Optimizing the Manufacturing and Efficacy of Gamma Delta T Cell Immunotherapies for the Treatment of Cancer Público

Abstract

Originally discovered in the 1980’s, gd T cells have become a frontline leader in the development of cellular cancer immunotherapies. Stimulating the in vivo expansion of gd T cells was initially the most common approach for immunotherapies utilizing gd T cells. However, when it became clear that off-target effects were common, interest in this method diminished. Instead, the field shifted towards investigating the ex vivo expansion of both autologous and allogeneic gd T cell therapies. The ability to use gd T cells in an allogeneic setting is both a unique and attractive property, as it allows for the development of an off-the-shelf cell therapy. The focus of this dissertation is to optimize the manufacturing process and improve the efficacy of allogeneic gd T cells as a cancer immunotherapy.

The cellular manufacturing process consists of multiple processes including donor selection, cellular isolation and expansion, and storage. In this work, we have identified exercise as being a critical parameter in donor selection. In comparison to cells from sedentary donors, gd T cells from exercise donors have greater expansion capabilities. Interestingly, modifying the expansion protocol to include an additional cytokine, IL-21, can rescue the expansion of gd T cells from sedentary donors. αβ T cell depletions are necessary step in the expansion of gd T cells to ensure that cells capable of promoting graft versus host disease are removed. Depleting αβ T cells on day 6 of expansion is the most efficient time-point to perform the depletion to minimize cell losses and remain cost effective. Additionally, we report the development of a novel and highly cytotoxic gd T cell immunotherapy in which gd T cells from multiple donors are mixed and expanded in culture together. The final step in the cellular manufacturing process, cryopreservation and storage, requires significant consideration in addition to donor selection and cellular expansion, because this step can drastically alter the final cell product. In fact, thawing gd T cells into their standard culture media results in poor viability and extreme cell losses. Thawing gd T cells into human serum albumin, offers protection to cell viability. Additionally, chromatin condensation prior to cryopreservation offers additional protection to gd T cell viability after thawing. However, mitochondria in gd T cells that have been cryopreserved exhibit high levels of membrane depolarization, suggesting that the mitochondria are critically damaged and further optimization is required.

Improving the tumor homing capabilities of gd T cells is critical to improving the efficacy of gd T cells against solid tumors. Currently, efficient tumor homing is a limitation reducing the efficacy of T cell therapies against solid tumors. We describe a novel dual-cell gMSC-gd T cell immunotherapy which utilizes the pairing between chemokines secreted from gMSCs and chemokine receptors expressed by gd T cells to increase tumor homing. gd T cells preferentially migrated to both conditioned media from gMSCs and gMSCs cells in vitro. When gMSCs were directly injected into neuroblastoma tumors, gd T cells preferentially migrated to the gMSCs tumors in vivo. Additionally, we provide evidence that direct cell contact between gMSCs and gd T cells does not impact gd T cell viability or cytotoxicity. Further development of the dual-cell gMSC-gd T cell immunotherapy provides promise for increasing T cell tumor homing efficiency.

Table of Contents

Abstract

Acknowledgements

Table of Contents

List of Figures and Tables

 

Chapter 1: Introduction to Cancer Immunotherapy………………………………………….1

1.1 Cancer Immunotherapies……………………………………………………………...2

A.  Early Cancer Immunotherapies……………………………………………….2

B.   Immune Checkpoint Therapy………………………………………………....3

C.   Cancer Vaccine Therapy……………………………………………………...6

D.  Antibody Therapy……………………………………………………………..9

E.   Cytokine Therapy…………………………………………………………….10

F.   Adoptive Cell Therapy……………………………………………………….12

1.2 Cellular Immunotherapies……………………………………………………………13

A.  Tumor Infiltrating Lymphocyte Therapy…………………………………….13

B.   Engineered TCR Therapy……………………………………………………14

C.   CAR T Cell Therapy…………………………………………………………15

D.  NK Cell Therapy……………………………………………………………..18

E.   gd T Cell Therapy……………………………………………………………19

1.3 Pharmacology of Cellular Therapies………………………………………………...20

A.  Development of a Cellular Therapy…………………………………………20

B.   Potency………………………………………………………………………21

C.   Biodistribution……………………………………………………………….21

D.  Toxicity……………………………………………………………………....22

1.4 gd T Cell Immunotherapies…………………………………………………………..23

A.  gd T Cell Subsets (Vd1 and Vd2) …..……………………………………….23

B.   In Vivo Expansio.…………………………………………………………….25

C.   Ex Vivo Expansion…...……………………………………………………....26

D.  Challenges that remain…………………………………………………….....27

Chapter 2: Characterization of donor variability for γδ T cell ex vivo expansion and development of an allogeneic γδ T cell immunotherapy……………………………………..28

            2.1 Abstract……………………………………………………………………………...29

            2.2 Introduction………………………………………………………………………….30

            2.3 Materials and Methods………………………………………………………………32

            2.4 Results……………………………………………………………………………….36

            2.5 Discussion…………………………………………………………………………...60

            2.6 Supplemental Information…………………………………………………………..66

Chapter 3: Human serum albumin and chromatin condensation rescue ex vivo expanded γδ T cells from the effects of cryopreservation………………………………………..…………75

            3.1 Abstract……………………………………………………………………………...76

            3.2 Introduction………………………………………………………………………….76

            3.3 Materials and Methods………………………………………………………………78

            3.4 Results……………………………………………………………………………….82

            3.5 Discussion………………………………………………………………………….102

Chapter 4: Increasing the efficiency of gd T cell tumor homing using a novel dual-cell gMSC-gd T cell cancer immunotherapy……………………………………………………108

            4.1 Abstract…………………………………………………………………………..109

            4.2 Introduction………………………………………………………………………109

            4.3 Materials and Methods…………………………………………………………...112

            4.4 Results……………………………………………………………………………115

            4.5 Discussion………………………………………………………………………..123

Chapter 5: General Discussion……………………………………………………………..127

            5.1 Summary of Results……………………………………………………………...128

            5.2 Implications of Findings…………………………………………………………131

            5.3 Limitations and Future Directions……………………………………………….133

            5.4 Conclusions………………………………………………………………………135

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Palavra-chave	Cancer Immunotherapy Cellular Therapies
Committee Chair / Thesis Advisor	Spencer, H. Trent, Emory University
Committee Members	Shanmugam, Mala, Emory University Doering, Christopher, Emory University Horwitz, Edwin, Emory University

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