CRISPR engineering of armored CAR T cells enables tumor-restricted payload delivery with enhanced safety and efficacy

The efficacy of chimeric antigen receptor (CAR) T cell therapy in solid tumors is limited by immunosuppression and antigen heterogeneity. To overcome these barriers, “armored” CAR T cells, which secrete proinflammatory cytokines, have been developed. However, their clinical application has been limited due to toxicities related to peripheral expression of the armoring transgene. Here, we developed a CRISPR knock-in strategy that leverages the regulatory mechanisms of endogenous genes to drive transgene expression in a tumor-localized manner. By screening endogenous genes with tumor-restricted expression, the NR4A2 and RGS16 promoters were identified to support the delivery of cytokines such as IL-12 and IL-2 directly to the tumor site, leading to enhanced anti-tumor efficacy and long-term survival of mice in both syngeneic and xenogeneic models. This was concomitant with improved CAR T cell polyfunctionality, activation of endogenous anti-tumor immunity, a favorable safety profile, and was applicable using CAR T cells from patients. Bulk RNA-seq profiling of human anti-LeY CAR T cells engineered to express IL-12 from the NR4A2 gene locus. Experiment includes n=2 Mock control, NR4A2/GFP control and NR4A2/IL12 CAR T cells.