Engineering novel chimeric antigen receptors (CARs) for T-cell malignancies using innate immune cells Open Access

Abstract

CAR T-cell therapy has successfully treated B-cell malignancies, however, there are many challenges translating these therapies for treatment of T-cell malignancies, including fratricide, T-cell aplasia, and product contamination. Approaches to address these challenges include targeting an antigen specific to a subset of T cells, disrupting target antigen expression on CAR-modified cells, non-viral delivery methods, safety mechanisms, and utilizing third party donor non-alloreactive cells or genome editing to prevent alloreactivity. This dissertation explores some of these approaches for the specific treatment of T-cell acute lymphoblastic leukemia (T-ALL) using CD5-CAR therapy. We evaluate the potential for i) CRISPR-Cas9 genome editing of CD5 to reduce fratricide and increase CAR expression, ii) NK-92 cells and γδ T cells as alternative effector cells within allogeneic settings to avoid product contamination, iii) AAV CAR-delivery to limit long-term expression to reduce concern of T-cell aplasia, as well as iv) a novel class of CARs, non-signaling CARs (NSCARs), to avoid fratricidal constraints.

Our studies show that disruption of CD5 expression in T cells increased CD5-CAR surface expression, however, this did not translate into enhanced CD5-specific cytotoxicity. Using a CD5-negative NK-derived lymphoma cell line, NK-92 cells, we demonstrated in an NSG xenograft model of T-ALL that mice treated with CD5-CAR-modified NK-92 cells, exhibited a survival advantage over control mice. However, due to rapid CD5 down-regulation, fratricide is not a primary concern for CD5-targeted therapy. Therefore, we used γδ T cells because NK-92 cells require irradiation to prevent expansion of the lymphoma cell line in vivo. AAV6 resulted in efficient modification of γδ T cells and as these cells exhibit limited persistence in vivo and because AAV is primarily non-integrating, the combination of these approaches can regulate CAR expression. While fratricide is of minimal concern for CD5-targeted CAR T-cell therapies, other T-cell antigens do not down-regulate rapidly and completely. Therefore, we generated NSCARs, which lack signaling domains and are only advantageous in cells with endogenous cytotoxicity mechanisms, such as γδ T cells. We demonstrate NSCAR-modified γδ T cells exhibited enhanced antitumor cytotoxicity in vitro. Herein, we assess novel approaches for CAR-modified T-cell generation for the treatment of T-cell malignancies.

Table of Contents

Abstract

Table of Contents

List of Figures and Tables

List of Abbreviations

Chapter 1 – Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Chimeric Antigen Receptor (CAR) T-cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

1.2 Translating CAR T-cell Therapy for Treatment of T-cell Malignancies . . . . . . . . . . . . . . . . . .6

1.3 “Off-the-shelf” CAR T-cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

Alternative Effector Cell Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

NK cells and NK-92 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Gamma Delta T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

1.4 Prevention of T-cell Memory Formation and T-cell Aplasia . . . . . . . . . . . . . . . . . . . . . . . . . 26

Non-viral Delivery Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Adeno-Associated Viral Vector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Suicide Genes and Safety Switches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

1.5 Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Chapter 2 – Development of chimeric antigen receptors targeting T-cell malignancies using two structurally different anti-CD5 antigen binding domains in NK and CRISPR-edited T cell lines . .38

2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

2.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

2.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

2.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

2.6 Supplemental Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71

Chapter 3 – CRISPR-Cas9 gene delivery and CD5-CAR modification of αβ T cells . . . . . . . . . . . . .80

3.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

3.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83

3.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

3.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Chapter 4 – γδ T cells as an alternative effector cell type for CAR T-cell therapy . . . . . . . . . . . . . 100

4.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

4.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .104

4.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Chapter 5 – Non-signaling chimeric antigen receptors (NSCARs) enhance antigen-directed killing by γδ T cells in contrast to αβ T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .136

5.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

5.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

5.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140

5.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

5.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

5.6 Supplemental Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .164

Chapter 6 – Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .170

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

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	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular
Keyword	Gene therapy T-cell leukemia CAR therapy
Committee Chair / Thesis Advisor	H. Trent Spencer, Emory University
Committee Members	Brian G. Petrich, Emory University Cassandra L. Quave, Emory University Christopher B. Doering, Emory University

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