Elucidating the Mechanism(s) of Action of Anti-Erythrocyte Antibodies in a Murine Model of Immune Thrombocytopenia by Ramsha Khan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto © Copyright by Ramsha Khan 2021 Elucidating the Mechanism(s) of Action of Anti-Erythrocyte Antibodies in a Murine Model of Immune Thrombocytopenia Ramsha Khan Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2021 Abstract Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder that causes thrombocytopenia (decreased platelet numbers in the blood) primarily due to the presence of antiplatelet autoantibodies. Anti-D therapy (treatment with an antibody against the Rhesus-D factor on erythrocytes) has been proven to ameliorate ITP but as a human-derived product, it is limited in quantity and carries the potential risk of transferring emerging pathogens. Additionally, the US Food and Drug Administration has issued a black box warning for potentially serious adverse effects in ITP patients, including anemia, fever and/or chills. Therefore, there is incentive present for developing a safe and effective recombinant replacement that mimics the therapeutic activity of anti-D. Thus far, monoclonal anti-D antibodies have only had limited clinical success and better knowledge of its mechanism is required to accelerate the development of anti-D alternatives. This thesis documents studies that evaluated multiple anti- erythrocyte antibodies (as surrogates for anti-D) in murine models of ITP in order to provide insight regarding their mechanism(s) of action. Results indicate that adverse events such as anemia and temperature flux, which can be indicative of inflammatory activity, are not required for ITP amelioration; thus demonstrating that certain toxicities associated with anti-D therapy are independent of therapeutic activity and have the potential to be minimized when developing ii and/or screening synthetic alternatives. Additionally, it was also shown that the ability of the anti-erythrocyte antibodies to ameliorate murine ITP in vivo is correlated with, and therefore can be predicted by, their ability to mediate erythrocyte phagocytosis and inhibit platelet phagocytosis in vitro. The data suggests that the inhibition of in vitro platelet phagocytosis may prove to be a valuable tool for testing the functionality of erythrocyte-specific antibodies and screening likely candidates for clinical use in ITP. Mechanistically, evidence is provided demonstrating that anti-erythrocyte antibodies do not lead to a decrease in the expression level of the activating receptor FcγRIIIA on splenic macrophages, but instead are likely exerting their therapeutic activity through a direct competitive blocking mechanism. The results provide substantial support for the mononuclear phagocytic system (MPS) blockade hypothesis as the primary mechanism of action in anti-erythrocyte antibody-mediated ITP amelioration. iii Acknowledgments I would like to start by thanking Dr. Alan Lazarus for first taking me on as a graduate student in his lab and then for the continuous mentoring and support that followed. Alan provided much guidance and assistance over the years that has helped me grow as a scientist. He was always around to help with any issue that arose and even during the COVID-19 pandemic, he ensured to help resolve any problem that may come up. I am truly grateful to have gained Alan as a mentor. I would also like to express my gratitude to Dr. Gregory Fairn, Dr. Gregory Hare and Dr. Gerald Prud’homme for serving as valuable committee members. Their constant questioning and advice have provided great insight into my project and helped me succeed in it in an improved manner. I would also like to acknowledge all my collaborators and facilitators. Previous members of the lab, Melissa Menard, Mark Jen, and Xi Chen, for their efforts on the phagocytosis project. Special thanks to CSL Behring for kindly providing the IgG1 and IgG2a variants of the TER-119 antibody, and to Dr. Heyu Ni and Guangheng Zhu for the anti-platelet antibodies (5C4, 10B5, 2B12 and 2G1). Thank you to all the vivarium staff at St. Michael’s hospital for providing animal care and to the research core facility – Dr. Christopher Spring for flow cytometry training and support, and Dr. Caterina Di Ciano-Oliveria for bioimaging training. Thank you to all members of the Lazarus lab. Your continuous camaraderie has kept the environment friendly and productive. Particularly: Dr. Yoelys Cruz-Leal, for both her valuable scientific and personal advice. Joan Legarda for all her administrative help. Dr. Lazaro Gil Gonzalez, for helping me with my experimental problems and training in new techniques. Dr. Yuexin Shan, for her professional insight and expertise. Andrew Crow and Danielle Marjoram, for all their help when I was just getting started in the lab. I would also like to thank Gurleen Kaur and Hanna Wabnitz for their lively and productive discussions related to just about anything and everything. Thank you to Peter Norris for his helpful scientific suggestions and to Hongkang Lin, Doyoon (Kevin) Won and Zoya Tawhidi for their support in the lab. I would like to extend a special thanks to all my family members for their continuous support and encouragement. Specifically, my parents for simultaneously motivating me to try my hardest but also not to get overwhelmed in the process. My sisters for their unique way of injecting amusement into my lab problems and providing me with hilarious (but doomed to fail) solutions. My grandmother, for always being available to listen. And finally, my husband for cheering me up through all my bad and stressful days and for his continuous support. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ................................................................................................................................. ix List of Figures ..................................................................................................................................x List of Appendices ....................................................................................................................... xiii Abbreviations ............................................................................................................................... xiv Chapter 1 ..........................................................................................................................................1 Immune Thrombocytopenia ........................................................................................................1 1.1 Overview ..............................................................................................................................1 1.1.1 Etiology ....................................................................................................................2 1.1.2 Pathophysiology of Primary ITP .............................................................................3 1.1.3 Secondary ITP ..........................................................................................................9 Chapter 2 ........................................................................................................................................10 Current Management of Immune Thrombocytopenia...............................................................10 2.1 Diagnosis............................................................................................................................10 2.2 Therapeutic Interventions for ITP ......................................................................................10 2.2.1 First line treatments................................................................................................11 2.2.2 Second line Treatments ..........................................................................................14 2.2.3 Emergency Treatment ............................................................................................18 Chapter 3 ........................................................................................................................................19 The role of Anti-D in ITP .........................................................................................................19 3.1 Anti-D production ..............................................................................................................19 3.2 Clinical Uses ......................................................................................................................19 3.3 Limitations .........................................................................................................................20 3.4 Potential Mechanisms of Action ........................................................................................21 v 3.4.1 Mononuclear Phagocytic System (MPS) Blockade ...............................................21 3.4.2 Cytokine Modulation .............................................................................................25 3.4.3 Anti-Idiotypic Antibodies ......................................................................................26 3.5 Monoclonal