ID #459 Computationally structurally optimized IL13Rα2 – B7-H3 tandem CAR T cells overcome antigen-heterogeneity mediated tumor escape in a brain tumor model

Abstract Chimeric antigen receptor (CAR) T cells have recently begun to be clinically evaluated for the treatment of brain tumors. This treatment has demonstrated promising efficacy and safety; however, one limitation is tumor antigen-heterogeneity and downregulation, which allows tumors to evade conventional, monospecific CAR T cells. One approach to overcome this tumor escape is by utilizing a tandem CAR that recognizes two antigens, but these tandem CAR constructs often require several optimizations to achieve cell surface expression and function. Here, we sought to design an IL13Rα2 – B7-H3 tandem CAR targeting two antigens widely expressed in brain tumors. Interestingly, our original tandem CAR failed to express on the cell surface, leading to a systematic evaluation of 24 constructs varying in their scFv positioning, linkers, and amino acids. We identified a ‘trouble region’ in the CAR and optimized it using computational approaches, rescuing surface expression and improving function compared to monospecific CAR T cells. Once functional, the tandem CAR T cells effectively recognized both target antigens, killed single-antigen and double-antigen expressing tumor cells in vitro, and did not kill non-antigen expressing tumor cells, indicating antigen-specificity. In a brain tumor model in vivo, the tandem CAR T cells effectively cleared tumors when both antigens were present. Further, in an in vivo model of antigen-heterogenous brain tumor, where only some of the tumor cells expressed each of the antigens, the tandem CAR T cells continued to eradicate tumors and had greater efficacy than single-antigen targeting CAR T cells. Our study demonstrates that structural components of the CAR can detrimentally affect surface expression and can be rescued by computational optimizations. Our study also highlights the feasibility of creating tandem CAR T cells for the treatment of brain tumors and demonstrates that these tandem CAR T cells can effectively overcome antigen-heterogeneity mediated tumor escape.