Abstract 860: BCMA CAR-T cells releasing mutant CST6 N137D proteins further improve therapeutic effects in multiple myeloma

Abstract Introduction: Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of abnormal monoclonal plasma cells and osteolytic bone disease. The latter is a key feature of MM, which is associated with worse clinical outcomes. We have found that high cystatin E/M (CST6) expression is linked to the absence of bone lytic lesions in human MM patients and CST6 suppresses the formation of osteolytic lesions by blocking osteoclast differentiation and function in MM. We have also developed the B cell maturation antigen (BCMA)- chimeric antigen receptor T cell secreting wild-type CST6 proteins (BCMA/CST6WT), which specifically kill MM cells and suppress osteolytic lesions in MM mouse models. However, we also found that CST6WT induced immunosuppression in MM, which is an unwanted effect of MM therapy. The objective of this study is to develop a BCMA-CAR-T engineering a mutant CST6, which still targets both MM cells and osteoclasts but without immunosuppressive function. Materials and Methods: The different functional mutants of CST6 proteins were purified to test their effects on both osteoclastogenesis and immune cells. The proliferation assays and osteoclast formation assays were conducted to test the mutant CST6 function on immunosuppression and osteoclast formation. the BCMA/CST6 N137D CAR vector contained BCMA-scFv and the mutant CST6 N137D was constructed. A P2Aself-cleaving peptide was inserted between CAR and CST6 N137D. The CBA assays were employed to test the concentration of the cytokines. The ELISA assays were conducted to test the concentration of CST6 protein. Results: We have generated 6 different CST6 mutants to determine the key domain(s) for the immunosuppressive effects of CST6. By determining CD8+ cell proliferation and performing osteoclast formation assays, we have identified that the non-glycosylated CST6 N137D proteins lack an immunosuppressive function but maintain the suppressive function of osteoclastogenesis. We further developed the BCMA/CST6 N137D CAR-T cells releasing a mutant CST6 and found that the BCMA/CST6 N137D CAR-T cells significantly increased the expression of both IFN-γ and TNF- α cytokines compared with the BCMA-CAR-T cells and BCMA/CST6 WT -CAR-T cells. The BCMA/CST6N137DCAR-T cells also had a stronger antitumor activity compared with the BCMA-CAR-T cells and BCMA/CST6 WT -CAR-T cells. Conclusion: In this study, we have identified a CST6N137D mutant, which could suppress osteoclast differentiation without immunosuppressive effects. Furthermore, we have also developed that the BCMA/CST6N137D CAR-T cells may have a better therapeutic effect by targeting both tumor cells and osteoclasts but without suppression of cytotoxic T cell proliferation in MM. Citation Format: Yamin Meng, Zijun Zhang, Yan Cheng, Hongwei Xu, Frits van Rhee, Guido Tricot, John D. Jr Shaughnessy, Fenghuang Zhan. BCMA CAR-T cells releasing mutant CST6 N137D proteins further improve therapeutic effects in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 860.