NOT-gated chimeric antigen receptor circuits in T and NK cells

Chimeric antigen receptor (CAR) T cells are powerful cancer immunotherapies, but the lack of tumor-specific single antigens for most cancers limits the therapeutic window. A NOT-gated CAR circuit, consisting of an activating CAR (aCAR) and an inhibitory CAR (iCAR), is particularly powerful because it enables new sets of antigens to be used in tumor targeting. We systematically evaluated more than 60 pairs of NOT-gated CAR circuits. Different inhibitory dose-response characteristics were observed depending on the CD3ζ signaling domain on the aCAR. Furthermore, LIR1-iCAR-expressing T cells display fewer exhaustion phenotypes. Mechanistic studies have shown that SHP-1 is the major intracellular regulator of LIR1-iCAR and the iCAR function in cis. Several of the NOT-gated circuits developed in T cells were functionally transferred into primary natural killer (NK) cells. Our NOT-gated CAR circuits provide a precise targeting strategy for safer and more effective cancer immunotherapies.