Phase separation of chimeric antigen receptor promotes immunological synapse maturation and persistent cytotoxicity

Major challenges of chimeric antigen receptor (CAR)-T cell therapy include poor antigen sensitivity and cell persistence. Here, we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of an engineered T cell receptor CD3ε motif, E_B6I, into the conventional 28Z or BBZ CAR induced self-phase separation through cation-π interactions. E_B6I CAR formed a mature immunological synapse with the CD2 corolla to transduce efficient antigen and costimulatory signaling, although its tonic signaling remained low. Functionally, E_B6I CAR-T cells exhibited improved signaling and cytotoxicity against low-antigen tumor cells and persistent tumor-killing function. In multiple primary and relapsed murine tumor models, E_B6I CAR-T cells exerted better antitumor functions than conventional CAR-T cells against blood and solid cancers. This study thus unveils a CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.