Abstract 46: Mechanisms and impact of bystander killing by CAR T cells

Abstract

Abstract While CD19-directed chimeric antigen receptor (CAR) T cell therapy has improved outcomes of patients with relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL), a majority will suffer disease relapse following CAR T cell infusion. Therefore, understanding mechanisms of CAR T cell killing is critical to enhance therapeutic efficacy. Cytolysis of CAR T cell targets presumably involves recognition of CD19 antigen by the CAR followed by activation of the granzyme/perforin axis. However, bystander elimination of CD19-negative lymphoma cells by CAR T cells has been reported, indicating that an alternative pathway may be required for optimal killing. Through in-depth phenotyping of a large panel of human DLBCLs and in vitro killing assays, we observed that CD19 expression levels on DLBCL cells and vulnerability to CAR T cell killing were weakly correlated. Conversely, cell surface expression of the death receptor, FAS, on DLBCL cells was strongly associated with susceptibility to CAR T cell killing. Although FAS expression across DLBCL cell lines varied, it could be broadly enhanced following exposure to IFNγ. Potent CAR T cell-mediated cytolysis of CD19-negative, Fas-positive murine DLBCL cells required the presence of CD19-positive lymphoma cells in the culture. CRISPR/Cas9 targeting was employed to delete FAS and/or CD19 in isogenic human DLBCL cells to generate CD19+FAS+, CD19+FAS−, CD19−FAS+, and CD19−FAS− subsets, which enabled us to define the extent to which CD19 engagement by the CAR was required to affect killing via FAS. When CAR T cells were co-cultured with equal numbers of the four gene-modified DLBCL cells described above, CD19+FAS+ DLBCL cells were killed most effectively, followed by CD19+FAS− DLBCL cells, and CD19−FAS+ DLBCL cells. Double-negative DLBCL cells were poorly killed. This result suggested that CD19 engagement by the CAR was required for optimal DLBCL cell killing via FAS, and that CD19 and FAS need not necessarily be engaged on the same target cell. Future studies will determine if Fas/FasL interactions can be exploited by overexpression of FasL in CAR T cells and examination of DLBCL killing in vitro and in vivo. Citation Format: Joanna Chorazeczewski, Lishi Xie, Xiufen Chen, Sidney Wang, Justin Kline. Mechanisms and impact of bystander killing by CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 46.