Sense-and-Respond Payload Delivery Using a Novel Antigen-Inducible Promoter Improves Suboptimal CAR-T Activation

[Image: see text] Chimeric antigen receptor (CAR)-T cell therapies demonstrate the clinical potential of lymphocytes engineered with synthetic properties. However, CAR-T cells are ineffective in most solid tumors, partly due to inadequate activation of the infused lymphocytes at the site of malignancy. To selectively enhance antitumor efficacy without exacerbating off-target toxicities, CAR-T cells can be engineered to preferentially deliver immunostimulatory payloads in tumors. Here, we report a novel antigen-inducible promoter for conditional payload expression in primary human T cells. In therapeutic T cell models, the novel NR4A-based promoter induced higher reporter gene expression than the conventional NFAT-based promoter under weakly immunogenic conditions, where payload expression is most needed. Minimal activity was detected from the inducible promoters in the absence of antigen and after withdrawal of stimulation. As a functional proof-of-concept, we used the NR4A-based promoter to express cytokines in an antimesothelin CAR-T model with suboptimal stimulation and observed improved proliferation compared to T cells engineered with the conventional NFAT promoter or CAR alone. Our system achieves CAR-directed payload expression under weakly immunogenic conditions and could enable the next generation of cell therapies with enhanced antitumor efficacy.