Developing novel cellular and gene therapies for pediatric malignancies Restricted; Files Only

Zoine, Jaquelyn (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/8s45q988k?locale=es
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Abstract

Alternative strategies for cancer therapy are necessary because existing therapeutics have not been universally effective and current treatments introduce major toxicities. In some cancers, immunotherapy, a therapy responsible for activating, suppressing, boosting, or modifying the immune system or its components, has shown tremendous promise in targeting tumor cells. Current strategies of immunotherapy include the administration of cancer vaccines, cytokines, monoclonal antibodies, checkpoint inhibitors, and adoptive cell therapy (ACT). ACT has emerged as a successful strategy for immunotherapy and focuses on the infusion of immune competent cells into a patient. The primary objectives of the studies presented in this dissertation were to explore unique strategies of ACT, including γδ T cells and the expansion of chimeric antigen receptor (CAR) T cells. γδ T cells have intrinsic anti-tumorgenicity through multiple mechanisms by their cell surface receptors NKG2D, FasL, and CD16. Peripheral blood contains between 1-5% of γδ T cells, so their expansion for therapy is essential to achieve robust responses. Our studies demonstrate an efficient method for expanding and storing γδ T cells from neuroblastoma patient-derived apheresis products. The expanded patient-derived γδ T cells were cytotoxic against neuroblastoma cell lines. Furthermore, low-dose temozolomide in combination with expanded γδ T cells and dinutuximab caused targeted killing of neuroblastoma xenografts in vivo, reducing tumor burden and prolonging survival. Additionally, we developed a novel CAR T cell, which are T cells engineered with a recombinant receptor consisting of an antigen binding domain, a costimulatory domain, and T cell activation domain. Major limitations of CAR T cells are the limited number of available tumor associated antigens and methods to target existing antigens. Therefore, we designed a ligand-based CAR targeting the thrombopoietin (TPO) receptor, MPL, is a critical survival signal for hematopoietic stem cells (HSCs) and leukemia stem cells. We designed a CAR against MPL using a fragment of TPO. The TPO-CAR targeted MPL+ leukemia cell lines in vitro and in vivo and selectively targeted MPL+ HSCs. This dissertation discusses the novel development and incorporation of γδ T cells and ligand-based CARs into immunotherapeutic strategies for pediatric malignancies.

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 History of Cancer Immunology…………………………………………………………………………2

           A. Overview………………………………………………………………………………………..2

B. Innate immune system’s response to cancer……………………………………………………..8

C. Adaptive immune system’s response to cancer………………………………………………...14

D. Cancer evasion of the immune response………………………………………………………16

1.2 Cancer Immunotherapy overview……………………………………………………………………...17

           A. Cytokines………………………………………………………………………………………17

           B. Monoclonal antibodies………………………………………………………………………....19

C. Immune Checkpoint Inhibitors………………………………………………………………...23

D. Cancer Vaccines……………………………………………………………………………….24

E. Adoptive Cell Therapy…………………………………………………………………………26

1.3 Challenges in Cancer Immunotherapy…………………………………………………………………30

           A. Immunosuppressive tumor microenvironment…………………………………………...……33

           B. T cell tolerance and exhaustion………………………………………………………………...34

           C. Biomarkers……………………………………………………………………………………..35

D. Neoantigens and antigen escape………………………………………………………………..36

1.4 Overcoming problems in immunotherapy……………………………………………………………..37

           A. Utilization of alternative cell sources…………………………………………………………..38

           B. Expanding the repertoire of antigen recognition………………………………………………38

Chapter 2: Ex vivo expanded patient-derived γδ T-cell immunotherapy enhances neuroblastoma tumor regression in a murine model...41

2.1 Abstract………………………………………………………………………………………………..42

2.2 Introduction……………………………………………………………………………………………42

2.3 Results…………………………………………………………………………………………………45

2.4 Discussion……………………………………………………………………………………………..61

2.5 Materials and Methods…………………………………………………………………………………64

2.6 Supplemental Figures, Tables, and Legends…………………………………………………………..70

Chapter 3: Novel ligand-based CAR targets MPL receptor in vitro and in mouse xenograft AML model ……………………………………………………………………………………………………..80

3.1 Abstract………………………………………………………………………………………………..82

3.2 Introduction……………………………………………………………………………………………82

3.3 Results…………………………………………………………………………………………………83

3.4 Discussion……………………………………………………………………………………………105

3.5 Materials and Methods………………………………………………………………………………..108

Chapter 4: General Discussion……………………………………………………………….………...118

4.1 Summary of Results………………………………………………………………………….……….119

4.2 Implications of Findings……………………………………………………………………………...121

4.3 Limitations and Future Directions…...……………………………………………………………….128

4.4 Conclusions…………………………………………………………………………………………..129

Literature Cited…………………………………………………………………………………………131

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