Engineering programmable CAR and antigen pairing via drug-gated light activation

Abstract

Abstract Chimeric antigen receptor (CAR) T-cell immunotherapy has achieved clinical success, but remains limited in solid tumors by antigen escape and tumor heterogeneity. Here, we engineer a high-affinity R-phycoerythrin (PE)-binding monobody to create a PE-programmable CAR toward user-defined antigens sequentially or simultaneously. To reduce off-tumor toxicity, we implement a drug-gated light-activation approach (DGLA) strategy to spatially confine CAR function. We further couple DGLA-controlled tumor antigen presentation with synNotch-mediated programmable antigen-targeting CAR (sPAT) to establish programmable CAR-antigen pairing (DGLA-sPAT). This system induces clinically validated antigens on tumor cells as local ‘training centers’ that recruit and activate sPAT CAR T cells, enabling elimination of entire tumor populations through broadly expressed tumor antigens using PE-conjugated antibodies. In vivo, DGLA-sPAT manifests local T cell activation and potent tumor suppression with minimal off-tumor toxicity. Thus DGLA-sPAT provides a modular and spatially controlled framework to overcome antigen escape and heterogeneity while improving safety in solid-tumor CAR-T therapy.