High-throughput single-cell screening of a cross-lineage CAR library for early-stage CAR design and candidate discovery.

Chimeric antigen receptor T cell therapy (CAR-T) has demonstrated promising efficacy in hematological malignancies, but translating that success to solid tumors remains challenging. Here, we construct a CAR library comprising approximately 1000 variants targeting prostate-specific membrane antigen by recombining transmembrane (TM), co-stimulatory, and activation domains from Natural Killer (NK) and T cell receptors. Single-cell screening identifies ICOSTM-containing variants with improved T cell activation; NK-derived activation domains, such as DAP10ζ, DAP12ζ, and FcRγζ, further augment the effector capacity. Gene regulatory network analysis reveals that CAR variants with elevated expression of T cell-activation-related transcription factors correlates with enhanced cell function. Overall, our study advances early-stage CAR design by expanding the repertoire of structural components from diverse immune cells, providing a scalable platform for identifying candidates with functional profiles comparable to clinical benchmarks.