Abstract 1777: A non-signaling CAR for gamma-delta (γδ) T cells to preserve healthy tissues
Authors: Ding Lei , Li Yanjie , Haak Mariska ter , Lamb Lawrence 
Published: April 2023

Abstract
Chimeric antigen receptor T cell (CAR-T) therapy for B cell leukemias and lymphomas have shown remarkable responses in the clinic. However, the elimination of all cells expressing target antigen including healthy tissues by CAR-T cells is a significant safety concern. Such on-target off-tumor toxicities are problematic for many types of cancer antigens and in some cases have resulted in patient deaths. There is a need to address these limitations and develop better and safer next generation CAR-T therapies as we move beyond these tumor types.
γδ T cells can directly identify and kill malignant cells through the recognition of tumor and/or stress antigens that are not generally expressed on normal healthy tissues. We hypothesized that we could demonstrate the potential of our non-signaling γδ CAR platform to kill tumors, while protect healthy cells against a validated target such as CD19. We developed a non-signaling CD19 CAR (ns19CAR) for γδ T cells by removing the CD3z signaling domain. In addition, IL-15 was co-expressed to enhance γδ T cell fitness and persistence.
We first demonstrated CAR expression in Jurkat T cells with either a signaling CD19 CAR (s19CAR) or ns19CAR lentiviral vectors. As expected, after co-cultured with CD19+ Raji cells, s19CAR-Jurkat cells demonstrated activation by strong CD69 expression, while ns19CAR-Jurkat cells showed no activation. We next tested transduction of the ns19CAR in expanded Vδ2+ γδ T cells. In experiments with γδ T cells from different donors (N=4), a high percentage (66-86%) of γδ T cells were ns19CAR positive as measured by flow cytometry. ns19CAR γδ T cells were shown to effectively kill CD19+ Nalm6 acute lymphoblastic leukemia (ALL) cells at low effector-to-target ratios (E:T=1:1 to 4:1) in 16hr and 48hr co-culture assays and demonstrated enhanced (&gt;1.5x) cytotoxicity compared to non-transduced control γδ T cells (NTC). After 48hr co-culture (E:T=2:1), the ns19CAR-γδ T cells killed 79.7% Nalm6 cells (SD=6.6%) compared to NTC’s cytotoxicity of 46.1% (SD=7.2%). In comparison, minimal cytotoxicity was observed for ns19CAR and NTC γδ T cells against B cells from healthy donor PBMC. At E:T=2:1, an average cytotoxicity of 5.2% (SD=6.6%) were observed with ns19CAR-γδ T cells compared to -4.6% (SD=7.7%) with NTC. There is no significant difference in cytotoxicity against CD19- K562 cells between the NTC or ns19CAR γδ T cells suggesting the enhanced cytotoxicity of ns19CAR-γδ T cells is specific to CD19+ target cells. Our results demonstrate that our nsCAR-γδ T cell platform may be able to target malignant cells while preserving healthy tissues where the antigen target may be widely expressed such as hematopoietic stem cells and solid tumor cancers.
Our unique combination of non-signaling CAR and γδ T cells is a promising candidate for next generation CAR-T therapies and provides insights towards the development of future CAR therapies against other types of hematological and solid tumor cancers.
Citation Format: Lei Ding, Yanjie Li, Mariska ter Haak, Lawrence Lamb. A non-signaling CAR for gamma-delta (γδ) T cells to preserve healthy tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1777.