Although BCMA-targeted CAR T-cell therapy has achieved notable success in treating multiple myeloma (MM), disease recurrence remains common. This is largely driven by T-cell dysfunction and the selection of BCMA-negative tumor variants. Identifying new antigens is essential to counteract antigen escape. We analyzed B7-H3 (CD276) surface expression on primary MM patient specimens. Nanobody-derived CAR T cells (nanoCARs) targeting B7-H3 were constructed and assessed for their ability to kill target cells and secrete cytokines in vitro, including against autologous patient myeloma cells. Therapeutic potential was further examined in two independent MM xenograft mouse models. In addition, dual-specificity BCMA/B7-H3 CAR T cells and a CARpooling approach (combined infusion of separate BCMA- and B7-H3-specific CAR T cells) were evaluated in models simulating antigen heterogeneity and escape. B7-H3 was found on malignant plasma cells in 60% of MM cases examined. The B7-H3 nanoCAR T cells demonstrated robust, antigen-specific killing and effector cytokine production against both MM cell lines and primary tumor cells. In vivo, treatment led to substantial tumor regression and extended overall survival. Dual BCMA/B7-H3 CAR T cells as well as the CARpooling strategy effectively cleared tumor populations displaying mutually exclusive expression of either BCMA or B7-H3. Collectively, these findings highlight the potential of combining BCMA and B7-H3 targeting to overcome antigen loss. B7-H3 emerges as an attractive candidate for immunotherapy in MM. Both single B7-H3-specific and dual-targeted nanobody-based CAR T cells offer a promising route to reduce antigen escape and achieve more durable remissions.
