CARPOOL: A library-based platform to rapidly identify next generation chimeric antigen receptors

CD19-targeted CAR therapies have successfully treated B cell leukemias, but many responders later relapse or experience toxicities. CAR ICDs are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domains that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications. Overall design: Single-cell RNA sequencing data of primary human T cells expressing three different CD19-targeted CAR variants following three tumor challenges with NALM6 leukemia cells. An unstimulated, untransduced control was also included. Two merged sample records of the following samples: Untransduced_unstimulated BBz Var1 Var3 as detailed in the sample_metadata.txt