Medicine, Faculty of
DSpace
UBCV
Law, Hin Ni
Master of Science - MSc
University of British Columbia
Cancer cell quiescence or dormancy is a reversible state whereby cells have undergone G0-G1 cell cycle arrest and are non-proliferating. These cells can recover the ability to self-renew with exposure to reopened or new vasculatures, and may be the founders of metastasis. One factor that may promote cancer cells to enter a dormant state is exposure to hypoxia, which not only limits cellular proliferation but also promotes drug resistance. In addition, some dormant cancer cells have been reported to express “stem-ness” markers that may provide these cells with stem cell-like characteristics to prolong survival. Thus, this thesis aims to describe work developing radioimmunoconjugates to target certain cancer stem cell and hypoxia-induced markers, specifically CD96 and carbonic anhydrase IX (CAIX), in efforts to potentially identify and eradicate dormant tumour cells, adding to the clinical arsenal against metastatic relapse and drug resistance in cancer patients. We hypothesize that leukemic stem cell- and hypoxia-specific markers can be targeted for cancer treatment and/or theranostic applications to help identify areas of tumor dormancy, aiding against drug resistance and patient relapse. Both panels of anti-CD96 and anti-CAIX radioimmunoconjugates showed specific tumour uptake in vivo for their respective target. The first aim is to evaluate radioimmunoconjugate candidates against CD96 for acute myeloid leukemia (AML) by positron emission tomography (PET) imaging, which was extensively investigated but resulted in decreasing tumour uptake over time in NRG/NSG mouse models and a loss of specific tumour uptake in the nude mouse model, suggesting a novel panel of mAbs may be required. The second aim is to screen mAbs targeting CAIX by PET imaging in renal cancer, colon cancer, and acute myeloid leukemia models, which resulted in three successful radioimmunoconjugate candidates for the solid cancers, and possible further study needed for expression of CAIX in AML. Overall, from this thesis, c2C7 emerged as a lead anti-CAIX mAb candidate that proved to have most potential as a radioimmunotherapy (RIT) agent, which warrants further therapeutic and possibly clinical studies.
USING RADIOIMMUNOCONJUGATES TO TARGET QUIESCENT TUMOUR CELLS VIA CANCER STEM CELL AND HYPOXIA-INDUCED MARKERS: AN APPROACH AGAINST METASTATIC DISEASE AND THERAPEUTIC RESISTANCE by Hin Ni (Teresa) Law B.Sc., The University of British Columbia, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Interdisciplinary Oncology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2021 © Hin Ni (Teresa) Law, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Using radioimmunoconjugates to target quiescent tumour cells via cancer stem cell and hypoxia-induced markers: an approach against metastatic disease and therapeutic resistance submitted by Hin Ni (Teresa) Law in partial fulfillment of the requirements for the degree of Master of Science in Interdisciplinary Oncology Examining Committee: Dr. François Bénard, Professor, Department of Radiology, Faculty of Medicine, UBC Supervisor Dr. Urs Häfeli, Professor, Faculty of Pharmaceutical Sciences, UBC Supervisory Committee Member Dr. Kevin Hay, Assistant Professor, Division of Hematology, Faculty of Medicine, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Paul Schaffer, Associate Professor, Department of Radiology, Faculty of Medicine, UBC Supervisory Committee Member iii Abstract Cancer cell quiescence or dormancy is a reversible state whereby cells have undergone G0-G1 cell cycle arrest and are non-proliferating. These cells can recover the ability to self-renew with exposure to reopened or new vasculatures, and may be the founders of metastasis. One factor that may promote cancer cells to enter a dormant state is exposure to hypoxia, which not only limits cellular proliferation but also promotes drug resistance. In addition, some dormant cancer cells have been reported to express “stem-ness” markers that may provide these cells with stem cell-like characteristics to prolong survival. Thus, this thesis aims to describe work developing radioimmunoconjugates to target certain cancer stem cell and hypoxia-induced markers, specifically CD96 and carbonic anhydrase IX (CAIX), in efforts to potentially identify and eradicate dormant tumour cells, adding to the clinical arsenal against metastatic relapse and drug resistance in cancer patients. We hypothesize that leukemic stem cell- and hypoxia-specific markers can be targeted for cancer treatment and/or theranostic applications to help identify areas of tumor dormancy, aiding against drug resistance and patient relapse. Both panels of anti-CD96 and anti-CAIX radioimmunoconjugates showed specific tumour uptake in vivo for their respective target. The first aim is to evaluate radioimmunoconjugate candidates against CD96 for acute myeloid leukemia (AML) by positron emission tomography (PET) imaging, which was extensively investigated but resulted in decreasing tumour uptake over time in NRG/NSG mouse models and a loss of specific tumour uptake in the nude mouse model, suggesting a novel panel of mAbs may be required. The second aim is to screen mAbs targeting CAIX by PET imaging in renal cancer, colon cancer, and acute myeloid leukemia models, which resulted in three successful radioimmunoconjugate candidates for the solid cancers, and possible further study needed for expression of CAIX in iv AML. Overall, from this thesis, c2C7 emerged as a lead anti-CAIX mAb candidate that proved to have most potential as a radioimmunotherapy (RIT) agent, which warrants further therapeutic and possibly clinical studies. v Lay Summary Antibodies that efficiently bind to proteins on tumour cells can be labeled with a radioactive material and serve imaging or treatment purposes. Some patients relapse with cancer that has spread to other tissues, which may be explained by quiescence, a reversible state whereby cells stop growing and spreading. Cancer cells may enter quiescence via exposure to low oxygen conditions (hypoxia), and some quiescent cancer cells have characteristics of stem cells (which make up the rest of the body) to prolong survival. Our goal was to make and test new radiolabelled antibodies that target two proteins that are abundant in cancer stem cells in leukemia, CD96, and in cancer cells under hypoxia, CAIX. The lead candidate with the best tumour uptake and image contrast was the anti-CAIX antibody c2C7 and will be evaluated as a therapeutic agent, and if successful, further evaluated in the clinic to aid against cancer relapse. vi Preface For Chapter 3, I performed part of each of the FACS evaluations, mAb-chelator conjugations, mAb radiolabelling and quality control processes, cell culture, tumour inoculations into mice, and biodistribution studies, as this was the project in which I was initially trained for the aforementioned experimental techniques. J Rousseau performed part of each of the above techniques with me as she trained me, and she was responsible for the 89Zr purifications. J Rousseau, H Merkens, J Zeisler, K Gitschtaler, and I performed the immunoreactivity assays. J Rousseau and N Colpo performed the radiotracer injections into mice and PET/CT and SPECT/CT image acquisitions. J Zeisler and I performed the internalization and retention assays. adMare Bioinnovations developed and provided the anti-CD96 mAb candidates. A version of Chapter 4 is currently being prepared for submission [HN Law, J Rousseau, PC McDonald, AE Lenferink, N Colpo, J Zeisler, R Tan, S Dedhar, KS Lin, F Bénard. Evaluation of novel anti-CAIX antibodies by PET imaging and biodistribution studies in a colorectal adenocarcinoma model]. In this work, I was the lead researcher on this project. I performed the FACS evaluations, mAb-chelator conjugations, part of the 89Zr purifications, 89Zr-mAb radiolabelling and quality control processes, cell culture, tumour inoculations into mice, and biodistribution studies. J Rousseau was responsible for part of the 89Zr purifications and performed the radiotracer injections into mice. J Rousseau and N Colpo performed the PET/CT image acquisitions. J Rousseau and R Tan aided in biodistribution studies. J Zeisler and R Tan performed the immunoreactivity assays. PC McDonald and AE Lenferink developed and provided the anti-CAIX mAb candidates. S Dedhar, KS Lin, and F Bénard were supervisory authors, with heavy input in experimental design. The manuscript is being drafted and will be finalized by me with critical input from J Rousseau, PC McDonald, AE Lenferink, S Dedhar, KS Lin, and F Bénard. vii For the CAIX in AML pilot study in Chapter 4, I performed the in vitro FACS experiments, tumour inoculations into mice, part of the ex vivo FACS assays, and part of the IHC studies. J Zeisler performed part of the ex vivo FACS assays. H Merkens performed part of the IHC studies. This research thesis was conducted under animal protocols A20-0113-002 (Preclinical Evaluation of Radiotracers for Cancer Imaging and Therapy) and A20-0114 (Radiolabelled Antibodies for Cancer Imaging and Therapy). These were approved by the Institutional Animal Care Committee of the University of British Columbia in compliance with the Canadian Council on Animal Care Guidelines. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................ v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................... xxi Dedication ................................................................................................................................. xxiv Chapter 1: Introduction ........................................................................................................... 1 1.1 Thesis Theme .................................................................................................................. 1 1.2 Nuclear Medicine ............................................................................................................ 2 1.2.1 Nuclear Imaging Modalities ....................................................................................... 2 1.2.1.1 Positron Emission Tomography .......................................................................... 2 1.2.1.2 Single Photon Emission Computed Tomography ............................................... 4 1.2.2 Radioimmunoconjugates............................................................................................. 5 1.2.2.1 Comparison of PET and SPECT for Radioimmunoconjugates .......................... 7 1.3 Targets of Interest ........................................................................................................... 8 1.3.1 CD96 as a Leukemic Stem Cell Marker ..................................................................... 8 1.3.2 Carbonic Anhydrase IX as a Tumour Hypoxia Marker .............................................. 9 ix 1.4 Hypothesis and Aims .................................................................................................... 11 Chapter 2: Materials and Methods ....................................................................................... 12 2.1 Monoclonal Antibodies ................................................................................................. 12 2.2 Monoclonal Antibody Radiotracers .............................................................................. 13 2.2.1 89Zr and 111In Chelator Conjugation to mAbs ........................................................... 13 2.2.2 89Zr and 111In Radiolabelling Processes for Chelator-Conjugated mAbs ................. 17 2.2.2.1 89Zr Production and Purification ....................................................................... 17 2.2.2.2 89Zr Radiolabelling of Chelator-Conjugated mAbs .......................................... 18 2.2.2.3 111In Radiolabelling of Chelator-Conjugated mAbs ......................................... 19 2.2.2.4 Quality Control Process for 89Zr- and 111In-labelled mAbs .............................. 19 2.3 Cell Culture ................................................................................................................... 21 2.4 In Vitro Evaluations ...................................................................................................... 23 2.4.1 Fluorescence-Activated Cell Sorting Assays ............................................................ 23 2.4.1.1 In Vitro Cell Preparation ................................................................................... 23 2.4.1.1.1 Additional Steps for CAIX in AML Pilot Study: Cell Line Screening ....... 23 2.4.1.2 Ex Vivo Cell Preparation .................................................................................. 23 2.4.1.2.1 Additional Steps for CAIX in AML Pilot Study: Analysis of Engrafted Tumours ..................................................................................................................... 24 2.4.1.3 Non-Fluorophore-Tagged mAbs of Interest ..................................................... 24 2.4.1.3.1 Additional Details for CD96 in Acute Myeloid Leukemia ......................... 25 2.4.1.3.2 Additional Details for CAIX in Renal and Colon Cancer ........................... 26 2.4.1.3.3 Additional Details for CAIX in AML Pilot Study: Cell Line Screening .... 26 x 2.4.1.3.4 Additional Details for CAIX in AML Pilot Study: Analysis of Engrafted Tumours ..................................................................................................................... 26 2.4.1.4 Fluorophore-Tagged mAbs of Interest.............................................................. 27 2.4.1.4.1 Additional Steps for CAIX in AML Pilot Study: Analysis of Engrafted Tumours ..................................................................................................................... 27 2.4.2 Immunoreactivity Assays.......................................................................................... 27 2.4.3 Internalization and Retention Assays........................................................................ 28 2.5 Immunohistochemistry Studies ..................................................................................... 29 2.6 In Vivo Evaluations ...................................................................................................... 32 2.6.1 Tumour Implantation ................................................................................................ 32 2.6.2 89Zr-PET/CT Imaging ............................................................................................... 33 2.6.3 111In-SPECT/CT Imaging ......................................................................................... 34 2.6.4 Biodistribution Studies.............................................................................................. 35 Chapter 3: Anti-CD96 mAbs for radioimmunotherapy of acute myeloid leukemia: evaluation of candidates by PET imaging ................................................................................ 36 3.1 Introduction ................................................................................................................... 36 3.2 Materials and Methods .................................................................................................. 36 3.3 Results and Discussion ................................................................................................. 36 3.4 Conclusion .................................................................................................................... 52 Chapter 4: CAIX as an additional target against tumour dormancy: radioimmunoconjugate screening in renal cancer, colon cancer, and acute myeloid leukemia models ............................................................................................................................... 53 4.1 Introduction ................................................................................................................... 53 xi 4.1.1 CAIX in Renal and Colon Cancer Models ............................................................... 53 4.1.2 CAIX in Acute Myeloid Leukemia Models – Pilot Study ........................................ 54 4.2 Materials and Methods .................................................................................................. 54 4.3 Results and Discussion ................................................................................................. 55 4.3.1 CAIX in Renal and Colon Cancer ............................................................................ 55 4.3.2 CAIX in Acute Myeloid Leukemia – Pilot Study ..................................................... 74 4.4 Conclusion .................................................................................................................... 80 Chapter 5: Conclusions .......................................................................................................... 82 5.1 Summary of Findings, Future Directions, and Significance ......................................... 82 5.2 Concluding Remarks ..................................................................................................... 83 Bibliography ................................................................................................................................ 85 xii List of Tables Table 2.1 Various cell lines used and their corresponding culture media. ................................... 22 Table 2.2 Primary antibodies applied for each tumour slice. ....................................................... 30 Table 2.3 Primary antibodies applied onto NSE1082 (CAIX-positive control) slide. ................. 30 Table 2.4 Summary of various cell lines and number of cells implanted into mice. .................... 33 Table 3.1 Immunoreactivity assay values for anti-CD96 tracers and corresponding in vivo biodistribution study tumour uptake values. ................................................................................. 39 Table 4.1 Immunoreactivity assay values for anti-CAIX tracers and corresponding in vivo biodistribution study tumour uptake values. ................................................................................. 57 Table 4.2 Summary of screening of 9 different AML cell lines by FACS. .................................. 74 xiii List of Figures Figure 1.1 Schematic representation of the detection of a generic PET radiotracer previously injected into a patient prior to entering scanner; 18F is an example radionuclide used to illustrate the positron-electron annihilation event.......................................................................................... 3 Figure 1.2 Schematic representation of the detection of a generic SPECT radiotracer in a patient; 111In is used as an example radioisotope to illustrate the detection of gamma rays. ...................... 5 Figure 1.3 Simplified depiction of radioimmunotherapy. .............................................................. 6 Figure 2.1 Schematic representation of the conjugation of the octadentate bifunctional chelator, mDFO*, to free thiol side chain groups of cysteine residues on an mAb, for subsequent 89Zr radiolabelling. ............................................................................................................................... 14 Figure 2.2 Schematic representation of the conjugation of the octadentate bifunctional chelator, BnDFO*, to free primary amino side chain groups of lysine residues on an mAb, for subsequent 89Zr radiolabelling. ........................................................................................................................ 15 Figure 2.3 Schematic representation of the conjugation of the octadentate bifunctional chelator, BnDTPA, to free primary amino side chain groups of lysine residues on an mAb, for subsequent 111In radiolabelling. ....................................................................................................................... 16 Figure 3.1 In vitro FACS results showed expression of CD96 target in cell lines of interest, HEK-CD96 and KG1α. .......................................................................................................................... 37 Figure 3.2 Preliminary PET imaging and biodistribution results for [89Zr]Zr-mDFO*-11F02 and [89Zr]Zr-mDFO*-OVA (anti-ovalbumin negative control mAb) in KG1α tumour-bearing NRG female mice. .................................................................................................................................. 41 xiv Figure 3.3 SPECT imaging and biodistribution results for [111In]In-BnDTPA-A070 and [111In]In-BnDTPA-Trastuzumab (anti-HER2 negative control mAb) in KG1α tumour-bearing NRG female mice. .............................................................................................................................................. 43 Figure 3.4 Biodistribution results 1 and 3 days after injection of [111In]In-BnDTPA-11F02, [111In]In-BnDTPA-9E05, or [111In]In-BnDTPA-14G11 in HEK-CD96 and HEK-WT tumor-bearing NRG female mice. ........................................................................................................... 44 Figure 3.5 Biodistribution results 1, 3, and 6 days after injection of [111In]In-BnDTPA-cCD96 (10.0 ± 1.2 µg and 1.6 ± 0.5 MBq per mouse) or [111In]In-BnDTPA-mIgG (negative control mAb, 11.8 ± 0.6 µg and 2.6 ± 2.8 MBq per mouse) in KG1α tumour-bearing NRG female mice. ....... 45 Figure 3.6 In vitro internalization assay results for 111In-labeled positive control mAbs and anti-CD96 mAbs of interest. ................................................................................................................ 47 Figure 3.7 In vitro retention assay results for 111In-labeled positive control mAbs and anti-CD96 mAbs of interest. ........................................................................................................................... 48 Figure 3.8 Ex vivo FACS (no injection) results showed expression of CD96 target in cell lines of interest. .......................................................................................................................................... 49 Figure 3.9 Ex vivo FACS (post-injection) results showed expression of CD96 target in AML cell line of interest. .............................................................................................................................. 50 Figure 3.10 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-11F02 and [89Zr]Zr-BnDFO*-Trastuzumab (anti-HER2 negative control mAb) in KG1α tumour-bearing nude female mice. .............................................................................................................................................. 51 Figure 4.1 In vitro FACS results showed expression of CAIX target in cell lines of interest, MDA-MB-231 CAIX+, SK-RC-52, and HT-29, and binding to mAbs of interest. ................................ 56 xv Figure 4.2 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*c11H9 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NRG female mice. ..................................................................... 59 Figure 4.3 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-c2C7 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NRG female mice. ..................................................................... 60 Figure 4.4 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-m4A2 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NRG female mice. ..................................................................... 61 Figure 4.5 SPECT imaging and biodistribution results for [111In]In-BnDTPA-c11H9 in HT-29 tumour-bearing NSG male mice, to try and replicate previous model results. ............................. 63 Figure 4.6 SPECT imaging and biodistribution results for [111In]In-BnDTPA-c11H9 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NSG male mice, to confirm previous 89Zr-mAb transfected model results. ................................................................................................................................ 64 Figure 4.7 PET imaging results for [89Zr]Zr-BnDFO*-Trastuzumab in SKOV3 tumour-bearing female nude† and NSG mice. ........................................................................................................ 66 Figure 4.8 Biodistribution results for [89Zr]Zr-BnDFO*-Trastuzumab in SKOV3 tumour-bearing female nude and NSG mice. ......................................................................................................... 67 Figure 4.9 PET imaging results for [89Zr]Zr-BnDFO*-c11H9 in HT-29 tumour-bearing male nude and NSG mice. .............................................................................................................................. 69 Figure 4.10 Biodistribution results for [89Zr]Zr-BnDFO*-c11H9 in HT-29 tumour-bearing male nude and NSG mice. ..................................................................................................................... 70 Figure 4.11 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-c11H9, -c2C7, and -hIgG1 in HT-29 tumour-bearing male nude mice. ....................................................................... 72 Figure 4.12 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-m4A2 in HT-29 tumour-bearing male nude mice. .................................................................................................. 73 xvi Figure 4.13 FACS results for HT-29 and the 3 most promising AML cell lines for CAIX expression. .................................................................................................................................... 75 Figure 4.14 Ex vivo FACS results for OCIAML3 and Molm13, with their in vitro counterparts for comparison. ................................................................................................................................... 76 Figure 4.15 Ex vivo FACS results for Molm13 and NOMO1, with their in vitro counterparts for comparison. ................................................................................................................................... 78 Figure 4.16 Immunohistochemistry results for CAIX expression in various slides. .................... 79 Figure 4.17 Immunohistochemistry results for pimonidazole and BrdU staining. ....................... 80 xvii List of Abbreviations %ID/g Percent injected dose per gram of tissue 2D 2-Dimensional 3D 3-Dimensional ACC Animal Care Committee ADC Antibody-drug conjugate AML Acute myeloid leukemia ARC Animal Resource Centre (at BCCRI) AUC Area under the curve BC Cancer British Columbia Cancer Centre (Vancouver location only in this thesis) BCCRI British Columbia Cancer Research Institute BnDFO* p-isothiocyanatobenzyl-deferoxamine-star BnDTPA 2-(p-isothiocyanatobenzyl)-cyclohexyldiethylenetriaminepentaacetic acid Bq Becquerel BrdU Bromodeoxyuridine BSA Bovine serum albumin CAIX Carbonic anhydrase IX CCAC Canadian Council on Animal Care cCD96 Commercialized anti-CD96 antibody CD96 Cluster of Differentiation 96 cG250 Girentuximab CPM Counts per minute CR Complete remission xviii CT Computed tomography DFO Deferoxamine DFO* Deferoxamine-star dH2O Deionized water DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide DTPA Diethylenetriaminepentaacetic acid EDTA Ethylenediaminetetraacetic acid eV Electron volt(s) FACS Fluorescence-activated cell sorting FBS Fetal bovine serum FDG Fluorodeoxyglucose FLT3 Fms-like tyrosine kinase 3 g Gram(s) GSC Canada’s Michael Smith Genome Sciences Centre h Hour(s) HCl Hydrochloric acid HiDAC High-dose cytarabine (also called cytosine arabinidose or Ara-C) HIF Hypoxia-inducible factor hIgG1 Human immunoglobulin G1 HPLC High performance liquid chromatography HSC Hematopoietic stem cell IHC Immunohistochemistry xix IMDM Iscove’s Modified Dulbecco’s Medium iTLC-SG Instant thin layer chromatography silica gel L1 Left ear punch LSC Leukemic stem cell M Molar mAb Monoclonal antibody mDFO* Maleimide-deferoxamine-star mIgG2b Mouse immunoglobulin G2b min Minute(s) mL Millilitre MRD Measurable residual disease MRI Magnetic resonance imaging NaOH Sodium hydroxide NH4OAc Ammonium acetate NP No ear punch NRC National Research Council of Canada NRG NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ NSG NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ OSEM3D 3-Dimensional ordered-subsets expectation maximization OVA Ovalbumin (used as an abbreviation for the anti-ovalbumin negative control mAb) pAb Polyclonal antibody PBS Phosphate-buffered saline (pH 7 unless otherwise stated) PET Positron emission tomography xx PG Proteoglycan pHi Intracellular pH p.i. Post-injection RCC Radiochemical conversion RCP Radiochemical purity RIT Radioimmunotherapy RL Right and left ear punch RPMI Roswell Park Memorial Institute Medium s Second(s) SCT Stem-cell transplantation SD Standard deviation SEC Size-exclusion chromatography SPECT Single photon emission computed tomography SUV Standardized uptake values TLC Thin layer chromatography UBC University of British Columbia VHL von Hippel-Lindau tumour suppressor gene WT Wildtype xxi Acknowledgements First and foremost, I would like to thank my supervisor, Dr. François Bénard, for providing me with the opportunity to pursue my graduate degree in such a wonderfully interdisciplinary research group with some of the best people I have had the pleasure of working with. I am so fortunate to have had such a kind and humble supervisor with such extensive knowledge and experience in the nuclear medicine field, as well as inspiring leadership as not just our Principal Investigator but also as the Vice President of Research at BC Cancer. Thank you for all your mentorship and guidance for the past three years. I would also like to thank the other members of my supervisory committee, Drs. Urs Häfeli and Paul Schaffer, for their expertise and advice at our many meetings, especially when my projects were not going as expected and needed troubleshooting. Your support throughout this research is highly appreciated. Moreover, I need to express my gratitude to all of our past and current Bénard-Lin lab members, who have become my work family in my past three years at BC Cancer: Firstly, I am so grateful that Dr. Julie Rousseau agreed to take me under her wing from Day 1 of my program to be my in-lab mentor. I have learned so much about research and also about life with the countless hours we have spent together in the past three years. Your positivity and encouragement have helped me through some trying times in this program. Thank you for all of your time and effort (often going above and beyond) throughout my degree to make this thesis happen. Secondly, I need to give thanks to the others who have made the work in this thesis possible: Thank you Nadine for acquiring many of the images in my data sets. Thank you Helen, xxii Jutta, Katrin, and Ruiyan for helping me perform many of the biological assays when I couldn’t be in two places at once during busy experimental days. Thank you Johnson, Chengcheng, and Aron for your advice and support in teaching me more about the chemical aspects of my projects. Thirdly, I would like to thank everyone else in our lab for their friendship and support. Graduate school is tough and requires relentless spirit, and it wouldn’t have been possible without our conversations and laughs during downtime and after work. I would also like to thank all of our collaborators at adMare, Dr. Steven Jones’ group at the Genome Sciences Centre, Dr. Shoukat Dedhar’s group at BC Cancer, Dr. Anne Lenferink at the National Research Council, and Dr. Florian Kuchenbauer’s group at BC Cancer. Without all their hard work, none of my projects would have been possible! Out of these groups, I would also like to specifically acknowledge Dr. Patrick Chan, Dr. Paul McDonald, and Leo Escano for personally providing me with some of the materials I needed for experiments so the data in this thesis could be generated! I would also like to acknowledge the members of the BCCRI Animal Resource Centre (ARC), Terry Fox Laboratory Flow Cytometry Core Facility, and BC Cancer (Vancouver) cyclotron facility for their support in making all my studies possible. Of course, my acknowledgements wouldn’t be complete without addressing my own family. Thank you to my parents for giving me everything I needed in my life to achieve this career milestone, and always being supportive of my aspirations. Also, thank you to my partner and best friend, Alex, for getting me through the pandemic and this degree in one piece – both incredibly challenging times! xxiii Lastly, I would like to acknowledge all the mice that were sacrificed in the name of science for this thesis. I was humbly reminded of this important message by a plaque hanging on the wall opposite to the air shower entrance of the ARC, dedicated to the mice whose lives were lost within its walls. It is seldom mentioned in published scientific work that the advancement of medicine in our society rests not only upon the shoulders of the scientists that came before us, but also on those of the animals that received the earliest versions of said medicine. Dr. Julie Rousseau gave me this lesson at the very beginning of my degree: The moment we stop feeling sad and remorseful when we euthanize these creatures, is the day we should stop performing our work. As scientists, we must never lose our ethics and conscience in pursuit of information. With this lesson in mind, I owe them a debt of gratitude in my journey to contribute knowledge to the field of medicine. xxiv Dedication To my parents – I would not have gotten this far in my career without all your sacrifices, your love, and your wonderful, delicious food. And to Alex, the love of my life – I would not have gotten through the pandemic and to the end of this degree without you. You kept me calm, sane, and most importantly, loved. 1 Chapter 1: Introduction 1.1 Thesis Theme The advancement of targeted therapy and immunotherapy has brought and continues to bring optimism to cancer patients and their families. Unfortunately, even after successful treatment and removal of the primary tumour in question, some patients present with metastatic relapse and further drug resistance. To put this problem into perspective, 90% of cancer-related deaths is due to metastatic disease1,2. This phenomenon of cancer recurrence could be explained by the concept of cancer cell dormancy or quiescence, two terms that have been used interchangeably in literature and will be used synonymously in this thesis. Cancer cell dormancy is a reversible state whereby cells have undergone G0-G1 cell cycle arrest and are non-proliferating3,4. These cells can recover the ability to self-renew with exposure to reopened or new vasculatures, and may be the founders of metastasis5. One factor that may promote cancer cells to enter a dormant state is exposure to hypoxia, which not only limits cellular proliferation but also promotes drug resistance6,7. In addition, some dormant cancer cells have been reported to express “stem-ness” markers that may provide these cells with stem cell-like characteristics to prolong survival8-13. Thus, this thesis aims to describe work developing radiotracers to target certain cancer stem cell and hypoxia-induced markers, in efforts to potentially identify and eradicate dormant tumour cells, adding to the clinical arsenal against metastatic relapse and drug resistance in cancer patients. 2 1.2 Nuclear Medicine Nuclear medicine is the medical specialty within which the work in this thesis resides. This field utilizes radiopharmaceuticals for both diagnostic and therapeutic purposes, the former of which will be the focus in following chapters14. For clarity, the terms radiopharmaceutical and radiotracer will be used interchangeably in this thesis, but otherwise the term radiopharmaceutical usually denotes being formulated for human use. The design of a radiotracer allows the pharmaceutical component to preferentially localize within certain tissues, while the radionuclide component emits radiation to either image or treat the targeted region14. 1.2.1 Nuclear Imaging Modalities There are two types of radioisotopes used in nuclear imaging: positron emitters (for positron emission tomography, PET) and single photon emitters (for single photon emission computed tomography, SPECT). Both PET and SPECT are non-invasive functional imaging techniques used in preclinical and clinical settings that allow real-time collection of physiological information from a living subject, providing expression and metabolic information that can be used to deduce and monitor disease progression15. The pairing of computed tomography (CT) functionality with either PET or SPECT provides anatomical information in image overlays to aid in diagnosis, staging, and therapy monitoring16. 1.2.1.1 Positron Emission Tomography Positron emission tomography (PET) is a nuclear imaging modality that utilizes radiopharmaceuticals containing a positron (β+)-emitting radionuclide. Commonly used PET isotopes include 15O, 13N, 11C, 18F, 68Ga, and 89Zr, the choice of which depends on compatibility between the half-life of the radioisotope and the biological half-life of the pharmaceutical 3 component15. When a PET radiotracer is injected into a patient, usually intravenously, there is a waiting period for the tracer to localize into the tissue of interest. Thereafter, the patient is transferred to a PET scanner, which is often paired with a CT or magnetic resonance imaging (MRI) scanner to provide helpful anatomical information. As the radionuclide decays, each emitted positron travels a short distance before encountering an electron within the patient, leading to an annihilation event that results in two 511 keV (gamma) photons simultaneously emitted at an approximate 180° angle from each other (Figure 1.1)17. The two photons are detected in coincidence by the detectors that surround the patient, and the signals are reconstructed by computer algorithms to create a 3D image17. In the clinical setting, PET is widely considered to be the most sensitive imaging modality, providing quantitative information such as activity per tissue volume (kBq/mL) versus time or standardized uptake values (SUV)18. Figure 1.1 Schematic representation of the detection of a generic PET radiotracer previously injected into a patient prior to entering scanner; 18F is an example radionuclide used to illustrate the positron-electron annihilation event. 4 The gold standard of PET radiopharmaceuticals is [18F]fluorodeoxyglucose ([18F]FDG), which is used for the majority of PET scans. As cancer cells have higher metabolic and glycolytic rates than normal cells, [18F]FDG utilizes the Warburg effect for tumour cell uptake19. However, the caveat is that [18F]FDG is not specific to any type of cancer and inflammatory cells also use glucose, lowering imaging specificity, leaving a niche for development of more targeted imaging probes20. 1.2.1.2 Single Photon Emission Computed Tomography Single photon emission computed tomography (SPECT) is a nuclear imaging modality that utilizes radiopharmaceuticals tagged with single photon (gamma, γ) emitting radioisotopes. Commonly used SPECT isotopes include 67Ga, 99mTc, 123I, and 111In, the choice of which, like PET, also reflects the pairing of the half-life of the radioisotope to the biological half-life of the pharmaceutical component14. Similar to the administration of PET tracers, SPECT tracers are also usually injected intravenously into patients prior to scanning. However, the detection of SPECT isotopes is remarkably different, as detailed below. The detector, called the Anger scintillation (gamma) camera, rotates around the patient to acquire SPECT images at defined points typically every 3-6 degrees (Figure 1.2)14. Unlike in PET, the tracers used in SPECT emit gamma radiation that is uniformly distributed over a spherical geometry14. Thus, a multi-hole aperture or a collimator is used to determine the directionality of these gamma rays before they interact with the scintillation crystal14. Unfortunately, this means that many of the gamma rays that do not travel in the direction of the collimator holes are lost. Approximately 1 out of every 100,000 photons is detected by the detector system, an inefficient process that contributes to its lower sensitivity in the clinical setting compared to PET14. 5 Figure 1.2 Schematic representation of the detection of a generic SPECT radiotracer in a patient; 111In is used as an example radioisotope to illustrate the detection of gamma rays. The resulting 2D images obtained by the gamma camera are then processed by a tomographic reconstruction algorithm to yield a 3D representation, also often coupled with CT for anatomical information14. 1.2.2 Radioimmunoconjugates The interest in using antibodies or antibody derivatives for diagnosis and radionuclide therapy of cancer has been revitalized due to recent developments of several high-affinity monoclonal antibodies (mAbs) as cancer therapeutics, the identification of novel cancer-associated proteins and splice-variants, mAb-cytotoxic drug conjugates, and novel isotope production and radiolabeling methods for PET imaging of antibodies21-25. In addition, the affinity of an antibody for its target protein makes radiolabeled antibodies a valuable tool for imaging studies, which can aid in the development of therapeutic mAbs such as radioimmunoconjugates or antibody-drug conjugates (ADCs). 6 One therapeutic strategy using antibodies is radioimmunotherapy (RIT), an innovative approach that aims to treat localized, metastatic, or diffuse tumors while sparing healthy tissues (Figure 1.3)26. Figure 1.3 Simplified depiction of radioimmunotherapy. RIT utilizes radioisotopes (α, β, or Auger electron emitters) to label mAbs, delivering damaging radiation to tumor cells by targeting specific tumor-associated antigens with high affinity26,27. The mAbs can also be labeled with positron- or γ-emitters for imaging prior to treatment in order to predict the efficacy of the therapeutic counterpart27-29. In this context, PET (using 89Zr) or SPECT (using 111In) can be used27-29. Imaging is also a powerful tool in preclinical studies to investigate potential targets and screen mAbs for potential therapeutic development, both as ADCs and for RIT28,29. 7 1.2.2.1 Comparison of PET and SPECT for Radioimmunoconjugates Due to the widespread availability of gamma cameras in hospitals, as well as cost and availability of SPECT isotopes, single photon emitters are widely used in the clinic. However, SPECT imaging results in low sensitivity and relatively poor spatial resolution due to the required collimation to create images from single photon emitters, decreasing small tumour detection rate. The radioisotope classically used for mAb labeling for SPECT is 111In, which has a half-life of 2.8 days, making it compatible with the biological half-life of antibodies30. In contrast, PET imaging results in higher spatial resolution and sensitivity due to coincidence detection of simultaneously emitted photons from positron emitting radioisotopes (almost exactly 180° apart), producing higher quality images that are superior to those from SPECT and improving diagnostic accuracy. The first positron emitting radioisotope for radiolabeling mAbs for PET imaging was 124I, leading to the development of mAb imaging agents such as 124I-cG250, but now 89Zr has emerged as a popular radioisotope for the same purpose31-38. The 3.3-day half-life of 89Zr is suitable with the biological half-life of antibodies, and its short positron emission range in tissues prior to annihilation leads to high image quality and spatial resolution. Compared to clinical imagers, preclinical SPECT scanners have significantly higher spatial resolution due to highly focused multi-pinhole collimators being used (0.4 mm spatial resolution for the MILabs USPECT/CT II scanner at the British Columbia Cancer Research Institute (BCCRI), versus 1.3 mm for the Inveon preclinical PET/CT scanner), and so both 111In and 89Zr bring valuable contributions to radiolabeled antibody development. 111In provides high resolution imaging in rodents, while 89Zr contributes to clinical translation into human studies, where PET imaging is superior to conventional clinical nuclear imaging. 8 1.3 Targets of Interest 1.3.1 CD96 as a Leukemic Stem Cell Marker The cancer of interest for targeting a cancer stem cell marker in this thesis is acute myeloid leukemia (AML), the most common type of acute leukemia39,40. The current standard of care is typically the “3 + 7” induction chemotherapy regimen, which is 3 days of daunorubicin and 7 days of cytarabine, with 5-year survival rates ranging from 10-45% depending on the age of the AML patient41. The addition of other approved agents in AML, such as gemtuzumab ozogamicin (anti-CD33 monoclonal antibody with a calicheamicin payload) for CD33+ AML or midostaurin (fms-like tyrosine kinase 3 or FLT3 inhibitor) for FLT3+ AML, helps increase long-term survival rates41. Also, the detection of measurable residual disease (MRD) is monitored for patients in complete remission (CR)41. After achieving CR, patients with a low risk of relapse can receive consolidation chemotherapy with high-dose cytarabine (also called cytosine arabinidose or Ara-C) or “HiDAC” to target remaining cancer cells41. Patients with a high risk of relapse (due to adverse cytogenetic abnormalities, high FLT3-mutation allelic ratio, or persistent MRD >0.1% in CR after first consolidation) are considered for allogeneic hematopoietic stem-cell transplantation (SCT)41. Salvage therapy is offered to patients who are refractory to initial induction chemotherapy, and once they achieve CR, they are immediately considered for SCT41. As part of pre-SCT conditioning in transplant-eligible patients, ongoing studies are evaluating AML surface antigen-targeting antibodies for RIT to achieve significant myeloablation41. Currently, the most clinically advanced radioimmunoconjugates are CD45-targeting antibodies (e.g. Iomab-B or 90Y-BC8-DOTA), for which Iomab-B is being evaluated in 9 a randomized phase III study against investigator choice salvage therapy prior to SCT for patients with relapsed-refractory AML (NCT02665065)41. However, the effective eradication of leukemic stem cells (LSCs), the self-renewing component of AML, continues to be a barrier to a permanent cure for most patients undergoing chemotherapy alone42-44. CD96 (Cluster of Differentiation 96), originally discovered in 1992 and named “T cell activation, increased late expression”, was identified as a cell surface marker that distinguishes LSCs from normal hematopoietic stem cells (HSCs)42,45-48. The functional role of CD96 in AML-LSC biology is largely unknown, but it has been detected to act upon interaction with CD155 as inhibitors of T and NK cells48. Furthermore, the inhibition of CD96 using mAbs has been shown to improve tumor control in mice49. In several studies, it was shown that increased expression of CD96 correlated with poor prognosis and enhanced resistance to chemotherapy, further supporting CD96 as a diagnostic marker48. In addition, CD96 is expressed on 74.0% of CD34+CD38- AML cells while only weakly expressed on 4.9% of cells in the normal HSC-enriched population, making this antigen a promising candidate for therapeutic targeting42. Therefore, a potential treatment strategy would be to separate CD96-expressing stem cells prior to autologous transplantation of AML patients48. Thus, development of RIT against CD96 has the potential to be a powerful, novel addition to the therapeutic arsenal for AML. 1.3.2 Carbonic Anhydrase IX as a Tumour Hypoxia Marker Another avenue to target against dormant cancer cells is tumour hypoxia, which is known to be a negative prognostic factor associated with increased resistance against radiation therapy and conventional chemotherapy50-52. When deprived of oxygen, hypoxia-inducible factor (HIF)-10 activated tumour cells undergo a change in metabolism via increased glycolytic rates, producing abnormally high levels of acidic metabolites that lower intracellular pH (pHi)53. To maintain pHi homeostasis for cell survival and proliferation, cancer cells overexpress a surface membrane protein called carbonic anhydrase IX (CAIX), upregulated by hypoxia and HIF-1α53-56. CAIX was discovered by Pastorekova et al. as a surface membrane protein in the HeLa human cervical carcinoma cell line and is one of 15 different zinc metalloenzymes in the α-family of carbonic anhydrases57,58. Out of these isoforms, CAIX is the one most strongly associated with cancer52,59. To enable cancer cell survival, CAIX catalyzes the reversible hydration of carbon dioxide to bicarbonate and hydrogen ions (H2O + CO2 ↔ HCO3- + H+)60. The HCO3- ions produced are carried into the cell via ion transporter systems to return the intracellular environment to a neutral or slightly alkaline pHi, while H+ ions acidify the tumour microenvironment to promote invasion and metastasis61. To further highlight its importance, CAIX is expressed in up to 60% of hypoxic areas in tumours without a mutation in the von Hippel-Lindau (VHL) tumour suppressor gene, which normally acts as a negative regulator of HIF-1α, and at >85% in tumours with the VHL mutation62-64. For some cancers, the loss of VHL functionality through mutations, loss of heterozygosity, or epigenetic silencing leads to constitutive HIF-1α activation and thereby CAIX expression, even without hypoxia65,66. CAIX is also an attractive RIT target to investigate while minimizing radiation exposure to healthy tissues, versus other hypoxia tracers (e.g. [18F]FMISO)67,68. CAIX is expressed at low levels in normal tissues with the exception of the small intestine, while overexpressed in many solid cancers including renal and colon, contributing to adhesion, migration, invasion, and metastasis64,69-77. Various molecules targeting CAIX (mAbs, peptides, small molecule inhibitors, 11 antibody mimetics) have been investigated for imaging and therapeutic purposes, notably the chimeric mAb girentuximab (cG250)78. While [177Lu]Lu-cG250 RIT has been shown to stabilize progressive disease, transient hematological toxicity was observed in patients78. Thus, there is still a necessity for novel anti-CAIX mAb candidates and strategies. 1.4 Hypothesis and Aims Hypothesis: Leukemic stem cell- and hypoxia-specific markers can be targeted for cancer treatment and/or theranostic applications to help identify areas of tumour dormancy, aiding against drug resistance and patient relapse. Aim 1: Evaluate radioimmunoconjugate candidates against CD96 for acute myeloid leukemia by PET imaging. Aim 2: Screen mAbs targeting CAIX by PET imaging in renal cancer, colon cancer, and acute myeloid leukemia models. 12 Chapter 2: Materials and Methods 2.1 Monoclonal Antibodies The mAbs described below were provided in Dulbecco’s Phosphate-Buffered Saline (PBS; Gibco, 14190-144) and used as-is unless otherwise stated. The novel anti-CD96 mAbs of interest, namely 11F02 (CDRD-0149-002; chimeric mouse/human or Ms/Hu), 9E05 (CDRD-0148-001; Ms/Hu), 14G11 (CDRD-0149-001; Ms/Hu), and A070 (CDRD-0178-001; humanized) were developed and provided by Dr. Peter Bergqvist’s team at adMare Bioinnovations (adMare; previously named CDRD) in collaboration with Dr. Steven Jones’ group at Canada’s Michael Smith Genome Sciences Centre (GSC). The anti-ovalbumin (OVA) negative control mAb (CDRD-0012-007, Ova4C6-12) was also obtained from adMare. The commercially available murine anti-CD96 mAb (cCD96) was obtained from R&D Systems (MAB6199). The humanized anti-CD33 mAb was purchased from BioVision (A1443) in PBS with 0.02% Proclin 300 preservative and was buffer-exchanged by adMare into PBS prior to usage. The novel anti-CAIX mAbs of interest, namely c11H9 (Ms/Hu), c2C7 (Ms/Hu), and m4A2 (murine) were developed and provided by Dr. Shoukat Dedhar’s group (BC Cancer) in collaboration with Dr. Anne Lenferink (National Research Council (NRC) of Canada). The cG250 mAb was kindly provided by Dr. Paul McDonald in Dr. Dedhar’s group. The isotype control human IgG1 mAb (hIgG1) used in the in vivo study in nude mice was purchased from BioLegend (403502), while the isotype control mouse IgG2b mAb (mIgG2b) to be used in a similar study was purchased from BioXCell (MPC-11). 13 Trastuzumab (common trade name Herceptin) was obtained from Genentech and required purification prior to usage. Approximately 100 mg of Trastuzumab was dissolved in 30 mL PBS, and filtered using Amicon Ultra 15mL 50 kDa molecular weight cut-off centrifugal filters (Millipore, UFC905024). 2.2 Monoclonal Antibody Radiotracers This section aims to describe the conjugation of 89Zr and 111In chelators to mAbs, followed by the process of 89Zr and 111In radiolabelling and quality control, to produce radioimmunoconjugates for either in vivo or in vitro studies in subsequent chapters. 2.2.1 89Zr and 111In Chelator Conjugation to mAbs Three different chelator types were used in the mAb experiments described in subsequent chapters, two of which were suitable for 89Zr complexation and the third for 111In complexation. To prepare for 89Zr radiolabelling, maleimide-deferoxamine-star (mDFO*; source undisclosed by adMare) or p-isothiocyanatobenzyl-deferoxamine-star (BnDFO*; Pharma Inventor Inc., PI-A-1003-31-3) chelators were conjugated to mAbs beforehand (Figure 2.1 and Figure 2.2). The maleimide group of mDFO* forms a thiosuccinimide bond with the free thiol side chains of cysteine residues on an mAb, while the isothiocyanate group of BnDFO* forms a thiourea bond with the free primary amino side chain groups of lysine residues. The 89Zr4+ metal ion prefers the formation of octadentate complexes and thereby is stably chelated by DFO* via four hydroxamate groups, which is one more hydroxamate group than its predecessor, deferoxamine (DFO)79. The stable complexation minimizes demetallation and bone accumulation in mice for [89Zr]Zr-immunoPET imaging80-85. 14 In preparation for 111In radiolabelling, the 2-(p-isothiocyanatobenzyl)-cyclohexyldiethylenetriaminepentaacetic acid (p-SCN-Bn-CHX-A”-DTPA or BnDTPA) chelator (Macrocyclics, B-355) was used for conjugation (Figure 2.3). The isothiocyanate group of BnDTPA reacts as described above with lysine residues. The 111In3+ metal ion forms very thermodynamically stable complexes with DTPA via full octadentate coordination in a square antiprismatic geometry. The stability is contributed by the cyclohexyl backbone of BnDTPA, providing rigidity and a degree of preorganization on the metal ion binding site83-87. Figure 2.1 Schematic representation of the conjugation of the octadentate bifunctional chelator, mDFO*, to free thiol side chain groups of cysteine residues on an mAb, for subsequent 89Zr radiolabelling. 15 Figure 2.2 Schematic representation of the conjugation of the octadentate bifunctional chelator, BnDFO*, to free primary amino side chain groups of lysine residues on an mAb, for subsequent 89Zr radiolabelling. 16 Figure 2.3 Schematic representation of the conjugation of the octadentate bifunctional chelator, BnDTPA, to free primary amino side chain groups of lysine residues on an mAb, for subsequent 111In radiolabelling. The mDFO* conjugation was performed by adMare with an undisclosed experimental method, and will not be described here. However, both BnDFO* and BnDTPA conjugations were performed at BCCRI, with a similar protocol using a 5:1 chelator-to-mAb molar ratio. Each mAb was added to PBS (brought to pH 8.9-9.1 using 1 M NaOH) for a 2 mg/mL final mAb concentration. Each chelator was resuspended from powder form in DMSO and added to the mAb-PBS solution (at <2% DMSO in total volume). The final mixture was then placed securely on a shaker in a 37°C incubator for 1 h. After incubation, the mixture was purified using 50 kDa molecular weight cutoff Amicon spin filters (Millipore, UFC505024) to remove any unconjugated chelator. The chelator-mAb conjugates were washed once with PBS, once with the appropriate radiolabelling buffer (5 mg/mL dihydrobenzoic acid in 0.25 M sodium acetate for 89Zr; 0.15 M ammonium acetate pH 6 for 111In), then resuspended in the same radiolabelling buffer for storage 17 at -20°C up to 7 days before radiolabelling. The concentrations of purified immunoconjugates were obtained using a Bradford assay kit according to the manufacturer’s recommendations (Sigma-Aldrich, St. Louis, MO, USA). 2.2.2 89Zr and 111In Radiolabelling Processes for Chelator-Conjugated mAbs This section aims to describe the different processes for 89Zr and 111In radiolabelling, with a detailed section dedicated to explaining the in-house 89Zr production and purification protocols. Despite their differences, both 89Zr and 111In radiolabelling utilize an almost identical quality control process prior to radioimmunoconjugate usage for both in vivo and in vitro studies. 2.2.2.1 89Zr Production and Purification At the British Columbia Cancer (BC Cancer; Vancouver) Cyclotron Facility, 89Zr was produced from proton irradiation on an 89Y-coated coin (Part ID TA-2167, Advanced Cyclotron Systems Inc. (ACSI) in Richmond, Canada) in a TR-19 cyclotron (ACSI) at 13 MeV. The coin was then transferred into a 50 mL Falcon tube and dissolved in 10 mL of 2 M hydrochloric acid (HCl) to incubate for 1 h at room temperature. During the incubation period, 100-150 mg of Zr resin (Triskem) was suspended in Milli-Q water and left under agitation (rotor) for at least 30 min. The suspended Zr resin was then transferred to a 4 mL reservoir containing a previously wetted frit and topped with a second frit. The Zr resin was then washed with 4 mL of Milli-Q water and conditioned with 4 mL of 2 M HCl in preparation for 89Zr purification. After incubation, the HCl containing dissolved 89Zr was retrieved in a syringe and loaded onto the previously described Zr resin. The resin was then washed with 10 mL of 2 M HCl and 10 18 mL of Milli-Q water. To elute the purified 89Zr, 0.5 mL of freshly prepared 0.05 M oxalic acid was loaded onto the resin and pushed to the bottom to incubate for 3 min. After incubation, the purified 89Zr in oxalic acid was collected into a 1.5 mL glass vial. The elution step with oxalic acid was repeated once to provide two collections of 89Zr. 2.2.2.2 89Zr Radiolabelling of Chelator-Conjugated mAbs The amounts of 89Zr and chelator-conjugated mAbs used for each radiolabelling varied depending on the amount of time points, mice, and mAbs available for each study. There is an expected 70% radiochemical conversion (RCC) from 89Zr-DFO* radiolabelling and 80% PD-10 purification column (GE Healthcare, GE17-0851-01) yield, so the amounts of 89Zr and chelator-conjugated mAbs used in the radiolabelling process were in excess of the amounts of radioimmunoconjugate needed for each study. Before radiolabelling, the previously eluted 89Zr in 0.05 M oxalic acid was neutralized with 0.1 M sodium carbonate (Na2CO3) at a 0.45:1 ratio of 0.1 M Na2CO3 to 0.05 M oxalic acid, and incubated for 3 min at room temperature80. The amount of chelator-conjugated mAb added depended on the minimum specific activity for 89Zr-PET imaging at 0.2 MBq/µg. After addition of chelator-mAb conjugate (suspended in 5 mg/mL dihydrobenzoic acid in 0.25 M sodium acetate to prevent radiolysis), the final volume of the reaction was reached by topping up with PBS at an average of 0.26 ± 0.10 mg/mL mAb. The resulting solution was incubated for 1 h at room temperature. 19 2.2.2.3 111In Radiolabelling of Chelator-Conjugated mAbs 111InCl3 in 0.1 M HCl was purchased from BWX Technologies Inc. and used as provided. The amounts of 111In and chelator-mAb conjugates used varied for the same reasons mentioned in section 2.2.2.2, with an expected RCC of 80% for 111In-DTPA radiolabelling. The chelator-conjugated mAb in 0.15 M ammonium acetate (NH4OAc) pH 6 buffer was added directly to the 111InCl3 in a 0.1 M HCl, at 0.5 MBq/µg, the minimum specific activity for 111In-SPECT imaging. The final volume of the reaction was reached by topping up with more 0.15 M NH4OAc pH 6 buffer at an average of 0.46 ± 0.12 mg/mL mAb. The resulting solution was then incubated for 1 h at room temperature. 2.2.2.4 Quality Control Process for 89Zr- and 111In-labelled mAbs To purify the radioimmunoconjugates from free radioisotope, the radiolabelling reaction solution was loaded onto PD-10 desalting columns (Sephadex G-25 M, 50 kDa, GE Healthcare, London, United Kingdom), which were previously conditioned with PBS prior to use. All solutions were then topped up to 2.5 mL with PBS. Generally, four fractions were eluted with the same buffer into separate 15 mL Falcon tubes: 0.5 mL for the first, 1 mL for the second, 0.5 mL for the third, and 1.5 mL for the fourth. This process usually yielded most of the radioimmunoconjugates in the second and third fractions. RCCs and RCPs were determined by instant thin layer chromatography silica gel (iTLC-SG, Agilent Technologies, Santa Clara, CA, USA) with either 50 mM DTPA pH 7 (for 89Zr-labelled mAbs) or 50 mM EDTA pH 5 (for 111In-labelled mAbs) as the eluent. The iTLC-SGs were read by a Raytest miniGita with Beta GMC detector radio-TLC plate reader, and the resulting chromatograms were analyzed by TLC control Mini Ginastar software. It was expected that 20 radiolabelled mAbs remained at baseline, while free radioisotope complexed as either 89Zr-DTPA or 111In-EDTA were eluted with the solvent front. Specifically, the software enabled the integration of areas under the curve (AUCs) for each peak to determine the total AUC. The fraction of total AUC for each peak was computed to give an RCC% or RCP%. To ensure that the system produced reliable signals (i.e. to test the solvent and to make sure future samples can run past baseline), pure radioisotope was first run on the iTLC-SG in the appropriate eluent and determined to be at the solvent front prior to running subsequent iTLC-SGs in the same eluent. This makes certain that if we only see signals at baseline for radioimmunoconjugate samples that it is truly >99% RCC or RCP. Afterward, iTLC-SGs were performed on the crude reaction and the eluted fractions from the PD-10 column. The specific activity of radiolabelled mAbs were determined by a size-exclusion chromatography (SEC) column (Phenomenex, BioSep-SEC-s-3000) on an Agilent High Performance Liquid Chromatography (HPLC) system equipped with a model 1200 quaternary pump, a model 1200 UV absorbance detector (set at 280 nm), and a Bioscan NaI scintillation detector (Washington, DC). The radiodetector was connected to a Bioscan B-FC-1000 flow-count system, and the output from the Bioscan flow-count system was fed into an Agilent 35900E interface, which converted the analog signal to a digital signal. Each 20 min run used an isocratic elution of 0.1 M sodium phosphate monobasic dehydrate, 0.1 M sodium phosphate dibasic dodecahydrate, 0.1 M sodium azide, and 0.15 M sodium chloride (pH 6.2-7.0). The radioimmunoconjugate was expected to elute between 8 and 10 min. A known amount of mAb was injected under the same conditions to determine detector response, from which the AUC of the radioimmunoconjugate could be converted into mass of mAb injected. Based on the 21 measurement of the HPLC syringe pre- and post-injection, the activity injected was determined, which combined with the mAb mass injected gives the specific activity (MBq/µg). 2.3 Cell Culture All cell lines were cultured and maintained in a humidified incubator (5% CO2, 37°C) in various media (Life Technologies, Table 2.1) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, Oakville, ON), 100 I.U./mL penicillin (Life Technologies), and 100 µg/mL streptomycin (Life Technologies). IMDM = Iscove’s Modified Dulbecco’s Medium (Gibco, 12440-053), DMEM = Dulbecco’s Modified Eagle Medium (Gibco, 10566-016), RPMI = Roswell Park Memorial Institute Medium (Gibco, 61870-036). For adherent cells, passaging was performed by washing cells with PBS, then using 0.25% Trypsin-EDTA (1X, Gibco, 25200-056) as a dissociation reagent to lift the cells, and subsequently quenched with the appropriate cell culture medium. In this thesis, 0.25% Trypsin-EDTA will be referred to as trypsin. 22 Table 2.1 Various cell lines used and their corresponding culture media. Target Protein Cell Line Cell Type Culture Medium CD96 KG1α Human acute myeloid leukemia IMDM CD96 HEK293-CD96 (HEK-CD96) Human embryonic kidney cell, CRISPR-modified to express CD96 DMEM Negative control HEK293 (HEK-WT) Human embryonic kidney cell DMEM CAIX MDA-MB-231 CAIX KO +CAIX Human breast adenocarcinoma, CRISPR-modified to express CAIX DMEM Negative control MDA-MB-231 CAIX KO Human breast adenocarcinoma, CRISPR-modified to remove CAIX expression DMEM CAIX HT-29 Human colorectal adenocarcinoma DMEM + 1% non-essential amino acid solution CAIX SK-RC-52 Human renal cell carcinoma RPMI HER2 SKOV3 Human ovarian adenocarcinoma DMEM Cell lines used in the pilot study for determining CAIX expression in acute myeloid leukemia were provided by Leo Escano from Dr. Florian Kuchenbauer’s research group. 23 2.4 In Vitro Evaluations 2.4.1 Fluorescence-Activated Cell Sorting Assays 2.4.1.1 In Vitro Cell Preparation Adherent cells were harvested using cell-dissociation buffer (Gibco, 13150-016) instead of trypsin to prevent cleavage of target proteins and quenched with their respective media as described in section 2.3. Suspension cells were harvested as-is in media. Cells were washed twice with fluorescence-activated cell sorting (FACS) buffer (PBS + 2% FBS + 0.02% sodium azide (NaN3)) and seeded at a 250,000-1,000,000 cells/200 µL concentration per well in a V-bottom 96-well polystyrene plate. 2.4.1.1.1 Additional Steps for CAIX in AML Pilot Study: Cell Line Screening For screening of 9 different AML cell lines for CAIX expression, additional steps were performed prior to harvesting cells for FACS. Two 10 cm dishes of each cell line were kindly provided by Leo Escano from Dr. Florian Kuchenbauer’s laboratory, and one dish was placed in a normoxic incubator (20% O2, 5% CO2), while the other was placed in a hypoxic chamber (1% O2, 5% CO2). Each cell line was incubated as such for 3 days, then prepared for FACS as described in sections 2.4.1.1 and 2.4.1.3. 2.4.1.2 Ex Vivo Cell Preparation All animal studies described in this section were performed in compliance with the Canadian Council on Animal Care (CCAC) guidelines and approved by the Animal Care Committee (ACC) of the University of British Columbia (UBC). When tumours in mice reached the desired volume for analysis, the mice were anesthetized with 2.5% isoflurane in oxygen and 24 then euthanized by CO2 inhalation. Tumours were harvested, washed in PBS, and blotted dry. In a dry culture dish, each tumour was cut into small pieces using forceps and a blade, and then transferred into collagenase in an approximate 1:1 volume ratio. This mixture was vortexed and placed into a humidified incubator (5% CO2, 37°C) for 1.5-2 h. After incubation, cells were treated with media (10:1 media to collagenase) and filtered through a cell strainer into a 50 mL Falcon tube. Cells were pelleted and resuspended in regular media (if waiting for other tumours to be processed as above) or directly in FACS buffer. Cells were then washed twice with FACS buffer and seeded at a 250,000-1,000,000 cells/200 µL concentration per well in a V-bottom 96-well polystyrene plate. 2.4.1.2.1 Additional Steps for CAIX in AML Pilot Study: Analysis of Engrafted Tumours For the CAIX in AML pilot study, additional steps were performed on the mice prior to euthanasia. Under anesthesia with 2.5% isoflurane in oxygen, each mouse was administered 200 µL of pimonidazole in PBS (7.5 mg/mL; Hypoxyprobe, HP2-100Kit) via intraperitoneal injection 2 h prior, and with 200 µL of bromodeoxyuridine (BrdU) in PBS (3 mg/mL; Sigma, B5002-5G) 1 h before euthanasia. 2.4.1.3 Non-Fluorophore-Tagged mAbs of Interest Each assay, for each cell line of interest, was designed with wells of unstained cells (x 2, a), secondary (fluorescent) mAb (x 1, b), primary mAb of interest with secondary mAb added (x number of mAbs of interest, c), and primary isotype control mAb with secondary mAb added (x 1, d). With supernatant aspirated off the cell pellets in each well, 25-50 µL of 5 µg/mL primary mAb was added to c and d, while the same volume of FACS buffer was added to a and b. Plates were kept on ice for 1 h. Afterward, each well was topped up to a final volume of 200 µL before 25 being pelleted. After aspirating off supernatant, cells were washed three times with FACS buffer to remove excess primary mAb. After pelleting cells and aspirating supernatant, 25-50 µL of 1-2 µg/mL secondary mAb secondary mAb was added to b, c, and d, while the same volume of FACS buffer was added to a. Plates were kept on ice in the dark for 30 min. Wells were then processed in the same manner as after primary mAb incubation. Cells were resuspended in 100-200 µL FACS buffer per well into tubes to be analyzed in the FACSCalibur Tangerine flow cytometer (BD Biosciences) at the Flow Cytometry Core Facility of the Terry Fox Laboratory at BCCRI. Data was acquired with BD CellQuestTM Pro software, and the resulting data was analyzed using FlowJoTM software. 2.4.1.3.1 Additional Details for CD96 in Acute Myeloid Leukemia For the in vitro FACS, the A070, 11F02, 14G11, 9E05, anti-OVA, and Trastuzumab mAbs were used at 25 µL of 5 µg/mL per well. The secondary mAb (25 µL of 2 µg/mL per well) for the above Fc human IgG1 primary antibodies was from Jackson ImmunoResearch Laboratories Inc. (109-605-098). For the ex vivo (no injection) FACS, the 9E05, 14G11, 11F02, A070, and anti-CD33 mAbs were used at the same volume and concentration as the above in vitro FACS. The same secondary mAb as above was used in the same manner. For the ex vivo (post-injection) FACS, both the PE-conjugated anti-CD96 mAb (BioLegend, 338406) and the isotype control mAb (BioLegend, 400112) were used at a concentration of 1.0 µg/100 µL per well. 26 2.4.1.3.2 Additional Details for CAIX in Renal and Colon Cancer The c2C7 mAb was used at 1 µg/mL per well, while the c11H9 and positive control cG250 mAbs were used at 5 µg/mL per well. The negative control OVA mAb (5 µg/mL per well) was from the leftover adMare stock used for the CD96 mAb project. The secondary mAb (1 µg/mL per well) was from Jackson ImmunoResearch Laboratories Inc. (109-605-098). The m4A2 mAb (5 µg/mL per well) and the negative control Fc mouse IgG2b polyclonal Ab (pAb; 5 µg/mL per well, Millipore, MABC006) were also provided by Dr. McDonald. The secondary pAb (2 µg/mL per well) for the above Fc mouse IgG2b primary antibodies was from Abcam (ab6785). 2.4.1.3.3 Additional Details for CAIX in AML Pilot Study: Cell Line Screening The cG250 (leftover from CAIX in Renal and Colon Cancer project) and anti-OVA negative control (leftover adMare stock) mAbs were used at 5 µg/mL per well. The secondary mAb (1 µg/mL per well) was from Jackson ImmunoResearch Laboratories Inc. (109-605-098). 2.4.1.3.4 Additional Details for CAIX in AML Pilot Study: Analysis of Engrafted Tumours The volumes and concentrations of primary mAbs were different from those in section 2.4.1.3. Both the anti-CAIX mAb (R&D Systems, MAB2188) and the isotype control mAb (either BioLegend, 400220, or R&D Systems, MAB003 if viability dye present) were used at 100 µL of 0.5 µg/mL per well. Except for the OCIAML3 ex vivo FACS, the secondary antibody (2 µg/mL, 50 µL/well; Abcam, ab6785) was incubated along with a 7AAD viability dye (5 µL (= 0.25 µg)/well; Biolegend, 420403). 27 2.4.1.4 Fluorophore-Tagged mAbs of Interest Each assay contained wells of unstained cells (x 2, e), mAb of interest (x number of mAbs of interest, f), and isotype control mAb with the same fluorophore as the mAb of interest (x 1, g) per cell line of interest. With supernatant aspirated off the cell pellets in each well, mAb of interest and isotype control mAb were added to f and g respectively as recommended by manufacturer, and the same volume of FACS buffer was added to a. Plates were kept on ice for 1 h in the dark. Cells were processed in the same manner as after mAb incubation, and samples were prepared and analyzed in the same process, as in section 2.4.1.3. 2.4.1.4.1 Additional Steps for CAIX in AML Pilot Study: Analysis of Engrafted Tumours Prior to addition of mAb of interest (10 µL (= 0.5 µg)/well; R&D Systems, FAB2188F) and isotype control mAb (10 or 20 µL (= 0.25 or 0.5 µg)/well; R&D Systems, IC003F), cells were pre-incubated with 30 µL of 10% mouse serum (Abcam, ab7486) per well for 10 minutes, and then 5 µL of 7-AAD viability dye (0.25 µg/well; Biolegend, 420403) per well for all wells except for e. 2.4.2 Immunoreactivity Assays Immunoreactivity assays were performed after each mAb radiolabeling to ensure there is still sufficient binding to cells of interest. The immunoreactive fractions of radioimmunoconjugates were estimated using the Lindmo cell-binding method using the appropriate target-expressing cell lines at various cell concentrations (Table 3.1, Table 4.1)88. For each antibody of interest, 0.45 µg of the radioimmunoconjugate was diluted in 5 mL of 1% bovine serum albumin (BSA) in PBS, and 50 µL of the diluted solution was added to each cell tube in duplicate. The solutions were incubated for 1 h at 37°C under gentle agitation. Cells were then 28 pelleted in a microcentrifuge and washed twice with PBS. The counts per minute (CPM) of each sample, including standards containing only radioimmunoconjugate, were determined using the Wallac WIZARD2 gamma counter with background and decay correction. The percentage of radiolabelled mAb bound to cells was determined as a percentage of the total added radioimmunoconjugates, which was represented by the standard samples. Immunoreactive fractions were estimated for conditions representing infinite antigen excess by linear regression analysis of a plot of total/bound activity against 1/[cell concentration]88. 2.4.3 Internalization and Retention Assays In vitro internalization assays were performed for 111In-labeled positive control mAbs and anti-CD96 mAbs of interest. Cells were seeded into 24-well poly-D-lysine coated plates at 100,000 cells per well, in triplicates per time point. Approximately 53 ng of radiolabeled mAb was added into each well with varying specific activities for each mAb. Cells were incubated with radiolabeled mAb for 0.5, 1, 2, 4, and 24 h at 37°C before being subjected to PBS and acid buffer (0.2 M acetic acid, 0.5 M NaCl, pH 2.6) washes for separation of the membrane bound and internalized fractions. In vitro retention assays were also performed for 111In-labeled positive control mAbs and anti-CD96 mAbs of interest. Cells and radiolabeled mAbs were prepared as previously described for internalization assays above. Cells were incubated with radiolabeled mAb for 1 h at 37°C before being subjected to PBS washes and replacement of reaction media, followed by further incubation for 0, 2-3, 24, and 72 h and separation of supernatant (secreted) from cells (retained). 29 2.5 Immunohistochemistry Studies To confirm the presence of CAIX expression in AML tumours previously analyzed by ex vivo FACS, specifically Molm13, OCIAML3, and NOMO1, histological tissue analysis was performed. Tumours were harvested from athymic nude mice, fixed in 10 mL of formalin (10% formaldehyde in PBS), and stored at 4°C for a minimum of 4 days. The tumour slices originated from the RL and NP mice for OCIAML3, the L1 mouse for NOMO1, and the NP mouse for Molm13 (RL = right and left ear punch, NP = no ear punch, L1 = left ear punch). Tissues were then put in cassettes and dehydrated through a set of graded ethanol solutions: 70%, 75%, 95%, and 100% (each step is 30-60 min). Next, tissues were embedded in paraffin, cut into 6 µm sections, and mounted onto Superfrost microscope slides (Fisherbrand, Ottawa, Canada) by Rebecca Wu from Dr. Yuzhuo Wang’s lab. There were two tumour slices mounted per slide, with a total of 4 slides per tumour, designed for application of primary antibodies as described in Table 2.2. All dilutions were made in blocking solution (1% BSA, 0.2% TritonX-100, and 0.05% Tween in PBS). An additional OCIAML3 RL slide was used as a blank control with only blocking solution applied. A CAIX-positive control slide, with two tumour slices mounted, was kindly provided by Oksana Nemirovsky from Dr. Shoukat Dedhar’s laboratory, called NSE1082 (from PK8 human pancreatic tumour). The NSE1082 slide was used to test the two anti-CAIX mAbs in this immunohistochemistry (IHC) study side-by-side on slices from the same tumour, as described in Table 2.3. 30 Table 2.2 Primary antibodies applied for each tumour slice. Slide # Left Tumour Slice Right Tumour Slice 1 Anti-CAIX†, 1:200 dilution of 1 mg/mL stock (M75, Bioscience; mouse IgG2b mAb) Isotype control mouse IgG2b, 1:200 dilution of 1 mg/mL stock (Abcam ab170192; mouse IgG2b kappa mAb) 2 Anti-Pimonidazole, 1:1500 dilution of 0.5 mg/mL stock (Hypoxyprobe, HP2-100Kit, FITC-MAb1; mouse IgG1 mAb) Anti-Pimonidazole, 1:100 dilution of 0.5 mg/mL stock (Hypoxyprobe, HP2-100Kit, FITC-MAb1; mouse IgG1 mAb) 3 Anti-CAIX, 2 µg/mL (Abcam ab107257, mouse IgG1 mAb) Isotype control IgG1, 2 µg/mL (Biolegend 400122, mouse IgG1 kappa mAb) 4 BrdU 1:5 dilution from stock (Abcam, ab8152, mouse IgG1 mAb) BrdU 1:10 dilution from stock (Abcam, ab8152, mouse IgG1 mAb) †Kindly provided by Oksana Nemirovsky from Dr. Shoukat Dedhar’s laboratory. Table 2.3 Primary antibodies applied onto NSE1082 (CAIX-positive control) slide. Left Tumour Slice Right Tumour Slice Anti-CAIX†, 1:200 dilution of 1 mg/mL stock (M75, Bioscience; mouse IgG2b mAb) Anti-CAIX, 2 µg/mL (Abcam ab107257, mouse IgG1 mAb) †Kindly provided by Oksana Nemirovsky from Dr. Shoukat Dedhar’s laboratory. On Day 1 of immunostaining, slides were incubated in a 37°C incubator for 30 minutes to soften paraffin. All slides were then subjected to de-paraffinization and rehydration by sequentially submerging the slides in a xylene bath (2x, 5 min each time), 100% ethanol (2x, 3 min each time), 95% ethanol (2x, 3 min each time), 70% ethanol (1x, 3 min), PBS (3x, 5 min each time on shaker), 0.2% TritonX-100 in PBS (1x, 10 min), PBS (3x, 5 min each time on shaker), 0.01 M citrate buffer pH 6 (in microwave on high for 10 min, cool at room temperature), PBS (3x, 5 min each time on shaker), 3% H2O2 (1x, 15 min on shaker), PBS (3x, 5 min each time on shaker), and blocking solution (1 h, room temperature on shaker). Then each slide was subjected to immunostaining by drying around the section before drawing a circle around the section with a diamond pen, applying approximately 30 µL primary antibody or blocking solution per tumour slice, as detailed previously, and promptly placing in a wet chamber to incubate at 4°C overnight. 31 On Day 2 of immunostaining, all slides were washed in PBS (3x, 15 min each time on shaker). The secondary antibody (Abcam ab64255, biotinylated goat anti-mouse IgG (H+L)) was applied to each slide as-is as described previously, except slides were incubated for 2 h at room temperature. All slides were then washed with 0.05% Tween (3x, 15 min each on shaker), then PBS (1x, 15 min on shaker). The Vector Laboratories Vectastain Elite ABC Kit was used to produce a biotin-avidin bond to the secondary antibody applied to each section, used as recommended by manufacturer and applied to each slide as previously described, except the slides were incubated for 30 min at room temperature. The slides were then washed in PBS (3x, 5 min each on shaker). The DAB Peroxidase Substrate Kit (SK-4100, Vector Laboratories) was used as recommended by manufacturer and applied to each slide as previously described, except the slides were incubated for 10 min each until brown colour appeared. The slides were then washed with PBS (1x, 5 min on shaker), then dH2O (3x, 5 min each on shaker). The slides were then counterstained with hematoxylin (cat 65065, EM Science) for exactly 30 s, and immediately washed under running tap water (3x, 5 min each time) afterwards. All slides were then dipped into Staining Blueing Reagent (cat 65354-85, EM Science; 15x, 2 s each), then washed under running tap water (3x, 5 min each). For the dehydration process, all slides were submerged into 95% ethanol (10x), 100% ethanol (10x), xylene (1x, 1 min), and a second jar of xylene (1x). One drop of permount was added per section and covered with cover slips, tapped gently with a pipette tip to remove bubbles. All slides were dried in a fume hood overnight. The stained sections were examined and analyzed with a Leica DM 2500 light microscope using the Leica Application Suite X software. 32 2.6 In Vivo Evaluations All animal studies described in this section were performed in compliance with the CCAC guidelines and approved by the UBC ACC. Both the female NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) mice and the female and male NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were obtained from in-house colonies, and the female and male NU/J (athymic nude) mice were obtained from The Jackson Laboratory (002019). 2.6.1 Tumour Implantation All experiments were performed using either NRG, NSG, or nude mice, as described in subsequent chapters. Mice were anesthetized briefly with 2.5% isoflurane in 2.0 L/min of oxygen during cell implantation. After wiping the skin surrounding the injection site with an alcohol prep pad, a 25-gauge needle (except SK-RC-52 cells, which required a 21.5-gauge needle to prevent shearing of the cells) was used to subcutaneously implant cells under either the left or right shoulder. The number of cells implanted for each cell line are summarized in Table 2.4, suspended in 100 µL of PBS and BD Matrigel Matrix at 1:1 ratio. Imaging and biodistribution studies were performed when tumours reached 100 mm3 in volume for all studies except for CAIX, which required 300-500 mm3 in volume to reach hypoxia. 33 Table 2.4 Summary of various cell lines and number of cells implanted into mice. Target Protein Cell Line Cell Type Number of Cells Implanted Per Mouse (million) CD96 KG1α Human acute myeloid leukemia 15 CD96 HEK293-CD96 (HEK-CD96) Human embryonic kidney cell, CRISPR-modified to express CD96 10 Negative control HEK293 (HEK-WT) Human embryonic kidney cell 5 CAIX MDA-MB-231 CAIX KO +CAIX Human breast adenocarcinoma, CRISPR-modified to express CAIX 5 Negative control MDA-MB-231 CAIX KO Human breast adenocarcinoma, CRISPR-modified to remove CAIX expression 5 CAIX HT-29 Human colorectal adenocarcinoma 5-8 CAIX SK-RC-52 Human renal cell carcinoma 5 HER2 SKOV3 Human ovarian adenocarcinoma 6 CAIX OCIAML3 Human acute myeloid leukemia 5 CAIX Molm13 Human acute myeloid leukemia 5 CAIX NOMO1 Human acute myeloid leukemia 5 2.6.2 89Zr-PET/CT Imaging When tumours reached a size of 100-150 mm3 (CD96 studies) or 300-500 mm3 (CAIX studies), mice were anesthetized with 2% isoflurane in oxygen and each injected via tail vein with various amounts (~5 MBq; specific activity varied depending on radiolabelling results) of 89Zr-radioimmunoconjugate, as described in subsequent chapters. Prior to each PET/CT image 34 acquisition, each mouse was given a subcutaneous injection of 10 µL/g of sterile saline for hydration. Mice were scanned while sedated with their body temperature maintained by a heating pad. PET/CT images were taken at specified time points post-injection using a Siemens (Knoxville, TN, USA) Inveon microPET/CT scanner. Each mouse underwent a CT scan for attenuation correction, then a 30-min static PET acquisition. Image reconstruction was performed using the three-dimensional ordered-subsets expectation maximization (OSEM3D, 2 iterations) followed by a fast maximum a priori algorithm (FastMAP: 18 iterations). The Inveon Workplace software (Siemens Medical Solutions USA, Inc.) was used to analyze and visualize the images. On the last time point of each study, the mice were imaged before being sedated and sacrificed for biodistribution analysis described in section 2.6.4. 2.6.3 111In-SPECT/CT Imaging When tumours reached sizes described previously, mice were anesthetized via inhalation of 2% isoflurane in oxygen and each injected via tail vein various amounts of 111In-radioimmunoconjugate, as described in following chapters. Prior to each SPECT/CT image acquisition, each mouse was administered sterile saline as described in section 2.6.2. Mice were sedated during each scan and their body temperature was maintained by heating pad. SPECT/CT images were taken at specified time points post-injection using a U-SPECT II/CT scanner (MILabs, Utrecht, Netherlands) with an ultra-high resolution multi-pinhole rat-mouse collimator (1 mm pinhole size). Each mouse underwent a 5 min baseline CT scan for localization and attenuation with voltage setting at 60 kV and current at 615 µA, then a single static emission scan. The data was acquired in list mode, reconstructed using U-SPECT II software, 35 and co-registered for alignment. Reconstruction of SPECT images were performed using maximum-likelihood expectation maximization (3 iterations), pixel-based ordered subset expectation maximization (16 subsets), and a post-processing filter (Gaussian blurring) of 0.5 mm centered at photopeaks 171 keV and 245 keV with a 20% window width. Using the Inveon Research Workplace software (Siemens Medical Solutions USA, Inc.), imaging data sets were decay-corrected to injection time and converted to DICOM data for visualization. On the last time point of each study, the mice were imaged before being sedated and sacrificed for biodistribution analysis described in section 2.6.4. 2.6.4 Biodistribution Studies Mice were anesthetized with 2.5% isoflurane in oxygen and then euthanized at specified time points post-injection by CO2 inhalation. Blood was promptly collected in syringes by cardiac puncture and dispensed into tared tubes for weighing. Tissues/organs were harvested, washed in PBS, blotted dry, and transferred to tubes for weighing as well. The radioactivity of the blood, tissues, and organs were measured using a Wallac WIZARD2 gamma counter with background and decay correction. Using a yearly updated calibration factor (in kBq/CPM, obtained using standards of known activity) for the gamma counter, the CPM of each sample is converted into MBq. The radioactivity uptake is then calculated, decay corrected, and expressed as a percentage of the injected dose per gram of tissue (%ID/g). 36 Chapter 3: Anti-CD96 mAbs for radioimmunotherapy of acute myeloid leukemia: evaluation of candidates by PET imaging 3.1 Introduction Prior to the start of my studies under Dr. Bénard, CD96 was identified as a promising target for antibody-based therapeutic strategies for acute myeloid leukemia (AML), in collaboration with adMare and Dr. Steven Jones’ group (GSC). Four anti-CD96 mAbs of interest were chosen and produced by adMare: three chimeric Ms/Hu mAbs called 11F02, 9E05, and 14G11, as well as the humanized version of 11F02 denoted A070. The addition of A070 highlights the intended direction of therapeutic mAb development from murine origin toward near-human forms, in attempts to reduce immunogenicity, increase efficacy, and decrease patient infusion reactions89. 3.2 Materials and Methods All materials and methods pertinent to this chapter are described in Chapter 2. 3.3 Results and Discussion All mAbs of interest were tested with a human Caucasian bone marrow AML cell line, KG1α, as well as a primary human embryonic kidney cell line stably transfected to express CD96 (HEK293-CD96, or HEK-CD96 for short). The HEK293-wildtype (HEK-WT) cell line was used as a negative control when paired with HEK-CD96. The HEK-CD96 and HEK-WT cell lines were used in addition to KG1α as we did not have a true chimeric Ms/Hu negative control mAb for 9E05, 14G11, and 11F02, while we used the humanized Trastuzumab as a negative control mAb for A070. The anti-OVA mAb was initially used as a negative control mAb for the chimeric mAbs 37 of interest when testing on KG1α cells, but was later retired as it was a chimeric rabbit/human (Rb/Hu) mAb and not a true isotype control. First, cell-surface CD96 expression and mAb binding of the above cell lines were confirmed by in vitro FACS (Figure 3.1). In the HEK-WT and HEK-CD96 graphs, it is clear that the anti-CD96 mAbs of interest, A070, 9E05, 14G11, and 11F02 only bind to the latter cell line, and do not show binding in the former. In the KG1α graphs, with the negative control mAbs on the left and the mAbs of interest on the right, there is clear CD96 expression in the KG1α cell line with binding to A070, 9E05, 14G11, and 11F02, and no binding of the anti-ovalbumin mAb and Trastuzumab as expected. This shows that not only are the HEK-CD96 and KG1α cell lines suitable for further CD96 investigations, but also that the mAbs of interest bind well and the negative control mAbs were chosen appropriately for further studies. Figure 3.1 In vitro FACS results showed expression of CD96 target in cell lines of interest, HEK-CD96 and KG1α. 38 Immunoreactivity assays were performed after each mAb radiolabeling with the intention to ensure there is still sufficient binding to cells of interest. As illustrated in Table 3.1 and Table 4.1 across both the CD96 and CAIX projects, the immunoreactive fraction percentage did not always reflect how the radioimmunoconjugate performed in vivo when compared against the corresponding biodistribution (tumour uptake) values. Firstly, it was found that each cell line specific to the target protein required different ranges of cell concentrations for the assay to work, and in some cases, the assay did not work (as shown by the low R2 values). Secondly, this assay has been performed by different operators, and it was found that this assay did not work consistently, so successful results did not depend on the operator. Lastly, the usage of two different radioisotopes, nine different antibodies, four different cell lines, three different mouse strains, and five different tumour cell lines further showed that successful results (i.e. >0.99 R2 values and >70% immunoreactive fraction) were inconsistently achieved for this assay and inaccurately predicted in vivo performance. Thus, these immunoreactivity values are reported in this thesis to show that each radioimmunoconjugate was tested for binding ability after radiolabelling, but overall do not reflect the success of the radiotracer shown in the imaging and biodistribution data that follows. 39 Table 3.1 Immunoreactivity assay values for anti-CD96 tracers and corresponding in vivo biodistribution study tumour uptake values. Figure Ref. No. Radio-isotope† Antibody Immunoreactivity Assay In vivo Biodistribution Study Cell Line Calibration Curve Immuno-reactive Fraction (%) Mouse Strain Tumour Cell Line Post-Injection Time Points Minimum Conc. Maximum Conc. R2 value Day 1 Day 3 Day 5 Day 6 (cells/mL) (%ID/g) 3.2 Zr-89 11F02 KG1a 1,803,000 54,636,364 0.9958 80.97 NRG HEK-CD96 - 8.54 - - 3.3 In-111 A070 KG1a 490,000 12,000,000 0.9880 41.72 NRG KG1a - 3.11 1.80 - 3.3 In-111 Trastuzumab SKOV3 490,000 12,000,000 0.9294 84.24 NRG KG1a - 3.24 1.22 - 3.4 In-111 11F02 KG1a 490,000 12,000,000 0.9652 99.00 NRG HEK-CD96 20.41 7.76 - - 3.4 In-111 9E05 KG1a 490,000 12,000,000 0.9937 99.00 NRG HEK-CD96 16.20 7.29 - - 3.4 In-111 14G11 KG1a 490,000 12,000,000 0.8863 99.00 NRG HEK-CD96 - 10.51 - - 3.5, 3.6B, 3.7B In-111 cCD96 KG1a 490,000 12,000,000 0.8469 10.12 NRG KG1a 4.43 9.03 - 8.82 3.6C, 3.7C In-111 11F02 KG1a 490,000 12,000,000 0.9966 52.67 - - - - - - 3.6D, 3.7D In-111 A070 KG1a 490,000 12,000,000 0.9989 51.27 NRG KG1a 3.15 1.67 - 1.05 3.10 Zr-89 11F02 KG1a 490,000 12,000,000 0.8942 27.16 Nude KG1a 3.20 3.70 - - †The only Zr-89 experiment that used mDFO* was the one shown in Figure 3.2. All other Zr-89 experiments used BnDFO*. All In-111 experiments used BnDTPA. 40 In order to select the best candidate for RIT studies, the pharmacokinetic behavior of each mAb was studied in vivo with the assistance of [89Zr]Zr-PET/[111In]In-SPECT imaging and biodistribution experiments. It is the goal of therapeutic mAb development to achieve consistent, specific and sustained tumour uptake over time in preclinical studies. Thus, it was to our surprise when the radiolabeled versions of the mAbs of interest exhibited decreasing tumour uptake over the course of 5-6 days, in immunodeficient NRG female mice bearing subcutaneous KG1α or HEK-CD96 tumours. To investigate this abnormal fast decrease of tumour uptake over time, multiple variables were tested, including two different radionuclides (89Zr, 111In) and two different tumour models. Each radionuclide above required either a DFO* or DTPA chelator, respectively, for complexation, and different chelator-mAb conjugation methods (maleimide- or isothiocyanatobenzyl-based) were investigated to assess in vivo stability of the conjugation itself. Based on the chelator and conjugation methods, the antibody extensions were denoted mDFO*, BnDFO*, or BnDTPA, as described in Section 2.2.1. The decrease in tumour uptake was first observed in a preliminary in vivo study for [89Zr]Zr-mDFO*-11F02 in the KG1α model, which showed significant and specific tumour uptake (~10%ID/g in PET image) at day 1 post-injection (p.i.) that was lost by day 7 p.i. (1.86 ± 0.54 %ID/g), as shown in Figure 3.2. The biodistribution study was only planned for day 7 p.i. in this preliminary experiment as the expectation was that the long biological half-life of radioimmunoconjugates would typically show the best tumour-to-background organ imaging contrast after 3-5 days p.i., and biodistribution data would provide confirmation for imaging data. Imaging was performed at day 1 p.i. to provide useful information regarding tumour uptake and pharmacokinetics. However, these results did not match expectations, so further experiments were performed to figure out the root cause of the problem. 41 Figure 3.2 Preliminary PET imaging and biodistribution results for [89Zr]Zr-mDFO*-11F02 and [89Zr]Zr-mDFO*-OVA (anti-ovalbumin negative control mAb) in KG1α tumour-bearing NRG female mice. (A) Mice were injected with either [89Zr]Zr-mDFO*-11F02 (5.83 µg, 3.75 MBq) or [89Zr]Zr-mDFO*-OVA (5.69 µg, 1.98 MBq). Images were acquired at different times after injection and decay corrected to injection time. Tumours are highlighted with arrows and results are shown as percentage of the injected dose per gram of tissue (%ID/g). (B) Biodistribution results 7 days after injection of [89Zr]Zr-mDFO*-11F02 (5.7 ± 0.3 µg and 3.7 ± 0.2 MBq per mouse) or [89Zr]Zr-mDFO*-OVA (5.7 ± 0.3 µg and 2.0 ± 0.1 MBq per mouse). No specific tumour uptake comparing 11F02 versus OVA was found (p = 0.15). Results were decay corrected to injection time and are expressed as %ID/g (mean ± standard deviation (SD), n = 4 mice per group). 42 To confirm whether it was a radioisotope or chelator issue, [111In]In-BnDTPA-A070 was also tested in the same model, yielding similar results (Figure 3.3). To verify whether it was an mAb or tumour model issue, [111In]In-BnDTPA-11F02, [111In]In-BnDTPA-9E05, and [111In]In-BnDTPA-14G11 were tested in the HEK-CD96 versus HEK-WT model, exhibiting the same pharmacokinetic behavior (Figure 3.4). However, it is worth noting that significant HEK-CD96 tumour uptake was observed at day 1 p.i. in the 111In studies for 11F02 (20.41 ± 9.47 %ID/g) and 9E05 (16.20 ± 13.14 %ID/g), albeit non-specific as compared to HEK-WT. For 14G11, the HEK-WT uptake was unexpectedly found to be significantly greater than HEK-CD96 uptake (p < 0.05 for both days). Since the quantity of injected mass of mAb impacts on mAb biodistribution, quantities closer to an optimal dose to increase tumour-to-background ratio were injected (~20 µg and ~50 µg, as found to be optimal in a previous study)90. For this study, the immunoreactivity of [111In]In-BnDTPA-11F02 was particularly low (17.25%). Nevertheless, no improvement tendency in the mAb biodistribution was observed. This indicated that even with a higher immunoreactive fraction, injecting higher activities may not have been the solution. 43 Figure 3.3 SPECT imaging and biodistribution results for [111In]In-BnDTPA-A070 and [111In]In-BnDTPA-Trastuzumab (anti-HER2 negative control mAb) in KG1α tumour-bearing NRG female mice. (A) Mice were injected with either [111In]In-BnDTPA-A070 (43.64 µg, 9.83 MBq) or [111In]In-BnDTPA-Trastuzumab (45.70 µg, 8.23 MBq). Tumours are highlighted with arrows and results are shown as %ID/g. (B) Biodistribution results 3 and 5 days after injection of [111In]In-BnDTPA-A070 (9.5 ± 1.3 µg and 2.1 ± 0.3 MBq per mouse) or [111In]In-BnDTPA-Trastuzumab (10.3 ± 0.2 µg and 1.8 ± 0.04 MBq per mouse). No specific tumour uptake comparing A070 versus Trastuzumab was found (p = 0.73 at Day 3 p.i. p = 0.07 at Day 5 p.i.). Results are presented as mean %ID/g ± SD (n = 4 mice per group on Day 3, n = 3 on Day 5). 44 Figure 3.4 Biodistribution results 1 and 3 days after injection of [111In]In-BnDTPA-11F02, [111In]In-BnDTPA-9E05, or [111In]In-BnDTPA-14G11 in HEK-CD96 and HEK-WT tumor-bearing NRG female mice. (A) [111In]In-BnDTPA-11F02 results, with 6.9 ± 0.2 µg and 1.8 ± 0.1 MBq injected per mouse. No specific tumor uptake was found (p = 0.08 at Day 1 p.i., p = 0.20 at Day 3 p.i.). Replicates: n = 4 mice per group on Day 1, n = 2-3 on Day 3. (B) [111In]In-BnDTPA-9E05 results, with 5.1 ± 1.8 µg and 1.3 ± 0.5 MBq injected per mouse. No specific tumor uptake was found (p = 0.60 at Day 1 p.i., p = 0.66 at Day 3 p.i.). Replicates: n = 4 mice per group on Day 1, n = 3 on Day 3. (C) [111In]In-BnDTPA-14G11 results, with 5.2 ± 0.2 µg and 3.1 ± 0.1 MBq injected per mouse. HEK-WT uptake was found to be significantly greater than HEK-CD96 uptake on both days (p = 0.0002 at Day 1 p.i., p = 0.02 at Day 3 p.i.). Replicates: n = 4 mice per group on Day 1, n = 2-3 on Day 3. 45 To assess if the in vivo behavior of these mAbs was due to the mAbs themselves or the biology of the targeted protein, a commercially available murine mAb (cCD96) was tested. In the KG1α model, [111In]In-BnDTPA-cCD96 was found to show specific and consistent tumour uptake over time compared to [111In]In-BnDTPA-mIgG (9.0 ± 1.2 %ID/g versus 4.7 ± 0.4 %ID/g, p < 0.001 on Day 3 p.i.; 8.8 ± 0.4 %ID/g versus 5.2 ± 0.3 %ID/g, p < 0.001 on Day 6 p.i.), suggesting that CD96 can be targeted in vivo (Figure 3.5). Figure 3.5 Biodistribution results 1, 3, and 6 days after injection of [111In]In-BnDTPA-cCD96 (10.0 ± 1.2 µg and 1.6 ± 0.5 MBq per mouse) or [111In]In-BnDTPA-mIgG (negative control mAb, 11.8 ± 0.6 µg and 2.6 ± 2.8 MBq per mouse) in KG1α tumour-bearing NRG female mice. Results are presented as mean %ID/g ± SD (n = 4 mice per group on all days). * = p < 0.05, ** = p < 0.01, *** = p < 0.001. 46 In vitro assays were also performed to determine what percentage of the radiolabeled mAbs were internalized, retained, or secreted after binding to the tumour cells91,92. Two positive control mAbs, Trastuzumab and cCD96, were also tested within the same assay parameters. Trastuzumab is a widely used humanized mAb in the clinic against HER2 in breast cancer patients, and so it was tested on SKOV3 (HER2+) cells. All mAbs were conjugated to BnDTPA and labeled with 111In. For the internalization assays, Trastuzumab was found to internalize slowly into SKOV3 cells up to 44% after 24h (Figure 3.6A), while cCD96 was internalized faster in KG1α cells at 72% after 30 min, dropping slightly to 69% after 24h (Figure 3.6B). Much like Trastuzumab, 11F02 was internalized slowly in HEK-CD96 cells up to 46% after 24h (Figure 3.6C), with comparable results (not shown) obtained for 9E05 and 14G11 on the same cell line. In contrast, A070 performed initially more like cCD96, being internalized quickly in KG1α cells at 82% after 30 min, but dropped to 56% after 24h (Figure 3.6D). 47 Figure 3.6 In vitro internalization assay results for 111In-labeled positive control mAbs and anti-CD96 mAbs of interest. (A) [111In]In-BnDTPA-Trastuzumab was internalized slowly into SKOV-3 cells up to 44.3 ± 3.3% after 24 h. (B) [111In]In-BnDTPA-cCD96 was internalized faster into KG1α cells at 72.4 ± 1.8% after 30 min, dropping slightly to 69.3 ± 1.8% after 24 h. (C) [111In]In-BnDTPA-11F02 was internalized slowly into HEK-CD96 cells up to 45.8 ± 1.3% after 24 h. (D) [111In]In-BnDTPA-A070 performed similarly to cCD96, being internalized quickly into KG1α cells at 82.1 ± 4.7% after 30 min, but dropping to 55.9 ± 8.0% after 24 h. For the retention assays, only 35% of Trastuzumab was secreted back into supernatant after 3 days (Figure 3.7A), while 52% of cCD96 was secreted after just 1 day, but stays relatively constant at 55% secretion after 3 days (Figure 3.7B). Similar to Trastuzumab, 50% of both 11F02 48 and A070 were secreted after 3 days (Figure 3.7C and Figure 3.7D), with similar results (not shown) obtained for other mAbs of interest. From both types of assays, the control mAbs showed similar behavior as that of the mAbs of interest, suggesting that the decrease in tumour uptake may not lie with internalization or retention of the mAbs in the cell lines of interest. Figure 3.7 In vitro retention assay results for 111In-labeled positive control mAbs and anti-CD96 mAbs of interest. Blue = retained, red = secreted. (A) [111In]In-BnDTPA-Trastuzumab on SKOV-3 cells, with 35.0 ± 1.4% secreted back into supernatant after 3 days. (B) [111In]In-BnDTPA-cCD96 on KG1α cells, with 52.1 ± 1.6% secreted back into supernatant after just 1 day, but stays relatively constant at 55.4 ± 9.9% secretion after 3 days. (C) [111In]In-BnDTPA-11F02 on HEK-CD96 cells, with 49.5 ± 4.7% of the bound activity secreted back into supernatant after 3 days. (D) [111In]In-BnDTPA-A070 on KG1α cells, resembling its chimeric counterparts with up to 50.3 ± 5.8% secreted back into supernatant after 3 days. 49 The in vivo expression of the target on grown KG1α and HEK-CD96 tumours was assessed by ex vivo FACS. Results showed that 100% of the tumour cells express CD96 and can still bind all mAbs of interest (Figure 3.8). The target was assessed and found to still be present at the cell surface 1 and 3 days after injection of [111In]In-BnDTPA-A070. All tumour cells expressed CD96, indicating that the target is still present and potentially targetable 3 days post-injection (Figure 3.9). Figure 3.8 Ex vivo FACS (no injection) results showed expression of CD96 target in cell lines of interest. NRG female mice were inoculated with either KG1α or HEK-CD96 tumour cells. When tumours reached ~100 mm3, tumours were harvested, digested with collagenase, filtered, and stained with mAbs of interest indicated. 50 Figure 3.9 Ex vivo FACS (post-injection) results showed expression of CD96 target in AML cell line of interest. NRG female mice were inoculated with KG1α tumour cells. When tumours reached ~100 mm3, mice were intravenously injected with either PBS (untreated), 111In-Trastuzumab, or 111In-A070. At day 1 and 3 p.i., tumours were harvested, digested with collagenase, filtered, and stained with a PE-labeled anti-CD96 mAb for FACS. While the mouse neonatal Fc receptor (FcRn, implicated in mAb recycling leading to increased biological half-life) is supposed to have high affinity for the human Fc protein, this may not hold true for some mouse strains, notably the NSG and NRG mice used in these experiments. Due to recently reported differences in Fc-mediated uptake between strains of mice, the use of the classical nude mice was explored93. [89Zr]Zr-BnDFO*-11F02 mAb was tested on athymic nude mice with subcutaneous KG1α tumours. In a preliminary study it was found that there was no specific tumour uptake of the 11F02 mAb compared to a negative control mAb in the same model 51 (11F02: 3.2 ± 0.3 %ID/g on Day 1 and 3.7 ± 2.3 %ID/g on Day 3; control: 6.2 %ID/g on Day 3; Figure 3.10). It is interesting to note that this radiotracer achieved a low 27.16% immunoreactivity result in vitro, and was mostly taken up by the spleen and liver, which may indicate that this particular batch of radiotracer did not perform well in this mouse model. However, more radioimmunoconjugate studies in this mouse strain were needed to conclude where the issue was – either in the radiotracer or the mouse model. Figure 3.10 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-11F02 and [89Zr]Zr-BnDFO*-Trastuzumab (anti-HER2 negative control mAb) in KG1α tumour-bearing nude female mice. (A) Mice were injected with either [89Zr]Zr-BnDFO*-11F02 (14.64 µg, 3.61 MBq) or [89Zr]Zr-BnDFO*-Trastuzumab (16.5 µg, 1.1 MBq). Tumours are highlighted with arrows and results are shown as %ID/g. (B) Biodistribution results 1 and 3 days after injection of either [89Zr]Zr-BnDFO*-11F02 (15.9 ± 0.9 µg and 1.5 ± 1.0 MBq per mouse) or [89Zr]Zr-BnDFO*-Trastuzumab (16.5 µg, 1.1 MBq). Results are presented as mean %ID/g ± SD (n = 3 mice on both days for 11F02, n = 1 for Trastuzumab). 52 3.4 Conclusion There were several achievements as well as shortcomings from the above investigation in developing anti-CD96 radioimmunoconjugates. On a positive note, cell-surface CD96 expression and mAb binding were confirmed by both in vitro and ex vivo FACS, and specific tumour uptake of the radioimmunoconjugates was achieved on Day 1 p.i. in NRG mice models. However, the downsides are that the radioimmunoconjugates exhibited decreasing tumour uptake over time in the same models, even with the use of 2 different radioisotopes, 2 chelator conjugation methods, 4 different mAbs of interest with multiple control mAbs, 2 tumour models, and internalization and retention assays showing similar patterns between mAbs of interest and control mAbs. Unfortunately, even with the switch to the athymic nude mice strain, the 11F02 mAb did not show specific (albeit now consistent) tumour uptake over time, which discouraged further investigation with the other three anti-CD96 mAbs. Further investigation into a different panel of anti-CD96 mAbs may be needed for future radioimmunoconjugate development for this target protein. 53 Chapter 4: CAIX as an additional target against tumour dormancy: radioimmunoconjugate screening in renal cancer, colon cancer, and acute myeloid leukemia models 4.1 Introduction 4.1.1 CAIX in Renal and Colon Cancer Models The development of this project in creating novel radioimmunoconjugates against CAIX began with Dr. Joseph Lau, a previous PhD graduate from our research group under Dr. François Bénard, in collaboration with Dr. Anne Lenferink (NRC) and Dr. Shoukat Dedhar (BC Cancer). Together, their preliminary research culminated in a leading imaging candidate: a murine anti-CAIX mAb, m11H9. This mAb was radiolabeled with 111In and tested in a preliminary study. It was shown to have high consistent tumour uptake over time in vivo (35.6%ID/g on day 7 p.i.) in an immunodeficient NSG mouse bearing a subcutaneous HT-29 (colorectal adenocarcinoma) tumour94. With the success of the m11H9 mAb, its chimeric mouse/human (Ms/Hu) version, c11H9, was later developed to further decrease risk of immunogenicity. Two additional mAbs were also generated and selected as new candidates: the chimeric Ms/Hu c2C7 and the murine m4A2. It is interesting to note that c11H9 binds the extracellular proteoglycan (PG)-like domain of CAIX (same binding site as cG250), while c2C7 and m4A2 bind the catalytic domain, allowing this investigation to cover different target areas of the same protein. The PG-like domain is known to be unique to CAIX compared to other isoforms of carbonic anhydrase, fully justifying the initial choice of targeting this domain for cG250 and our m11H9/c11H9 candidates for selective cancer treatment78. However, the development of mAbs targeting the catalytic domain of CAIX 54 introduces the advantage of specifically disrupting the catalytic activity of the enzyme itself, affecting its tumorigenic functions including pH regulation59. Thus, the three mAb candidates introduced for this chapter’s radioimmunoconjugate development offer a potentially powerful, cross-domain arsenal against CAIX-expressing cancers. 4.1.2 CAIX in Acute Myeloid Leukemia Models – Pilot Study In parallel with the radioimmunoconjugate screening in renal and colon cancers, a pilot study in collaboration with Dr. Florian Kuchenbauer at BC Cancer was initiated to explore CAIX expression in AML cell lines. This was in search of a novel avenue to target AML using this well-known protein for potential radio-imaging studies, which has been seldom investigated to date. In solid tumours, it is well established that hypoxia is a negative prognostic factor associated with increased resistance against radiation therapy and conventional chemotherapy50-52. Since leukemia is not considered a “solid” tumour, the availability of oxygen in this disease has been considered as inconsequential and therefore overlooked. However, this perspective has recently been revised as hypoxia has been shown to promote leukemic cell proliferation, differentiation, and resistance to chemotherapy95. In addition, it has been found that a small fraction of leukemic cells can survive in hypoxic areas of the bone marrow, where they can elude treatment and lead to relapse96. Thus, the preliminary investigation into targeting CAIX in AML for future radiotracer studies was deemed to have possibly undiscovered potential for drug development. 4.2 Materials and Methods All materials and methods pertinent to this chapter are described in Chapter 2. 55 4.3 Results and Discussion 4.3.1 CAIX in Renal and Colon Cancer The c11H9, c2C7, and m4A2 mAbs were tested with three cell lines of interest. A human Caucasian breast adenocarcinoma called MDA-MB-231, previously CRISPR-modified to become CAIX knockout (KO) (CAIX-) and CAIX KO +CAIX (CAIX+) cells, served as negative and positive control cell lines, respectively. A human Caucasian colon adenocarcinoma, HT-29, and a mediastinum metastatic human renal cell carcinoma, SK-RC-52, were also included as cell lines that inherently expressed CAIX. FACS was performed on all three cell lines prior to any further studies to confirm CAIX expression and cold mAb binding (Figure 4.1). As expected, the HT-29, SK-RC-52, and MDA-MB-231 CAIX+ cells showed CAIX expression with the indicated mAbs of interest and positive control cG250 mAb bound, while the MDA-MB-231 CAIX- cells showed no CAIX expression with none of the aforementioned mAbs bound. cG250 was only included with c2C7 and c11H9 plots as their isotypes were the same and so required the same secondary mAb and analysis channel on the flow cytometer; m4A2 required a different secondary mAb that was only visible on a different channel. Immunoreactivity assays were also performed, and as discussed in the previous chapter, the immunoreactivity values did not always reflect performance in vivo (Table 4.1). 56 Figure 4.1 In vitro FACS results showed expression of CAIX target in cell lines of interest, MDA-MB-231 CAIX+, SK-RC-52, and HT-29, and binding to mAbs of interest. cG250 = Phase III clinical trial anti-CAIX mAb. 57 Table 4.1 Immunoreactivity assay values for anti-CAIX tracers and corresponding in vivo biodistribution study tumour uptake values. Figure Ref. No. Radio-isotope† Antibody Immunoreactivity Assay In vivo Biodistribution Study Cell Line Calibration Curve Immuno-reactive Fraction (%) Mouse Strain Tumour Cell Line Post-Injection Time Points Minimum Conc. Maximum Conc. R2 value Day 1 Day 3 Day 5 Day 6 Day 7 (cells/mL) (%ID/g) 4.2 Zr-89 c11H9 MDA-MB-231 CAIX KO +CAIX 490,000 12,000,000 0.8588 49.65 NRG MDA-MB-231 CAIX KO +CAIX - 5.48 - 1.97 - 4.3 Zr-89 c2C7 MDA-MB-231 CAIX KO +CAIX 490,000 12,000,000 0.9183 80.97 NRG MDA-MB-231 CAIX KO +CAIX - 17.75 - 5.37 - 4.4 Zr-89 m4A2 MDA-MB-231 CAIX KO +CAIX 490,000 12,000,000 0.9953 43.49 NRG MDA-MB-231 CAIX KO +CAIX - 8.71 - 4.50 - 4.5 In-111 c11H9 HT-29 408,333 10,000,000 0.9497 24.62 NSG HT-29 - 19.35 - - 6.49 4.6 In-111 c11H9 MDA-MB-231 CAIX KO +CAIX 490,000 12,000,000 0.5543 8.45 NSG MDA-MB-231 CAIX KO +CAIX - 11.88 - - 3.03 4.7, 4.8 Zr-89 Trastuzumab SKOV3 250,000 2,500,000 0.9900 53.15 Nude SKOV3 8.95 16.80 - 12.71 - NSG SKOV3 14.54 16.06 - 5.72 - 4.9, 4.10 Zr-89 c11H9 HT-29 490,000 12,000,000 0.6103 7.60 Nude HT-29 12.19 15.13 - 15.88 - NSG HT-29 13.55 9.58 - 3.08 - 4.11 Zr-89 c2C7 HT-29 490,000 12,000,000 0.0506 0.49 Nude HT-29 - 18.88 - 20.22 - 4.12 Zr-89 m4A2 MDA-MB-231 CAIX KO +CAIX 490,000 12,000,000 0.0611 32.43 Nude HT-29 - 15.28 - 10.31 - †All Zr-89 experiments used BnDFO*. All In-111 experiments used BnDTPA. 58 All three mAbs were tested in the MDA-MB-231 CAIX-/CAIX+ model in NRG female mice to assess the specific tumour uptake of the mAb. Specific uptake of [89Zr]Zr-BnDFO*-c11H9 in the CAIX+ tumours as compared to CAIX- tumours was observed by imaging over time and was confirmed by biodistribution studies (day 3: 5.48 ± 0.42 %ID/g, p < 0.001; day 6: 1.97 ± 0.15 %ID/g, p < 0.005). Similar observations were noticed for [89Zr]Zr-BnDFO*-m4A2 (8.71 ± 1.15 %ID/g, p < 0.005 at day 3; 4.50 ± 0.44 %ID/g, p < 0.005 at day 6). Surprisingly, [89Zr]Zr-BnDFO*-c2C7 showed higher but non-specific CAIX+ tumour uptake on day 3 p.i. (17.75 ± 4.71 %ID/g, p = 0.9) as compared to the CAIX- tumour uptake. However, all three mAbs showed decreasing tumour uptake over time in vivo, similar to the results obtained for the anti-CD96 mAbs in the same mouse strain (Figure 4.2 to Figure 4.4). Despite the fact that m4A2 is a murine mAb, it does not exhibit the same consistent tumour uptake like cCD96 and m11H9 as previously discussed. 59 Figure 4.2 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*c11H9 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NRG female mice. (A) This imaging mouse was injected with 20.87 µg (11.08 MBq) and showed decreasing tumour uptake over time in vivo. Tumours are highlighted with arrows and results are shown as %ID/g. Tumours: left shoulder = CAIX KO, right shoulder = CAIX KO +CAIX. (B) Biodistribution results 3 and 6 days after injection of 20.5 ± 0.4 µg (10.9 ± 0.2 MBq) per mouse, showing decreasing tumour uptake over time in vivo. Results are presented as mean %ID/g ± SD (n = 4 mice on both days). 60 Figure 4.3 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-c2C7 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NRG female mice. (A) This imaging mouse was injected with ~20 µg (3.22 MBq) and showed decreasing tumour uptake over time in vivo. Tumours are highlighted with arrows and results are shown as %ID/g. Tumours: left shoulder = CAIX KO, right shoulder = CAIX KO +CAIX. (B) Biodistribution results 3 and 6 days after injection of ~20 µg (1.0 ± 0.04 MBq) per mouse, showing decreasing tumour uptake over time in vivo. Results are presented as mean %ID/g ± SD (n = 4 on both days). 61 Figure 4.4 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-m4A2 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NRG female mice. (A) This imaging mouse was injected with ~17 µg (2.01 MBq) and showed decreasing tumour uptake over time in vivo. Tumours are highlighted with arrows and results are shown as %ID/g. Tumours: left shoulder = CAIX KO, right shoulder = CAIX KO +CAIX. (B) Biodistribution results 3 and 6 days after injection of ~17 µg (2.0 ± 0.1 MBq) per mouse, showing decreasing tumour uptake over time in vivo. Results are presented as mean %ID/g ± SD (n = 3 mice on both days). 62 In order to replicate the preliminary m11H9 study, a similar experiment using the HT-29 tumour model was performed. Since the murine mAb was not available, its chimeric version was used instead: [111In]In-BnDTPA-c11H9, in immunodeficient NSG mice bearing subcutaneous HT-29 tumours. A significant tumour uptake of 19.35 ± 2.08 %ID/g was observed on Day 3 p.i., but decreased to 6.49 %ID/g on Day 7 p.i., exhibiting the same decreasing tumour uptake over time in vivo as seen in the previous experiments discussed (Figure 4.5). A parallel experiment using MDA-MB-231 CAIX-/CAIX+ model in the same mouse strain was also performed, showing similar results (Figure 4.6). No specific tumour uptake comparing CAIX+ versus CAIX- tumours was found on Day 3 p.i. (11.88 ± 1.75 %ID/g versus 9.13 ± 2.86 %ID/g, p = 0.2). 63 Figure 4.5 SPECT imaging and biodistribution results for [111In]In-BnDTPA-c11H9 in HT-29 tumour-bearing NSG male mice, to try and replicate previous model results. (A) This imaging mouse was injected with 17.53 µg (10.49 MBq) and showed decreasing tumour uptake over time in vivo. Tumours are highlighted with arrows and results are shown as %ID/g. (B) Biodistribution results 3 and 7 days after injection of ~15 µg (6.3 ± 0.2 MBq) per mouse. Decreasing tumour uptake was still observed over time in vivo. Results are presented as mean %ID/g ± SD (n = 3 on Day 3, n = 1 on Day 7). 64 Figure 4.6 SPECT imaging and biodistribution results for [111In]In-BnDTPA-c11H9 in MDA-MB-231 CAIX-/CAIX+ tumour-bearing NSG male mice, to confirm previous 89Zr-mAb transfected model results. (A) This imaging mouse was injected with 16.54 µg (9.90 MBq) and showed decreasing tumour uptake over time in vivo. Tumours are highlighted with arrows and results are shown as %ID/g. Tumours: left shoulder = CAIX KO, right shoulder = CAIX KO +CAIX. (B) Biodistribution results 3 and 7 days after injection of ~15 µg (6.4 ± 0.2 MBq) per mouse. Decreasing tumour uptake was still observed over time in vivo. Results are presented as mean %ID/g ± SD (n = 3 on Day 3, n = 1 on Day 7). 65 Since the [89Zr]Zr-BnDFO*-11F02 in KG1α tumour-bearing nude mice experiment (from Chapter 3) did not succeed in explaining whether the issue resided with the mAbs of interest or the mouse strain used, our next hypothesis was that the use of a validated mAb could shed light on the question. In this context, an experiment using FDA-approved Trastuzumab labeled with 89Zr in SKOV-3 tumour-bearing mice was performed. Imaging and biodistributions were compared side by side between the two strains of mice (NSG vs nude mice) using the same batch of radiolabeled mAb (Figure 4.7 and Figure 4.8). The NSG mice showed decreasing tumour uptake over time in vivo as expected (14.54 ± 1.42 %ID/g on day 1 p.i.; 16.06 ± 2.85 %ID/g on day 3 p.i.; 5.72 ± 3.13 %ID/g on day 6 p.i.), while the nude mice showed relatively consistent tumour uptake (8.95 ± 2.13 %ID/g on day 1 p.i.; 16.80 ± 6.80 %ID/g on day 3 p.i.; 12.71 ± 2.26 %ID/g on day 6 p.i.), indicating that the nude mice offered a promising model that could be successful with mAbs of interest for the first time in both the CD96 and CAIX experiments discussed thus far. In addition, it is worth noting that the retention of the radioimmunoconjugate in the blood was significantly higher in nude mice versus NSG mice, which is consistent with the expected biological half-life of this type of radiotracer. This observation is reinforced with the significantly lower uptake in the excretory organs (spleen, liver, small and large intestines) in the nude mice, with an additional positive characteristic of lower bone uptake (less demetallation). 66 Figure 4.7 PET imaging results for [89Zr]Zr-BnDFO*-Trastuzumab in SKOV3 tumour-bearing female nude† and NSG mice. These imaging mice were injected with 27.66 ± 1.27 µg (4.93 ± 0.23 MBq). The NSG mouse showed decreasing tumour uptake over time in vivo, while the nude mouse showed consistent tumour uptake. Tumours are highlighted with arrows and results are shown as %ID/g. Tumour: left shoulder = HT-29. †Day 1 vertical signal is in abdominal aorta; Day 6 mouse moved during CT scan. 67 Figure 4.8 Biodistribution results for [89Zr]Zr-BnDFO*-Trastuzumab in SKOV3 tumour-bearing female nude and NSG mice. Biodistribution results 1, 3, and 6 days after injection of 25.35 ± 1.44 µg (1.59 ± 1.14 MBq) per mouse. NSG mice showed decreasing tumour uptake over time in vivo, while nude mice showed increasing tumour uptake. Results are presented as mean %ID/g ± SD (n = 4-6 mice on both days). * = p < 0.05, ** = p < 0.01, *** = p < 0.001. 68 With the success of the [89Zr]Zr-BnDFO*-Trastuzumab in nude mice, a similar study using the [89Zr]Zr-BnDFO*-c11H9 in HT-29 tumour-bearing mice was also performed (Figure 4.9 and Figure 4.10). This mAb was selected as this was the only mAb that provided some expected and promising results when using the murine constructs. Analogous to the previous study with [89Zr]Zr-BnDFO*-Trastuzumab, the NSG mice showed decreasing tumour uptake over time in vivo (13.55 ± 0.84 %ID/g on day 1 p.i.; 9.58 ± 1.80 %ID/g on day 3 p.i.; 3.08 ± 0.35 %ID/g on day 6 p.i.), while the nude mice showed consistent tumour uptake (12.19 ± 1.67 %ID/g on day 1 p.i.; 15.13 ± 1.38 %ID/g on day 3 p.i.; 15.88 ± 1.08 %ID/g on day 6 p.i.). The same background organ trends and differences between the nude and NSG mice were observed for [89Zr]Zr-BnDFO*-c11H9 as previously discussed for [89Zr]Zr-BnDFO*-Trastuzumab. 69 Figure 4.9 PET imaging results for [89Zr]Zr-BnDFO*-c11H9 in HT-29 tumour-bearing male nude and NSG mice. These imaging mice were injected with 19.20 ± 0.12 µg (4.60 ± 0.03 MBq). The NSG mouse showed decreasing tumour uptake over time in vivo, while the nude mouse showed consistent tumour uptake. Tumours are highlighted with arrows and results are shown as %ID/g. Tumour: left shoulder = HT-29. 70 Figure 4.10 Biodistribution results for [89Zr]Zr-BnDFO*-c11H9 in HT-29 tumour-bearing male nude and NSG mice. Biodistribution results 1, 3, and 6 days after injection of 21.84 ± 1.18 µg (1.55 ± 0.85 MBq) per mouse. NSG mice show decreasing tumour uptake over time in vivo, while nude mice show increasing tumour uptake. Results are presented as mean %ID/g ± SD (n = 4-5 mice on both days). * = p < 0.05, ** = p < 0.01, *** = p < 0.001. 71 With the consistent tumour uptake over time successfully shown by [89Zr]Zr-BnDFO*-c11H9 in HT-29 tumour-bearing male nude mice, there was reason to believe that similar results could be achieved by [89Zr]Zr-BnDFO*-c2C7 and [89Zr]Zr-BnDFO*-m4A2 in the same model. This hypothesis was validated by the results shown in Figure 4.11, in which the chimeric c11H9 and c2C7 mAbs are compared side-by-side with their isotype control human IgG1 mAb (hIgG1). The validated results for the m4A2 mAb are shown in Figure 4.12, with its isotype control mouse IgG2b mAb (mIgG2b) to be determined in an upcoming study. The leading radioimmunoconjugate candidate appears to be [89Zr]Zr-BnDFO*-c2C7, with tumour uptake values reaching 18.88 ± 3.71 %ID/g on Day 3 p.i., and 20.22 ± 1.50 %ID/g on Day 6 p.i. (n = 5 on both days). 72 Figure 4.11 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-c11H9, -c2C7, and -hIgG1 in HT-29 tumour-bearing male nude mice. (A) Mice were injected with 22.3 ± 0.4 µg (5.0 ± 2.2 MBq, n = 1 per mAb) and showed consistent tumour uptake over time for c11H9 and c2C7. The c2C7 Day 1 image was not available due to equipment issues. Tumours are highlighted with arrows and results are shown as %ID/g. Tumour: left shoulder = HT-29. (B) Biodistribution results 3 and 6 days after injection of 19.7 ± 0.8 µg (1.6 ± 0.1 MBq) per mouse, showing consistent tumour uptake over time in vivo for c11H9 and c2C7. Results are presented as mean %ID/g ± SD (n = 5 mice on both days per mAb). * = p < 0.05, ** = p < 0.01, *** = p < 0.001. 73 Figure 4.12 PET imaging and biodistribution results for [89Zr]Zr-BnDFO*-m4A2 in HT-29 tumour-bearing male nude mice. (A) This imaging mouse was injected with 19.16 µg (8.00 MBq) and showed robust tumour uptake over time in vivo Tumours are highlighted with arrows and results are shown as %ID/g. Tumour: left shoulder = HT-29. (B) Biodistribution results 3 and 6 days after injection of 18.64 ± 0.65 µg (1.86 ± 0.07 MBq) per mouse, showing robust tumour uptake over time in vivo. Results are presented as mean %ID/g ± SD (n = 5 mice on both days). * = p < 0.05, ** = p < 0.01, *** = p < 0.001. 74 4.3.2 CAIX in Acute Myeloid Leukemia – Pilot Study A screening of nine different acute myeloid leukemia cell lines was undertaken by FACS, using the HT-29 cell line as the control, for CAIX expression under normoxia and hypoxia (Table 4.2). It was expected that a higher level of protein expression would be observed at hypoxia compared to normoxia. Table 4.2 Summary of screening of 9 different AML cell lines by FACS. Name Cancer type Level of CAIX expression observed† Normoxia Hypoxia Positive control HT-29 Colorectal adenocarcinoma ++ +++ Cell lines of interest, ranked from most to least promising for in vivo study2 Molm13 AML + +/++ OCIAML3 AML ++ ++ NOMO1 AML +/++ +/++ OCIAML5 AML + + UCSD AML1 AML + + MUTZ3 AML + + Kasumi1 AML -/+ -/+ HNT34 AML -/+ -/+ MOLM1 AML - - †“-” denotes no CAIX expression; “+” denotes slight CAIX expression, ~1 log shift from negative controls; “++” denotes moderate CAIX expression, ~2 log shifts from negative controls; “+++” denotes high CAIX expression, ~3 log shifts from negative controls. 75 From these studies, 3 cell lines, Molm13, OCIAML3, and NOMO1 seemed to prove most promising in CAIX expression under these expectations (Figure 4.13), and were engrafted in nude mice (n = 4 for each of Molm13 and OCIAML3, n = 3 for NOMO1) for further studies. Figure 4.13 FACS results for HT-29 and the 3 most promising AML cell lines for CAIX expression. Level of CAIX expression is denoted in the same manner as in Table 4.2. Nude mice were pre-treated with pimonidazole and BrdU prior to euthanization for tumour excision; half of each tumour was dedicated for ex vivo FACS, and the other half for immunohistochemistry (IHC) studies. During IHC, tumours were sliced and stained for CAIX, pimonidazole (hypoxia), and BrdU (proliferation). During the ex vivo FACS, it was unfortunately found that due to the negative control mAb used during the cell line screening process, there was a false negative signal as it was not a true 76 isotype control mAb. This conclusion was made when true isotype controls were used in the ex vivo FACS assays for OCIAML3 and Molm13, with their in vitro normoxic counterparts added to the assays for parallel comparisons (Figure 4.14). The expected result was to see only the anti-CAIX mAb signal shift, with the unstained/secondary only/isotype control populations remaining at baseline. However, it appears that non-specific binding of both isotype control and secondary mAb occurred for the ex vivo OCIAML3 and Molm13 cells, and non-specific binding of isotype control occurred for the in vitro OCIAML3 cells. Only the Molm13 in vitro normoxic cells achieved the expected result. Figure 4.14 Ex vivo FACS results for OCIAML3 and Molm13, with their in vitro counterparts for comparison. * = stained with 7AAD viability dye and gated for confirmed live cells only. 77 It was then thought that if there was non-specific binding, perhaps this issue could be solved by replacing the need of the secondary mAb with fluorophore-conjugated anti-CAIX and isotype control mAbs, with the addition of 10% mouse serum prior to antibody incubation. The mouse serum was thought to saturate any Fc receptors that may be present in the cells or cellular matrix, which may have been causing the non-specific binding97. The ex vivo FACS results for Molm13 and NOMO1 were thus drastically different, as shown in Figure 4.15, with no CAIX-positive signals observed. It seems that the addition of the mouse serum proved that all signal shifts observed in past FACS data were due to non-specific binding, including the anti-CAIX mAb signals, and the mouse serum appeared to have saturated Fc receptors that have otherwise been binding the added mAbs. 78 Figure 4.15 Ex vivo FACS results for Molm13 and NOMO1, with their in vitro counterparts for comparison. Note that there were no more OCIAML3 tumours available for analysis at this point in time, and so no OCIAML3 ex vivo data under these conditions are shown. The isotype control mAb was added at both manufacturer-recommended volumes at the time to ensure effectiveness. These FACS results were confirmed by the IHC results performed on the other halves of the tumours used for the ex vivo FACS assays, with non-specific staining appearing across all tumour slices (Figure 4.16 and Figure 4.17). It appears that non-specific staining also occurred for anti-pimonidazole and anti-BrdU mAbs. 79 Figure 4.16 Immunohistochemistry results for CAIX expression in various slides. Positive CAIX-expressing control sections stained with (A) Oksana’s anti-CAIX mAb and (B) Abcam anti-CAIX mAb; OCIAML3 NP sections stained with (C) Oksana’s anti-CAIX mAb and (D) isotype control; OCIAML3 NP sections stained with (E) Abcam anti-CAIX mAb and (F) isotype control; Molm13 NP sections stained with (G) Abcam anti-CAIX mAb and (H) isotype control; NOMO1 L1 sections stained with (I) Abcam anti-CAIX mAb and (J) isotype control. Similar results were obtained for Molm13 and NOMO1 sections for Oksana’s anti-CAIX mAb vs. isotype control and are not shown. 80 Figure 4.17 Immunohistochemistry results for pimonidazole and BrdU staining. OCIAML3 NP sections stained with anti-pimonidazole mAb at (A) 1:1500 dilution and (B) 1:100 dilution, and with anti-BrdU mAb at (C) 1:5 dilution and (D) 1:10 dilution. Similar results were obtained for Molm13 and NOMO1 sections and are not shown. Due to the positive signals that were shown by the original FACS screening being disproven by the ex vivo FACS and IHC results, at this point there does not seem to be CAIX expression in any AML cell lines tested under the conditions attempted. Further investigation may be needed under different hypoxia-inducing conditions. 4.4 Conclusion For the CAIX in renal and colon cancer study, it was shown that for either 89Zr- or 111In-labelled anti-CAIX mAbs of interest in NRG/NSG mice, the tumour uptake was not consistent over the course of each study. However, all of the 89Zr-labelled c11H9, c2C7, and m4A2 mAbs showed high specific tumour uptake with good contrast over time in athymic nude mice, as 81 compared to the 89Zr-hIgG1 isotype control. The success in the nude mice strain could be attributed to a higher level of endogenous mAbs that could saturate FcRn or Fc gamma receptor-mediated mAb sequestration in non-target organs, allowing radioimmunoconjugates to remain in the body longer and providing more time to bind to the tumour of interest, while avoiding uptake by excretory organs for the duration of the study. In particular, the leading PET imaging candidate appears to be 89Zr-c2C7, which had the highest uptake and contrast compared to the other two antibodies. The next step from this investigation would be to evaluate the performance of the c2C7 mAb as a therapeutic agent when conjugated with beta and/or alpha emitting radioisotopes. For the CAIX in AML pilot study, there did not seem to have been definitive CAIX expression in OCIAML3, Molm13, and NOMO1 cells in the conditions tested. Further investigations into either alternate hypoxia-inducing conditions or different testing methods (e.g. Western blot) may be needed to confirm whether CAIX could be induced in these cells. 82 Chapter 5: Conclusions 5.1 Summary of Findings, Future Directions, and Significance The investigations discussed in Chapters 3 and 4 have overall shed new light into the field of radioimmunoconjugate development to aid in identifying areas of tumour dormancy. The almost parallel undertaking of both anti-CD96 and anti-CAIX radiolabelled mAb experiments in this thesis led to the discovery that while previous mAb candidates in our research group have succeeded in NRG/NSG mouse models for imaging screening, the mAbs of interest discussed herein exhibited an issue of decreasing tumour uptake over time that was not previously encountered in similar models with the same mouse strains. For the leukemic stem cell marker CD96, the novelty of this target resulted in an enthusiastic venture (for everyone involved in our collaborations) to figure out exactly what parameters we could improve on and until what point we could make conclusions about the investigation. The initial success of the specific tumour uptake of radiolabelled anti-CD96 mAbs brought hope that this cell-surface protein could be imaged, and it is this hope that should be carried forward in future radiotracer investigations of this protein. The downstream implications of a successful anti-CD96 radioimmunoconjugate candidate for RIT will open up a new avenue of treatment for AML patients that would be LSC-specific, targeting the self-renewing component of this aggressive disease. The hypoxia marker CAIX is a well-studied target in radioimmunotherapeutic research but there is still room for improvement on anti-CAIX radiotracers thus far. The switch to athymic nude mice from NRG/NSG strains for the radioimmunoconjugate in vivo investigations proved to be a wise choice, resulting in high and specific tumour uptake with good contrast over time for all three 83 novel mAb candidates involved. The higher retention of radioimmunoconjugates in the blood in nude mice may be due to higher levels of endogenous mAbs that could saturate FcRn or Fc gamma receptor-mediated mAb sequestration in non-target organs, enabling longer circulation time for the radiotracer to bind to the tumour while avoiding the excretory routes for antibodies. Due to this observation, it is recommended that nude mice be chosen for mouse models over NRG/NSG strains for future screening of radiolabelled antibodies that have human Fc portions. The lead candidate, c2C7, will move on to the next phase of radioimmunoconjugate development to be labelled with beta and/or alpha emitters for RIT studies. The aspired end goal would be introducing c2C7 into the clinic as a novel targeted diagnostic and therapeutic approach for patients that do not respond to conventional treatment for aggressive cancers such as renal and colon cancers. At the time of these findings, whether or not CAIX expression is present in AML cell lines has not yet been determined. It may be the presence of Fc receptors in the leukemic cell lines that could explain why controls exhibited positive signals in these cells, in both the FACS and IHC experiments, but there is a need for further research into the biology of these cells for a complete understanding97. Given that the multiple anti-CAIX mAbs used in this pilot study produced similar results to their corresponding controls, it is concluded that under the conditions used to induce hypoxia that no CAIX expression was found. 5.2 Concluding Remarks In this thesis, we hypothesized that leukemic stem cell- and hypoxia-specific markers can be targeted for cancer treatment and/or theranostic applications to help identify areas of tumor dormancy, aiding against drug resistance and patient relapse. As both panels of anti-CD96 and anti-CAIX radioimmunoconjugates showed specific tumour uptake in vivo for their respective 84 target, this hypothesis can be accepted. The first aim was to evaluate radioimmunoconjugate candidates against CD96 for acute myeloid leukemia by PET imaging, which was extensively investigated but yielded undesirable results for the selected panel of candidates, suggesting a novel panel of mAbs may be required. The second aim was to screen mAbs targeting CAIX by PET imaging in renal cancer, colon cancer, and acute myeloid leukemia models, which identified three radioimmunoconjugate candidates for the tumour models studied, and possible further study needed for expression of CAIX in AML. Overall, from this thesis, c2C7 emerged as a lead anti-CAIX mAb candidate that proved to have most potential as an RIT agent, which warrants further therapeutic and possibly clinical studies. 85 Bibliography 1 Lambert, A. W., Pattabiraman, D. R. & Weinberg, R. A. Emerging Biological Principles of Metastasis. Cell 168, 670-691, doi:10.1016/j.cell.2016.11.037 (2017). 2 Dillekås, H., Rogers, M. S. & Straume, O. Are 90% of deaths from cancer caused by metastases? Cancer Med 8, 5574-5576, doi:10.1002/cam4.2474 (2019). 3 Phan, T. G. & Croucher, P. I. The dormant cancer cell life cycle. Nature Reviews Cancer 20, 398-411, doi:10.1038/s41568-020-0263-0 (2020). 4 Recasens, A. & Munoz, L. Targeting Cancer Cell Dormancy. Trends in Pharmacological Sciences 40, 128-141, doi:10.1016/j.tips.2018.12.004 (2019). 5 Butturini, E., Carcereri de Prati, A., Boriero, D. & Mariotto, S. Tumor Dormancy and Interplay with Hypoxic Tumor Microenvironment. International journal of molecular sciences 20, 4305, doi:10.3390/ijms20174305 (2019). 6 Carcereri de Prati, A. et al. Metastatic Breast Cancer Cells Enter Into Dormant State and Express Cancer Stem Cells Phenotype Under Chronic Hypoxia. Journal of cellular biochemistry 118, 3237-3248, doi:10.1002/jcb.25972 (2017). 7 Fluegen, G. et al. Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments. Nature cell biology 19, 120-132, doi:10.1038/ncb3465 (2017). 8 Sosa, M. S. et al. NR2F1 controls tumour cell dormancy via SOX9- and RARβ-driven quiescence programmes. Nature Communications 6, 6170, doi:10.1038/ncomms7170 (2015). 9 Patel, P. & Chen, E. Cancer stem cells, tumor dormancy, and metastasis. 3, doi:10.3389/fendo.2012.00125 (2012). 10 Giancotti, Filippo G. Mechanisms Governing Metastatic Dormancy and Reactivation. Cell 155, 750-764, doi:10.1016/j.cell.2013.10.029 (2013). 11 Lin, W.-C., Rajbhandari, N. & Wagner, K.-U. Cancer Cell Dormancy in Novel Mouse Models for Reversible Pancreatic Cancer: A Lingering Challenge in the Development of Targeted Therapies. Cancer Research 74, 2138, doi:10.1158/0008-5472.CAN-13-3437 (2014). 12 Zhou, N. et al. Stem cell characteristics of dormant cells and cisplatin‑induced effects on the stemness of epithelial ovarian cancer cells. Mol Med Rep 10, 2495-2504, doi:10.3892/mmr.2014.2483 (2014). 13 Plaks, V., Kong, N. & Werb, Z. The Cancer Stem Cell Niche: How Essential Is the Niche in Regulating Stemness of Tumor Cells? Cell Stem Cell 16, 225-238, doi:10.1016/j.stem.2015.02.015 (2015). 14 Khalil, M. M. & SpringerLink ebooks, M. Basic sciences of nuclear medicine. (Springer, 2011). 15 Gambhir, S. S. Molecular imaging of cancer with positron emission tomography. Nature Reviews Cancer 2, 683-693, doi:10.1038/nrc882 (2002). 16 Townsend, D. W. Positron emission tomography/computed tomography. Seminars in nuclear medicine 38, 152-166, doi:10.1053/j.semnuclmed.2008.01.003 (2008). 17 Lau, J. et al. Insight into the Development of PET Radiopharmaceuticals for Oncology. Cancers (Basel) 12, 1312, doi:10.3390/cancers12051312 (2020). 86 18 Acton, P. D., Zhuang, H. & Alavi, A. Quantification in PET. Radiologic clinics of North America 42, 1055-1062, viii, doi:10.1016/j.rcl.2004.08.010 (2004). 19 Liberti, M. V. & Locasale, J. W. The Warburg Effect: How Does it Benefit Cancer Cells? Trends in biochemical sciences 41, 211-218, doi:10.1016/j.tibs.2015.12.001 (2016). 20 Griffeth, L. K. Use of PET/CT scanning in cancer patients: technical and practical considerations. Proceedings (Baylor University. Medical Center) 18, 321-330, doi:10.1080/08998280.2005.11928089 (2005). 21 Majeti, R. Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells. Oncogene 30, 1009-1019, doi:10.1038/onc.2010.511 (2011). 22 Kim, J. S. Combination Radioimmunotherapy Approaches and Quantification of Immuno-PET. Nuclear medicine and molecular imaging 50, 104-111, doi:10.1007/s13139-015-0392-7 (2016). 23 Song, I. H. et al. Immuno-PET Imaging and Radioimmunotherapy of 64Cu-/177Lu-Labeled Anti-EGFR Antibody in Esophageal Squamous Cell Carcinoma Model. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 57, 1105-1111, doi:10.2967/jnumed.115.167155 (2016). 24 Yoshimoto, M., Kurihara, H. & Fujii, H. Theragnostic imaging using radiolabeled antibodies and tyrosine kinase inhibitors. TheScientificWorldJournal 2015, 842101, doi:10.1155/2015/842101 (2015). 25 Freise, A. C. & Wu, A. M. In vivo imaging with antibodies and engineered fragments. Molecular immunology 67, 142-152, doi:10.1016/j.molimm.2015.04.001 (2015). 26 Pouget, J.-P. et al. Clinical radioimmunotherapy—the role of radiobiology. Nature Reviews Clinical Oncology 8, 720-734, doi:10.1038/nrclinonc.2011.160 (2011). 27 Navarro-Teulon, I., Lozza, C., Pèlegrin, A., Vivès, E. & Pouget, J. P. General overview of radioimmunotherapy of solid tumors. Immunotherapy 5, 467-487, doi:10.2217/imt.13.34 (2013). 28 McKnight, B. N. & Viola-Villegas, N. T. (89) Zr-ImmunoPET companion diagnostics and their impact in clinical drug development. Journal of labelled compounds & radiopharmaceuticals 61, 727-738, doi:10.1002/jlcr.3605 (2018). 29 Marik, J. & Junutula, J. R. Emerging role of immunoPET in receptor targeted cancer therapy. Current drug delivery 8, 70-78, doi:10.2174/156720111793663589 (2011). 30 Hnatowich, D. J. Recent developments in the radiolabeling of antibodies with iodine, indium, and technetium. Seminars in nuclear medicine 20, 80-91 (1990). 31 Wilson, C. B. et al. Quantitative measurement of monoclonal antibody distribution and blood flow using positron emission tomography and 124iodine in patients with breast cancer. International Journal of Cancer 47, 344-347, doi:10.1002/ijc.2910470305 (1991). 32 Verel, I. et al. High-quality 124I-labelled monoclonal antibodies for use as PET scouting agents prior to 131I-radioimmunotherapy. European journal of nuclear medicine and molecular imaging 31, 1645-1652, doi:10.1007/s00259-004-1632-8 (2004). 33 Divgi, C. R. et al. Preoperative characterisation of clear-cell renal carcinoma using iodine-124-labelled antibody chimeric G250 (124I-cG250) and PET in patients with renal masses: a phase I trial. The Lancet. Oncology 8, 304-310, doi:10.1016/s1470-2045(07)70044-x (2007). 34 Meijs, W. E. et al. A facile method for the labeling of proteins with zirconium isotopes. Nuclear medicine and biology 23, 439-448 (1996). 87 35 Perk, L. R. et al. Preparation and evaluation of (89)Zr-Zevalin for monitoring of (90)Y-Zevalin biodistribution with positron emission tomography. European journal of nuclear medicine and molecular imaging 33, 1337-1345, doi:10.1007/s00259-006-0160-0 (2006). 36 Holland, J. P., Sheh, Y. & Lewis, J. S. Standardized methods for the production of high specific-activity zirconium-89. Nuclear medicine and biology 36, 729-739, doi:10.1016/j.nucmedbio.2009.05.007 (2009). 37 Vosjan, M. J. et al. Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine. Nature protocols 5, 739-743, doi:10.1038/nprot.2010.13 (2010). 38 Wadas, T. J., Wong, E. H., Weisman, G. R. & Anderson, C. J. Coordinating radiometals of copper, gallium, indium, yttrium, and zirconium for PET and SPECT imaging of disease. Chem Rev 110, 2858-2902, doi:10.1021/cr900325h (2010). 39 O'Donnell, M. R. et al. NCCN Clinical Practice Guidelines Acute myeloid leukemia. Journal of the National Comprehensive Cancer Network : JNCCN 10, 984-1021, doi:10.6004/jnccn.2012.0103 (2012). 40 Williams, B. A. et al. Antibody Therapies for Acute Myeloid Leukemia: Unconjugated, Toxin-Conjugated, Radio-Conjugated and Multivalent Formats. Journal of clinical medicine 8, doi:10.3390/jcm8081261 (2019). 41 Kantarjian, H. et al. Acute myeloid leukemia: current progress and future directions. Blood Cancer Journal 11, 41, doi:10.1038/s41408-021-00425-3 (2021). 42 Hosen, N. et al. CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia. Proceedings of the National Academy of Sciences of the United States of America 104, 11008-11013, doi:10.1073/pnas.0704271104 (2007). 43 Jin, L., Hope, K. J., Zhai, Q., Smadja-Joffe, F. & Dick, J. E. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med 12, 1167-1174, doi:10.1038/nm1483 (2006). 44 Yabushita, T. et al. Expression of multiple leukemic stem cell markers is associated with poor prognosis in de novo acute myeloid leukemia. Leukemia & lymphoma 59, 2144-2151, doi:10.1080/10428194.2017.1410888 (2018). 45 Chavez-Gonzalez, A. et al. Expression of CD90, CD96, CD117, and CD123 on different hematopoietic cell populations from pediatric patients with acute myeloid leukemia. Archives of medical research 45, 343-350, doi:10.1016/j.arcmed.2014.04.001 (2014). 46 Coustan-Smith, E. et al. Universal monitoring of minimal residual disease in acute myeloid leukemia. JCI insight 3, doi:10.1172/jci.insight.98561 (2018). 47 Ding, Y., Gao, H. & Zhang, Q. The biomarkers of leukemia stem cells in acute myeloid leukemia. Stem cell investigation 4, 19, doi:10.21037/sci.2017.02.10 (2017). 48 Georgiev, H., Ravens, I., Papadogianni, G. & Bernhardt, G. Coming of Age: CD96 Emerges as Modulator of Immune Responses. 9, doi:10.3389/fimmu.2018.01072 (2018). 49 Blake, S. J., Dougall, W. C., Miles, J. J., Teng, M. W. & Smyth, M. J. Molecular Pathways: Targeting CD96 and TIGIT for Cancer Immunotherapy. Clinical cancer research : an official journal of the American Association for Cancer Research 22, 5183-5188, doi:10.1158/1078-0432.Ccr-16-0933 (2016). 50 Harris, A. L. Hypoxia--a key regulatory factor in tumour growth. Nature reviews. Cancer 2, 38-47, doi:10.1038/nrc704 (2002). 88 51 McDonald, P. C., Winum, J.-Y., Supuran, C. T. & Dedhar, S. Recent developments in targeting carbonic anhydrase IX for cancer therapeutics. Oncotarget 3, 84-97, doi:10.18632/oncotarget.422 (2012). 52 Supuran, C. T. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nature reviews. Drug discovery 7, 168-181, doi:10.1038/nrd2467 (2008). 53 Parks, S. K., Chiche, J. & Pouyssegur, J. pH control mechanisms of tumor survival and growth. Journal of cellular physiology 226, 299-308, doi:10.1002/jcp.22400 (2011). 54 Boyer, M. J. & Tannock, I. F. Regulation of intracellular pH in tumor cell lines: influence of microenvironmental conditions. Cancer Res 52, 4441-4447 (1992). 55 Brahimi-Horn, M. C. & Pouyssegur, J. Hypoxia in cancer cell metabolism and pH regulation. Essays in biochemistry 43, 165-178, doi:10.1042/bse0430165 (2007). 56 Pastorekova, S., Zavadova, Z., Kostal, M., Babusikova, O. & Zavada, J. A novel quasi-viral agent, MaTu, is a two-component system. Virology 187, 620-626 (1992). 57 Alterio, V., Di Fiore, A., D'Ambrosio, K., Supuran, C. T. & De Simone, G. Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? Chem Rev 112, 4421-4468, doi:10.1021/cr200176r (2012). 58 Pastoreková, S., Závadová, Z., Kostál, M., Babusíková, O. & Závada, J. A novel quasi-viral agent, MaTu, is a two-component system. Virology 187, 620-626, doi:10.1016/0042-6822(92)90464-z (1992). 59 McDonald, P. C., Winum, J. Y., Supuran, C. T. & Dedhar, S. Recent developments in targeting carbonic anhydrase IX for cancer therapeutics. Oncotarget 3, 84-97, doi:10.18632/oncotarget.422 (2012). 60 Hilvo, M. et al. Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes. The Journal of biological chemistry 283, 27799-27809, doi:10.1074/jbc.M800938200 (2008). 61 Kessenbrock, K., Plaks, V. & Werb, Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141, 52-67, doi:10.1016/j.cell.2010.03.015 (2010). 62 Huizing, F. J. et al. CAIX-targeting radiotracers for hypoxia imaging in head and neck cancer models. Scientific Reports 9, 18898, doi:10.1038/s41598-019-54824-5 (2019). 63 Muglia, V. F. & Prando, A. Renal cell carcinoma: histological classification and correlation with imaging findings. Radiol Bras 48, 166-174, doi:10.1590/0100-3984.2013.1927 (2015). 64 Stillebroer, A. B., Mulders, P. F., Boerman, O. C., Oyen, W. J. & Oosterwijk, E. Carbonic anhydrase IX in renal cell carcinoma: implications for prognosis, diagnosis, and therapy. European urology 58, 75-83, doi:10.1016/j.eururo.2010.03.015 (2010). 65 Herman, J. G. et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proceedings of the National Academy of Sciences of the United States of America 91, 9700-9704, doi:10.1073/pnas.91.21.9700 (1994). 66 Shuin, T. et al. Frequent somatic mutations and loss of heterozygosity of the von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas. Cancer Res 54, 2852-2855 (1994). 67 Bruehlmeier, M., Roelcke, U., Schubiger, P. A. & Ametamey, S. M. Assessment of hypoxia and perfusion in human brain tumors using PET with 18F-fluoromisonidazole and 15O-H2O. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 45, 1851-1859 (2004). 89 68 Couturier, O. et al. Fluorinated tracers for imaging cancer with positron emission tomography. European journal of nuclear medicine and molecular imaging 31, 1182-1206, doi:10.1007/s00259-004-1607-9 (2004). 69 Choschzick, M. et al. Overexpression of carbonic anhydrase IX (CAIX) is an independent unfavorable prognostic marker in endometrioid ovarian cancer. Virchows Archiv : an international journal of pathology 459, 193-200, doi:10.1007/s00428-011-1105-y (2011). 70 Haapasalo, J. A. et al. Expression of carbonic anhydrase IX in astrocytic tumors predicts poor prognosis. Clinical cancer research : an official journal of the American Association for Cancer Research 12, 473-477, doi:10.1158/1078-0432.Ccr-05-0848 (2006). 71 Hoogsteen, I. J. et al. Colocalization of carbonic anhydrase 9 expression and cell proliferation in human head and neck squamous cell carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 11, 97-106 (2005). 72 Hutchison, G. J. et al. Hypoxia-inducible factor 1alpha expression as an intrinsic marker of hypoxia: correlation with tumor oxygen, pimonidazole measurements, and outcome in locally advanced carcinoma of the cervix. Clinical cancer research : an official journal of the American Association for Cancer Research 10, 8405-8412, doi:10.1158/1078-0432.Ccr-03-0135 (2004). 73 Ilie, M. et al. High levels of carbonic anhydrase IX in tumour tissue and plasma are biomarkers of poor prognostic in patients with non-small cell lung cancer. Br J Cancer 102, 1627-1635, doi:10.1038/sj.bjc.6605690 (2010). 74 Klatte, T. et al. Carbonic anhydrase IX in bladder cancer: a diagnostic, prognostic, and therapeutic molecular marker. Cancer 115, 1448-1458, doi:10.1002/cncr.24163 (2009). 75 Korkeila, E. et al. Expression of carbonic anhydrase IX suggests poor outcome in rectal cancer. Br J Cancer 100, 874-880, doi:10.1038/sj.bjc.6604949 (2009). 76 Lou, Y. et al. Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors. Cancer Res 71, 3364-3376, doi:10.1158/0008-5472.Can-10-4261 (2011). 77 Saarnio, J. et al. Immunohistochemistry of carbonic anhydrase isozyme IX (MN/CA IX) in human gut reveals polarized expression in the epithelial cells with the highest proliferative capacity. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 46, 497-504, doi:10.1177/002215549804600409 (1998). 78 Lau, J., Lin, K. S. & Bénard, F. Past, Present, and Future: Development of Theranostic Agents Targeting Carbonic Anhydrase IX. Theranostics 7, 4322-4339, doi:10.7150/thno.21848 (2017). 79 Chomet, M. et al. Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical 89Zr-immuno-PET. European journal of nuclear medicine and molecular imaging 48, 694-707, doi:10.1007/s00259-020-05002-7 (2021). 80 Vosjan, M. J. W. D. et al. Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine. Nature protocols 5, 739-743, doi:10.1038/nprot.2010.13 (2010). 81 Rousseau, J. et al. Design, synthesis and evaluation of novel bifunctional tetrahydroxamate chelators for PET imaging of 89Zr-labeled antibodies. Bioorganic & Medicinal Chemistry Letters 27, 708-712, doi:https://doi.org/10.1016/j.bmcl.2017.01.052 (2017). 82 Vugts, D. J. et al. Comparison of the octadentate bifunctional chelator DFO*-pPhe-NCS and the clinically used hexadentate bifunctional chelator DFO-pPhe-NCS for (89)Zr-90 immuno-PET. European journal of nuclear medicine and molecular imaging 44, 286-295, doi:10.1007/s00259-016-3499-x (2017). 83 Morais, M. & Ma, M. T. Site-specific chelator-antibody conjugation for PET and SPECT imaging with radiometals. Drug discovery today. Technologies 30, 91-104, doi:10.1016/j.ddtec.2018.10.002 (2018). 84 Price, E. W. & Orvig, C. Matching chelators to radiometals for radiopharmaceuticals. Chemical Society Reviews 43, 260-290, doi:10.1039/C3CS60304K (2014). 85 Wadas, T. J., Wong, E. H., Weisman, G. R. & Anderson, C. J. Coordinating radiometals of copper, gallium, indium, yttrium, and zirconium for PET and SPECT imaging of disease. Chemical reviews 110, 2858-2902, doi:10.1021/cr900325h (2010). 86 Teresa Albelda, M., Garcia-España, E. & Frias, J. C. Visualizing the atherosclerotic plaque: a chemical perspective. Chem Soc Rev 43, 2858-2876, doi:10.1039/c3cs60410a (2014). 87 Price, E. W. et al. A comparative evaluation of the chelators H4octapa and CHX-A″-DTPA with the therapeutic radiometal (90)Y. Nuclear medicine and biology 43, 566-576, doi:10.1016/j.nucmedbio.2016.06.004 (2016). 88 Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J. & Bunn, P. A., Jr. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. Journal of immunological methods 72, 77-89, doi:10.1016/0022-1759(84)90435-6 (1984). 89 Doevendans, E. & Schellekens, H. Immunogenicity of Innovative and Biosimilar Monoclonal Antibodies. Antibodies (Basel, Switzerland) 8, doi:10.3390/antib8010021 (2019). 90 Dias, G. M. Evaluation of cyclotron produced radiometals for radiolabeling of immuno- and bio-conjugates for nuclear imaging, University of British Columbia - Dissertation, (2017). 91 Naruki, Y. et al. Differential cellular catabolism of 111In, 90Y and 125I radiolabeled T101 anti-CD5 monoclonal antibody. International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology 17, 201-207, doi:10.1016/0883-2897(90)90148-t (1990). 92 Zhang, C. et al. Preclinical Melanoma Imaging with (68)Ga-Labeled α-Melanocyte-Stimulating Hormone Derivatives Using PET. Theranostics 7, 805-813, doi:10.7150/thno.17117 (2017). 93 Sharma, S. K. et al. Fc-Mediated Anomalous Biodistribution of Therapeutic Antibodies in Immunodeficient Mouse Models. Cancer Res 78, 1820-1832, doi:10.1158/0008-5472.Can-17-1958 (2018). 94 Lau, J. C. C. Targeting tumour microenvironment : Development of carbonic anhydrase IX nuclear imaging agents, University of British Columbia - Dissertation, (2016). 95 Deynoux, M., Sunter, N., Hérault, O. & Mazurier, F. Hypoxia and Hypoxia-Inducible Factors in Leukemias. Front Oncol 6, 41-41, doi:10.3389/fonc.2016.00041 (2016). 96 Chen, F., Rogozea, A., Sandusky, G., Ivan, M. & Konig, H. Inhibition of Carbonic Anhydrase-IX As a Novel Strategy to Overcome Hypoxia Mediated Drug Resistance in FLT3/ITD Mutated Acute Myeloid Leukemia. Blood 130, 2521-2521, doi:10.1182/blood.V130.Suppl_1.2521.2521 %J Blood (2017). 97 Liesveld, J. L., Abboud, C. N., Looney, R. J., Ryan, D. H. & Brennan, J. K. Expression of IgG Fc receptors in myeloid leukemic cell lines. Effect of colony-stimulating factors and cytokines. Journal of immunology (Baltimore, Md. : 1950) 140, 1527-1533 (1988).
Thesis/Dissertation
eng
Interdisciplinary Oncology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Bénard, François
Using radioimmunoconjugates to target quiescent tumour cells via cancer stem cell and hypoxia-induced markers : an approach against metastatic disease and therapeutic resistance
Text