Evaluating Efficacy of CD26+ CAR T Cells in an Orthotopic, Immunocompetent Mouse Model of Pancreatic Ductal Adenocarcinoma 公开
Karpovsky, Isaac (Spring 2023)
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an extremely difficult cancer to treat, with an abysmal 12% 5-year survival rate. At present, chemotherapy confers little benefit, targeted therapies are limited in success, and surgery is considered the only curative approach. Clearly, there is an urgent need to pioneer new treatment strategies against this malignancy. Modern developments in immunotherapy research have produced new treatment options for both hematological and solid tumor malignancies, yet efficacy in PDAC remains low due to roadblocks such as identifying tumor-specific antigen targets, overcoming poor T cell persistence and viability, and improving trafficking of immune cells into the disease site. We hypothesized that enriching chimeric antigen receptor (CAR) T cells with a novel subpopulation of memory T cells characterized by high expression of the CD26 cell surface protein would bolster therapeutic persistence and improve antitumor ability when targeting the mesothelin antigen. We further speculated that treating our CAR T cells with a phosphatidylinositol kinase-3 gamma delta (PI3Kδ/γ) inhibitor ex vivo would enhance their stem-like phenotype and improve therapeutic capabilities. Herein, we investigate our orthotopic, immunocompetent mouse model’s ability to recapitulate the human PDAC tumor microenvironment (TME) and optimize our ability to produce therapeutic CAR T cells. Next, we conduct in vivo trials to test the efficacy of our novel immunotherapy and the utility of PI3Kδ/γ inhibition. Our preclinical studies find that CD4+CD26+ CAR T cells produce a therapeutic response and reduce tumor burden in immunocompetent, PDAC-bearing mice. Although a number of challenges remain, our preclinical studies produce bedrock data for the development of a new CAR T cell-mediated therapeutic approach against pancreatic cancer.
Table of Contents
INTRODUCTION 3
Pancreatic Ductal Adenocarcinoma and the Oppressive Microenvironment 4
Immunocompetent, Orthotopic Murine Models of Pancreatic Cancer 7
Chimeric Antigen Receptor T Cell Therapy: Benefits and Roadblocks 8
CD26: A Multifunctional Protein of Interest 12
Phosphatidylinositol Kinase-3 δ/γ Inhibition Enhances Therapeutic Function 13
Scope of Scientific Work 14
MATERIALS AND METHODS 15
Cell Lines and Reagents 15
Western Blot 16
T Cell Transduction 16
Luciferase Transduction 17
In Vivo Studies 17
Flow Cytometry 18
Immunohistochemistry (IHC) 18
Statistical Analysis 19
RESULTS 19
Chapter One: Desmoplasia and Therapeutic Infiltration in Orthotopic Tumor Models 19
Mesothelin is Expressed on Mouse-Derived PDAC Cell Lines 20
Syngeneic Models Differentially Affect Mouse Survival and Tumor Histology 21
Orthotopic PDAC Tumors Recapitulate the TME of GEMM Tumors 24
Desmoplastic Stroma Obstructs CAR T Cell Infiltration into the TME 26
Chapter Two: CD26 mesoCAR Development and in vitro Characterization 29
Optimized CAR T Cell Product Development 29
The in vitro Effect of Duvelisib on CD26 CAR T Cells 31
Chapter Three: Results and Limitations of Novel CD26-high mesoCAR Therapy 32
Pilot Study Shows CD26+ mesoCAR T Cell Efficacy but Fails to Surpass Control 33
Design Complications and Confounding Factors 35
DISCUSSION 38
FUTURE DIRECTIONS 45
REFERENCES 47
APPENDIX I: Protocols 52
T Cell Harvest Protocol 52
Mouse T Cell Transduction Protocol 54
Orthotopic Surgery Protocol 57
Single-Cell Clone Isolation, Expansion, and Selection Protocol: 61
APPENDIX II: Antibodies for Flow and IHC 62
CD26+ mesoCAR T Cell Panel 62
TME Infiltration Study Panel 62
IHC Antibodies 63
About this Master's Thesis
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