[摘 要] 目的：探讨基于新型单载体SynNotch系统构建的局部分泌IL-2的CAR-T细胞（Syn.CD19.IL-2 CAR-T细胞）对传统 CD19 CAR-T细胞功能的影响。方法：基于本课题组前期构建的单载体SynNotch系统，将CD19特异性FMC63抗体与IL-2表达 模块整合，通过自失活逆转录病毒载体转导T细胞制备Syn.CD19.IL-2 CAR-T细胞。诱导验证实验分为Syn.CD19.IL-2 CAR-T 细胞组和未转导T细胞组，通过ELISA检测抗原刺激后IL-2分泌水平。采用CFSE染色法检测在Syn.CD19.IL-2 CAR-T细胞存 在时，CD19 CAR-T细胞与肿瘤Raji-Luc或SW620-CD19-Luc细胞共培养时，IL-2的分泌对CD19 CAR-T细胞增殖的影响。流式 细胞术检测CD69表达，观察在Syn.CD19.IL-2 CAR-T细胞分泌IL-2的情况下，CD19 CAR-T细胞与Raji-Luc细胞共培养时的激 活情况。结果：成功构建自失活逆转录病毒载体Syn.CD19.IL-2 CAR，制备出Syn.CD19.IL-2 CAR-T细胞，病毒滴度 > 1×107 拷贝/mL，转导效率达37.1%。抗原刺激后，SynNotch CAR-T细胞IL-2分泌量显著高于未转导T细胞（P < 0.001）。在Syn.CD19. IL-2 CAR-T细胞分泌IL-2时，CD19 CAR-T细胞具有更强的增殖能力和更高的活化水平（均P < 0.001）。结论：成功构建的Syn. CD19.IL-2 CAR-T细胞能显著增强CD19 CAR-T细胞的增殖和活化能力。

[Abstract] Objective: To investigate the impact of locally IL-2-secreting CAR-T cells (Syn.CD19.IL-2 CAR-T cells) constructed using a novel single-vector SynNotch system on the function of conventional CD19 CAR-T cells. Methods: Building upon our previously established single-vector SynNotch system, the CD19-specific FMC63 antibody was integrated with an IL-2 expression module to prepare Syn.CD19.IL-2 CAR-T cells through T cell transduction using a self-inactivating retroviral vector. For validation experiments, cells were divided into the Syn.CD19.IL-2 CAR-T cell group and the untransduced T cells group. ELISA was used to detect IL-2 secretion levels after antigen stimulation. The CFSE staining assay was implemented to evaluate the effects of IL-2 secretion on the proliferation of CD19 CAR-T cells when CD19 CAR-T cells were co-cultured with Raji-Luc or SW620-CD19-Luc cells in the presence of Syn. CD19. IL-2 CAR-T cells. Flow cytometry was employed to detect CD69 expression and monitor the activation status of CD19 CAR-T cells when co-cultured with Raji-Luc cells in the condition of Syn.CD19.IL-2 CAR-T cells secreting IL-2. Results: The self-inactivating retroviral vector Syn.CD19.IL-2 CAR were successfully constructed and Syn.CD19.IL-2 CAR-T cells with a viral titer >1 × 10? copies/mL and transduction efficiency of 37.1% were generated. After antigen stimulation, SynNotch CAR-T cells demonstrated significantly elevated IL-2 secretion compared with the untransduced T cell group (P < 0.001). When Syn. CD19.IL-2 CAR-T cells secreted IL-2, the CD19 CAR-T cells exhibited enhanced proliferative capacity and elevated activation level (all P < 0.001). Conclusion: Successfully constructed Syn.CD19.IL-2 CAR-T cells can significantly enhance the proliferative capacity and activation ability of conventional CD19 CAR-T cells.

王建勋高层次人才科研启动经费（No. 90011451310032）