Chimeric Antigen Receptors (CARs) are the first successful synthetic immune receptors for adoptive T cell therapy. Although CD28 and 41BB costimulatory domains have shown clinical success in 2nd-generation CARs, using different costimulatory domains of the CAR can elicit distinct T cell functions including cytokine secretion, cytotoxicity, and overall persistence in the patient. Determining the most effective intracellular signals will extend CAR therapeutic success, including its application to solid tumors and treatment opportunities beyond cancer.
Here, we have developed a high-throughput approach for phenotyping novel CAR signaling domains using synthetic DNA libraries. To enable rapid characterization of specific T cell functions across this library, we employed a pooled screen in primary human T cells, which combines FACS sorting and deep sequencing of DNA-barcoded CAR variants. We used this approach to characterize over 40 CAR intracellular signaling domains, some of which alter cytokine secretion, improve proliferative capacity, and confer resistance to exhaustion, relative to the standard CD28 and 41BB CARs. Finally, we performed further in-depth phenotyping (both in vitro and in vivo) on a small number of domains, confirming their improved short-term killing dynamics, increased long-term cytotoxic potential, and reduced exhaustion compared to the state-of-the-art CAR architectures. This integrated strategy to identify optimized CAR signaling domains will both improve T cell immunotherapy and expand our general understanding of T cell receptor signaling.