Modular construction of bispecific antibodies through bioconjugation for T cell-based immunotherapy

Bispecific antibodies (bsAbs) represent a groundbreaking advance in antibody engineering, overcoming therapeutic limitations of monoclonal antibodies through dual-targeting capabilities. However, their clinical translation is often hindered by structural and manufacturing complexities. In this study, we developed a modular bsAb platform utilizing the SpyCatcher-SpyTag site-specific coupling system for T cell-based immunotherapy. We successfully assembled functional bsAbs by conjugating tumor-targeting SpyCatcher-fused antibodies (anti-CD19, FMC63-Fc-SC or anti-HER2, ZHER-SC) with SpyTag-fused anti-CD3 domains (7G03-ST). These bsAbs effectively killed CD19-expressing leukemic cells and HER2-positive solid tumor cells. Furthermore, through substituting the anti-CD3 domain (e.g., OKT3-ST), we readily generated bsAbs targeting CD19, HER2, PD-L1 and EGFR, all of which specifically engaged their respective tumor antigens. More importantly, bsAbs constructed via this modular strategy significantly enhanced the cytotoxicity of CAR-T cells. In conclusion, this flexible bioconjugation platform offers a reliable and efficient technical solution for the rapid development of diverse bispecific antibodies.