In vivo double knockout CAR-T screen identifies synergistic gene pairs that enhance anti-tumor immunity [crispr_screen]

Mechanistically, these DKOs enhance calcium influx, phospho-ERK, NF-κB and NFAT/AP-1 signaling. Among these hits, NR4A1_SOCS3 DKO has multiple favorable immunological features and showcases the most robust phenotypes. NR4A1_SOCS3 DKO CAR-T cells show potent in vivo anti-tumor efficacy and markedly enhanced infiltration as compared to both single gene knockouts, without increase in safety risk.

Whole-transcriptome profiling and single cell RNA sequencing of tumor-infiltrating CAR-T cells reveal that NR4A1_SOCS3 DKO specifically enhanced metabolic fitness of CAR-T cells, rendering them exceptionally potent in long-term tumor control in an orthotopic model, superior to both single gene knockouts and the PD1_CTLA4 dual-checkpoint DKO benchmark. These data together demonstrate the efficacy of high-throughput DKO screening to rapidly uncover critical genetic interactions within T cells, and discover NR4A1_SOCS3 as promising joint intracellular checkpoints to engineer high-performance CAR-T therapies against solid tumors.