Abstract: Objective To construct CAR-T cells harboring distinct intracellular signaling domains, thereby addressing the core limitations of conventional CAR-T cells, including premature exhaustion, insufficient antitumor efficacy, and uncontrolled cytokine release syndrome caused by excessive intracellular activation signals. Methods The intracellular signaling domain CD3ζ of the conventional CAR construct was replaced with the CD3ε signaling domain containing only one immunoreceptor tyrosine-based activation motif （ITAM）, to generate a novel BCMA-E-CAR-T cell product. Flow cytometry was employed to assess the cytotoxicity against target cells, activation status, exhaustion profile, central memory phenotype differentiation, proliferative capacity, and cytokine secretion levels of the engineered CAR-T cells. Results BCMA-E-CAR-T cells exhibited significantly enhanced cytotoxicity against target cells, accompanied by reduced activation levels, alleviated exhaustion, diminished cytokine release, elevated differentiation toward a central memory phenotype, and stronger proliferative potential. Conclusion This study successfully constructed novel BCMA-E-CAR-T cells harboring CD3ε as the intracellular activation signaling domain, which exhibited significantly enhanced antitumor activity and improved therapeutic safety in tumor models.

Key words: CAR-T, B-cell maturation antigen, CD3ε, immune receptor tyrosine activation motif, exhaustion, cytokine release syndrome