RESUMEN
@#[摘 要] 目的：制备双特异性CAR-T（bsCAR-T）细胞，观察其对表达表皮生长因子Ⅲ型突变阳性（EGFRvⅢ+，简称vⅢ+）和CD133+胶质瘤干细胞的靶向杀伤作用。方法：基于前期研制的vⅢ/CD133双特异性微抗体和二代CAR构建的双特异性CAR（bsCAR），制备慢病毒载体转染人外周血T细胞，FCM和WB法检测bsCAR转染效率和表达水平。bsCAR-T细胞和vⅢ+/CD133+ U87胶质瘤干细胞共培养，乳酸脱氢酶（LDH）释放实验、IFN-γ分泌实验检测其特异性杀伤作用和对IFN-γ分泌的促进作用。制备裸鼠vⅢ+/CD133+ U87干细胞移植瘤模型检测bsCAR-T细胞对移植瘤生长的抑制作用。结果：vⅢscFv和CD133scFv通过重叠PCR无缝连接入二代CAR表达框（S-vⅢscFv/CD133scFv-Hinge-TM-CD137-CD3z）中，然后克隆入pCDH-MSCV-MCS-EF1-copGFP载体的EcoRⅠ和BamHⅠ位点（pbsCAR）。3种质粒（pVSV-G、pCMV-dR8.9和pbsCAR）共转染HEK293T细胞制备慢病毒载体，转染外周血T细胞，FCM检测bsCAR表达率为71.1%，WB法结果显示bsCAR表达正确。bsCAR-T细胞和vⅢ+/CD133+ U87干细胞共培养检测结果显示，bsCAR-T细胞对胶质瘤干细胞具有特异性杀伤作用，与效靶比呈正比；IFN-γ分泌量为（2 350.6±92） pg·mL-1，明显高于对照组（P<0.01）。裸鼠移植瘤动物模型显示，bsCAR-T细胞在体内具有明显的移植瘤抑制作用（P<0.01）。结论： bsCAR-T细胞能够特异性靶向杀伤vⅢ+/CD133+胶质瘤干细胞，实验结果为促进实体瘤的细胞免疫治疗提供了实验依据。
RESUMEN
@#[Abstract] 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 detected by 51Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xenograft 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 5×106 TU/ml. Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51Cr 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 secretion was (1 836±148.2) pg/ml, which was significantly different from that of NTT and GFP+ T cells (P<0.01). The specific killing activity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tumor 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.