Developing Receptor-Directed Synthetic Antagonists against the IL-23 Signal Transduction Pathway Deelaka Wellappili A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Deelaka Wellappili 2019 Developing Receptor-Directed Synthetic Antagonists against the IL-23 Signal Transduction Pathway Deelaka Wellappili Master of Science Department of Molecular Genetics University of Toronto 2019 Abstract IL-23 is a pro-inflammatory cytokine that initiates and stabilizes the TH17 lineage of T helper cells, often associated with autoimmune inflammatory conditions such as inflammatory bowel disease (IBD), psoriasis, and arthritis. Several antibodies targeting IL-23 have been developed but only one monoclonal antibody has been developed to target IL-23 signaling at the level of the receptor. Using in vitro selections against recombinant IL-23R, we have isolated one antibody from a phage-displayed synthetic antibody library which bound the receptor with high affinity, and interacts with ectopically-expressed cell surface receptors. This antibody was also shown to block the in vitro ligand-receptor interaction and antagonize IL-23-induced STAT3 signalling in cells. These results represent the first report of a recombinant antibody against IL-23R that antagonizes downstream cytokine-induced signalling. Further development and characterization of such antibodies would be beneficial since antibodies that block IL-23 signals at the level of the receptor may possess therapeutic value. ii Acknowledgments First and most importantly, I would like to thank my parents for their continued support and understanding over the course of my project. I would like to thank my mother for introducing me to the world of science, and thanks to my father for driving me to be my best and always showing interest in my graduate work. A heartfelt thank you to both of them for immigrating to a new and foreign country to make sure that I had the best possible opportunities to succeed, leaving behind their friends and family. A special thanks to my father for taking me wherever I needed for school or extracurricular activities all throughout my school years, an opportunity that allowed me to focus on learning and bettering myself. I would also like to acknowledge all my friends, who kept me sane and optimistic during the hard times while also celebrating my successes. I want to sincerely thank my supervisor, Dr. Sachdev Sidhu, who showed me the importance of always moving forward with my research, and not getting stuck on any pitfalls. He was available via emails at all times, sometimes even at 3 am, and gave valuable thoughts and feedback about my work. Thanks to my graduate committee – Drs. Eleanor Fish, Michael Moran and Jason Moffatt, whose insight and often challenging questions gave me focus and improved my presentation skills immensely. A very special thanks to Dr. Shane Miersch, who while not being an official committee member, was instrumental in accelerating my project forwards. He worked one-on-one with me, planning all experiments as well as improving my presentations and reports, making sure I was accurate and concise with my work. Finally, I would also like to thank all the members of the Sidhu Lab who were enthusiastic in helping me overcome the hurdles of my project and graduate school, and for all the laughs and stories throughout my time there. I particularly want to thank Hamed Shaykhalishahi, Paknoosh Pakarian, Gino Gallo, Bradley Yates, Levi Blazer, Gianluca Veggiani, Mart Ustav, and Greg Martyn for their invaluable help and for an unforgettable experience. iii Table of Contents Acknowledgments .................................................................................................................... iii Table of Contents ..................................................................................................................... iv List of Tables ........................................................................................................................... vi List of Figures ......................................................................................................................... vii List of Abbreviations ............................................................................................................. viii Introduction ...........................................................................................................................1 1.1 Immune system and cytokines .......................................................................................1 1.1.1 T cells, differentiation and immunity .................................................................1 1.1.2 Interleukin 23 .....................................................................................................2 1.1.3 Pathology associated with IL-23 ........................................................................5 1.2 Antibodies ......................................................................................................................6 1.2.1 Antibody structure and function ........................................................................6 1.2.2 Current therapeutic antibodies targeting the IL-23 signaling pathway ..............7 1.3 Phage Display ................................................................................................................8 1.4 Thesis Overview ............................................................................................................8 Developing receptor-directed synthetic antagonists against the IL-23 signal transduction pathway ...............................................................................................................................10 2.1 Statement of Contribution ............................................................................................10 2.2 Materials and Methods .................................................................................................10 2.2.1 Plasmid constructs ...........................................................................................10 2.2.2 Antibodies and other proteins ..........................................................................10 2.2.3 Protein production and purification .................................................................11 2.2.4 Enzyme Linked Immunosorbent Assays (ELISAs) .........................................12 2.2.5 Analysis of binding kinetics by biolayer interferometry .................................14 2.2.6 STAT3 signaling assay by western immunoblotting .......................................14 iv 2.2.7 Flow cytometry detection of cell surface IL-23R expression ..........................16 2.2.8 RT-qPCR validation of cell lines .....................................................................17 2.3 Results ..........................................................................................................................17 2.3.1 ELISAs .............................................................................................................17 2.3.2 BLI Kinetic Analysis .......................................................................................20 2.3.3 Flow cytometry detection of cell surface IL-23R binding ...............................21 2.3.4 Development of HeLa cells for the assay ........................................................22 2.3.5 The effect of antibodies on IL-23 induced p-STAT3 signals ..........................23 2.4 Discussion ....................................................................................................................25 Conclusion and Future Perspectives ...................................................................................30 3.1 Development of antibodies targeting cytokine receptors .............................................30 3.2 Conclusion ...................................................................................................................31 References ...........................................................................................................................32 v List of Tables Table 1: Primers for qPCR ............................................................................................................ 17 Table 2: Estimate of binding kinetics for antibody:IL-23R interactions ...................................... 20 vi List of Figures Figure 1: IL-23 binds to IL-23R and IL-12Rb1 .............................................................................. 3 Figure 2: IL-23 signal transduction pathway .................................................................................. 5 Figure 3: Structure of an IgG .......................................................................................................... 6 Figure 4: Sequences of the CDR regions of each antibody clone ................................................. 18 Figure 5: Multipoint ELISA of antibody binding to IL-23R ........................................................ 18 Figure 6: Fab 11189 blocks IL-23:IL-23R interaction ................................................................. 19 Figure 7: 11188 and 11189 bind overlapping epitopes on IL-23R ............................................... 19 Figure 8: Both Fab and IgG 11189 block IL-23:IL-23R interaction ............................................ 21 Figure 9: 11189 Fab and IgG binds specifically to cells expressing IL-23R ...............................