RESUMEN
The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function-particularly at the molecular level-are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures. Informed by these observations, we functionally characterize transcriptionally distinct ICD variants across various contexts to build comprehensive maps from ICD composition to phenotypic output. We identify a unique tonic signaling signature associated with a subset of ICD architectures that drives durable in vivo persistence and efficacy in liquid, but not solid, tumors. Our findings work toward decoding CAR signaling design principles, with implications for the rational design of next-generation ICD architectures optimized for in vivo function.
RESUMEN
Chimeric antigen receptor therapies have demonstrated potent efficacy in treating B cell malignancies, but have yet to meaningfully translate to solid tumors. Here, we utilize our pooled screening platform, CARPOOL, to expedite the discovery of CARs with anti-tumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.3×10 6 3 rd generation CARs targeting IL13Rα2, a cancer testis antigen commonly expressed in glioblastoma. Selections were performed for cytotoxicity, proliferation, memory formation, and persistence upon repeated antigen challenge. Each enriched CAR robustly produced the phenotype for which it was selected, and one enriched CAR triggered potent cytotoxicity and long-term proliferation upon in vitro tumor rechallenge. It also showed significantly improved persistence and comparable antigen-specific tumor control in a microphysiological human in vitro model and a xenograft model of human glioblastoma. Taken together, this work demonstrates the utility of extending CARPOOL to diseases beyond hematological malignancies and represents the largest exploration of signaling combinations in human primary cells to date.