Phase Separation of Chimeric Antigen Receptor Promotes Immunological Synapse Maturation and Persistent Cytotoxicity

Tumor relapse, a major challenge of CAR-T cell therapy, can result from immune evasion by antigen-low tumor and poor persistence of CAR-T cells. Here we report a solution to these issues by exploiting CAR phase separation. We found that incorporation of a positively-charged CD3ε motif to the conventional 28Z CAR induced self-phase separation, promoting immunological synapse formation but also CAR endocytosis. Through protein engineering, we created an EB6I28Z CAR construct with endocytosis selectively inhibited but the phase separation ability preserved. Notably, this novel CAR can form a mature immunological synapse that integrates exogenous CAR signaling and endogenous costimulation signaling within segregated zones. Functionally, EB6I28Z CAR-T cells exhibited improved cytotoxicity against antigen-low tumor and maintained robust killing efficacy during chronic tumor exposure. In a murine model mirroring antigen-low tumor relapse, EB6I28Z CAR-T cells exerted long-term control over tumor progression. This study thus unveils a new CAR engineering strategy to improve CAR-T cell immunity by leveraging molecular condensation and signaling integration.