Systematic functional screening of immunoreceptor tyrosine-based inhibitory motif domains identifies potent inhibitory modules for chimeric antigen receptor-T.

Background: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies but remains limited by on-target, off-tumor toxicity and the lack of precise mechanisms to restrain aberrant activation. Inhibitory CARs (iCARs) offer a logic-gated strategy to suppress unwanted immune responses, yet optimization of inhibitory signaling modules has been hindered by an incomplete understanding of immunoreceptor tyrosine-based inhibitory motif (ITIM) diversity. Methods: We established a systematic screening platform to evaluate ITIM-containing intracellular domains (ICDs) from 35 candidate receptors. Using nuclear factor of activated T cells (NFAT) reporter assays across multiple activation contexts, we quantified the ability of each domain to counteract iimmunoreceptor tyrosine-based activating motif (ITAM)-mediated signaling. Intracellular truncated analyses tested the requirement of intact cytoplasmic motifs, and the most potent ITIMs were incorporated into iCAR architectures to assess their capacity to inhibit CAR-driven effector responses. Results: We identified 12 ITIM domains that strongly suppressed NFAT signaling across diverse activation states. Their inhibitory activity required intact ITIM motifs. When integrated into iCAR constructs, these domains conferred markedly greater suppression of CAR-induced activation compared with conventional programmed cell death protein 1 (PD-1)-based designs. Among them, the SIGLEC9-derived ITIM exhibited the most potent inhibition, significantly reducing CAR-T effector function and protecting target cells from cytotoxicity Conclusions: We identified distinct intracellular modules that effectively suppress CAR-mediated activation, with the SIGLEC9-derived ITIM exhibiting the strongest inhibition. Leveraging domain-specific inhibitory strength enables precise and modular control of T-cell activation, thereby improving the safety, specificity, and tunability of next-generation cellular immunotherapies.