Studying the combination of CAR T cells and ICI for treatment of Melanoma Open Access
Woo, June (Summer 2023)
Abstract
Melanoma proves to be the most aggressive form of skin cancer due to its metastatic ability. Both CAR T cell therapy and immune checkpoint inhibitor (ICI) have seen great progress with the treatment for melanoma, but both therapies have toxicities related to treatment. Due to the limitations of both CAR T cell and ICI therapy, we aim to increase the efficacy of CAR T cell therapy by combining both treatments. Because Muc16CD is a suitable TAA and the anti-Muc16CDCAR has been validated in combination with anti-PD-1 in an ovarian cancer, we decided to use the same combination treatments in a melanoma model. Thus, we hypothesize that the combination of CAR T and ICI treatments would address the limitations of CAR and ICI therapies: CAR T cells would be able to direct an immune response against cold tumors while ICI could mount a response against the suppressive TME. Furthermore, we demonstrated this treatment in a clinically relevant model of melanoma called the YUMM cell line with a Braf/Pten mutations. We investigated CAR T cell function by testing its cytotoxic function, cytokine levels, proliferating and activating capabilities against YUMM tumors. We also tested the in vitro and in vivo characteristics of the YUMM cells that were taken from a GEMM model with a Braf activation and Pten inactivation mutations, mimicking melanoma seen in clinic. In in vitro assays, antigen-stimulated CAR T cells displayed an increase in cytokine levels, such as IFN-γ, and degranulation factors, such as CD107a and granzyme B compared to unstimulated CAR T cells. The anti-Muc16CD CAR was also able to specifically kill Muc16CD+ tumors in vitro compared to Muc16CD- tumor. And mouse CAR T cells were also more activated and proliferated when cultured with antigen-expressing cells. Because immunotherapy is a constantly growing field, combination of CAR T cell and ICI should be further studied in melanoma and solid tumors.
Table of Contents
Table of Contents
Introduction 1
Melanoma 2
ICI therapies and limitations 3
Adoptive Cell Therapy 6
Tumor-infiltrating Lymphocyte Therapy 6
Chimeric Antigen Receptor T Cell Therapy and its Limitations 7
Muc16CD as a target for CAR T cell therapy 10
ACT and ICI Therapies 11
Mouse Melanoma Models 14
Materials and Methods 16
Generation of retroviral vectors 17
Cell Culture 17
Flow Cytometry 18
Mouse splenocyte isolation and transduction 18
Cytotoxicity Assays 19
LDH Assays 19
Cytokine and Degranulation Assays (IFN-γ, Granzyme B, CD107α) 20
Activation and Proliferation and Assays (CD69 and CFSE) 21
Tumor Injection and Imaging 21
Measuring tumors and evaluating tumor burden scores 22
Statistical Analysis 22
Results 23
Retrovirally transduced YUMM cells express Muc16CD, GFP, and luciferase 24
Exposure to IFN-gamma leads to the upregulation of PD-L1 on YUMM
Muc16CD-GFPffluc cells 25
Mouse T cells can be retrovirally transduced to express anti-Muc16CD CAR 25
Anti-Muc16CD CAR show specific killing to Muc16CD+ tumor cells 27
Anti-Muc16CD CAR shows elevated expression of degranulation, activation, and
proliferation markers 28
CAR T cell treatment group shows prolonged survival over the combination treatment
group in vivo 31
Future Directions 35
Discussion 38
References 44
Appendix I: Protocols 51
Appendix II: Plasmid 56
Appendix III: Supplementary Figures 58
Table of Figures
Figure 1- PD-1 and PD-L1 checkpoint proteins 3
Figure 2- Current Strategies for CAR Design 6
Figure 3- Anti-Muc16CD CAR has been validated in Muc16CD+ tumors 10
Figure 4: Muc16CD as a relevant tumor associated antigen 11
Figure 5: CAR Construct 12
Figure 6: Combination of ICI and CAR T Cell Therapies as a Strategy to Overcome Roadblocks13
Figure 7: Schematic of how LDH Assays Work 14
Figure 8: LDH Assay Setup 17
Figure 9: Transfection and Transduction of CAR Construct and Packaging Cells 20
Figure 10: Transducing Mouse T cells with CAR Packaging Cells 21
Figure 11: How YUMM cells were cultured 22
Figure 12: Muc16CD, GFP, and luciferase expression on retrovirally transduced YUMM cells 24
Figure 13a: Diagram of when T cells release IFN-γ, PD-L1 expression upregulates 25
Figure 13b: YUMM-Muc16CD-GFPffluc cells in different concentrations of IFN-γ 25
Figure 14a: Transduction efficiency of Phoenix Eco packaging cells of anti-Muc16CD construct26
Figure 14b: Transduction efficiency of anti-Muc16CD+ mouse CAR T cells 26
Figure 15a: Flow strategy for gating CAR-positive mouse T cells. 26
Figure 15b: Transduction efficiency of mouse CAR T cells 26
Figure 16a: Average transduction efficiency of anti-Muc16CD CAR T cells 27
Figure 16b: Average viability of mouse CAR T cells 27
Figure 17: 4H11-28z CAR shows more cytotoxicity to Muc16CD+ tumor cells than Muc16CD- cells 27
Figure 18: Cytokine and degranulation factors tested on unstimulated and stimulated mouse CAR T cells 28
Figure 19: Degranulation and activation factors were tested on stimulated and unstimulated CAR T cells 29
Figure 20: Preliminary data of activation of mouse CAR T cells between unstimulated and stimulated conditions 30
Figure 21: Preliminary data of proliferation markers on stimulated and unstimulated mouse CAR T cells 31
Figure 22: In vivo experiment timeline 32
Figure 23: Bioluminescence Imaging of mice seven and fourteen days post CAR T cell treatment32
Figure 24: Survival Curve of in vivo study
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