Prosthetic Antigen Receptors As a Platform for Solid Tumor Immunotherapy
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PROSTHETIC ANTIGEN RECEPTORS AS A PLATFORM FOR SOLID TUMOR IMMUNOTHERAPY A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Jacob Richard Petersburg IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ADVISOR: Dr. Carston R Wagner December, 2017 © Jacob R. Petersburg, 2017 ACKNOWLEDGMENTS The following dissertation may be an individual work, but it couldn’t have been accomplished without the help, support and guidance of several individuals. First and foremost, I would like to thank Dr. Carston Wagner for instilling in me the necessary qualities to be a good scientist and researcher. His infectious enthusiasm and unbreakable optimism have been a significant driving force throughout my graduate career. I would also like to thank my committee members, Dr. Daniel Harki, Dr. Barry Finzel, and Dr. William Pomerantz for their support in preparing and completing this dissertation work. I would like to acknowledge all the past and current members of the Wagner Laboratory for their help on my projects. The opportunity to learn from and work with everyone over the last several years has been an honor. Specifically, I would like to thank Dr. Kari Gabrielse, Dr. Amit Ganger and Dr. Sidath Kumarapperuma for their direction and mentorship when I first joined the lab. I would also like to thank all the members of the Fife lab, especially Dr. Brian Fife and Dr. Justin Spanier, who showed me how much there was to learn in the field of Immunology. I need to give a big thanks to my fellow graduate students and colleagues at the University of Minnesota. I would especially like to thank Cody Lensing, Trent West, Cliff Csizmar for always being there to either celebrate or grab a drink when the time required. Lastly, I would like to thank all my friends and family who’ve supported me over the last 5 years. I would like to thank my girlfriend Tenley Brown for her endless support and patience as I finished up my Ph.D. I would like to thank my siblings, Brandon and i Steph, for always keeping me grounded and motivated. Finally, I would like to thank my mother and father, Sheryl and Rick, for their unwavering love and support while providing me with every opportunity for success throughout all the stages of my life. ii DEDICATION This dissertation is dedicated to my parents, Sheryl and Rick, who have never failed to encourage me in life and inspired me to become the person I am today. I am truly grateful for everything you have done. iii ABSTRACT The ability to engineer and reprogram cell surfaces has significant potential for enabling the use of cell based therapies in cancer treatment. Unfortunately, a majority of current strategies utilize either genetic engineering or chemical modifications which have a number of significant drawbacks. To address these concerns our lab developed Prosthetic Antigen Receptors (PARs), a non-genetic system, to direct selective cell-cell interactions. PARs are formed by engineering fusion proteins that contain a scFv fused to two E. Coli dihydrofolate reductase (DHFR2) molecules which spontaneously assemble into octomeric chemically self-assembled nanorings (CSANs) upon the addition of a chemical dimerizer, bis-methotrexate (Bis-MTX). This thesis provides the field with foundational work addressing the functional effects of PARs in a solid tumor model, both in vitro and in vivo. Chapter 2 initially addresses the in vivo stability, circulation and tissue distribution of CSANs using a radiolabeled construct affording the direct visualization of in vivo tissue localization and ex vivo organ biodistribution by microPET/CT imaging and tissue-based gamma counting, respectively. As anticipated, CSANs displayed an in vivo profile between that of rapidly clearing small molecules and slow clearing antibodies. In Chapter 3 we discuss both the in vitro and in vivo development of anti-EpCAM PARs which are then applied to an in vivo orthotopic Breast Cancer Model. Our results demonstrated that anti-EpCAM/anti-CD3 PARs were found to stably bind T-cells for >4 days, and treating EpCAM+ MCF-7 breast cancer cells with anti-EpCAM/anti-CD3 PAR- functionalized T-cells resulted in the induction of IL-2, IFN-γ and MCF-7 cytotoxicity. iv Furthermore, an orthotopic breast cancer model validated the ability of anti-EpCAM/anti- CD3 PAR therapy to direct T-cell lytic activity towards EpCAM+ breast cancer cells in vivo leading to tumor eradication. Following the in vivo success of anti-EpCAM PAR therapy we chose to explore, Chapter 4, the use of both anti-EpCAM/anti-CD3 and anti- CD133/anti-CD3 CSANs in conjunction. Notably, when applied to a triple negative breast cancer model we found a synergistic effect from targeting EpCAM and CD133; in fact, full tumor eradication was only elicited when both antigens were simultaneously targeted. Due to the growing need of a more modifiable CSAN platform we developed monovalent streptavidin (mSA)-DHFR2 fusion proteins. When incorporated into bispecific CSANs, Chapter 5, we were able to rapidly analyze the activation and directed cell lysis of several targeting constructs simultaneously. Additionally, in Chapter 6 we further adapted mSA CSANs into a universal cell membrane labeling technique. This was accomplished by hydrophobically inserting phospholipids conjugated to biotin into the cell membrane. Heterobifunctional CSANs containing mSA are then stably bound to the biotin moieties. PAR therapy has several unique innovations, such as the capability of quickly reprogramming T cell membranes in hours rather than in days which is typically seen with standard CAR therapy. Additionally, our approach has the capability to remove the PARs from T-cells by incubation with the FDA approved antibiotic trimethoprim, at clinically relevant concentrations, allowing the pharmacological deactivation of T cells. Collectively, our results demonstrate PAR modified T-cells have the potential to be a viable cancer immunotherapy targeting solid tumors. v TABLE OF CONTENTS Abstract .............................................................................................................................. iv Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv List of Schemes ................................................................................................................ xix List of abbreviations ......................................................................................................... xx CHAPTER 1: ...................................................................................................................... 1 INTRODUCTION – IMMUNOTHERAPY IN CANCER ................................................ 1 1.1 CANCER BIOLOGY ............................................................................................... 2 1.2 CONTEMPORARY IMMUNOTHERAPY ............................................................. 3 1.2.1 Cancer Specific Overexpression ........................................................................ 3 1.2.2 Development of Therapeutic Monoclonal Antibodies ....................................... 6 1.2.3 Development of Antibody Fragments for Therapeutic Use ............................. 11 1.2.4 Development of BiTEs .................................................................................... 14 1.2.5 Development of Chimeric Antigen Receptor (CAR) T cells ........................... 21 1.3 UTILIZING PROSTHETIC ANTIGEN RECEPTORS IN CANCER THERAPY 25 1.3.1 Chemically Self-Assembled CSANs ............................................................... 25 1.3.2 Development of Prosthetic Antigen Receptors (PARs) ................................... 29 1.4 CONCLUSION ....................................................................................................... 32 CHAPTER 2: .................................................................................................................... 34 vi EVALUATING THE IN VIVO STABILITY AND BIODISTRIBUTION OF CHEMICALLY SELF-ASSEMBLED NANORINGS (CSANS) .................................... 34 2.1 INTRODUCTION .................................................................................................. 35 2.1.1 Principles of Positron Emission Tomography (PET)....................................... 35 2.1.2 Application of Immuno-Positron Emission Tomography (Immuno-PET) in Cancer Therapy ......................................................................................................... 38 2.1.3 Utilizing Immuno-PET to Improve Clinical Translation ................................. 41 2.1.4 Development of radiolabeled anti-EGFR CSANs ........................................... 43 2.2 RESULTS AND DISCUSSION ............................................................................. 46 2.2.1 Synthesis of bis-MTX-DOTA [64Cu]............................................................... 46 2.2.2 Preparation of anti-EGFR-CSANs ................................................................... 49 2.2.3 Preparation of PEGylated anti-EGFR CSANs ................................................. 52 2.2.4 In vitro modeling of RES uptake with mouse macrophages............................ 60 2.2.5 Characterization of anti-EGFR-CSANs