靶向B细胞成熟抗原的嵌合抗原受体T细胞的构建及其对肿瘤细胞的杀伤

Construction of anti-BCMA chimeric antigen receptor(CAR-BCMA) modified T cells and its cytotoxicity against tumor cells

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。

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 of43%. 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.