[摘要] 目的：探索通过嵌合抗原受体（chimeric antigen receptor，CAR）-T 细胞靶向B 细胞成熟抗原（B cell maturation antigen，BCMA）以治疗多发性骨髓瘤（multiple myeloma，MM）的方法。方法：构建基于鼠源BCMA scFv 的CAR-BCMA分子,包装为慢病毒载体并感染健康人T 细胞构建CAR-BCMA-T 细胞；构建BCMA阳性细胞系A549-BCMA、A549-BCMAOFP和K562-BCMA作为靶细胞。将CAR-BCMA-T细胞与构建的靶细胞和人骨髓瘤细胞U266 共孵育，CCK-8 法和流式细胞术检测其对BCMA阳性肿瘤细胞的杀伤能力。构建MM患者来源CAR-BCMA-T细胞并检测其杀伤靶细胞A549-BCMA的能力，并采用ELISA和流式细胞术检测CAR-BCMA-T细胞IFN-γ 的释放水平。结果：健康人来源的CAR-BCMA-T经过11 d 培养扩增300 倍，阳性率达到43%；成功构建BCMA阳性靶细胞。在5:1 效靶比下，CAR-BCMA-T对A549-BCMA、K562-BCMA和U266 细胞的杀伤率分别在80%、60%和80%左右，显著高于对BCMA阴性细胞的杀伤率，且杀伤力与靶细胞的BCMA表达强度相关。在效靶比20:1 时，MM患者来源CAR-BCMA-T细胞对靶细胞A549-BCMA的杀伤率达到95%以上，并且大量分泌IFN-γ。结论：本研究成功构建了健康人及MM患者来源的靶向BCMA的CAR-T细胞，其能够有效特异杀伤BCMA阳性的肿瘤细胞。

[Abstract] Objective：To explore a novel chimeric antigen receptor (CAR)-T cell treatment to treat Multiple Myeloma (MM) via target B cell maturation antigen (BCMA). Methods：A CAR-BCMA molecular was constructed based on mouse originated BCMA scFv, and was packaged into lentiviral vector and transfected into T cells from healthy donors to construct CAR-BCMA-T cells. The BCMA positive cell lines A549-BCMA, A549-BCMAOFP and K562-BCMA were constructed as target cells. Then, the CAR-BCMA-T cells were co-incubated with the constructed target cells and human myeloma U266 cells, and the cytotoxic effects of CAR-BCMA-T cells were evaluated via CCK-8 and FACS. Finally, the CAR-BCMA-T cells originated from MM patients were constructed, and its cytotoxicity against A549-BCMA were examined; in addition, the IFN-γ release level in CAR-BCMA-T cells was evaluated by ELISA and FACS.Results: After 11 days’incubation, the CAR-BCMA-T cells originated from healthy donors amplified 300 times with a positive rate of 43%. The BCMA positive target cell lines were constructed successfully. Under an effector : target ratio of 5:1, the killing rates of CARBCMA-T cells against A549-BCMA, K562-BCMA and U266 were about 80%, 60%, and 80%, respectively, which were significantly higher than those against BCMA negative cells; and the cytotoxicity was related to the BCMA expression level in target cells. What’s more, at the effector : target ratio of 20:1, the CAR-BCMA-T cells originated from MM patients were demonstrated to exhibit a killing rate of more than 95% against A549-BCMA positive cells, and produced large amount of IFN-γ. Conclusion: CAR-BCMA-T cells originated from both healthy and MM donors were successfully constructed, and they can effectively and specifically kill BCMA positive tumor cells.

江苏省社会发展-临床前沿技术项目（No. BE2016809），南京市科技发展计划项目（No. 201503011），江苏省中医院院级课题（No.Y17014）