The development and functional testing of cellular therapies against protein tyrosine kinase 7 (PTK7) to treat high-risk neuroblastoma Open Access

Abstract

New therapies for high-risk neuroblastoma (NB) are needed as it’s the most common extracranial pediatric solid tumor with an alarming survival rate of <50%. Immunotherapies utilize cells or proteins of the immune system to treat diseases such as cancer. A form of immunotherapy that’s at the forefront of treating previously difficult to treat cancer is chimeric antigen receptor (CAR) T-cell therapy which has been largely successful for hematological malignancies but has not had the same success in solid tumors. Another variety of immunotherapies are monoclonal antibodies, a form of targeted therapy utilizing lab-engineered antibodies. In recent years, a monoclonal antibody against a disialoganglioside, GD2, has significantly improved survival for high-risk NB patients. However, toxicities can occur and not all patients respond, underpinning the need to identify additional targets. Protein tyrosine kinase 7 (PTK7) has been identified as a promising NB target in a model that represents the post-chemotherapy refractory/recurrent NB patient population. The primary aim of the research presented in this dissertation was to determine the feasibility of targeting PTK7 through cellular-based immunotherapies. We engineered anti-PTK7 CARs and found PTK7 CAR T cells specifically target and kill PTK7-expressing NB cell lines. In vivo, PTK7 CAR T cells regress an aggressive NB metastatic model and improve survival. The primary T cells utilized in these studies were of an alpha-beta (𝜶β) subtype, the subtype choice for the CAR T-cell therapies currently tested in humans, largely due to the abundance and ease of cell isolation and expansion. Unlike 𝜶β T cells, gamma-delta (𝜸𝛿) T cells lie on a bridge between both innate and adaptive immunity and therefore have an intrinsic ability of inducing potent cytotoxicity against cancer cells without priming. Our lab has previously shown that non-modified ex vivo expanded 𝜸𝛿 T cells are cytotoxic against NB cells. To further enhance the effectiveness of this cell-based immunotherapy, we modified 𝜸𝛿 T cells to express PTK7 CAR and show early evidence of preclinical utility in vitro and in vivo. This work supports ongoing studies to further optimize these cellular therapies against PTK7 to treat NB and other cancers.

Table of Contents

Table of Contents

Abstract 

Acknowledgments

Table of Contents 

List of Figures and Tables

Chapter 1: Introduction

1.1 Neuroblastoma

1.2 Cancer Immunotherapies

1.2.a. History

1.2.b. Monoclonal antibodies and immune checkpoint inhibitors

1.2.c. Cytokine therapies

1.2.d. Cancer vaccines

1.2.e. Adoptive cell therapies

1.2.f. 𝜸𝛿 T-cell therapies

1.2.g. Immunotherapies for neuroblastoma

1.3 PTK7 identification, expression, and function in neuroblastoma

Chapter 2: Engineering and characterization of anti-PTK7 chimeric antigen receptor (CAR)

2.1 Abstract

2.2 Introduction

2.3 Results

2.4 Discussion

2.5 Materials and Methods

2.6 Supplemental Figures, Tables, and Legends

Chapter 3: Method development of introducing PTK7 CAR expression in primary 𝜶β T cells

3.1 Abstract

3.2 Introduction

3.3 Results

3.4 Discussion

3.5 Materials and Methods

Chapter 4: PTK7-targeting 𝜶β CAR T cells induce NB cell death in vitro and in mouse metastasis NB model

4.1 Abstract

4.2 Introduction

4.3 Results

4.4 Discussion

4.5 Materials and Methods

Chapter 5: PTK7-targeting 𝜸𝛿 CAR T cells and bispecific T-cell engagers enhance NB cell death in vitro and in mouse metastasis NB model.

5.1 Abstract

5.2 Introduction

5.3 Results

5.4 Discussion

5.5 Materials and Methods

5.6 Supplemental Figures, Tables, and Legends

Chapter 6: General Discussion

6.1 Summary of Results

6.2 Implication of Findings

6.3 Limitations and Future Directions

6.4 Conclusions

Citations

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Cell
Keyword	neuroblastoma PTK7 protein tyrosine kinase 7 CAR T cell bispecific engager cell therapy gamma-delta T cells
Committee Chair / Thesis Advisor	H. Trent Spencer, Emory University
Committee Members	Haydn T. Kissick, Emory University Christopher Doering, Emory University Kelly C. Goldsmith, Emory University Sarwish Rafiq, Emory University

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