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。

Objective: To prepare bsCAR-T cells and observe its targeted killing effect on epidermal growth factor variant Ⅲ(EGFRvⅢ+,simplified as vⅢ+) and CD133+ glioma stem cells. Methods: Based on the previously generated vⅢ/CD133 minibody and second-generation CAR, bispecific CAR (bsCAR) was constructed. Lentiviral bsCAR were prepared for the transfection of human peripheral blood T cells. Flow cytometry (FCM) and Western blot test were used to detect the transfection efficiency and expression of bsCAR. After bsCAR-T cells were co-cultured with vⅢ+/CD133+ U87 glioma stem cells, their killing effects were detected by LDH release test and cytokine IFN-γ secretion. vⅢ+/CD133+ U87 stem cell transplantation tumor model of nude mouse was established to test the inhibition of bsCAR-T cells on transplanted tumors. Results: vⅢscFv and CD133scFv were joined seamlessly by over-lap PCR with CAR expression cassette (S-vⅢ/CD133scFv-Hinge-TM-CD137-CD3z). The above bsCAR construct was then cloned into EcoRⅠand BamHⅠsites of pCDH-CMV-MCS-EF1-copGFP (pbsCAR). Lentiviral bsCAR were prepared by co-transfection of three plasmid (pVSV-G, pCMV-dR8.9 and pbsCAR) into HEK293T cells and later transfected human peripheral blood T cells. The expression of bsCAR detected by flow cytometry was 71.1%.Western blot analysis showed correct expression of bsCAR. The co-culture assay of bsCAR-T cells and vⅢ+/CD133+ U87 stem cells showed that bsCAR-T cells had specific killing effect on glioma stem cells, which was proportional to the effector-target ratio. IFN-γ secretion was (2 350.6±92) pg?mL-1, which was significantly higher than that of the control group (P<0.01). Nude mice transplantation tumor model demonstrated the transplantation tumor inhibition effect of bsCAR-T cells in vivo (P<0.01). Conclusion: bsCAR-T cells can kill specifically vⅢ+/CD133+ glioma stem cells, which provides experimental basis for cell immunotherapy of solid tumors.

国家自然科学基金（No. 81772670）