Strategies for engineering γδ T cells to treat pediatric cancers Restricted; Files Only
Becker, Scott (Summer 2023)
Abstract
Developing novel treatments to fight cancer is essential for improving patient outcomes and safety. Cancer immunotherapy has become an attractive alternative to chemotherapy that harnesses the immune system to target cancer cells. Adoptive cell therapy (ACT) is a therapeutic technique where immunocompetent cells are isolated from a patient or healthy donor, expanded and engineered ex vivo, and then administered to the patient. Traditionally, ACT utilizes autologous αβ T cells that are stably engineered using viral vectors, such as lentivirus. One of the most common engineering strategies is the expression of chimeric antigen receptors (CARs) that are specific to an antigen on cancer cells. While CAR T cell therapy has seen promising clinical results, there are several limitations that have been identified during clinical testing. Transient engineering of γδ T cells offers a promising alternative to the traditional stable engineering of αβ T cells and can overcome some of the obstacles with CAR T cell therapy. γδ T cells are a small subset of lymphocytes that bridge the gap between the adaptive and innate immune systems, which makes them the ideal candidate for ACT. They recognize antigens in an MHC-independent manner and have several inherent cytotoxic mechanisms that allow them to recognize infected and cancerous cells. Therefore, the goal of this dissertation was to develop a transient engineering platform for γδ T cells through the use of recombinant AAV and mRNA electroporation. Despite limited gene transfer and poor viability with AAV6 techniques, mRNA electroporation resulted in high transfection efficiency and CAR expression up to 60-70%. We also show that mRNA electroporation can be used to express a secreted bispecific T cell engager (sBite) in γδ T cells. The sBite-modified γδ T cells were specific and improved the cytotoxicity of modified and unmodified cells. The CAR- and sBite-modified γδ T cells not only exhibited increased cytotoxicity in vitro, but also reduced tumor burden and improved survival in two in vivo cancer models. Overall, we show that transient engineering of γδ T cells can be an effective platform to develop the next generation of cancer therapeutics.
Table of Contents
Abstract
Acknowledgments
Table of Contents
List of Figures and Tables
List of abbreviations
Chapter 1: Introduction to Cancer Immunotherapy ………………………………………………........1
1.1 Cancer immunotherapy overview ………………………………………………………………………2
A. Adoptive cell therapy……………………………………………………………..……………..2
B. CAR T cell therapy…………………………………………………………………….………..4
C. CD19 CARs and clinical landscape……………………………………………………………...5
1.2 Challenges in traditional CAR T cell therapy……………………………………………………………9
A. Graft-versus-host disease……………………………………………………………..…………9
B. On-target-off-tumor toxicity……………………………………………………………...……13
C. Cytokine release syndrome……………………………………………..……………………...15
D. Antigen escape…………………………………………………………..……………………..17
1.3 Transient engineering for cancer therapeutics…………………………………………….……………19
A. Gene delivery techniques………………………………………………………………....……19
B. Genetic engineering of TCRs………………………………………………...………………...25
C. Expression of CARs……………………………………………………………..……………..27
D. Expression of cytokines and other recombinant proteins.……………………….……………..32
1.4 γδ T cells……………………………………………………………………………………….………37
A. Classification, characterization, and functions……………………….………………………..37
B. Mechanisms of antigen recognition ……………………………………………………………39
C. Vγ9Vδ2 T cells for ACT…………………………………………………………...…………..42
Chapter 2: Engineering γδ T cells using AAV6 and microfluidics devices … ………...………………43
2.1 Abstract………………………………………………………………………………………………..44
2.2 Introduction……………………………………………………………………………………………44
2.3 Results…………………………………………………………………………………………………46
2.4 Discussion……………………………………………………………………………………………..64
2.5 Materials and Methods…………………………………………………………………………………67
2.6 Supplemental Figures, Tables, and Legends……………………………………………….…………..70
Chapter 3: Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific antibodies ……………………………………………………...………………..74
3.1 Abstract………………………………………………………………………………………………..75
3.2 Introduction……………………………………………………………………………………………75
3.3 Results…………………………………………………………………………………………………77
3.4 Discussion……………………………………………………………………………………..………99
3.5 Materials and Methods………………………………………………………………………………..102
3.6 Supplemental Figures, Tables, and Legends…………………………………………………….……107
Chapter 4: General Discussion……………………………………………………………….………...115
4.1 Summary of Results………………………………………………………………………….……….116
4.2 Implications of Findings……………………………………………………………………………...122
4.3 Limitations and Future Directions…...……………………………………………………………….126
4.4 Conclusions…………………………………………………………………………………………..129
Literature Cited…………………………………………………………………………………………130
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File download under embargo until 16 August 2025 | 2023-07-05 13:35:29 -0400 | File download under embargo until 16 August 2025 |
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