Universal Protein Trans-Splicing-Based CAR System Enabling CAR-T Cells with Reduced Exhaustion and Enhanced Efficacy

Adapter switches are commonly developed to control the activation process of CAR-T cells. However, these affinity-based adapter switches cannot control the exhaustion level of CAR-T cells, which leads to a reduction of antitumor activity. To overcome this hurdle, we developed a CAR system based on split intein-mediated protein trans-splicing. In this system, a split C-intein-mediated adapter switch (SIMAS) containing a CD19 antibody splices with an N-intein motif engineered on T-cell receptors to incorporate CD19 antibodies into T-cell receptors site-specifically, which generates protein trans-splicing-based integrated CAR-T (protinCAR-T) cells. Importantly, trans-splicing does not activate CAR-T cells, thus reducing exhaustion level. Only the binding of protinCAR-T cells to tumor cells and cell motility activate protinCAR-T cells, which have good antitumor activity in vivo. Generally, we developed a novel CAR system that enables tuning of CAR-T-cell activity, which provides solutions to address the safety and efficacy barriers of CAR-T-cell therapy.