双特异性CAR-T细胞对EGFRvⅢ^(+)/CD133^(+)胶质瘤干细胞的靶向杀伤  

Targeted killing effect of bispecific CAR-T cells on EGFRvⅢ^(+)/CD133^(+)glioma stem cells

作　　者：刘亚丹 谢甲贝 朱琼琼 卢文杰 丁辉 韩双印 LIU Yadan;XIE Jiabei;ZHU Qiongqiong;LU Wenjie;DING Hui;HAN Shuangyin(Department of Gastroenterology,The Second People's Hospital of Henan Province,Zhengzhou 451191,Henan,China;Department of Gastroenterology,People's Hospital of Zhengzhou University,Zhengzhou 450066,Henan,China)

机构地区：[1]河南省第二人民医院消化内科,河南郑州451191 [2]郑州大学人民医院消化内科,河南郑州450003

出　　处：《中国肿瘤生物治疗杂志》2023年第4期296-301,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金(No.81772670)。

摘　　要：目的:制备双特异性CAR-T(bs CAR-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(bs CAR),制备慢病毒载体转染人外周血T细胞,FCM和WB法检测bs CAR转染效率和表达水平。bs CAR-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bs CAR-T细胞对移植瘤生长的抑制作用。结果:vⅢsc Fv和CD133sc Fv通过重叠PCR无缝连接入二代CAR表达框(S-vⅢscFv/CD133scFv-Hinge-TM-CD137-CD3ζ)中,然后克隆入p CDH-MSCV-MCS-EF1-copGFP载体的Eco RⅠ和Bam HⅠ位点(pbs CAR)。3种质粒(p VSV-G、p CMV-d R8.9和pbs CAR)共转染HEK293T细胞制备慢病毒载体,转染外周血T细胞,FCM检测bs CAR表达率为71.1%,WB法结果显示bs CAR表达正确。bs CAR-T细胞和vⅢ^(+)/CD133^(+)U87干细胞共培养检测结果显示,bs CAR-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(2350.6±92)pg·m L^(-1),明显高于对照组(P<0.01)。裸鼠移植瘤动物模型显示,bs CAR-T细胞在体内具有明显的移植瘤抑制作用(P<0.01)。结论:bs CAR-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 variantⅢ(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)andWestern 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-γsecretion.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-CD3ζ).The above bsCAR construct was then cloned into EcoRⅠand BamHⅠsites of pCDH-CMV-MCSEF1-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-γsecretion was(2350.6±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.

关 键 词：表皮生长因子Ⅲ型突变体 CD133 双特异性CAR vⅢ^(+)/CD133^(+)U87胶质瘤干细胞 肿瘤干细胞 

分 类 号：R392[医药卫生—免疫学] R730.51[医药卫生—基础医学]