Overcoming therapy resistance with multi-targeted CAR-T cells secreting bispecific antibody (BsAb) for the treatment of B-cell malignancies

Abstract Adoptive T-cell immunotherapies have revolutionized the treatment landscape for aggressive B-cell malignancies. Despite high rates of initial complete remission, more than half of patients eventually relapse. While T cell-mediated cytotoxicity remains a powerful therapeutic mechanism, recent attention has turned toward targeting innate immune checkpoints as an additional strategy. In particular, blocking “don't eat me” (DEM) signals—such as CD47 and CD24—has been shown to enhance macrophage-mediated phagocytosis and exert potent anti-tumor effects in B-cell malignancies. In this study, we found that CD24 is abundantly expressed on B-cell lymphoma and leukemia cells and identified it as a novel immunologic vulnerability that can be leveraged to activate both innate and adaptive immune responses. We developed a CD24-targeting CAR-T cell therapy engineered to express CXCR3 (CD24-CXCR3 CAR-T), which significantly improves T-cell trafficking and function within the tumor microenvironment. CD24-CXCR3 CAR-T cells demonstrated strong anti-tumor activity by simultaneously promoting cytotoxic T lymphocyte (CTL) responses and enhancing phagocytosis through blockade of DEM signaling. Furthermore, we rationally designed a “dual specificity, dual functionality” CD19-targeted CAR-T cell capable of secreting CD24-targeting components, thereby allowing simultaneous targeting of both CD24 and CD19 antigens on malignant B cells. This strategy addresses tumor antigen heterogeneity and achieved superior efficacy in preclinical models of aggressive B-cell lymphoma and acute lymphoblastic leukemia, including those resistant to conventional CD19 CAR-T therapy. Collectively, our findings support the development of CD24-directed adoptive T cell therapies as a promising approach to harness both innate and adaptive immunity for the treatment of B-cell malignancies.