用于高危多发性骨髓瘤的异体BCMA/CD70双CAR-T细胞的临床前评估

Off-the-shelf allogeneic CAR T cells manufactured from healthy donor T cells could potentially address some of the limitations of autologous therapies by providing a more consistent product, immediate availability, and the convenience of scalable manufacturing. However, premature rejection of allogeneic CAR T cells may limit persistence and clinical responses. We previously reported the development of an anti-rejection CD70 CAR that can selectively eliminate CD70 + alloreactive T cells and overcome rejection. Notably, recent studies reported high CD70 expression in high-risk multiple myeloma (MM), supporting the evaluation of CD70-targeted therapies in this patient population. Here we evaluated the ability of allogeneic CAR T cells targeting BCMA and CD70 to overcome rejection and BCMA antigen loss in preclinical models of multiple myeloma. Human T cells were engineered with CRISPR gene-editing technology to knock out the TRAC locus and to allow site-specific integration (SSI) of a BCMA/CD70 Dual CAR construct. Following expansion, TRAC KO Dual CAR T cells were characterized by flow cytometry and through various in vitro assays including cytotoxicity, cytokine release, target-mediated expansion and mixed lymphocyte reactions (MLR). In vivo efficacy and expansion of Dual CAR T cells were evaluated in mice engrafted with MOLP-8 MM cells. Allogeneic BCMA/CD70 Dual CAR T cells produced from multiple donors exhibited high efficiency of TRAC KO (98-99%), sustained expression of the BCMA and CD70 CARs, and a balanced effector and memory phenotype. In MLR assays with previously-primed T cells, BCMA/CD70 Dual CAR T cells showed resistance to allorejection and expanded > 5-fold, whereas control cells not expressing the CD70 CAR were rapidly eliminated. In response to target cells, BCMA/CD70 Dual CAR T cells produced high levels of IFN gamma, TNF alpha, and IL-2, and exhibited sustained killing and expansion following repeated stimulation. In addition, BCMA/CD70 Dual CAR T cells displayed high efficacy and expansion in a xenograft model of multiple myeloma. Compared with single targeting BCMA CAR T cells, BCMA/CD70 Dual CAR T cells showed improved cytotoxic activity and expansion in an in vitro model of antigen-escape. Taken together, these results demonstrate that BCMA/CD70 Dual CAR T cells produced using site-specific integration show specific cytotoxic activity, rejection avoidance, and the ability to eliminate tumor cells that have downregulated BCMA. Our findings underscore the promise of allogeneic BCMA/CD70 Dual CAR T cells as an accessible, off-the-shelf treatment option for high-risk multiple myeloma patients and those who have progressed after BCMA-directed therapies, with the potential to extend beyond multiple myeloma toward broader applications in autoimmune disorders.