University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Development Of Canine Chimeric Antigen Receptor T Cell Therapy For Treatment & Translation Mohammed Kazim Panjwani University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Allergy and Immunology Commons, Immunology and Infectious Disease Commons, Medical Immunology Commons, and the Oncology Commons Recommended Citation Panjwani, Mohammed Kazim, "Development Of Canine Chimeric Antigen Receptor T Cell Therapy For Treatment & Translation" (2017). Publicly Accessible Penn Dissertations. 2513. https://repository.upenn.edu/edissertations/2513 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2513 For more information, please contact [email protected]. Development Of Canine Chimeric Antigen Receptor T Cell Therapy For Treatment & Translation Abstract Chimeric antigen receptor (CAR) T cell therapy has had remarkable success targeting B cell leukemias in human patients, but unexpected toxicities and failures in other disease demonstrate the need for more predictive pre-clinical animal models than the murine ones currently used. Dogs develop spontaneous malignancies similar to humans in their tissues of origin, gene expression profiles, treatments, and disease courses, and have long been used as models for immunotherapy. I hypothesize that the development of CAR T cell therapy for dogs with spontaneous disease and that the treatment of these canine patients will recapitulate the observations found in human patients, and provide new insights into the safety and efficacy of this eakthrbr ough therapy. To achieve this, I first established methods for growing primary canine T cells from healthy and disease-bearing donors to clinically relevant scale, developed RNA electroporation protocols to transiently express a CAR targeting the canine tumor- associated antigen CD20, demonstrated its function in vitro, and treated a relapsed canine B cell lymphoma patient with autologous CAR T cells as a proof of feasibility. I then developed methods to permanently express a second-generation cCD20-8-28-ζ CAR in canine T cells using lentiviral transduction, showed in vitro antigen-specific function and proliferation of CAR T cells, and treated four canine B cell lymphoma patients with CAR T cells. Based on my observations from those patients, I made iterative improvements to the T cell culture system and CAR construct, and treated a canine B cell lymphoma patient with cCD20-8-BB-ζ CAR T cells, whose tumor cells lost target antigen expression to avoid immune pressure. These results prove that it is not only possible to generate canine CAR T cell therapy, but that it recapitulates observations found until now only in human patients. In addition, novel findings egarr ding the recovery of T cells during ex vivo culture and the host immune response to the CAR demonstrate that this model can already inform human medicine. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Immunology First Advisor Nicola J. Mason Keywords chimeric antigen receptor, comparative oncology, immunotherapy, pre-clinical animal model Subject Categories Allergy and Immunology | Immunology and Infectious Disease | Medical Immunology | Oncology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2513 DEVELOPMENT OF CANINE CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY FOR TREATMENT & TRANSLATION Mohammed Kazim Panjwani A DISSERTATION in Immunology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation ________________________ Nicola J. Mason , BVetMed, PhD, DACVIM Associate Professor of Clinical Studies and Pathobiology Graduate Group Chairperson ________________________ David M. Allman, PhD Associate Professor of Pathology & Laboratory Medicine Dissertation Committee Christopher A Hunter, PhD, Professor of Pathobiology (Chair) Martha S. Jordan, PhD, Research Assistant Professor of Pathology & Laboratory Medicine Michael C. Milone, MD, PhD, Assistant Professor of Pathology & Laboratory Medicine Robert H. Vonderheide, MD, DPhil, Professor of Medicine DEVELOPMENT OF CANINE CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY FOR TREATMENT & TRANSLATION COPYRIGHT 2017 Mohammed Kazim Panjwani This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/ DEDICATION To my father, for telling me to keep working both harder and smarter. To my mother, for listening to my daily complaints – even when they were about genetic cloning – just to make sure I was alive each morning. To my brother, for suggesting I pursue something other than Latin for my career path. To my sister-in-law, for being in Philadelphia at the right time and place. To my grandfather, for a well-stocked library. And to the USPS – you know what you did. iii ACKNOWLEDGMENTS Before this thesis even begins, I would like to acknowledge my mentor, Dr. Nicola J. Mason. Even if I were not your first and, so far, only graduate student, I certainly have been your most difficult. Nevertheless, you gave me the rarest of opportunities for a PhD student: to take the science from the bench to the bedside, and back again – and again, and again. We are almost there, Nicky – Today is the day! This work would not have been possible without my two stalwart lab companions. Dr. Josephine S. Gnanandarajah worked with such efficiency that it still amazes me to this day. You also got me into the habit of eating lunch semi-regularly, even if it was a candy bar from the library vending machine at 4PM. Dr. Martha A. MaloneyHuss has never slept, and yet has been willing to answer even the most rudimentary questions about patient care for me at any hour. I could not have hoped for a better friend to get a second opinion about the cells in tissue culture. Treatment of canine patients would not have gotten off the ground without the mRNA CAR collaboration with Dr. Daniel Powell, Jr., and his then-graduate student Dr. Jenessa Smith. Resources generously provided by Dr. Michael Milone, Dr. Carl June, Dr. Avery Posey, Jr., the Translational and Correlative Studies Laboratory, and eventually the Parker Institute for Cancer Immunotherapy have been key in achieving what we have so far. I want to thank the Immunology Graduate Group and the broader immunology community here at University of Pennsylvania for the counsel, equipment, and reagents that they have shared over the years, especially those that they were not yet aware of. I iv particularly would like to thank Dr. Karla Wiehagen, my mentor during my third rotation, for teaching how to do 14-color flow cytometry and how to design fail-proof Matryoshka doll experiments so that valuable data can be gained even if it 99% of the experiment fails; I look forward to the day that I can apply the former skill in canine immunology – the latter skill has already been invaluable for the same. I cannot express my gratitude enough to the owners of our patients for the enormous trust that they have put in us, and to the staff of the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania for their assistance in these trials. There remain three people who deserve acknowledgment here. Walter Muller taught me how to read journal articles in my sophomore year of high school and pushed me into lab science; I have spent nearly half my life so far pursuing immunology, and this is all his fault. Mrs. Weinblatt took a chance on the high school student living down the hall, and took her son by the ear and had him pass around his resume; may her memory be a blessing. Dr. Matthew S. Perzanowski of Columbia University spent some of his earliest days as a newly-appointed assistant professor teaching a 17-year old how to pipette and, for some reason, kept on taking him back summer after summer, year after year; he taught me that most protocol is voodoo, but actually discovering the mechanism is magic. v ABSTRACT DEVELOPMENT OF CANINE CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY FOR TREATMENT & TRANSLATION Mohammed Kazim Panjwani Nicola J. Mason, BVetMed, PhD, DACVIM Chimeric antigen receptor (CAR) T cell therapy has had remarkable success targeting B cell leukemias in human patients, but unexpected toxicities and failures in other disease demonstrate the need for more predictive pre-clinical animal models than the murine ones currently used. Dogs develop spontaneous malignancies similar to humans in their tissues of origin, gene expression profiles, treatments, and disease courses, and have long been used as models for immunotherapy. I hypothesize that the development of CAR T cell therapy for dogs with spontaneous disease and that the treatment of these canine patients will recapitulate the observations found in human patients, and provide new insights into the safety and efficacy of this breakthrough therapy. To achieve this, I first established methods for growing primary canine T cells from healthy and disease-bearing donors to clinically relevant scale, developed RNA electroporation protocols to transiently express a CAR targeting the canine tumor-associated antigen CD20, demonstrated its function in vitro, and treated a relapsed canine B cell lymphoma patient with autologous CAR T cells as a proof of