Construction of Switch Modules for CAR-T Cell Treatment Using a Site-Specific Conjugation System

Chimeric antigen receptor T-cell (CAR-T cell) therapy has become a promising treatment option for B-cell hematological tumors. However, few optional target antigens and disease relapse due to loss of target antigens limit the broad clinical applicability of CAR-T cells. Here, we conjugated an antibody (Ab) fusion protein, consisting of an Ab domain and a SpyCatcher domain, with the FITC-SpyTag (FITC-ST) peptide to form a bispecific safety switch module using a site-specific conjugation system. We applied the safety switch module to target CD19, PDL1, or Her2-expressing tumor cells by constructing FMC63 (anti-CD19), antiPDL1, or ZHER (anti-Her2)-FITC-ST, respectively. Those switch modules significantly improved the cytotoxic effects of anti-FITC CAR-T cells on tumor cells. Additionally, we obtained the purified CD8