University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-5-2018 MUC4 Based Immunotherapy for Pancreatic Cancer Kasturi Banerjee University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, Biology Commons, Disease Modeling Commons, Medical Cell Biology Commons, Medical Immunology Commons, Molecular Biology Commons, Nanomedicine Commons, and the Translational Medical Research Commons Recommended Citation Banerjee, Kasturi, "MUC4 Based Immunotherapy for Pancreatic Cancer" (2018). Theses & Dissertations. 283. https://digitalcommons.unmc.edu/etd/283 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. i MUC4 BASED IMMUNOTHERAPY FOR PANCREATIC CANCER By KASTURI BANERJEE A DISSERTATION Presented to the Faculty of The University of Nebraska Graduate College In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Biochemistry and Molecular Biology Graduate Program Under the Supervision of Associate Professor Dr. Maneesh Jain University of Nebraska Medical Center Omaha, Nebraska April 2018 Supervisory Committee: Surinder K. Batra, Ph.D. Rakesh Singh, Ph.D. Kaustubh Datta, Ph.D. Joyce Solheim, Ph.D. ii DEDICATION I dedicate my PhD dissertation to my father Mr. Chandan Banerjee, my mother Mrs. Sarbasree Banerjee, my younger sister Ms. Sanchari Banerjee And my late grandmother Mrs. Kalyani Banerjee For providing me constant love and support. iii ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my PI Maneesh Jain (PhD) for the continuous support of my PhD study and related research, for his patience, motivation, and immense knowledge. His faith in me to work on a cancer immunotherapy project that is out of the norm of our lab had provided me with confidence to understand research on a deeper level. Besides my advisor, I would like to thank the rest of my thesis committee: Dr. Surinder K. Batra, Dr. Joyce Solheim, Dr. Rakesh Singh and Dr. Kaustubh Datta, for their insightful comments and encouragement, but also for the hard questions which incented me to widen my research from various perspectives. My sincere thanks also go to Dr. Shailendra Gautam, Dr. Prakash Kshirsagar, and Dr. Mohd Wasim Nasser, for their precious support, without which it would not be possible to conduct this research. I want to sincerely thank Dr. Sushil Kumar for mentoring me through the MUC4 nanovaccine project, and Dr. Sukhwinder Kaur for guiding me in the MUC4 autoantibody project. I will take this opportunity to thank our collaborator at Iowa State University: Dr. Balaji Narasimhan, Dr. Kathleen Ross and Dr. Michael Wannemuehler, for synthesizing and providing us with the nanoparticles and being very responsive to my queries and emails. I thank my fellow lab mates and friends: Saswati, Koelina, Rahat, Pranita and Sanchita; for the stimulating discussions, for the sleepless nights we were working together before deadlines, and for all the fun we have had in the last four and a half years. iv I want to thank my Alma Mater, IISER Mohali and all the faculties there who taught me and developed my eagerness in scientific research. I want to especially thank my school friend Abhishek Banerjee, who forced me to apply for KVPY fellowship and that entirely changed my professional life and I will be forever grateful to him for being an awesome friend. I owe to my friends Dr. Matharishwan Naganbabu and Neelam Singh for constantly being my support during my PhD application days and still valuing the meaning of friendship even when we live miles apart. I want to thank Justin Garrison and Dr. Matthew Ingersoll for being valuable friend and support throughout the initial stages of my PhD journey. I am grateful to all of you for having faith in me and encouraging me to pursue my career in research. A special thanks to Linda Bernadt for adopting me into her life as a granddaughter and taking care of me emotionally, especially after I lost my late grandmother Mrs. Kalyani Banerjee. I will forever be grateful to you for showering me with unconditional love and support and making Omaha feel like home, away from my home in India. Last but never the least, I would like to thank my family: my parents and my sister for supporting me spiritually throughout writing this thesis and my life in general. v ABSTRACT MUC4 based immunotherapy for pancreatic cancer Kasturi Banerjee, Ph.D. University of Nebraska Medical Center, 2018 Supervisor: Maneesh Jain, PhD. Pancreatic Cancer (PC) is a lethal disease claiming approximately 45000 lives in the US in 2018, and it establishes an elaborate immunosuppressive tumor microenvironment that aids in disease pathogenesis. Immunotherapy has emerged as a strategy to target tumor cells by reprogramming patient’s immune system. Challenges present in PC immunotherapy are: i) identifying a tumor-associated antigen that could be targeted, ii) identifying adjuvants that could efficiently deliver antigens, iii) eliciting robust anti-tumor responses and iv) overcoming peripheral tolerance and immunosuppression elicited by the tumor. Firstly, we detected circulating autoantibodies to MUC4 present in PC patients and observed that IgM autoantibodies to MUC4 peptides significantly correlate with overall PC patient survival, thus suggesting that MUC4 could potentially be targeted for PC immunotherapy. Our group is the first to successfully purify recombinant MUC4β protein and characterize its immunogenic activity. We addressed the challenge of protein delivery by encapsulating MUC4β in novel polyanhydride nanoparticles (MUC4 nanovaccine). In the second part of the dissertation, we characterized MUC4 nanovaccine in both in vitro and in vivo system. Our studies showed that MUC4 nanovaccine could robustly activate dendritic cells (DCs) and induce secretion of Th1 cytokines in vitro. High levels of Th1 IgG2b anti-MUC4β antibodies were detected in MUC4 nanovaccine-immunized mice. vi As described in the third part of the dissertation, we observed that ex vivo T-cells activated by MUC4 nanovaccine-pulsed DCs showed enhanced cytotoxic killing of miniMUC4 tumor cells, when compared to soluble MUC4β mixed with empty nanoparticles (MUC4+NP). We validated our data in an in vivo subcutaneous PC tumor mouse model, and observed enhanced immune cells infiltration and corresponding levels of necrosis in miniMUC4 tumors corroborated with low tumor volume of miniMUC4 tumor (in comparison to contralateral vector control tumor) in MUC4-immunized mice. Furthermore, the presence of PD-L1 surface expression on miniMUC4 tumor cells indicated active immunosuppression lodged by tumor cells in response to IFNγ-secreting infiltrating cytotoxic T-cells. Taken together, studies in this dissertation demonstrate that MUC4 nanovaccine could serve as a potential strategy for PC immunotherapy. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................... iii ABSTRACT ................................................................................................... v TABLE OF CONTENTS ...................................................................................... vii LIST OF TABLES .............................................................................................. xiii LIST OF FIGURES ............................................................................................. xiv LIST OF ABBREVIATIONS ............................................................................... xvi CHAPTER 1A: INTRODUCTION ......................................................................... 1 1. Synopsis ............................................................................................... 2 2. Introduction to Immunotherapy .......................................................... 3 3. Immunoediting and Immunosuppressive Microenvironment .......... 3 4. PC Microenvironment and Immune Suppression ............................. 5 5. Immunotherapy Based Approaches ................................................... 8 5.1 Passive Immunotherapeutic Strategies ................................ 8 5.1.1 Antibody-Mediated Passive Immunotherapy ................... 8 5.1.2 Passive T-cell-Mediated Immunotherapy ....................... 12 5.2 Active Immunotherapeutic Strategies ................................. 16 5.2.1 Cancer Vaccines ............................................................... 16 6. Challenges in Pancreatic Cancer (PC) Immunotherapy ................. 22 viii 6.1 Identification of Tumor Associated Antigens for PC Immunotherapy ........................................................................... 22 6.2 Generation of Anti-Tumor Responses Against PC Microenvironment ....................................................................... 23 6.3 Overcoming Immunosuppression in PC Microenvironment ................................................................................................................. 24 CHAPTER 1B: MUCINS-BASED VACCINES AND MUC4 IN ............................. PANCREATIC CANCER.......................................................... 57 1. Synopsis ............................................................................................