目的： 制备靶向EGFRvⅢ的第三代CAR-T(EGFRvⅢ/3CAR-T)，检测其在体外和体内对EGFRvⅢ + U87胶质瘤细胞的 特异性杀伤效应。 方法： 用磷酸钙沉淀法将三质粒共转染HEK293T细胞包装EGFRvⅢ/3CAR慢病毒（LV-EGFRvⅢ/3CAR），感 染健康人CD3 + T细胞，Western blotting和流式细胞术检测T细胞中EGFRvⅢ/3CAR的表达水平， 51 Cr释放法检测EGFRvⅢ/3CAR- T对EGFRvⅢ + U87细胞的体外杀伤效应，ELISA法检测EGFRvⅢ/3CAR + T的IFN-γ分泌水平。构建裸鼠异种胶质瘤移植模型， 检测EGFRvⅢ/3CAR-T对移植瘤的体内杀伤活性。 结果： EGFRvⅢ/3CAR慢病毒包装成功，滴度值为5×10 6 TU/ml。Western blotting在相对分子质量58 000处检测到EGFRvⅢ/3CAR表达，未转导组无相同分子量蛋白表达。流式细胞术检测结果显示EG- FRvⅢ/3CAR转导效率平均为52.3%， 51 Cr释放法检测EGFRvⅢ/3CAR-T特异性杀伤作用与E∶T值（E∶T为4∶1、为8∶1、16∶1、32∶ 1）呈正比关系。ELISA法检测到细胞因子IFN-γ分泌量为(1 836±148.2) pg/ml，与NT T和GFP + T细胞相比差异有统计学意义 （ P ＜0.01）。EGFRvⅢ/3CAR + T细胞的特异性杀伤活性及IFN-γ分泌均依赖于EGFRvⅢ在U87细胞中的表达水平。体内肿瘤生 长检测结果显示，注射后3周EGFRvⅢ/3CAR + T组肿瘤体积较GFP + T细胞和PBS组差异有统计学意义（ P< 0.01）。 结论： EGFRv Ⅲ/3CAR-T在体外和体内均能发挥靶向杀伤EGFRvⅢ + 脑胶质瘤细胞的作用，为后续临床试验提供依据。

Objective:To prepare the third generation CAR-T cells targeting EGFRvⅢ (EGFRvⅢCAR-T) and to detect its specific killing effect against EGFRvⅢ + U87 cells in vitro and in vivo. Methods: Human CD3 + T cells were transfected with lentiviral EGFRv Ⅲ/3CAR, which was generated by calcium phosphate co-precipitation of three plasmids. The expression of EGFRvⅢ/3CAR in T cells was detected by Western blotting and flow cytometry. In vitro killing effect of EGFRvⅢ/3CAR-T cells on EGFRvⅢ + U87 cells was de- tected by 51 Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xeno- graft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3CAR-T cells on xenograft tumor. Results: The EGFRvⅢ/ 3CAR lentivirus was successfully packaged with an average titer of 5×10 6 TU/ml. Western blotting showed that a protein band of ap- proximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indi- cated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51 Cr release assay showed that the specific killing effect of EGFRvⅢ/3CAR-T cells was positively correlated with E/T ratio (E∶T=4∶1, 8∶1, 16∶1, 32∶1). ELISA showed that cytokine IFN-γ se- cretion was (1 836±148.2) pg/ml, which was significantly different from that of NT T and GFP + T cells (P<0.01). The specific killing ac- tivity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tu-mor growth monitoring results showed that the tumor volume of EGFRvⅢ/3CAR-T cell group was significantly different from that of GFP + T cell group and PBS group around 3 weeks after injection (P<0.01). Conclusion: EGFRvⅢ/3CAR-T cells demonstrated specific antitumor effectagainstEGFRvⅢ + U87cellsbothinvitro and in vivo, providing basis for immunotherapyofgliomainfuture clinical use.

国家自然科学基金资助项目（No.81372405, No.81772670）