[摘 要] 目的：构建了一种靶向成纤维细胞活化蛋白（FAP）且能分泌CTLA-4单链抗体（scFv）的CAR-T细胞，并探讨其联合血管靶向药物阿帕替尼对胶质母细胞瘤（GBM）的抗肿瘤效果及机制。方法：构建FAP-CTLA-4 CAR过表达载体，转导至T细胞中制备FAP-CTLA-4 CAR-T细胞。流式细胞术检测FAP-CTLA-4 CAR-T细胞表面分子CD69表达，ELISPOT检测其分泌IFN-γ 的能力，细胞增殖实验分析FAP-CTLA-4 CAR-T细胞的增殖情况，杀伤实验分析其联合阿帕替尼处理对人脑星形胶质母细胞瘤U-87 MG细胞治疗效果的影响。建立小鼠U-87 MG细胞异种移植瘤模型。分别用FAP-CTLA-4 CAR-T细胞单独治疗或联合阿帕替尼治疗荷瘤小鼠，命名为FAP-CTLA-4 CAR-T组、阿帕替尼组和FAP-CTLA-4 CAR-T + 阿帕替尼组，观察各组小鼠肿瘤生长情况，免疫组化、TUNEL染色与免疫荧光法检测各组肿瘤组织中Ki-67、CD31表达及细胞凋亡情况，流式细胞术分析FAP- CTLA-4 CAR-T细胞在荷瘤小鼠体内的存活状况，H-E染色和血清生化分析评估联合治疗的安全性。结果：与FAP-CTLA-4 CAR-T组、阿帕替尼组相比，FAP-CTLA-4 CAR-T + 阿帕替尼组中CAR-T细胞的活化水平显著提高，细胞增殖能力明显增强， IFN-γ分泌细胞比例及对靶细胞的杀伤效率均提高（P < 0.01或P < 0.05）。在荷瘤小鼠模型中，联合治疗显著抑制肿瘤生长并延长小鼠生存时间（P < 0.01或P < 0.05），同时抑制肿瘤细胞增殖、降低微血管密度、促进肿瘤细胞凋亡，并延长CAR-T细胞在肿瘤组织中的存活时间（P < 0.01或P < 0.05），且未对主要组织器官造成毒性损伤。结论：FAP-CTLA-4 CAR-T细胞与阿帕替尼联合应用在GBM治疗中显示出协同增效作用与良好的安全性。

[Abstract] Objective: To evaluate the anti-tumor efficacy of FAP-CTLA-4 CAR-T cells targeting fibroblast activation protein (FAP) and secreting anti-CTLA-4 single-chain antibody (scFv) combined with apatinib in the treatment of glioblastoma (GBM), and to explore the underlying mechanisms. Methods: A FAP-CTLA-4 CAR overexpression vector was constructed and transduced into T cells to generate FAP-CTLA-4 CAR-T cells. In vitro functions of CAR-T cells were evaluated by flow cytometry (CD69 expression) and ELISPOT assay (IFN-γ secretion). Proliferation assays were used to assess the proliferation capacity of FAP-CTLA-4 CAR-T cells, and cytotoxicity assays were performed to evaluate the anti-tumor effect of the combination treatment with apatinib on U-87 MG cells. A mouse U-87 MG xenograft tumor model was established, and tumor-bearing mice were assigned to the monotherapy group (FAP- CTLA-4 CAR-T or apatinib alone) and the combination therapy group (FAP-CTLA-4 CAR-T plus apatinib). Tumor growth was monitored; tumor tissue Ki-67/CD31 expression was detected by immunohistochemistry and immunofluorescence; apoptosis was assessed by TUNEL staining. In vivo CAR-T persistence was analyzed by flow cytometry; treatment safety was assessed via H-E staining and serum biochemistry. Results: Compared with the monotherapy group, the combination therapy group showed significantly · · 661 [[PAGE_INDEX=12 FILE=7f0b8458-32b0-497d-b515-1deca828e6eb.pdf]] 中国肿瘤生物治疗杂志, 2026, 33(6) increased CAR-T cell activation, enhanced proliferation capacity, a higher proportion of IFN-γ-secreting cells and superior cytotoxicity against target cells (P < 0.05 or P < 0.01).In the xenograft mouse model, the combination therapy markedly suppressed tumor growth and prolonged survival (P < 0.05 or P < 0.01), inhibited tumor cell proliferation and microvessel density, promoted tumor cell apoptosis, and extended the survival of CAR-T cells within tumor tissues (P < 0.05 or P < 0.01), without causing toxic damage to major organs. Conclusion: The combination of FAP-CTLA-4 CAR-T cells and apatinib exhibits synergistic anti-tumor effects in GBM, enhancing therapeutic efficacy while maintaining safety, and may represent a promising novel strategy for the treatment of this disease.

[基金项目] 贵州省科技厅基金（ZK2024-264）；贵州省科技厅基金（ZK2022-602）；贵州省卫生健康委员会（gzwkj2024-273）；遵义医科大学博士 （F-ZH-09）；遵义医科大学创新团队（ZHTD2024-1）；贵州省大学生创新创业（S202210661013）；遵义医科大学珠海校区大学生创新创业（ZHCX2023034；ZHCX2023070；X202410661021；X202410661022）