Optimization of γδ T Cell Expansion and Function in an Osteosarcoma Model: Laying the Groundwork for Future Therapeutic Strategies Restricted; Files Only

Abstract

The application of cellular immunotherapies (CI) for osteosarcoma (OS) has largely focused on autologous αβ T cells, which to date have shown minimal clinical benefit. γδ T cells, specifically allogeneic Vγ9Vδ2 T cells, offer distinct advantages, including multi-modal tumor killing and potential for off-the-shelf use. However, conventional large-scale expansion protocols generate more differentiated γδ T cells with limited in vivo persistence, reducing therapeutic impact. To address this, we tested a manufacturing strategy incorporating transforming growth factor beta (TGF-β) to produce a more central memory-like γδ T cell phenotype, hypothesizing that such cells would persist longer and sustain anti-tumor activity. We further leveraged activation mechanisms to enhance γδ T cell cytotoxicity. First, conditioning OS cells with zoledronate (ZOL) to potentially enhance recognition by the γδ TCR, which significantly boosted tumor killing. Second, administering ifosfamide (IFO), a clinically relevant chemotherapy for relapsed and refractory OS, upregulated stress antigens that engage the γδ T cell NKG2D receptor. Third, γδ T cells were engineered to secrete a PTK7-CD3 bispecific T cell engager (sBiTE), enabling for better targeting of the OS tumor cells. In vitro, ZOL pre-treatment significantly increased target cell death in both TGF-β expanded and PTK7-CD3 sBiTE γδ T cells, with engineered cells demonstrating superior killing compared to their mock controls. In vivo, administration of TGF-β expanded γδ T cells or PTK7-CD3 sBiTE cells combined with ZOL and IFO significantly extended survival in OSbearing lung metastasis mice, correlating with improved persistence in the TGF-β expanded γδ T cells and higher cytotoxicity in the PTK7-CD3 sBiTE γδ T cells. These findings establish a flexible γδ T cell platform that couples enhanced persistence with multi-pronged tumor targeting and lays the essential groundwork for future γδ T cell-based immunotherapies. 

Table of Contents

Table of Contents

Abstract…………………………………………………………………………………………...iv

Acknowledgements……………………………………………………………………………….vi

Table of Contents………………………………………………………………………………...vii

List of Figures……………………………………………………………………………………. ix

List of Tables……………………………………………………………………………………..xii

List of Abbreviations……………………………………………………………………………xiii

Chapter 1: Introduction………………………………………………………………………….1

1.1 Introduction to pediatric Osteosarcoma…………...…………………………………...1

1.2 Introduction to γδ T cells………………………………...…………………………...14

1.3 Using γδ T cells for cancer immunotherapy…...……….……….…………………….19

1.4 Summary, scope, and goals for this project…………………………………………...32

Chapter 2: Central Memory-enriched Vγ9Vδ2 γδ T cells via TGF-β expansion demonstrate

enhanced in vivo efficacy against metastatic osteosarcoma…………………………..………33

2.1 Abstract………………………………………………………………………………34

2.2 Introduction…………………………………………………………………………..35

2.3 Results………………………………………………………………………………..38

2.4 Discussion……………………………………………………………………………62

2.5 Materials & Methods…………………………………………………………………69

2.6 Supplemental Figures….……………………………………………………………..76

Chapter 3: PTK7-CD3 BiTE secreting γδ T cells demonstrate superior in vivo efficacy

against metastatic osteosarcoma……………………………………………………………….89

3.1 Abstract………………………………………………………………………………90

3.2 Introduction…………………………………………………………………………..91

3.3 Results………………………………………………………………………………..94

3.4 Discussion…………………………………………………………………………..107

3.5 Materials & Methods………………………………………………………………..113

3.6 Supplemental Figure.………………………………………………………………..119

Chapter 4: General Discussion and Closing Remarks………………………………………120

4.1 Summary of Results…………………………………………………………………120

4.2 Implications of Findings…………………...….……………………………………125

4.3 Limitations and Future Studies……..………………………………………………127

4.4 Closing Remarks……………………………………………………………………132

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Health Sciences, Oncology Health Sciences, Immunology Biology, Cell
Keyword	Gamma Delta T cells Osteosarcoma
Committee Chair / Thesis Advisor	Harold Trent Spencer, Emory University
Committee Members	Kelly Goldsmith, Emory University Jason Yustein, Emory University Sunil Raikar, Emory University Christopher Doering, Emory University

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