Engineering CXCL13 Secreting CAR T Cells for Ovarian Cancer Público

Abstract

As one of the leading causes of death in gynecological malignancies, ovarian cancers is mostly diagnosed in the late stage due to the lack of early detection tests and early symptoms^1,2. By the time of diagnosis, majority of patients with ovarian cancer have developed metastasis in peritoneal cavity³. And even after standard surgery and chemotherapy, drug resistance and relapse are still very common⁴. Hence, developing new treatments, such as immune checkpoint inhibitors and cellular therapy, are urgently needed. In this study, we developed a novel CAR T cell therapy for ovarian cancer. High-grade serous ovarian cancer cells express elevated levels of tumor-associated antigen, Muc16CD⁵. We used anti-Muc16CD CAR to target the ectodomain of Muc16 for antigen recognition and tumor clearance. To engage the endogenous immune system and create a pro-inflammatory tumor microenvironment in ovarian cancers, we co-modified CAR T cells to express the B cell chemoattractant CXCL13 as armor on anti-Muc16CD CAR hoping to induce tertiary lymphoid structure formation at local tumor site. We propose that tertiary lymphoid structures help to select and activate tumor antigen-specific antibody-secreting cells and potentially attract other CXCR5+ endogenous immune cells. In this study, we engineered, validated, and tested the function of a novel CXCL13 expressing armored CAR T cells in vitro and have begun in vivo study to investigate the potential anti-tumor roles of CXCL13 and to compare CAR T cell functions between armored-CAR and CAR in a syngeneic mouse ovarian cancer model.

Table of Contents

TABLE OF CONTENTS

 

1.    INTRODUCTION............................................................................................................ 1

1.1 CAR T Cell Therapy................................................................................................. 1

1.2 αMuc16CD CAR T Cell Therapy in Ovarian Cancer.............................................. 3

1.3 CXCL13 Chemokine and Its Cognate Chemokine Receptor CXCR5..................... 5

1.4 Tertiary Lymphoid Structure in Cancer................................................................ 6

1.5 Engineered CXCL13-secreting CAR in Ovarian Cancer........................................ 9

2.    MATERIALS AND METHODS...................................................................................... 10

2.1 Molecular Cloning................................................................................................. 10

2.2 H29 Transfection and Phoenix-ECO Transduction in Mouse T Cells................ 11

2.3 Flow Cytometry Analysis...................................................................................... 12

2.4 Mouse Splenocyte Isolation and T Cell Expansion and Transduction.............. 12

2.5 Luciferase-based Cytotoxicity Assay................................................................... 13

2.6 Intracellular Staining Flow Cytometry................................................................ 13

2.7 Cytokine Assay...................................................................................................... 14

2.8 Activation and Proliferation Assay...................................................................... 14

2.9 B Cell Migration Assay.......................................................................................... 14

2.10 Tumor Injection and Imaging............................................................................ 15

3.    RESULTS........................................................................................................................... 15

3.1 Construct Design and Molecular Cloning............................................................ 15

3.2 DNA Sequencing.................................................................................................... 20

3.3 Transduction of Phoenix-ECO Cells..................................................................... 20

3.4 Validating the CAR T Cells.................................................................................... 22

3.5 Luciferase-based Cytotoxicity Assay................................................................... 22

3.6 Cytokine Assay...................................................................................................... 24

3.7 CD69 Stimulation Assay....................................................................................... 25

3.8 Ki-67 Proliferation Assay..................................................................................... 26

3.9 Validation of CXCL13 Expression and Function by Stable Packaging Cells and CAR T Cells          28

3.10 In vivo Study........................................................................................................ 32

 

4.    DISCUSSION..................................................................................................................... 35

 

5.    FUTURE DIRECTIONS..................................................................................................... 39

 

 

REFERENCES............................................................................................................................... 42

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Health Sciences, Immunology Health Sciences, Oncology Biology, General
Palabra Clave	CAR T Cell Therapy Immunology Ovarian Cancer
Committee Chair / Thesis Advisor	Rafiq, Sarwish, Emory University
Committee Members	Kong, Rui, Emory University Ghosn, Eliver, Emory University Antia, Rustom, Emory University

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