[摘 要] 目的：构建靶向CD38和CD138分子抗原的双靶点嵌合抗原受体基因修饰T淋巴细胞（CD38/CD138 CAR-T细胞）， 探讨其对多发性骨髓瘤（MM）细胞的体外杀伤作用。方法：利用CAR-T细胞技术，基于MM细胞高表达CD38和CD138抗原，分 别构建靶向CD38、CD138的CD38 CAR-T与CD138 CAR-T细胞，以及同时靶向CD38与CD138的CD38/CD138 CAR-T细胞，实 验分为未处理T、CD38 CAR-T、CD138 CAR-T和CD38/CD138 CAR-T细胞组。采用流式细胞术检测CAR-T细胞的表型，利用 LDH释放法检测各种CAR-T细胞对MM细胞RPMI8226和U266的体外杀伤作用。结果：成功构建CD38 CAR-T、CD138 CAR-T 和CD38/CD138 CAR-T细胞。CD38/CD138 CAR-T细胞倾向于向记忆表型分化，表达较高水平的增殖分子（CD25）、激活分子 （CD27）和较低水平的耗竭分子（PD-1、CTLA-4、TIM-3）（均P < 0.001），而且CD38/CD138 CAR-T细胞不易于耗竭和衰老，且表达 较低水平的 r-H2AX、p-p53、p21 和 p16 蛋白（均 P < 0.01）。在不同效靶比条件下，CD38/CD138 CAR-T 细胞较 CD38 CAR-T、 CD138 CAR-T细胞对RPMI8226和U266细胞具有更强的杀伤作用（均 P < 0.001）。结论：靶向CD38和CD138治疗MM的 CD38/CD138 CAR-T 细胞在体外具有较优表型及较强的抗肿瘤功能。

[Abstract] Objective: To construct dual-targeting (CD38 and CD138) chimeric antigen receptor (CAR) gene-modified T cells (CD38/ CD138 CAR-T cells) and explore their in vitro cytotoxicity against multiple myeloma (MM) cells. Methods: Based on the high expression of CD38 and CD138 antigens in MM cells, CD38 CAR-T cells and CD138 CAR-T cells targeting CD38 and CD138 respectively, and CD38/CD138 dual-targeted CAR-T cells targeting both CD38 and CD138 were constructed using CAR-T cell technology. The experimental groups included untreated T cells, CD38 CAR-T, CD138 CAR-T, and CD38/CD138 CAR-T cells. The phenotype of CAR-T cells was detected by flow cytometry. The cytotoxicity of various CAR-T cells against MM cells (RPMI8226 and U266) was assessed using the LDH release assay. Results: Three types of CAR-T cells, CD38 CAR-T, CD138 CAR-T, and CD38/ CD138 CAR-T cells, were successfully constructed. The CD38/CD138 CAR-T cells tended to differentiate into a memory phenotype, expressing higher levels of proliferation marker (CD25), activation marker (CD27), and lower levels of exhaustion markers (PD-1, CTLA-4, TIM-3) (all P < 0.001). Moreover, CD38/CD138 CAR-T cells were less prone to exhaustion and senescence, and expressed lower levels of r-H2AX, p-p53, p21, and p16 proteins (all P < 0.01). Under different effector-to-target cell ratios, CD38/CD138 CAR-T cells exhibited stronger cytotoxic effects against RPMI8226 and U266 cells compared to CD38 CAR-T and CD138 CAR-T cells (all P < 0.001). Conclusion: CD38/CD138 CAR-T cells targeting both CD38 and CD138 demonstrate an optimal phenotype and enhanced anti-tumor activity in vitro, offering promising potential for immunotherapy in multiple myeloma.

山东省自然科学基金（No. ZR2023QC179）