNA is vital. Discrimination is generally attributed to a combination of factors including CpG suppression (20 fold lower frequency in mammalian compared to bacterial DNA; (Bird, Taggart et al. 1987; Krieg, Yi et al. 1995; Klinman, Yamshchikov et al. 1997; Chen, Lenert et al. 2001), and context (occurrence of eukaryotic CpG motifs within "immunosuppressive" flanking sequences) (Krieg, Yi et al. 1999; Krieg 2002). In addition, methylation of CpG DNA plays a major role in determining the immunostimulatory activity of CpG motifs. Since methylation has been extensively described to inactivate CpG motifs (Krieg, Yi et al. 1995; Hemmi, Takeuchi et al. 2000; Bauer, Kirschning et al. 2001), the unmethylated nature of pathogenic viral and bacterial CpG DNA (vs eukaryotic DNA in which the >70% of cytosine residues are methylated) (Bird, Taggart et al. 1987; Hergersberg 1991) is considered to be an essential characteristic for immunostimulatory activity. Finally, it has been recently proposed that intracellular localization/sequestration of TLR9 itself regulates DNA immunostimulatory potential (Barton, Kagan et al. 2006). While it is generally accepted that eukaryotic DNA is relatively inactive, more recent studies suggest that it should not be considered immunologically inert (Boule, Broughton et al. 2004; Tian, Avalos et al. 2007). Contrary to generally accepted immunological dogma, the present study demonstrates that methylated CpG ODN (mCpG ODN) sequences are actually immunologically active and that encapsulation within liposomal nanoparticles endows them with 68  potent immunostimulatory activity which, surprisingly, is similar or superior to that induced by the equivalent, encapsulated unmethylated form. Furthermore, this enhanced activity translates to superior anti-tumour efficacy in animal models. Finally, abrogation of activity in TLR9 knockout animals, confirms that mCpG ODN exert their activity through the same TLR9 pathway as their unmethylated counterparts. In summary, these studies show that mCpG DNA is able to interact with TLR9 and initiate potent immune responses that mediate effective antitumour activity. Furthermore, these results implicate an as yet unidentified, upstream mechanism regulating the activity of free methylated vs unmethylated CpG ODN, which is effectively bypassed when ODNs are delivered in liposomal nanoparticles.  69  3.2 Materials and methods 3.2.1 Animals and cell lines Six- to 8-week-old female C57BL/6 and ICR mice were obtained from Charles River Laboratories (Saint-Constant, PQ, Canada) or Harlan (Indianapolis, IN) and quarantined for 3 weeks prior to use. TLR9-K0 mice (Kaisho and Akira 2001) were obtained from Oriental Biosciences Inc., and backcrossed to C57BL/6 mice over 8 generations. Mice were held in a pathogen-free environment and all procedures involving animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care. All cells were obtained from the American Type Culture Collection (Manassas, VA). EL4 and E.G7-OVA thymoma (Moore, Carbone et al. 1988) cells were cultured in complete media (CM), consisting of RPM! 1640 medium supplemented with penicillin G (100 U/ml), streptomycin sulphate (100 jig/ml), 13-mercaptoethanol and 10% heat inactivated foetal bovine serum (FBS). RAW264.7 cells (Ralph and Nakoinz 1977) were cultured in DMEM supplemented with 10% FBS, Lglutamine, penicillin G and streptomycin sulphate. Tissue culture media and supplements were Obtained from Invitrogen (Burlington, ON, Canada).  3.2.2 Preparation of liposomal ODN Distearoylphosphatidylcholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, AL) and cholesterol from Sigma (St. Louis, MO). 1,2-dioleyloxy-3-N,Ndimethylaminopropane (DODMA) and polyethylene glycol-dimyristol glycerol (PEG-DMG) were provided by Tekmira Pharmaceuticals Corporation (Burnaby, BC, Canada). These studies evaluated the immunostimulatory activity of the phosphorothioate ODNs INX-6295 (5'TAACGTTGAGGGGCAT-3'), INX-5001 (5'-AACGTT-3'), CpG-2006 (5'TCGTCGTTTTGTCGTTTTGTCGTT-3') ^and^CpG-1826^(5'70  TCCATGACGTTCCTGACGTT-3') containing either an unmethylated or methylated cytosine nucleotide within the CpG motif(s) indicated in bold. All ODN were synthesized by Trilink Biotechnologies (San Diego, CA) and encapsulated into LN-CpG ODN using methodology previously described (Maurer, Wong et al. 2001; de Jong, Chikh et al. 2007; Wilson, Raney et al. 2007) Particle size was 100 ± 25 nm in diameter as determined by quasi-elastic light scattering using a NICOMP submicron particle sizer (model 370, Santa Barbara, CA).  3.2.3 Analysis of innate and adaptive immunopotency  The ability to induce innate immune responses was evaluated after s.c. administration of 5 mg/kg free and encapsulated CpG and mCpG ODN to ICR mice. Adaptive immune parameters were assessed in C57BL/6 mice immunized s.c. with 20 lag OVA adjuvanated with 100 i.tg of free or encapsulated CpG and mCpG ODN. The terms CpG ODN and mCpG ODN refer to studies with unmethylated and methylated forms of INX-6295 unless otherwise noted.  3.2.3.1 Immune cell activation  For cell activation analysis, mice were terminally anaesthetized with ketamine/xylazine (3.2%/0.8%, v/v), and spleens and lymph nodes were collected and processed to single cells. These were analyzed for stimulation (as judged by activation marker expression) of specific immune cell populations (as determined by phenotype analysis) by flow cytometry. Cell suspensions were labelled with combinations of FITC-conjugated anti- CD11c, H-21e, PEconjugated anti- IL-12R, CD1 lb, CD69, CD86 and allophycocyanin (APC)-conjugated antiCD1 1 c, DX5, CD86 antibodies and analyzed using either a FACSort or LSRII flow cytometer with CellQuest Pro v4.0.1 or FACSDiva v4.1 software, respectively (BD Biosciences, San Jose, CA). For the latter, data were analyzed with FlowJo flow cytometry analysis software v7.2.2 71  (Ashland, OR). All fluorescently labelled antibodies were obtained from BD Biosciences. Propidium iodide was used to exclude dead cells and 150,000 and 80,000 events were collected to analyze DCs or NK cells and macrophages, respectively.  3.2.3.2 Plasma cytokine levels For plasma cytokines, mice were terminally anaesthetized as previously described and blood was collected via cardiac puncture. Plasma was isolated by centrifugation and frozen at — 20°C until assayed. Plasma concentrations of IL-6, IL-10, IFNy and MCP-1 were determined using a cytometric bead array kit (BD Biosciences), as per manufacturer's instructions.  3.2.3.3 MHC tetramer assay The frequency of OVA-specific CD8 T-lymphocytes was determined by MHC-tetramer assay using PE-coupled H-2K' MHC tetramers containing the immunodominant peptide of OVA (SIINFEKL; Beckman-Coulter, Immunomics, San Diego, CA) and FITC-labelled anti-mouseCD8 and PE-cyanin-labelled anti-TCRP phenotype antibodies (BD Biosciences) prior to analysis on a flow cytometer as previously described. At least two hundred and fifty thousand events were collected to analyze the frequency of OVA-specific CD8 T-lymphocytes in immunized animals.  3.2.3.4 Cytotoxicity assay The ability of cells from immunized animals to lyse target cells in an Ag-specific manner was assessed in splenocytes after 5 days in vitro restimulation with mitomycin-treated E.G7OVA cells with recombinant human IL-2. Ovalbumin-specific cytotoxicity was assessed using a standard 4 h 51chromium (5ICr) release assay in which splenocytes were mixed in various 72  effector:target ratios with 51Cr-loaded parental EL4 or OVA-transfected E.G7 cells. The cellular cytotoxicity was calculated on the basis of 51 Cr released to the supernatant using the formula: % lysis =[(experimental CPM — spontaneous CPM) / (maximal CPM -spontaneous CPM)] x 100, where CPM represents counts per minute, maximal CPM was achieved by complete lysis of 51Cr-labeled targets in 10% Triton X 100, spontaneous CPM was determined by incubating labelled targets in media and Ag-specific killing was determined by comparison of cytotoxicity of51Cr-labeled OVA-expressing and non-expressing E.G7 and EL4 cells, respectively.  3.2.3.5 Cytokine secretion assay The IFNy secretion assay (Miltenyi Biotec Inc., Auburn, CA) was performed according to the manufacturer's instructions to enumerate CD8 T-lymphocytes secreting IFNy in response to Ag stimulation. Briefly, splenocytes were restimulated with OVA-expressing APCs prior to incubation with a bispecific antibody designed to bind to activated T-lymphocytes and capture secreted IFNy. The frequency and phenotype of cells actively secreting cytokines were determined by flow cytometry as described previously using fluorescently labelled anti-IFNy and phenotype antibodies.  3.2.3.6 Antigen-specific anti-tumour activity The syngeneic E.G7-OVA C57BL/6 tumour model was used to determine the efficacy of encapsulated ODN as a vaccine adjuvant to induce Ag-specific anti-cancer immune responses. For this model, mice were immunized prophylactically, once per week for three weeks, with OVA mixed with free and encapsulated, unmethylated and methylated CpG ODN. One week later, mice were challenged with E.G7-OVA tumour cells (2.5 x 106 cells) by s.c. hind flank injection and monitored for tumour growth. Tumour volumes were calculated using the formula 73  (length x width2)/2. Animals that cleared their tumours were re-challenged 3 wks later (approximately 6 wks following initial tumour challenge) and tumour growth was monitored as described above.  3.2.4 Role of TLR9 in immunostimulatory activity of mCpG ODN 3.2.4.1 Upregulation of TLR9 To assess the effects of LN-mCpG ODN on TLR9 expression, RAW264.7 cells were incubated for 4 h with 10 p.g/m1 free or encapsulated, methylated or unmethylated CpG ODN, washed, harvested and then fixed and permeabilized using BD Cytofix/Cytoperm Plus kit (BD Biosciences) according to the manufacturer's instructions. Cells were then incubated with biotinylated mouse mAb to TLR9 (Hycult, Uden, The Netherlands) followed by incubation with Streptavidin-PE (BD Biosciences) prior to analysis by flow cytometry. MFI of cells incubated with Streptavidin-PE only were subtracted from the data.  3.2.4.2 Nitric oxide production To assess relative NO production induced by LN- CpG and mCpG ODN, RAW264.7 cells were incubated for 12, 24 or 48 h with 10 Kg/m1 encapsulated methylated or unmethylated CpG ODN. Supernatants were harvested and concentrations of NO determined by Greiss reaction (Nitric Oxide Quantitation Kit, ActiveMotif, Carlsbad, California) according to the manufacturer's instructions.  3.2.4.3 Inhibition by chloroquine To assess the effects of chloroquine, an inhibitor of endosomal maturation, on LN-mCpG ODN immunostimulation, bone marrow from ICR mice were differentiated to DCs by culture in 74  CM supplemented with 300 ng each of IL4 and GM-CSF for 7 days, typically resulting in >85% CD11c+ cells. These cells were incubated with 10 ii,g/m1 chloroquine prior to treatment with 10 lig/m1 LN-mCpG ODN or LN-CpG ODN and then stained with combinations of antibodies against activation and phenotype markers prior to analysis by flow cytometry.  3.2.4.4 TLR9-K0 studies To confirm a role for TLR9 in LN-mCpG ODN immunostimulatory activity, C57BL/6 WT and TLR9 deficient (TLR9-/-) mice were injected s.c. with encapsulated CpG or mCpG ODN or the equivalent lipid dose of empty liposomal nanoparticles. Animals were euthanized at various time points and lymph nodes, spleens and blood harvested to assess the effect on cell activation and plasma cytokine levels as described above (24 h data shown).  3.2.5 Statistical analyses All statistical analyses were performed using SPSS Ver 14.0. Initially, a one-way ANOVA was used to statistically evaluate the differences between treatment groups. In the case of statistically significant results, the differences between treatment groups were assessed through the use of Bonferroni adjusted t tests, a post-hoc test which controls for error rate. Probability (p) values less than .05 were considered significant.  75  3.3 Results 3.3.1 Methylated CpG ODN induces potent innate and adaptive immune responses when delivered in liposomal nanoparticles  While in vitro assays have been widely used to assess the immunostimulatory capacity of free CpG ODN, they have proven to be poor indicators of the immunostimulatory capacity of encapsulated ODN, indicating that encapsulation significantly reduces immunopotency, in spite  of in vivo data to the contrary. However, in vitro analyses were able to reveal that while free, mCpG ODN is immunologically inactive, encapsulated mCpG ODN is actually able to induce immune responsiveness, albeit at a low level. Based on these and other preliminary data, a series of in vivo studies were undertaken to evaluate the immunostimulatory activity and anti-tumour efficacy of encapsulated mCpG ODN. To assess the effect of encapsulation on the immunopotency of mCpG ODN, the expression of the activation markers CD69 and CD86 was monitored in CD11b+ macrophages, CD11c+ dendritic cells and DX5+ NK cells from spleen and lymph nodes (Fig 3.1). In addition, the plasma levels of TH 1 and TH2 cytokines (IFN7 and IL-6, IL-10, respectively) as well as MCP-1 (a macrophage chemokine) were assessed (Fig 3.2). These data were collected over a 72 hr time period following administration to ICR mice of either free or encapsulated unmethylated or methylated CpG ODN. The data from the 24 hr time point are presented and are representative of at least ten independent experiments.  76  g 3 2  IIBS mCpG OCIN GpG OCN LN-mCpG L14-CpG OCN^ODN  FES rrepG ODN GpG OON LN-mCpG LN-CpG CCN^OCN  CD1M-LN 16 14 12 1 10 8 6 4 2  1-1E3S mCpG CON CpG CON LN-rtCpG LN-CpG CON^CCN  CD1U-SP  I-ES rrC.pG ODN CpG ODN LIN,rrCpG LN-CpG OCN^OON  CD110-LN 4 3.5 3 1 2.5 2  g'  0.5 0  FES mGpG OON CpG OCN LN-mCpG LN-CpG CON^CON  -I^ --^IES oCpG CCN CpG OCN LN-rnCpG LN-CpG ODN^OON  CDVIo-SP 3 ^ 2.5 /I.^2  0.5  FES mCpG CON CpG OON^rrCpG LN-CG CON^CCN  1-18S  rrCpG OCN CpG CCN  LN-rre,.pG OCN  LN-CpG CON  LN-rrCpG CON  LN-CpG OCN  DX6 LN 60 50 11.^40 30  10  rnGpG CCN CpG ODN LN-n-CpG LN-CpG 00N^ODN  —  1.-BS  mCpG CON  CpG OCN  Fig 3.1 Methylated CpG ODN induces potent immune cell activation when encapsulated in liposomal nanoparticles Five mg/kg of free and encapsulated, unmethylated and methylated CpG ODN was administered s.c. to ICR mice (4 animals/group). After 24 h, animals were euthanized and spleens harvested. Splenocytes were analyzed for expression of the CD69 and CD86 cell surface activation markers (% of total cell population ± SD) in conjunction with phenotype markers by flow cytometry as outlined in the Materials and Methods. Data presented here is representative of at least 10 separate experiments.  77  400 350 3 00 2 50 "R 200 ...I •  150 100 50  12^24^36^48  60^72^0^12^24^36^48  Time (hrs)  60  72  60  72  Time (hrs)  900  350  800  300  700 250  600 5C0 -a a  200  o 400  :3  •  U.  300  150 100  200 100 0 0^12^24^36^48 Time (hrs)  60  72  0  12^24^36^48 Time (hrs)  Fig 3.2 Encapsulated methylated CpG ODN induces elevated plasma cytokine levels Five mg/kg of free or encapsulated CpG ODN was administered s.c. to ICR mice (4 animals/group). Blood was collected from animals by cardiac puncture, processed to collect plasma and cytokine levels (pg/ml ± SE) were determined by ELISA or cytometric bead array. Data presented here is representative of at least 10 separate experiments and each data point represents an average of 4 animals.  An ANOVA was performed on each of the data sets shown in Figure 3.1 and revealed a statistically significant difference between treatment groups at the p<.001 level. Consistent with the previously reported literature (Krieg, Yi et al. 1995; Hemmi, Takeuchi et al. 2000; Bauer, Kirschning et al. 2001), Bonferroni t tests revealed that free mCpG ODN is not found to be immunoactive, inducing little or no upregulation of activation marker expression compared to control animals. Free CpG ODN induces minor upregulation of activation markers in CD1 1 b+ and CD1 1 c+ cells in spleen and lymph node compartments compared to both control (HBS treated) and mCpG ODN treated animals. As previously reported (Mui, Raney et al. 2001), encapsulation of CpG ODN dramatically enhances immunostimulatory activity compared to both free CpG and mCpG ODN, inducing in a statistically significant 3- to 4-fold (p<.005) and 4- to 78  6-fold (p<.001) increase in activation marker expression, respectively. The upregulation of MHC class I and IL12R on CD1 1 c+ cells and CD8 T-lymphocytes respectively was also found to be statistically significant, when compared with free methylated and unmethylated CpG ODN (data not shown). Importantly, these data confirm preliminary observations of immunostimulatory activity of LN-mCpG ODN. Encapsulated CpG ODN, regardless of methylation state, induces activation that is significantly greater than free CpG ODN in all cell types and activation markers examined with no significant difference detected between animals receiving encapsulated, CpG- and mCpG ODN. These data are also reflected in plasma cytokine levels. While no or modest increases in cytokine levels over 72 h are observed in animals following administration of free mCpG or free CpG ODN, respectively (Fig 3.2), compared to control animals, encapsulated CpG ODN, regardless of methylation state, is able to exert a dramatic effect. The peak plasma cytokine concentrations occurred within 24 h of administration and resulted in significant enhancement above baseline levels for all cytokines examined. While baseline levels of IL-6 were largely undetectable, animals treated with LNCpG and mCpG ODN exhibited levels exceeding 200 pg/ml by 7 to 24 h following treatment. Similarly, IL-10 was enhanced approximately 40-fold, and IFNy and MCP-1 enhanced approximately 10-15-fold above control levels at 24 h post administration. The kinetics and plasma cytokine levels were very similar for LN-CpG ODN and LN-mCpG ODN (Fig 3.2). Liposomal nanoparticulate mCpG ODN was also found to be immunoactive based on adjuvanation of adaptive, cell-mediated immune responses against the model Ag, OVA. Cellular immune responses were assessed quantitatively by enumerating CD8 T-lymphocytes specifically targeted against the immunodominant OVA peptide SIINFEKL using MHC tetramer analysis, (Fig 3.3A) and functionally by assessing the number of cells actively secreting IFNy following in vitro Ag stimulation (Fig 3.3B) and the cytotoxic activity of immune cells from immunized 79  animals against OVA-expressing tumour cells (Fig 3.3C). LN-mCpG ODN generated 9-10- fold more OVA-specific CD8 T-lymphocytes than control animals as compared to a 2-fold enhancement with free CpG ODN. Interestingly, in this assay, LN-mCpG ODN induced an approximate 2.5-fold higher frequency of Ag-specific CD8 T-lymphocytes compared with LNCpG ODN (Fig 3.3A), a trend that was observed for all encapsulated CpG/mCpG ODNs tested (Table 3.1) including the hexameric sequence INX-5001, the primate optimized CpG-2006 and the murine optimized CpG-1826.  HBS  HBS  ^  ^  CpG ODN^LN CpG ODN mCpG ODN LN-mCpG ODN  CpG ODN  ^  LN CpG ODN mCpG ODN LN-mCpG ODN  80  30  20  co 10  0 HBS  ^  CpG ODN^LN CpG ODN^rnCpG ODN LN-mCpG ODN  Fig 3.3 Liposomal nanoparticulate methylated CpG ODN adjuvanates potent, antigen-specific adaptive cellular immune responses after s.c. immunization with OVA C57BL/6 mice (4 animals/group) were immunized s.c. with 20 tig OVA adjuvanated with 100 pg free or encapsulated CpG or mCpG ODN.  Panel A Frequency of antigen-specific OVA-MHC tetramer+ CD8+ T cells  Splenocytes were isolated from immunized mice, incubated with an OVA-specific PE-labelled H-2Kb-SIINFEKL MHC tetramer in conjunction with fluorescently labelled anti- CD8 and TCRft antibodies and analyzed by flow cytometry to quantitate the frequency of OVA-specific CD8 T-lymphocytes in animals following immunization. These data are derived from 5 separate studies and expressed as a percentage of control levels + SD.  Panel B Frequency of antigen-specific, IFNy secreting CD8+ cells Splenocytes were isolated from immunized animals and activated as described in the Materials and Methods. Briefly, IFNy secreting ability of CD8+ cells was assessed after 8 h in vitro restimulation with OVA expressing E.G7 cells as determined by cytokine secretion assay. These data are derived from 5 separate studies and expressed as a percentage of control levels ± SD.  Panel C Antigen-specific cytolytic activity of splenocytes against E.G7-OVA cells Splenocytes were isolated as described in the Materials and Methods and used after 5 days in vitro restimulation as effector cells in a standard 51Cr release assay. The percentage of chromium released from radiolabelled E.G7 (for specific lysis) and EL-4 (for non-specific lysis) targets, after 4 h co-incubation with isolated splenocytes, was used to calculate specific cytolytic activity ± SD. Effector cells and target cells were plated at a variety of ratios; the 100:1 effector-target ratio is shown above. Data presented here is representative of 5 separate studies.  In addition to a quantitative assessment, the ability of LN-mCpG ODN to generate functional Ag-specific CTLs was assessed using the IFNy secretion assay as an indicator of TH1 response. While no detectable increase in frequency of IFNy secreting splenocytes was observed following immunization with free CpG ODN, encapsulated CpG ODN (unmethylated or methylated) induced 4- to 6- fold greater frequency of CD8+ IFNy producing cells compared to control or free CpG ODN treated animals (Fig 3.3B). Consistent with MHC-tetramer results, LN-mCpG ODN appeared to be more potent than the equivalent unmethylated ODN (0.37% vs 81  •• 0.23%). In addition, functional assessment of Ag-specific cytotoxic T-lymphocyte responses following in vitro restimulation using a standard cytotoxicity assay show that while effector cells from animals treated with free CpG ODN exhibited minor enhancement of target cell lysis compared to control animals, immunization with encapsulated CpG and mCpG ODN resulted in a 4- and 7- fold increase, respectively (Fig 3.3C).  3.3.2 Adaptive immune responses mediated by immunization with LN-mCpG ODN mediates effective anti-tumour activity  To determine the potential therapeutic relevance of these results, the anti-tumour efficacies of LN-CpG and LN-mCpG ODN-induced immune responses were evaluated in the well-defined, syngeneic EG.7 — C57BL/6 thymoma tumour model in which animals immunized against OVA as described above, were challenged with OVA-expressing E.G7 tumour cells. Tumour growth over 18 days (Fig 3.4A) post-challenge was monitored to determine the ability of the anti-OVA immune response to inhibit tumour growth and development. Results from these studies directly reflect the results seen in the ex vivo cellular immune response. Animals immunized with free A  3000  2500  E 20°0 ^ z  1500  O  1000  t  500  0 ^ — 0^2^4^6^8^10^12^14  16^18^20  Time Post Tumour Challenge (Days)  82  • •  3000  2500  E 2000 O 1500 o 1000 500  0 0  ^ ^ ^ 25^30 5 10^15^20  Time Post Tumour Challenge (Days)  Fig 3.4 Liposomal nanoparticulate methylated CpG ODN induces potent, antigen-specific anti-tumour activity following prophylactic immunization with OVA adjuvanated with free or encapsulated CpG ODN in a E.G7-OVA syngeneic tumour model C57BL/6 mice were immunized prophylactically s.c. with OVA adjuvanated with saline (closed diamonds), empty liposomes (closed squares), or 100 ug free and LN- (open and closed, respectively) CpG and mCpG ODN (triangles and circles, respectively). Panel A One week following the last vaccination, mice were challenged s.c. with 2.5 x 106 E.G7-OVA cells and tumour growth was monitored. Tumour volume was calculated using the formula V=(LxW2)/2. Each data point represents the mean and standard deviation of a group of 5 animals. Panel B A number of animals immunized with OVA adjuvanated with LN-CpG or mCpG ODN were able clear their initial tumour challenge. These mice were re-challenged s.c. with 2.5 x 106 E.G7-OVA cells and tumour growth was monitored as above.  mCpG ODN exhibited minimal inhibition of tumour growth compared to free CpG ODN (1700 vs 1050 mm3, respectively on day 15) while animals immunized with either LN-CpG or LNmCpG ODN exhibited significant tumour growth inhibition within the 18 days after tumour implantation (400 and 125 mm3, respectively). These data demonstrate the enhanced potency of unmethylated compared to methylated CpG ODN in free form and confirm the ability of liposomal encapsulation to either increase (as in the case of unmethylated) or endow (as in the case of methylated) CpG ODN immunostimulatory potency. To further evaluate and 83  discriminate potency, the immunological memory in mice that had cleared the initial EG7 tumour challenge was assessed. Figure 3.4B shows tumour growth in mice that had cleared the initial tumour challenge and been re-challenged. These data show that mice initially immunized with OVA and LN-mCpG ODN maintained almost complete tumour rejection at day 26 post rechallenge compared to those initially immunized with OVA adjuvanated with LN-CpG ODN. As expected, both exhibited superior anti-tumour responses compared to neve animals. Taken together, these results demonstrate that encapsulation endows methylated CpG ODN that are essentially inactive in their free form with potent immunostimulatory activity. Particularly surprising is the fact that encapsulated mCpG ODN exhibits enhanced potency over the encapsulated unmethylated form. Importantly, these effects are not sequence specific and extend to other ODNs. Anti-tumour activity analysis of encapsulated synthetic CpG ODN INX-5001, CpG-2006 and CpG-1826, yield similar results (Table 3.1) in regards to anti-tumour activity. Table 3.1 Encapsulation in liposomal nanoparticles confers immunostimulatory activity on a variety of methylated CpG oligodeoxynucleotides  % of CD8- SIINFEKL tetramer positive  Efficacy Day 21 Tumour Volume (mm)  Free 5001 LN 5001 Free 5001m LN 5001m  0.17 0.62 0.1 0.98  >2000 781 >2000 419  Free 2006 LN 2006 Free 2006m LN 2006m  0.18 0.66 0.26 0.98  1328 nd >2000 nd  Free 1826 LN 1826 Free 1826m LN 1826m  0.22 0.24 0.14 0.42  1000 128 1563 169  84  3.3.3 LN-mCpG ODN induces its immunostimulatory activity through TLR9  Preliminary analysis of a number of parameters, previously reported in the literature to be implicated in TLR9-mediated responses, found that LN-mCpG ODN induced responses similar to the unmethylated CpG ODN, thus suggesting both act through TLR9. As previously reported, analysis by intracellular flow cytometry showed that TLR9 is upregulated by free CpG ODN but not mCpG ODN (Bourke, Bosisio et al. 2003). However, expression levels were significantly increased by treatment with both LN-CpG and LN-mCpG ODN (p<.01) (Fig 3.5A). A  120 100 80  LT- 60 40 20  HBS  ^  mCpG CpG ODN LN-mCpG LN-CpG ODN^ODN^ODN  9 8 7  E 6 cn 5  04 3 2 1 0  12 hr  ^  24 hr  ^  48 hr  85  150 140 -A. 130 +5 c 120 15 110 100 90 80  Ell - Chloroquine + Chloroquine - Chloroquine + Chloroquine CD11b+ Macrophages^CD1 1 c+ DCs  Fig 3.5 Liposomal nanoparticulate, unmethylated and methylated CpG ODN induce similar responses Panel A Liposomal nanoparticulate, methylated CpG ODN induces TLR9 upregulation One million RAW264.7 murine macrophage cells/ml were incubated with 10 Ag/m1 of free and LN- mCpG ODN and CpG ODN for 1.5 h. Cells were harvested, fixed and permeabilized and subjected to intracellular flow cytometry analysis for TLR9 expression as described in the Materials and Methods section. Panel B Liposomal nanoparticulate, methylated CpG ODN induces nitric oxide production One million RAW264.7 murine macrophage cells/ml were incubated with 10 tg/m1 of encapsulated unmethylated (white bars) and methylated (black bars) CpG ODN for 12, 24 and 48 h. Supernatants were harvested and assessed for NO by Greiss reaction as described in the Materials and Methods section. NO level in control cells (12 h after incubated with saline) is indicated by dashed line Panel C Immune activation by liposomal nanoparticulate, methylated CpG ODN is inhibited by chloroquine Bone marrow derived DCs from ICR mice with 1L4 and GM-CSF were incubated with 10 pg/m1 chloroquine prior to treatment with 10 i.ig/m1 LN-CpG ODN (white bars) LN-mCpG ODN (black bars). Cells were analyzed for activation of immune cell populations by flow cytometry as described in the Materials and Methods section, specifically CD86 expression by CD11c+ DCs and CD11b+ macrophages. Data is expressed as % of expression on cells from control animals.  Similarly, analysis of NO levels, as with free unmethylated CpG ODN (He and Kogut 2003) found that both LN-CpG and LN-mCpG ODN induced increased NO production. Levels were found to be significantly elevated at 24 and 48 h and 12, 24 and 48 h for LN- CpG and mCpG ODN, respectively (p<.05) compared to cells treated with saline (Fig 3.5B) or free CpG ODN (data not shown) although no significant differences were observed between LN-CpG and LNmCpG ODN at any time point assessed. Finally, as previously reported for free CpG ODN, both LN-CpG and LN-mCpG ODN were inhibited by chloroquine, (Hacker, Mischak et al. 1998; Yi, 86  Tuetken et al. 1998; Ishii, Takeshita et al. 2002) due to either inhibition of endosomal maturation (Hacker, Mischak et al. 1998) and/or competition for TLR9 binding (Macfarlane and Manzel 1998; Rutz, Metzger et al. 2004) (Fig 3.5C). These data are consistent with an involvement of TLR9 in mediating the immune activity of LN-mCpG ODN. To confirm the hypothesis of a seminal role for TLR9 in mediating activity of LNmCpG ODN, studies in TLR9-K0 animals were undertaken. As expected, while wild-type animals upregulated CD69 and/or CD86, both in regards to number of cells (Fig 3.6A) as well as on a per cell basis (data not shown) on a variety of immune cell populations (Mac3+ macrophages, CD1 1 c+ DCs, CD45R/B220+ B-lymphocytes, CD8+ T-lymphocytes, DX5+ NK cells). In contrast, administration of LN-mCpG ODN to TLR9-K0 mice failed to induce any immune responsiveness, with cells exhibiting only background levels of activation marker expression. This was consistent in both splenic and lymph node (data not shown) cell populations. Similarly, evaluation of IL-10, MCP-1 and IFN7 plasma levels indicated that while wild-type animals displayed rapid statistically significant (p<.001) increases in plasma cytokine/chemokine levels following treatment with LN-mCpG ODN, however, control animals, animals receiving the equivalent lipid dose of empty liposomal nanoparticles, and TLR9-K0 animals failed to respond (Fig 3.6B). Bonferroni t tests also revealed that there were no significant differences between control animals, those receiving empty liposomal nanoparticles or TLR9-K0 animals.  87  A  80  60  60  20 -  1  i  1.  ‘.7.1111V1,^A4111, fie 0^ 100^101^102^103^1 0"  100  COlic - C069  101^102^103 CO1ic - C086  80 -  x 10-  *  20  10G  101^102^103^,o4 Mac 3 - C086  It,!, ^  80 -  . 60  et  * • • *U1 0^ 100^101^102^ , 04 DX5 . C069  -  40 -  20  •  1/.  106^101^102 8220 - C069  104  88  ^  20  60 -  18 50  16 14  cz,- 40  12  scl)  10  .  30  it 8 — 20  6 4  10  2 [17-li1 , Ctrl^ KO  Errp  0  ^ Uri^WT^KO  Ernp  7000 6000 5000 g  4000 3000  2  2000 1000  Ctrl^WI^KO^Errp  Fig 3.6 Liposomal nanoparticulate, methylated CpG ODN mediates immune cell activation through TLR9 Five mg/kg encapsulated, methylated CpG ODN was administered s.c. to wild-type and TLR9-K0 animals (3 animals/group). After 24 h, animals were euthanized and spleens and blood harvested. Panel A Splenocytes were analyzed for expression of the CD69 and CD86 cell surface activation markers (% of total cell population ± SD) in conjunction with phenotype markers by flow cytometry as outlined in the Materials and Methods. Flow cytometry histograms of CD69 and CD86 expression on macrophage, dendritic cells and CD69 expression on B-lymphocyte and natural killer cell populations in wild-type control (grey histogram) are shown in comparison to LN-mCpG ODN-treated wild-type (solid line) and TLR-KO (dotted line) animals. Panel B Blood was collected from animals by cardiac puncture, processed to plasma and frozen at -20°C until analysis. Cytokine levels (pg/ml + SE) in plasma isolated from wild-type animals treated with either LN-mCpG ODN or empty nanoparticles (Emp) were compared to TLR9-K0 animals treated with LN-mCpG ODN were determined by cytometric bead array as outlined in the Materials and Methods.  89  3.4 Discussion  Encapsulation within liposomal nanoparticles protects CpG ODN from degradation and avoids the disadvantages associated with protective backbone modification which can include toxicities following IV administration (Agrawal 1999) and reduced immunopotency (unpublished data) (Ballas, Rasmussen et al. 1996; Mui, Raney et al. 2001). Furthermore, encapsulation also functions to improve pharmacokinetic (such as avoiding rapid elimination from circulation and degradation by serum proteases) (Mui, Raney et al. 2001) and biodistribution characteristics (enhancing delivery to target immune tissues and cells), provides a depot effect upon s.c. administration to prolong bioavailability, (Gursel, Tunca et al. 1999; Wilson, Raney et al. 2007) and enhances cellular uptake. As a result, liposomal encapsulation has the potential to increase CpG ODN potency as it has been previously shown with respect to a number of innate and adaptive immune parameters and anti-tumour activity (Mui, Raney et al. 2001; de Jong, Chikh et al. 2007; Wilson, Raney et al. 2007). Consistent throughout many of these studies has been the use of ODN containing unmethylated CpG motifs in deference to the significant body of research indicating that methylation abrogates immunostimulatory activity (Krieg, Yi et al. 1995; Klinman, Yi et al. 1996; Gursel, Verthelyi et al. 2002; Klinman, Currie et al. 2004). However, it is reported here that when delivered within stabilized lipid nanoparticles, mCpG ODN are potent immunomodulators, exhibiting equivalent or often superior activity compared to the equivalent unmethylated form based on a number of innate and adaptive immune parameters as well as anti-tumour efficacy in animal models. CpG motifs are known to act through TLR9 to trigger potent innate immune responses and to this point, it has been largely assumed that TLR9 specifically recognizes unmethylated CpG motifs. Conversely, CpG ODN bearing modifications such as methylation that deviate it from the "conserved molecular motif', have been assumed to prevent recognition/binding by 90  TLR9 and abrogate immunostimulatory activity. However, it is reported here that LN-mCpG ODN induce potent immune responses equal, and often superior, to the equivalent encapsulated unmethylated ODN. Insomuch as these observations represent a deviation from accepted dogma regarding a major mechanism by which pathogenic and eukaryotic DNA is distinguished, this conclusion was predicated on an assumption that LN-mCpG ODN mediates its effect through the TLR9 signalling pathway rather than a novel pathway as confirmed by studies of TLR9 expression, NO production, chloroquine inhibition and immune response in TLR9-knockout animals. While an extensive body of research exists to show that mCpG motifs exhibit little or no immune activity, there is limited data to demonstrate differential binding of methylated and unmethylated ODN to TLR9, or a causal relationship with immunopotency. Researchers have used surface plasmon resonance (Biacore) biosensor technology and NF-KB assays in TLR9transfected cells (Comelie, Hoebeke et al. 2004; Rutz, Metzger et al. 2004) to evaluate CpGTLR9 interaction, and while these data support an effect of CpG methylation on TLR9 binding, the assay systems are highly artificial and the results are somewhat ambiguous. In fact, direct binding of CpG ODNs to TLR9 in vivo/in vitro has not yet been demonstrated, although confocal microscopy studies show that TLR9, MyD88 and CpG co-localize in late endosomes and require endosome maturation for signalling (Takeshita, Leifer et al. 2001; Ahmad-Nejad, Hacker et al. 2002). The studies reported here extend preliminary observations first made by our group (Raney, Sekirov et al. 2004) and later confirmed by Yasuda and colleagues (Yasuda, Yu et al. 2005; Yasuda, Rutz et al. 2006), that mCpG ODN is, in fact, capable of initiating TLR9mediated immune responses when delivered in lipid particles. However, while Yasuda et al. reported immunostimulation following mCpG ODN delivery in cationic lipid complexes, they 91  observed responses that were significantly lower than observed for free CpG ODN. The authors hypothesized that inefficient natural uptake restricts endosomal accumulation of CpG ODN, allowing only ligands with high affinity (such as unmethylated CpG ODN) and not those with low affinity (such as mCpG ODN) to initiate immune responses. They therefore, postulated that delivery in lipid complexes achieves sufficiently high levels to overcome the low affinity of mCpG ODN for TLR9 and enable immunostimulatory activity. In contrast, our results suggest that mCpG ODN is fully capable of triggering TLR9-dependent signalling as (or more) effectively than the equivalent unmethylated ODN, inducing responses that often exceed those found with equivalent amounts of either free or encapsulated unmethylated CpG ODN over a range of doses (unpublished data). The basis for the discrepancy between the data reported here and previously reported work is unclear, but one possibility is potential toxicity following i.v. administration of cationic complexes (Dass 2002; Yew and Scheule 2005). Results from our studies raise questions regarding mechanisms that regulate CpG DNA/TLR9 immunostimulatory activity. If TLR9 is in fact able to effectively bind and mediate immune responses to both unmethylated and methylated ODN, then what is the true mechanism responsible for differentiating between free CpG and mCpG ODN and how is it overcome by nanoparticulate delivery? Furthermore, if both unmethylated and methylated ODN mediate their activity through TLR9, what is the basis for the observation that encapsulated methylated ODN are often more potent that the unmethylated counterpart, particularly for adaptive immune responses. Ultimately, these data indicate that the mammalian immune system is not inherently inert to eukaryotic DNA. As noted above, immune cells are constantly exposed to self DNA as a result of cellular processes during normal development, growth and maintenance. This implies that methylated DNA from apoptotic/necrotic self cells must be subjected to other regulatory 92  mechanisms that promote self-tolerance in healthy individuals that may be inactive in individuals prone to autoimmune diseases. This is consistent with reports demonstrating that genomic DNA released by necrotic cells can induce the maturation/activation of APCs (Ishii, Suzuki et al. 2001). Based on these findings, we hypothesize that discrimination between methylated and unmethylated ODN occurs upstream of TLR9 and the lack of mCpG ODN immunological activity is due to an inability of mCpG ODN to access rather than bind the TLR9 receptor. This could be mediated by differences in uptake (i.e., discrimination by the plasma membraneresident "uptake" receptor) or trafficking (i.e., differential trafficking to appropriate intracellular compartments) that are overcome by encapsulation, thus providing mCpG ODN access to TLR9 by altering the uptake and/or trafficking pathways. Work is currently underway in our laboratory to elucidate the potential roles of mCpG ODN uptake and trafficking in regulating CpG ODN/TLR9 mediated immune responses In regards to the relative potency of encapsulated CpG vs mCpG ODN, since liposomal nanoparticulate delivery is able to overcome a primary barrier to mCpG ODN activity, it may be expected that previously unobserved aspects of the nature or character of the CpG/mCpGmediated immune response (such as potency) may manifest themselves. Therefore, allowing both CpG and mCpG ODN access to the TLR9-containing compartment by delivery in liposomal nanoparticles has revealed differences in immunostimulatory potency. Although the basis for this difference is unknown, one possibility is that mCpG may actually bind TLR9 more effectively than unmethylated motifs since TLR9 has been reported to share homology with methyl-CpG-binding proteins and DNA methyltransferases (Krieg 2002; Rutz, Metzger et al. 2004), containing the motifs responsible for the methylated DNA binding activity of these proteins.  93  This study provides evidence for the potent activity of LN-mCpG motifs, acting through TLR9 to mediate vigorous immune responses. While this work is contrary to immunological dogma that methylation of CpG motifs abrogates immunostimulatory activity, it does support a model in which sequestration regulates TLR9-CpG immune activity. Furthermore, it raises several important questions as to how TLR9/CpG interaction is controlled, implicating a mechanism by which relative immunostimulatory activity of methylated and unmethylated CpG is based on differential access to TLR9. Elucidating this process has implications in understanding both the natural process of self-tolerance as well as designing effective prophylactic and therapeutic intervention strategies with nucleic acid based drugs such as adjuvants, vaccines, genes and siRNA.  94  CHAPTER 4: Lipid Encapsulation Promotes Subcellular co-localization of Methylated CpG ODN and TLR9: a new model for the immunostimulatory activity of CpG DNA*  4.1 Introduction  The mammalian immune system has evolved highly conserved pathogenic recognition receptors (PRRs), such as the Toll-Like Receptor family, that recognize specific molecular patterns expressed by a diverse group of infectious microorganisms as danger signals of infection and trigger potent, protective immune responses (Yamamoto, Yamamoto et al. 1992; Krieg, Yi et al. 1995; Klinman, Yi et al. 1996; Yi, Chace et al. 1996; Yi, Klinman et al. 1996; Van Uden, Tran et al. 2001). Inherent to this "detection" system is the ability to distinguish pathogen-associated patterns from those encountered during benign or beneficial self and environmental interactions. While this can be easily conceptualized for TLRs 4, 2/6 and 5, all of which recognize structurally complex and unique ligands such as lipopolysaccharide, peptidylglycan and flagellin, it is somewhat less intuitive for TLR9 which specifically recognizes CpG motifs (Dalpke, Zimmermann et al. 2002). Distinguishing pathogenic from eukaryotic DNA is a multifactorial process, based on physical sequestration of TLR9 to the endosomal compartment, suppression of CpG frequency in eukaryotic DNA, occurrence of eukaryotic CpGs within immunosuppressive flanking sequences and methylation status (Krieg, Yi et al. 1995; Takeshita, Leifer et al. 2001; Rutz, Metzger et al. 2004). With regard to methylation status, TLR9 has been shown to specifically respond to unmethylated CpG motifs, such as those present in bacterial DNA as compared to eukaryotic CpGs of which 70-80% are methylated (Bird, Taggart et al. 1987). Furthermore, it has been *A version of this chapter has been submitted for publication de Jong, S., Basha, G., Wilson K., Kazem, K., Jefferies, W., Cullis, P., and Y. Tam (2007) Lipid Encapsulation Promotes Subcellular co-localization of Methylated CpG ODN and TLR9: a new paradigm for the immunostimulatory activity of CpG DNA  95  demonstrated that methylation of immunostimulatory CpG DNA abrogates activity, which is attributed to the specificity of TLR9 for unmethylated motifs and its inability to effectively bind methylated sequences. Direct evidence for this comes from studies using surface plasmon resonance technology that have reported superior interaction of TLR9 with both unmethylated CpG ODN and CpG-containing plasmid DNA compared with the methylated form (Comelie, Hoebeke et al. 2004; Rutz, Metzger et al. 2004). Conversely, it has been demonstrated that when expressed on the cell surface a chimeric TLR9 is capable of responding to self-DNA (Barton, Kagan et al. 2006) and our group and others have observed that mCpG-ODN possesses TLR9—mediated immunostimulatory potential when delivered in a lipid carrier system (Yasuda, Ogawa et al. 2005; Yasuda, Rutz et al. 2006). Specifically, we have recently reported that lipid nanoparticulate (LN) delivery endows mCpG ODN with immunostimulatory activity similar to or greater than the equivalent unmethylated ODN, through a TLR9-mediated mechanism. The ability of mCpG DNA to induce TLR9-mediated immune responses suggests that methylation status determines the immunostimulatory activity of CpG DNA by mechanisms other than by affecting binding properties. It is shown here that while both methylated and unmethylated CpG ODN in free form are taken up and traffic to the endosome similarly, only unmethylated ODN promotes effective trafficking of TLR9 to co-localize with its CpG ligand in the late endosomal compartment. Interestingly, administration of both methylated and unmethylated CpG ODN in LN, as well as empty LN, promotes co-localization of TLR9 to the late endosomal/lysosomal compartment. It is demonstrated that following exposure to both free unmethylated CpG ODN and LN, co-localization occurs through a src-family kinase (SFK) mediated signalling pathway. These results suggest that the lack of immunological activity for  96  free methylated ODN is largely due to a failure to properly localize with TLR9 in the late endosomal compartment rather than an inability to bind TLR9. The ability to differentiate between pathogenic and eukaryotic DNA represents a vital element of the eukaryotic immune system, promoting rapid and vigorous immune responses to protect against pathogenic attack, while avoiding inappropriate and pathologic immune responses to self DNA during normal processes such as development, growth and maintenance. Based on these data, a new model is proposed where the differential immunopotency of unmethylated and methylated CpG ODN and, by extension, pathogenic and self DNA, is determined by their relative ability to effectively induce mobilization of TLR9 and to co-localize with the receptor in the endosomal compartment rather than by their differing affinity for TLR9. It is suggested that discrimination between free methylated and unmethylated CpG ODN occurs upstream of TLR9 by recognition of the methylation status of free CpG-ODN, triggering a SFKsignalling pathway that induces TLR9 mobilization to the late endosomal compartment and colocalization with CpG ODN. Furthermore, it is observed that particulate delivery also triggers TLR9 mobilization effectively bypassing this "discrimination" process and allowing for colocalization of the nucleic acid payload with TLR9 irrespective of methylation status.  97  4.2 Materials and methods 4.2.1 Reagents  Phosphorothioated (PS) unmethylated and methylated (5' cytosine) INX-6295 (5'TAACGTTGAGGGGCAT-3'), both unlabelled and 5' carboxyfluorescein (FAM)-labelled were synthesized by Trilink BioTechnologies. Tissue culture media, L-glutamine, foetal bovine serum (FBS), penicillin G, streptomycin sulphate were from Invitrogen and recombinant mouse IL-4 and GM-CSF were from Cedarlane. Antibodies for enzyme linked immunosorbent assays (ELISA), cytometric bead array sets and all antibodies for flow cytometry were purchased from BD Biosciences. Poly-D-lysine was from Sigma, Ficoll-Paque from Amersham and collegenase D obtained from Roche Applied Sciences. The SFK inhibitor, PP2, was obtained from Calbiochem and anti-CD 11c MACS beads were from Miltenyi Biotech. Goat anti-mouse EEA1, rat anti-mouse LAMP1 were from Santa Cruz and rabbit anti-mouse to TLR3, TLR7 and TLR9 were obtained from Abcam. Texas Red conjugated dextran, DiICI8 (1,1'dioctadecy13,3,3',3'tetramethylindocarbocyanine perchlorate) and all secondary antibodies were purchased from Molecular Probes.  4.2.2 Mice and cell lines  Eight to 10-week-old female ICR and C57BL/6 mice were obtained from Charles River Laboratories and held in a pathogen-free environment. All procedures were approved by an institutional animal care committee and performed in accordance with the guidelines established by the Canadian Council on Animal Care. The murine macrophage cell line, RAW264.7, was obtained from the American Type Culture Collection and cultured in DMEM supplemented with penicillin G (100 U/ml), streptomycin sulphate (100 lAg/m1), 2 mM L-glutamine and 10% FBS. Bone marrow derived dendritic cells (BMDC) were derived from bone marrow cells collected 98  from the long bones of ICR mice cultured with IL-4 and GM-CSF for 7 d in compete medium (CM) consisting of RPM1 1640 supplemented with penicillin G (100 U/ml), streptomycin sulphate (100 Ag/m1), 2 mM L-glutamine and 10% FBS. Resultant cells were found to be greater than 85% CD1 1 c positive and displayed a myeloid phenotype (CD11cP0s, CD1 1 bP', Mac310) as assessed by flow cytometry.  4.2.3 Preparation of liposomal nanoparticles  ODN were encapsulated in lipid nanoparticles containing an ionisable aminolipid using an ethanol dialysis procedure, as previously described (Semple, Klimuk et al. 2001). DiIC18 (0.5 mol%) was used in the formulation of empty lipid nanoparticles. ODN concentrations were determined by UV spectroscopy (260 nm) on a Beckman DU 640 spectrophotometer and lipid concentrations were determined using an inorganic phosphorus assay after separation of the lipids from the ODN by a Bligh and Dyer extraction (Bligh and Dyer 1959). The ODN-to-lipid ratio was typically 0.10-0.13 (w/w) with a particle size of 100 ± 25 nm, as determined by quasielastic light scattering using a NICOMP Model 370 submicron particle sizer. .  4.2.4 Cell uptake and immune response  For in vitro analysis, RAW264.7 or BMDC were incubated for 1, 4, 12 and 24 h with 250 tig/m1 fluorescently labelled free or encapsulated, CpG ODN or mCpG ODN. Cell were harvested, washed and then analyzed for uptake using flow cytometry. For ex vivo assessment, ICR mice were injected s.c. with 5 mg/kg of fluorescently labelled free or encapsulated CpG ODN or mCpG ODN. Spleens and/or lymph nodes were obtained from mice 1, 4, 7 and 24 h post administration. To demonstrate the role of SFK signalling in the immunostimulatory activity of CpG ODN, LN-CpG ODN and LN-mCpG ODN, C57BL/6 mice were treated 99  intraperitoneally (i.p.) with 1 mg/kg PP2 twice per day for 7 d. Mice were injected i.v. with 20 mg/kg free or encapsulated, fluorescently labelled CpG ODN or mCpG ODN and tissue samples were collected 12 h post administration. Cells were processed to single cell suspensions as previously described (de Jong, Chikh et al. 2007). For plasma cytokine analysis, blood was collected and processed to plasma by centrifugation and frozen at —20°C until assayed. Plasma concentrations of IL-6, MCP-1 and IFN-y were determined using ELISA or cytometric bead array as per manufacturer's instructions.  4.2.5 Flow cytometry  Cell uptake (as judged by intensity of the fluorescently labelled ODN on a per cell basis) was assessed in specific immune cell populations (as determined by phenotype analysis; cell suspensions were stained with phycoerythrin [PE]-conjugated anti-CD 1 lb or allophycocyanin [APC]-conjugated anti-CD 11c antibodies) using a 4-colour FACSort flow cytometer and CellQuest Pro software (BD Biosciences). For a determination of immune activation, cell suspensions were stained with either fluorescein isothiocyanate (FITC)- or APC-labelled phenotype antibodies (CD1 1 b, CD1 1 c, Mac3, CD8 and B220/CD45R) in combination with PEconjugated antibodies directed against the activation markers CD69 or CD86. Cell activation was assessed using a LSRII flow cytometer and FACS Diva software (BD Biosciences).  4.2.6 Endosomal trafficking and localization  Endosomal localization of free and encapsulated CpG ODN and mCpG ODN was assessed by incubating RAW264.7 cells with 101.1g/m1 free or encapsulated CpG ODN or mCpG ODN for 4 h prior to the addition of 1 mg/ml of the sub-cellular compartmental marker, Texas Red conjugated dextran 10,000 MW, for an additional 2 h. BMDC were grown on Poly-D100  Lysine pre-coated coverslips and incubated with 5 f1g/m1 free or encapsulated CpG ODN or mCpG ODN for 4 h. To examine the possibility of preferential trafficking of unmethylated CpG ODN specifically to TLR9 containing endosomes thus allowing for interaction with TLR9 and immunostimulatory activity compared to mCpG ODN, C57BL/6 mice were injected i.v. with 20 mg/kg free or encapsulated, methylated or unmethylated fluorescently labelled CpG ODN or 150 mg/kg DiI labelled empty LN. To demonstrate the role of SFK signalling in the co-localization of CpG ODN, LN-CpG ODN and LN-mCpG ODN, C57BL/6 mice were initially injected i.p. with 1 mg/kg of the specific SFK inhibitor PP2 twice per day for seven days. Following the last treatment, mice were injected i.v. with 20 mg/kg free or encapsulated, methylated or unmethylated fluorescently labelled CpG ODN. After 4 h all mice were euthanized and spleens were disrupted, dendritic cells isolated and processed for visualization as outlined previously.  4.2.7 Immunofluorescence  Following incubation, RAW264.7 cells were washed and incubated in Optimem prior to live cell visualization. BMDC were treated with 2% BSA in PBS, fixed in 2% paraformaldehyde and permeabilized with 0.1% saponin and 2% BSA in PBS. Cells were stained with rat anti-mouse LAMP1 followed by Alexa-647 conjugated rabbit anti-rat antibody as a detection reagent. For ex vivo studies, spleens were dissociated by injection of 1 ml RPMI containing 5% FBS, 1 mg collagenase D and incubated for 30 min at 37°C. Subsequently, DCenriched cell populations were obtained by centrifugation of cell suspensions on Ficoll-Paque gradients. DCs were then purified by positive selection with anti-CD1 1 c MACS beads with the resulting population being > 98% CD1 lc. Splenic DCs were resuspended in CM and grown on Poly-D-Lysine pre-coated coverslips for 3 h. Attached DCs were then treated with 2% BSA in PBS followed by fixation with 2% paraformaldehyde. The cells were then permeabilized with 101  0.1% saponin and 2% BSA in PBS followed by incubation with goat anti-mouse EEA1 or rat anti-mouse LAMP 1. Secondary Alexa-647 conjugated rabbit anti-goat antibody and goat antirat Alexa-647 were used respectively, as a detection reagent. To examine the co-localization of EEA1 and LAMP1+ compartments with TLR3, TLR7 or TLR9, mouse anti-mouse or rabbit antimouse TLR3, TLR7 or TLR9 polyclonal antibody followed by rabbit anti-mouse and goat antirabbit both coupled to Alexa 568 were used, respectively.  4.2.8 Confocal microscopy  For both RAW264.7 and BMDC, sections 0.1 Jim in thickness were captured using a BioRad Radiance 2000 laser scanning or Nikon C 1 immunofluorescent confocal microscope. Data were analyzed using ImageJ v1.37 to select images with a total thickness of 0.2-0.3 p.m and processed with Adobe Photoshop CS2. For ex vivo studies, sections of 0.15 tim were captured with a Nikon-C1, TE2000-E immunofluorescent confocal microscope. Data were analyzed as described previously and stacks with a total thickness of 0.6 urn were processed with Adobe Photoshop CS2. Isotype control antibodies were used in all confocal microscopy experiments to confirm the specificity of antibody staining. Thirty to 50 images were collected for each treatment. The percentage of co-localization of combinations of free or encapsulated CpG ODN or mCpG ODN, TLR9 and LAMP 1, for all studies, was assessed in OpenLab.  4.2.9 Statistical analyses All statistical analyses were performed using SPSS Ver 14.0. Initially, a one-way  analysis of variance (ANOVA) was used to statistically evaluate the differences between treatment groups. In the case of statistically significant results, the differences between treatment groups were assessed through the use of Bonferroni adjusted t-tests unless otherwise 102  stated, a post-hoc test which controls for family-wise error rate. Probability (p) values less than .05 were considered significant.  103  4.3 Results We have previously reported that mCpG ODN, when delivered in LN, are able to induce similar and often superior immune responses compared to the equivalent, unmethylated sequence over a range of doses in a TLR9 dependent manner (manuscript submitted). These and other data (Yasuda, Yu et al. 2005; Yasuda, Rutz et al. 2006) demonstrate that, contrary to the current thinking, mCpG ODN is able to effectively interact with TLR9 to trigger immune responses, and suggests that the immunostimulatory activity of free unmethylated and methylated CpG motifs is controlled by a mechanism other than TLR9 binding which is effectively bypassed by LN delivery. Here, we examine whether the relative immunostimulatory activities of free CpG and mCpG ODN are based on differential uptake and/or trafficking to the TLR9 containing compartment, allowing access to unmethylated but not methylated CpG ODN. Furthermore, the hypothesis that LN delivery overcomes this differential uptake and/or trafficking and promotes delivery of CpG ODN to the TLR9-containing endosomal compartment, regardless of methylation status, is tested.  4.3.1 The methylation status of CpG ODN does not affect ODN uptake and intracellular trafficking by immune cells whether presented in free or LN-encapsulated form The uptake and trafficking characteristics of unmethylated and methylated CpG ODN were assessed to clarify their roles as determinants of the immunostimulatory activity. Results from in vitro uptake studies of fluorescently-labelled ODN in cultured macrophage cells and BMDCs demonstrate that, regardless of the duration or concentration (data not shown) of administration, free mCpG ODN and CpG ODN are taken up in a similar manner in both cultured macrophage and BMDC (Fig 4.1).  104  A  800 -  600 500 400 300 200 100 J 0 4^8^12^16^20^24  Time (h)  Fig 4.1 The uptake of free and LN-encapsulated CpC ODN by RAW264.7 cells and BMDC is not influenced by the methylation status of the ODN RAW264.7 murine macrophage [Panel A] and BMDC [Panel B] were incubated with free fluorescently-labelled (5PAM) mCpG ODN (closed circles) free CpG ODN (closed squares), LN-mCpG ODN (open circles) or LN-CpG ODN (open squares). Cells were analyzed for uptake of the ODN (as judged by mean fluorescence intensity or MFI) by flow cytometry. Levels of uptake at 4C and background fluorescence levels and were subtracted from the data. Data presented here are representative of 3 separate experiments  Likewise, in vivo analysis of uptake following s.c. administration of labelled ODN show that both free unmethylated and methylated CpG ODN are taken up similarly by antigenpresenting cells as demonstrated in CD11b+, CD1 lc, and to a lesser extent in B220+ cells (data not shown) from both lymph nodes and spleen (Fig 4.2). As may be expected, similar uptake for both encapsulated unmethylated and methylated ODN is also observed in cells from spleen and lymph nodes following s.c. administration and in cultured cells.  105  A  14  •  20  E. 12  O  2 0 0  4^8^12^16^20^24  4^8^12^16^20^24  140 -  2  50  120 -  40  100 80 -  30 0. • 20 z  •  60 40  bo  20  0 0^4^8^12^16^20^24^4^8^12^16^20^24  Time (h)  ^  Time (h)  Fig 4.2 The uptake of free and LN-encapsulated CpG ODN by immune cells in spleen and lymph nodes following s.c. administration is not influenced by the methylation status of the ODN Fluorescently-labelled (5-FAM) free mCpG ODN (closed circles) free CpG ODN (closed squares), LN-mCpG ODN (open circles) or LN-CpG ODN (open squares) was administered s.c. to mice (4 animals/group). Mice were euthanized at the indicated time points and spleens [Panels A and B] and lymph nodes [Panels C and D] were harvested and processed to single cells. Samples were analyzed for uptake of the ODN (as judged by mean fluorescence intensity or MFI) by specific cell types (as judged by expression of the phenotype markers CD1 lb [Panels A and C] and CD11c [Panels B and D] by flow cytometry. Background fluorescence levels were subtracted from the data. Data presented here are representative of at least 3 independent experiments.  Intracellular trafficking was also evaluated to assess if differential trafficking to the endosomal compartment could be responsible for the relative immunostimulatory activities of CpG and mCpG ODN. Endosomal trafficking was initially assessed in RAW264.7 cells using the fluid phase sub-cellular compartmental marker Texas-Red conjugated dextran in conjunction with fluorescently labelled ODN. As demonstrated in Fig 4.3, both mCpG and CpG ODN efficiently localize to the late endosomal compartment in either free (Panels A and B) or lipid nanoparticulate (Panels C and D) form, as shown by co-localization with Texas-Red dextran.  106  A  Fig 4.3 The trafficking of free and LN-encapsulated CpG ODN following uptake into RAW264.7 cells is not influenced by the methylation status of the ODN Fluorescent confocal micrographs showing CpG ODN trafficking to endosomal compartments in RAW264.7 cells. RAW264.7 murine macrophage cells were incubated with fluorescently-labelled free mCpG ODN [Panel A] free CpG ODN [Panel B], LN-mCpG ODN [Panel C] or LN-CpG ODN [Panel D] followed by the addition of the fluid phase marker Texas-Red conjugated dextran. Cells were analyzed by confocal microscopy for trafficking of CpG ODN to the endosomal compartment. Images represent Z-compressions of 1-2 sequential sections.  Examination of the trafficking of mCpG ODN and CpG ODN was also conducted in BMDC (Fig 4.4). As with RAW264.7 cells, both free mCpG and CpG ODN (Panels A and B, respectively) and LN-mCpG and LN-CpG ODN (Panels C and D, respectively) efficiently colocalize in LAMP1+ compartments of BMDCs, which further confirms that the ability of CpG ODN, in either free or encapsulated form, to traffic to endosomal compartments is not influenced by methylation status.  107  A  Fig 4.4 The trafficking of free and LN-encapsulated CpG ODN following uptake into BMDC is not influenced by the methylation status of the ODN Fluorescent confocal micrographs showing CpG ODN trafficking to endosomal compartments in BMDCs. BMDC were incubated with fluorescently-labelled free mCpG ODN [Panel A], free CpG ODN [Panel B], LN-mCpG ODN [Panel C] or LN-CpG ODN [Panel D]. After fixation, LAMP containing endosomes were stained with an antibody to the late endosomal marker protein LAMP1 prior to imaging by confocal microscopy for trafficking of CpG ODN to the LAMP1+ endosomal compartment. Images represent Z-compressions of 1-2 sequential sections.  4.3.2 Free CpG, LN-CpG and LN-mCpG ODN co-localize with TLR9 in the late endosomal compartment but free mCpG ODN does not The similar internalization and trafficking characteristics reported in the previous section indicates that the differential immunostimulatory activity of unmethylated and methylated CpG ODN cannot be attributed to differences in uptake or endosomal trafficking. Therefore, to further elucidate potential impacts of intracellular trafficking on immunostimulatory activity, an  ex vivo examination of the relative abilities of free CpG and mCpG ODN to co-localize with TLR9 in the endosomal compartment was undertaken. Specifically, the co-localization of CpG ODN with TLR9 in early and late endosomes was assessed in DCs isolated from the spleens of mice injected with fluorescently labelled free unmethylated or methylated CpG ODN. Consistent with immunostimulatory activity, cells from mice treated with free CpG ODN show co-localization of CpG ODN with TLR9 in LAMP1+ compartment (Fig 4.5 - Panel B). 108  Conversely, very little co-localization is observed for free methylated CpG ODN and TLR9 in LAMP1+ compartments (Fig 4.5 — Panel A), consistent with a lack of immunostimulatory activity.  A  a-TLR9  ODN CL A  Fig 4.5 Free CpG ODN, LN-mCpG-ODN and LN-CpG ODN co-localize with TLR9 in LAMP1+ compartments in vivo but free mCpG ODN does not Fluorescent confocal micrographs showing CpG-TLR9 co-localization in LAMP I+ endosomes of splenic DCs. Fluorescently-labelled free mCpG ODN [Panel A], free CpG ODN [Panel B], LN-mCpG ODN [Panel C] or LNCpG ODN [Panel D] was administered iv. to mice (5 animals/group). Animals were euthanized and spleens were harvested and CDI lc+ cells isolated and permeabilized. After fixation cells were stained directly with anti-TLR9 and anti-LAMP I antibodies and imaged by confocal microscopy for co-localization of CpG ODN with TLR9 in LAMM+ endosomes. Images represent Z-compressions of 3-4 sequential sections. Data presented here are representative of at least 3 independent experiments.  Administration of CpG ODN as lipid nanoparticles results in effective co-localization with TLR9 in the late endosomal compartment regardless of methylation status (Fig 4.5 - Panels C and D, respectively), which is also consistent with previously reported immunostimulatory activity. Of significance is the finding that LN uptake itself, regardless of payload, appears to be a sufficient trigger since administration of empty LN is able to induce endosomal co-localization of the nanoparticle with TLR9 in LAMP1+ endosomes (Fig 4.6 — Panel A). Therefore, these data 109  show that the ability of free CpG ODN to co-localize with TLR9 in late endosomes is dependent on methylation status and that delivery in particulate form overcomes this discrimination and allows for co-localization regardless of the methylation status of the payload. However, LNinduced co-localization is specific for TLR9 as no evidence of TLR3 and TLR7 co-localization is observed (data not shown).  A  +PP2  Dil-LN  a-TLR9 ct-LMAX  5 tun  Fig 4.6 Empty LN co-localize with TLR9 but PP2 inhibits the localization TLR9 to endosomes containing empty LN and LAMP! Fluorescent micrographs showing co-localization of empty LN with TLR9 in LAMP I- endosomes of splenic DCs. C57BL/6 mice (5 animals/group) were initially treated i.p. with PP2. Following final treatment, Dil labelled empty LN were administered i.v. to control [Panel Al and PP2-treated [Panel B] mice. Animals were euthanized and spleens were harvested and CDI1c+ cells isolated and permeabilized. After fixation cells were stained directly with anti-TLR9 and anti-LAMP1 antibodies and imaged by confocal microscopy for co-localization of empty LN with TLR9 in LAMP] endosomes. Images represent Z-compressions of 3-4 sequential sections. Data presented here are representative of 3 separate experiments.  Quantitative analyses show that free CpG ODN as well as LN-CpG and LN-CpG ODN effectively co-localize with TLR9 in the late endosome while free mCpG ODN does not (Fig 4.7). Statistical analysis of the percentage of co-localization confirms that free mCpG ODN shows a significantly lower co-localization with TLR9 in LAMP1+ compartments than CpG ODN (7.3 ± 3.2% compared with 16.8 ± 4.0%, t(53)=-7.829, p=.000), LN CpG-ODN (24.1 ± 6.8%, t(61)=-10.728, p=.000) and LN mCpG-ODN (22.3 ± 6.6%, t(51)=-7.277, p=.000). Of note, both LN-mCpG ODN and LN-CpG ODN resulted in significantly greater co-localization with TLR9 in LAMP1+ compartments than free CpG ODN [t(23)=-2.411, p=.043 and t(33)110  3.941, p=.001, respectively], and no significant differences in co-localization were observed between the two encapsulated forms. The empty DiI-labelled LN (17.2 ± 5.7%) also showed statistically significantly greater co-localization with TLR9 in LAMP1+ compartments than free mCpG ODN [454)=-8.152, p=.000].  35 30 ets  25  c..) 20  0 7  ° 15 4.•  10 0  5  rriCpG ODN  ^  CpG ODN LN-mCpG ODN LN-CpG ODN  Fig 4.7 Co-localization of CpG ODN with TLR9 in LAMPE endosomes of spier& DCs  Cells were imaged using confocal microscopy. Co-localization of fluorescently-labelled ODN, TLR9 and LAMP] (percent co-localization +SD) was quantified using OpenLab. Data presented here are representative of 3 separate experiments.  Consistent with previous reports of TLR9 signalling from late endosomes, while both free and LN CpG ODN, regardless of methylation status, were found to distribute to both early (EEA1+) and late (LAMP1+) endosomes, co-localization of free CpG ODN, LN-CpG and LNmCpG ODN with TLR9 was observed predominantly in the late endosomal compartment, with relatively little seen in early endosomes (data not shown). Independent t-tests of free CpG ODN and both LN-mCpG ODN and LN-CpG ODN show significantly increased co-localization to TLR9 containing LAMP1+ endosomes rather than EEA1+ endosomes [t(23)=-3.469, p=.001, 432)=-2.440, p=.011 and 042)=-3.427, p=.001, respectively].  111  4.3.3 Co-localization with TLR9 and immunostimulatory activity is mediated via a SFK signalling cascade It has been reported that CpG induces a sequence-specific, TLR9-independent, SFK signalling cascade upstream of TLR9 at the plasma membrane that is ultimately required for CpG engagement and activation of TLR9-MyD88 (Sanjuan, Rao et al. 2006). It was therefore investigated whether SFK signalling could mediate the co-localization of free CpG ODN (and potentially, LN-CpG and LN-mCpG ODN) with TLR9 in the LAMP1+ compartment. To demonstrate the role of SFK signalling in co-localization, mice were pre-treated with PP2, a SFK specific inhibitor, prior to the administration of fluorescently labelled free or encapsulated CpG ODN and mCpG ODN. As shown in Fig 4.8, pre-treatment with PP2 effectively abolishes the co-localization of free CpG ODN (Panel B) with TLR9 in the endosomal compartment compared to untreated controls (Panel A) (2.6 ± 3.2% compared with 16.1 ± 8.1% [474)=-9.189, p=.001]). Similarly, PP2 is effective in preventing co-localization of LN-CpG ODN (Panel C) (4.7 ± 5.4%) and LNmCpG ODN (Panel D) (2.7 ± 1.7%) with TLR9 in LAMP1+ compartments (Figs. 4.8, 4.9) as well as with empty liposomes (Fig 4.6 — Panel B) (17.3 ± 5.7%) compared to the non-PP2 treated control (Fig 4.6— Panel A) (1.3 ± 1.7%). Quantitative data is shown in Fig 4.9.  112  A  Fig 4.8 Src family kinase inhibitor PP2 inhibits the localization of TLR9 to LAMP1 containing endosomes Fluorescent confocal micrographs showing inhibition of CpG ODN and TLR9 co-localization in LAMP1+ endosomes of splenic DCs by PP2. C57BL/6 mice (5 animals/group) were treated i.p. with PP2. Following the final treatment, mice were injected i.v. with fluorescently-labelled free CpG ODN [Panels A and B], LN-mCpG ODN [Panel C] or LN-CpG ODN [Panel D] in control [Panel A] or PP2 treated [Panels B-D]. Animals were euthanized and spleens were harvested and CDI lc' cells isolated and permeabilized. After fixation cells were stained directly with anti-TLR9 and anti-LAMP I antibodies and imaged by confocal microscopy for co-localization of CpG ODN with TLR9 in LAMP1 endosomes. Images represent Z-compressions of 3-4 sequential sections. Data presented here are representative of 2 separate experiments.  113  C  0  30 25  Ts 20  8  15  4.6. 10  5 0 CpG ODN CpG ODN + LN-CpG ODN LN-mCpG PP2^+ PP2^ODN + PP2  Fig 4.9 Inhibition of CpG ODN and TLR9 co-localization in LAMM+ endosomes of splenic DCs by PP2 Cells were imaged by confocal microscopy and co-localization of fluorescently-labelled ODN, TLR9 and LAMP I (percent co-localization ±SD) was quantified using OpenLab. Data presented here are representative of 2 separate experiments.  CD11c  450  ^  B220 160 140  400  120  350 -  100  300  80 .16) 250 -  0  60 -  200  40  150  20  100  0  Mac3  45  70  35 U.  2  CD8  80  40  60 -  30  50-  25  40  20 0 15 10 5  CpG ODN  LN-mCpG ODN  Ii  LN-mCpG ODN + PP2  30 20 10  CpG ODN^LN-mCpG ODN^LN-mCpG ODN + PP2  Fig 4.10 CpG-mediated immune activation is a SFK dependent process  Inhibition of CpG-mediated cell activation marker expression on APCs by PP2. C57BL/6 mice (4 animals/group) were treated i.p. with PP2. Following the final treatment, free or LN-CpG-ODN was administered i.v. to mice. After 12 h, animals were euthanized and spleens harvested. Splenocytes were analyzed for expression of the CD69 and CD86 cell surface activation markers in conjunction with phenotype markers by flow cytometry. Data presented here are representative of 3 separate experiments.  114  Concomitant with a significant reduction in the co-localization of CpG ODN, LN-CpG ODN and LN-mCpG ODN with TLR9 in the late endosomal compartment following treatment with PP2, a reduction in both immune cell activation (Fig 4.10) and cytokine secretion (Fig 4.11) was observed. The most notable effects of SFK suppression resulting from the administration of PP2 were the reduction in the upregulation of CD86 and CD69 from both Mac3+ and CD1 1c cells. As shown in Fig 4.10, a significant reduction was observed in both the expression of CD69 in Mac3+ [t(5)=-5.958, p=.002], CD8+ [t(5)=2.788, p=.025] and CD11c+ cells [t(5)=4.611, p=.005 — data not shown] and the expression of CD86 in B220+, [t(5)=4.743, p=.005], and CD1 1c cells [t(5)=2.776, p=.025]. Similarly, a significant suppression in the secretion of cytokines was also observed (Fig 4.11) with an almost 10-fold reduction in IFNI', a 4-fold reduction in IL-6 and a 50% reduction in MCP-1 following the administration of PP2.  115  CpG ODN  ^  LN-mCpG ODN  ^  LN-mCpG ODN+ PP2  Fig 4.11 Plasma cytokine induction is a SFK dependent process Inhibition of CpG-mediated plasma cytokine levels by PP2. C5713L/6 mice (4 animals/group) were treated i.p. with PP2. Following the final treatment, free or LN-CpG-ODN was administered i.v. to mice. After 12 h, animals were euthanized and blood was collected by cardiac puncture, processed to collect plasma and cytokine levels were determined by cytometric bead array. Data presented here are representative of 3 separate experiments.  116  4.3.4 Free CpG ODN and encapsulated CpG and mCpG ODN enable co-localization by inducing TLR9 mobilization to LAMP1+ endosomes  While the preceding data implicates SFK-mediated differential co-localization as a factor in determining the immunostimulatory activity of methylated and unmethylated, free and encapsulated CpG ODN, they do not resolve whether the differential co-localization is due to free CpG ODN and LN-CpG and LN-mCpG ODN trafficking specifically to TLR9-containing endosomes or conversely, induction of TLR9 to mobilize to the late endosomal compartment To investigate these possibilities the trafficking behaviour of TLR9 and ODN to the endosomal compartment was assessed. Results indicate that SFK signalling directs TLR9 to mobilize and localize within LAMP1+ compartments following treatment with CpG-ODN (23.5 ± 6.0%), LN-CpG ODN (39.6 ± 6.0%) and LN-mCpG ODN (43.1 ± 10.5%) (Fig 4.12 — Panel B) but not of free mCpG ODN (12.3 ± 8.0%) (Fig 4.12— Panel A) [t(53)-4.663,p.000, 45 1)-10.606, p=.000 and t(60)=-13.776,p=.000, respectively). Quantitative data is shown in Fig 4.13. Importantly, no significant differences are observed in the trafficking of methylated vs unmethylated CpG ODN, in either free or encapsulated forms, to the late endosomal compartment with these treatments (Fig 4.14). These data are consistent with results from SFK inhibition studies where, as previously noted, pre-treatment of mice with PP2 results in a significant decrease in co-localization of free CpG ODN and encapsulated CpG and mCpG ODN with TLR9 within LAMP1+ compartments (Fig 5 — Panels B-D). SFK inhibition results in a concomitant decrease in TLR9 mobilization to late endosomes (Figs. 4.12) (free CpG ODN — Panel C, t(53)-8.255,p.000, LN-mCpG ODN — Panel E, t(33)=-10.867, p=.000, and LN-CpG ODN — Panel G, t(45)=-16.037, p=.000) while no significant effect on CpG ODN trafficking to the late endosomal compartment is noted (Fig 4.14). Interestingly, empty LN also promote the localization of TLR9 in LAMP1+ compartments (30.4 ± 6.3%) (Fig 4.12 — Panel H) which is inhibited by PP2 (5.3 ± 3.7%) (Fig 4.12 — Panel I), 117  suggesting that these empty particles affect TLR9 trafficking in a similar manner as LN-CpG ODN.  A  Fig 4.12 Co-localization is mediated by TLR9 mobilization and trafficking to the LAMP1+ compartment Fluorescent confocal micrographs showing TLR9 mobilization to LAMP I+ endosomes of splenic DCs which is inhibited by PP2. C57BL/6 mice (5 animals/group) were treated i.p. with PP2 [Panels C,E,G,I]. Following the final treatment free mCpG ODN [Panel Al, free CpG ODN [Panels B, C], LN-mCpG ODN [Panels D, E], LN-CpG ODN [Panels F, G], or Dil LN [Panels H, I] was administered iv. to mice. Animals were euthanized, spleens were harvested and CD1 lc cells isolated and permeabilized. After fixation cells were stained directly with anti-TLR9 and anti-LAMP1 antibodies and imaged by confocal microscopy for TLR9 mobilization to LAMM+ endosomes. Images represent Z-compressions of 3-4 sequential sections. Data presented here are representative of 2 separate experiments.  118  60 7 50 40 30  mCpG ODN^CpG ODN  LN-mCpG ODN LN-CpG ODN  LN-Dil  Fig 4.13 Mobilization of TLR9 to LAMP1+ endosomes of splenic DCs in control and PP2 treated mice  Cells were imaged by confocal microscopy and co-localization of immunostained TLR9 and LAMP! (percent colocalization +SD) was quantified using OpenLab. Solid bars represent data from control animals and open bars are data from animals pretreated with PP2. Data presented here are representative of 2 separate experiments.  40 ,  03 30 N  C.)  0  0 C.) 4E1 8,-  20  10 -1  mCpG ODN CpG ODN LN-mCpG^LN-CpG ^ ODN ODN  ^  LN-Dil  Fig 4.14 Trafficking of CpG ODN to the LAMPE' endosomes of splenic DCs in control and PP2 treated mice  Cells were imaged by confocal microscopy and co-localization of fluorescently-labelled ODN and LAMP] (percent co-localization +SD) was quantified using OpenLab. Solid bars represent data from control animals and open bars are data from animals pretreated with PP2. Data presented here is representative of 2 separate experiments.  119  It is notable that LN-CpG and LN-mCpG ODN induce significantly greater mobilization of TLR9 to the endosomal compartment compared to an equivalent dose of free CpG ODN ([43.1 ± 10.5% vs 23.5 ± 6.0%, t(33)-7.578,p.000] and [39.6 ± 6.0% vs 23.5 ± 6.0%, t(23)=5.709, p=.000], respectively). While we have previously attributed the enhanced immunostimulatory activity of encapsulated CpG ODN to improved delivery and uptake by APCs, increased levels of TLR9 in the late endosomal compartment may be another contributing factor. These data indicate that administration of free CpG ODN as well as particulate CpG and mCpG ODN promotes a mobilization of TLR9 to LAMP1+ compartments but does not influence the trafficking of the CpG ODN itself.  120  4.4 Discussion  Eukaryotic organisms have evolved systems to rapidly elaborate protective immune responses to combat pathogen invasion based on pattern recognition receptors that recognize specific, highly conserved molecular patterns associated with pathogens. While most bind ligands that are structurally complex and unique, TLR9 recognizes CpG motifs within pathogen DNA and synthetic ODN. Distinguishing pathogenic sequences from eukaryotic DNA has been proposed to be a multifactorial process based, in part, on methylation status and it is generally accepted that TLR9 specifically recognizes unmethylated CpG motifs and methylated DNA is non-immunostimulatory due to its inability to interact with TLR9 (Krieg, Yi et al. 1995; Takeshita, Leifer et al. 2001; Comelie, Hoebeke et al. 2004; Latz, Schoenemeyer et al. 2004; Rutz, Metzger et al. 2004). Recently however, several separate lines of research have indicated that methylated CpG are in fact, able to induce immune responses via TLR9 (Yasuda, Ogawa et al. 2005; Barton, Kagan et al. 2006; Yasuda, Rutz et al. 2006) including our own work demonstrating that LN delivery endows mCpG ODN with immunostimulatory potential, through a TLR9-dependent mechanism. These data show that methylated CpG ODN can act through TLR9 to stimulate an immune response and indicate that the immunostimulatory activity of unmethylated vs methylated CpG ODN is regulated by a mechanism that does not involve differential TLR9 affinity. The data presented here show that the dependence of immunopotency on methylation status arises from the ability of free unmethylated CpG ODN (and the inability of free methylated) to induce TLR9 mobilization and co-localization in the late endosomal compartment via a SFK-signalling cascade. In contrast, nanoparticulate delivery, allows effective CpG ODNTLR9 co-localization in the late endosome regardless of methylation status, also via a SFKsignalling pathway, resulting in immunostimulatory activity. 121  While the localization of TLR9 in the endoplasmic reticulum of resting APCs and its rapid trafficking to the endosomal/lysosomal compartments upon cellular activation has been well described (Latz, Schoenemeyer et al. 2004), the mechanisms that control TLR9 trafficking are poorly understood. The data presented here demonstrating differential trafficking of TLR9 in response to free unmethylated and methylated CpG ODN points to a cellular mechanism which can distinguish CpG methylation status and trigger TLR9 mobilization, thus allowing colocalization of CpG ODN and TLR9 in the endosomal compartment, subsequent binding and immunogenic signalling. Sanjuan and colleagues (Sanjuan, Rao et al. 2006) have recently reported on a CpG-dependent, TLR9-independent SFK signalling pathway that induces cytoskeletal reorganization and ultimately is required for TLR9 engagement and immunostimulatory activity initiated by a plasma membrane bound, sequence-specific receptor at the plasma membrane upstream of TLR9. While a definitive answer awaits further studies, we propose that the SFK-mediated, methylation-dependent mobilization and migration of TLR9 described here and the pathway proposed by Sanjuan et al., may represent a common pathway initiated by a yet to be identified surface receptor capable of distinguishing methylated vs nonmethylated and CpG vs non-CpG ODN. It is significant that LN uptake, regardless of payload, also acts via a SFK-signalling cascade to trigger TLR9 mobilization to the endosomal compartment, thus allowing both methylated and unmethylated CpG ODN to co-localize with and engage TLR9. Indeed, although not capable of initiating an immune response (Li, Bally et al. 2002; de Jong, Chikh et al. 2007), LN carrying no payload also efficiently co-localize with TLR9 in LAMP1+ compartments in a SFK-dependent manner. While receptor mediated and macropinocytotic/phagocytic uptake of free and LN CpG ODN, respectively, represent distinct and divergent events, it appears likely that both processes converge through a common SFK 122  signalling cascade that results in TLR9 mobilization from the ER to the late endosome. This induction of endosomal localization appears to be specific for TLR9 since TLR3 and TLR7 are not found to undergo similar mobilization. The results presented here suggest a new model whereby the relative immunopotency of unmethylated and methylated CpG and, by extension, self and pathogenic DNA is determined. In this model, free self DNA released as a result of certain pathological conditions would fail to mobilize TLR9 and induce immune responsiveness while advanced infections resulting in the release of free pathogen DNA would induce DNA-TLR9 co-localization and allow immunostimulatory activity. Importantly, in the early stages of pathogen infection where the primary exposure to pathogen DNA would likely be through ingested bacterial and viral particles, phagocytic uptake would result in TLR9 mobilization and co-localization with pathogenic DNA, allowing for immunostimulatory responses. However, since a common route of exposure to self DNA is through phagocytosis of apoptotic cells, additional mechanisms that specifically dampen immune responses to self DNA acquired by phagocytosis are likely to be involved and could include receptors for lipid components unique to apoptotic cells such as phosphatidylserine. In summary it has been shown that TLR9-mediated recognition of bacterial DNA is more regulated than previously thought, with TLR9 being sequestered within the ER until activated by a stimulus provided by either free unmethylated CpG DNA or particulate uptake. The results presented indicate that the SFK-dependent migration of TLR9 from the ER to the late endosomal/lysosomal compartment is a pivotal step for determining the immunostimulatory activity of free methylated and unmethylated CpG-ODN, suggesting that the ability to distinguish self from non-self DNA does not reside within TLR9 itself but rather through strict regulation of its subcellular distribution. Similarly, particulate delivery, regardless of payload, 123  primes APCs for immunostimulatory activity by promoting the SFK-dependent mobilization and trafficking of TLR9 from the ER to late endosomal compartments. Overall, these findings are consistent with recent work showing the importance of intracellular localization in regulating TLR9 activity and specificity (Barton, Kagan et al. 2006), unify a number of concepts regarding TLR9-mediated immunostimulatory activity and provide insight into the mechanisms and processes that regulate TLR9 localization and signalling.  124  CHAPTER 5: Concluding Remarks  5.1 Summary and significance of research  With the development of immunotherapy as a viable treatment for various pathological conditions including cancer, there is a need for safe but potent immunostimulatory agents that can be delivered as a stand-alone agent, as an adjunct therapy and co-delivered with diseaseassociated antigens to boost and re-programme the immune system to recognize and target pathogens or tumour cells. Bacterial DNA and synthetic unmethylated CpG ODNs are potent immunostimulatory agents that may contribute to the therapeutic options for many diseases and specifically have shown promise in the treatment of cancer (Krieg 2003). However, due to the sub-optimal stability, delivery and uptake to target tissues of CpG ODN, there has been great interest in developing strategies for prolonging their bioavailability and duration of action in order to improve their therapeutic efficacy. Using a nanoparticulate lipid-based delivery system with low surface charge, small homogeneous size and effective protection of the ODN payload, the enhanced immunopotency and therapeutic efficacy of LN-CpG ODN compared with free forms is demonstrated in Chapter 2. Specifically, it is shown that encapsulation specifically targets CpG ODN for uptake by immune cells likely providing the basis for the enhanced immunopotency of encapsulated CpG ODN, resulting in potent innate and adaptive in vivo immune responses and promoting the generation of TAA-specific immune responses. Finally in efficacy studies, delivery in lipid nanoparticles is shown to enhance the ability of CpG ODN to adjuvanate adaptive immune responses and generate protective immunity against tumour challenge in both highly and poorly immunogenic, xenogeneic and syngeneic tumour models. Thus encapsulation of CpG ODN within these particles provides an attractive strategy for enhancing the biological responses natural to CpG ODN and improving its therapeutic activity in the treatment of cancer. 125  Although it is generally accepted that mCpG ODN is immunologically inert, in Chapter 3 it is revealed that the encapsulation of mCpG ODN within liposomal nanoparticles induces potent innate and adaptive responses, typically more potent than unmethylated counterparts. Furthermore, encapsulated mCpG ODN is an effective adjuvant in xenogeneic animal models of cancer. As these results were based on the initial assumption that LN-mCpG ODN mediates its effects through the same signalling pathway as CpG ODN rather than a novel pathway, it was necessary to confirm that encapsulated methylated CpG sequences induce immune stimulation through the TLR9 pathway. Preliminary data from several in vitro studies, later confirmed using TLR9 KO animals, showed that both methylated and unmethylated CpG ODN act through TLR9. This research raised several important questions regarding the mechanism regulating CpG ODN/TLR9 immune stimulatory activity: Since both methylated and unmethylated ODN are both able to mediate immune responses through TLR9, what is the mechanism responsible for differentiating between them and how does encapsulation overcome the barrier to the immunostimulatory potential of free methylated CpG ODN? Furthermore, why is LN-mCpG ODN often more potent than its encapsulated unmethylated counterpart? The results from this chapter suggest that the vertebrate immune system is not as inert to self DNA as previously thought and that the discrimination between methylated and unmethylated sequences occurs upstream of TLR9 and is possibly related to differences in uptake or trafficking. Such a mechanism, based on differential access to TLR9, implicates TLR9 sequestration as playing a major role in regulating CpG activity and has implications in understanding the processes involved in self tolerance as well as the design of effective nucleic acid based therapeutics. As a direct consequence of the discoveries and suppositions made in the previous chapter, in Chapter 4, the mechanisms and the cellular regulation by which CpG ODN stimulates the immune system are investigated. It is shown that while both free methylated and 126  unmethylated CpG ODN are internalized and traffic to endosomes in a similar manner, only unmethylated CpG ODN promotes the effective trafficking of TLR9 to co-localize with its CpG ligand in the late endosomal compartment. Furthermore, it is shown that encapsulated forms as well as empty LN promote the co-localization of TLR9 to endosomal compartments. Finally it is revealed that the co-localization of TLR9 occurs via a SFK-mediated signalling pathway. From these results it can be concluded that the lack of immunological activity for methylated CpG ODN is largely due to a failure of TLR9 to localize to the late endosomes. Thus, a new paradigm is proposed whereby the differential immunostimulatory activity of unmethylated and methylated CpG ODN (and pathogenic and self-DNA) is determined by their disparate abilities to mobilize TLR9 and to co-localize with the receptor in the late endosomal compartment rather than their differing affinities for TLR9 and that discrimination between methylated and unmethylated CpG ODN occurs upstream of TLR9 via a SFK signalling pathway that induces TLR9 mobilization. These finding show the importance of the intracellular localization of TLR9, unify several key concepts regarding TLR9 mediated activity and provide insight into the mechanisms and processes that regulate TLR localization and signalling. An understanding of the mechanisms involved may provide insight into means by which this pathway could be stimulated or inhibited with respect to potential applications in pre- clinical and clinical settings.  5.2 Future work While the specific mechanism of ODN uptake is poorly understood, and a specific receptor for free CpG ODN has yet to be identified, data indicates a receptor mediated process of endocytosis (Dalpke and Heeg 2004). Similarly, the specific mechanism for uptake of lipid particles is not completely resolved although it has been suggested, with respect to complexes, to 127  likewise involve a receptor-mediated process (Semple, Klimuk et al. 2000; Li and Ma 2001). In an attempt to elucidate the mechanism of uptake for both free and encapsulated ODN, a series of competition studies were conducted in vitro using combinations of non-labelled or labelled, free or encapsulated, methylated or unmethylated CpG ODN; preliminary results showed that although free mCpG ODN and CpG ODN appear to be taken up by the same mechanism, encapsulated ODN is taken up by a different method. These differences in uptake were further examined through a more in-depth series of studies using various chemical inhibitors of uptake which confirmed that the uptake of free CpG ODN was receptor mediated while uptake of LNCpG ODN was likely by macropinocytosis (unpublished data). Although these in vitro results point to a non-receptor mediated mechanism of uptake, they need to be confirmed in other cell types and preferably, if possible, in vivo. In Chapter 4, it is suggested that a co-factor or co-receptor may exist near or at the plasma membrane which differentiates between unmethylated and methylated CpG ODN and possibly influences the migration of TLR9 to the late endosome. Future work should try to identify this co-receptor and any possible link it may have with autoimmune diseases such as systemic lupus erythematosus. Furthermore, although it is shown that the translocation of TLR9 from the ER to the LE is dependent on SFKs, there is a need to assess the activation (phosphorylation) states of various signalling intermediates following treatment with free and encapsulated CpG ODN to determine if induction of TLR9 translocation occurs through an identical, or distinct pathways. Future experimental methods should include an intracellular flow analysis of the phosphorylation states of these proteins of interest in primary cells, and/or the use of knock out or knock down mice. Although this work has been initiated in vitro, a suitable model for experimentation has yet to be determined.  128  Most animal studies are performed in young healthy animals and intervention typically occurs before or during the early stages of a disease. However, the medium age of patients undergoing cancer treatment is usually in excess of 50 with a significant percentage of this clinical population elderly with many secondary complications, such as diabetes, systemic vascular disease, high blood pressure, age related and disease related immune suppression, including diminished cytokine response, reduction in clonal expansion and Ag-specific T and B cells, and a decline in Ag-presenting cell function (Renshaw, Rockwell et al. 2002). Additionally age may be a factor in response rate as older patients are generally not able to tolerate high dose therapies and are more susceptible to toxicity. The recent discovery that all TLRs, but particularly TLR9, are significantly down-regulated in older animals (Renshaw, Rockwell et al. 2002; Letiembre, Hao et al. 2007) suggests a correlation with age related decline of immune function. It would be clinically relevant, therefore, to assess the potential enhancement due to encapsulation of CpG ODN on innate and adaptive immune responses, its adjuvanticity and anti-tumour efficacy in aged mice.  129  REFERENCES Agrawal, S. (1999). "Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides." Biochim Biophys Acta 1489(1): 53-68. Agrawal, S., J. Temsamani, W. Galbraith and J. Tang (1995). "Pharmacokinetics of antisense oligonucleotides." Clin Pharmacokinet 28(1): 7-16. Ahmad-Nejad, P., H. Hacker, M. Rutz, S. Bauer, R. M. Vabulas and H. Wagner (2002). "Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments." Eur J Immunol 32(7): 1958-68. Appay, V., D. E. 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Pisetsky (2001). "The role of the macrophage scavenger receptor in immune stimulation by bacterial DNA and synthetic oligonucleotides." Immunology 103(2): 226-34. Zimmermann, S., 0. Egeter, S. Hausmann, G. B. Lipford, M. Rocken, H. Wagner and K. Heeg (1998). "CpG oligodeoxynucleotides trigger protective and curative Thl responses in lethal murine leishmaniasis." J Immunol 160(8): 3627-30. Zwaveling, S., S. C. Ferreira Mota, J. Nouta, M. Johnson, G. B. Lipford, R. Offringa, S. H. van der Burg and C. J. Melief (2002). "Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides." J Immunol 169(1): 350-8.  149  APPENDIX  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A04-0319 Investigator or Course Director: Pieter R. Cuills Department: Biochemistry & Molecular Biology Animals:  Mice BalbIc 258 Mice AU 76 Mice C57BL/6 (TLR9-/-) Mice C57BL/6 127  Start Date:^October 1, 2002  Approval Date:  December 7, 2006  Funding Sources: Funding Agency: Funding Title: Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Investigations on liposome-encapsulated nucleic acid based therapeutics  Canadian Institutes of Health Research (CIHR) Liposomal Systems for Delivery of Conventional and Genetic Anti-Cancer Drugs  Unfunded title: itla  The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the ('('AC and some valuing agencies. A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102. 6190 Agronomy Road. Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  150  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE BREEDING PROGRAMS  Application Number: A04 0320 -  Investigator or Course Director: Pieter R. Cullis Department: Biochemistry & Molecular Biology Animals:^Mice C57BL,6 TLR9 KO 208  Mice C57BL/6 208  Approval Date: December 7, 2006 Funding Sources: Funding Agency: Funding Title:  Canadian Institutes of Health Research Investigations on liposome-encapsulated nucleic acid based therapeutics  Unfunded title: N/A  The Animal Care Committee has examined and approved the use of animals for the above breeding program. This certificate is valid for one year from the above approval date provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102. 6190 Agronomy Road. Vancouver. BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  151  

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