Engineering detours around the biologic barriers to allogeneic, iPSC-derived CAR T immunotherapy

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PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR) T cell therapy has produced remarkable results in otherwise treatment- refractory hematological malignancies. Currently, the process of manufacturing autologous CAR T cells is challenging due to the need for de novo generation of each individualized therapy and the inherent variability in T cell biology between patients, leading to unpredictable and inconsistent clinical responses. As a result, there is a growing interest in generating CAR T cells in vitro from an infinitely renewing, allogeneic source of human pluripotent stem cells (hPSCs). One advantage of this approach is that hPSCs are highly amenable to genetic editing, providing multiple avenues to manipulate the function of the final T cell product. However, there are several biologic barriers to generating CAR T cells from hPSCs. First, expression of the CAR transgene early in T cell differentiation diverts development towards the innate lymphoid pathway instead of the conventional T lineage. Second, the removal of the endogenous T cell receptor (TCR) to prevent alloreactivity leads to a block in development, as maturing T cells can no longer undergo positive selection. This proposal seeks to overcome these challenges to generating non-alloreactive, stem cell-derived CAR T cells by deploying an innovative stage-specific expression strategy and a novel in vitro method to induce the differentiation of mature conventional T cells, the Artificial Thymic Organoid (ATO) system. The ATO is a first-in-class, in vitro method for efficiently generating mature, single positive (SP) CD8+ and CD4+ T cells from multiple stem cell sources, including hPSCs. Improving upon previously established systems, the ATO can robustly support positive selection and maturation to the SP stage. Our preliminary studies have already demonstrated that innate fate diversion can be overcome by achieving delayed, stage-specific expression of the CAR transgene that is limited to mature T cells. In this proposal, Specific Aim 1 will build upon the delayed CAR expression model and evaluate how the disruption of the endogenous TCR affects CAR T cell development in the ATOs. Specific Aim 2 will evaluate two different methods of delivering exogenous positive selection signals to rescue development in the absence of the endogenous TCR. These methods include expression of a tonically signaling CAR or transiently expressing an exogenous TCR at the physiologically appropriate double positive (DP) T cell stage. The approaches developed in this proposal will enable the generation of hPSC-derived, non-alloreactive T cell therapy, ultimately reducing the cost and increasing the access to treatment for more patients in need.