Objective To construct chimeric antigen receptor T (CAR-T) cells targeting multiple myeloma-specific antigen-1 (MMSA-1) and evaluate their antitumor activity and preliminary safety in multiple myeloma (MM).

Methods Flow cytometry was used to detect MMSA-1/ZDHHC9 expression in K562 cells, MM cell lines (MM.1S, U266, and RPMI-8226), and primary MM cells. MMSA-1-specific single-chain variable fragments were screened and used to construct second-generation CAR-T cells. CAR expression, cell expansion, cytotoxic activity against RPMI-8226 cells, and effector molecule release after target-cell stimulation were evaluated in vitro. The in vivo efficacy of CAR-T cells was assessed in an RPMI-8226 xenograft mouse model. Preliminary safety was evaluated using public database analysis, hematological and biochemical parameters, and H&E staining.

Results MMSA-1/ZDHHC9 expression was detectable in MM.1S, U266, RPMI-8226, and primary MM cells, whereas K562 cells showed only low-level background signals. The CAR-positive rate of MMSA-1 CAR-T cells was approximately 52.2%, and MMSA-1 CAR-T cells showed favorable expansion in vitro. At effector-to-target ratios of 5:1, 3:1, and 1:1, MMSA-1 CAR-T cells significantly reduced the proportions of residual RPMI-8226 cells compared with conventional T cells group. In vivo experiments showed that MMSA-1 CAR-T cells reduced tumor burden and prolonged survival in mice, preliminary safety observations revealed no obvious sustained body weight loss, hematological abnormalities, liver or kidney dysfunction, or pathological changes in major organs.

Conclusion MMSA-1 CAR-T cells exhibit preliminary anti-MM activity, suggesting that MMSA-1 may serve as a candidate complementary target for CAR-T therapy in MM. However, the applicable patient population and tissue safety still require further validation.

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