摘要目的 以靶向HER2的人源化单克隆抗体H1-2 scFv为基础构建第三代嵌合抗原受体(CAR),并探讨CAR修饰的T淋巴细胞(CAR-T)对HER2高表达肿瘤的杀伤作用.方法 运用分子克隆方法将CAR的表达框和H1-2 scFv装入慢病毒表达载体,利用慢病毒载体将H1-2 CAR基因转导至T淋巴细胞构建CAR-T细胞,通过酶联免疫吸附测定检测细胞因子白细胞介素2的表达,通过LDH释放实验检测CAR-T细胞杀伤肿瘤细胞的效率.最后用NOD/SCID小鼠移植瘤模型来检测CAR-T细胞在体内杀伤肿瘤细胞的能力.结果 成功构建了靶向HER2的第三代嵌合抗原受体H1-2 CAR表达载体,该载体转染的H1-2 CAR-T细胞能与HER2+的肿瘤细胞结合并提高了细胞因子白细胞介素2的释放.在本研究中H1-2 CAR-T细胞与HER2高表达的肿瘤细胞相互作用时白细胞介素2分泌量可达到1000μg/L以上,而外周血单个核细胞与肿瘤细胞相互作用时基本上不分泌白细胞介素2.在体外肿瘤细胞培养体系中,H1-2 CAR-T细胞对HER2高表达肿瘤细胞的杀伤率明显高于HER2低/不表达的细胞.当效靶比为20:1时,H1-2 CAR-T细胞对HER2高表达的乳腺癌细胞SK-BR-3的杀伤率可以达到(90.1±2.8)%,而H1-2 CAR-T对HER2阴性的乳腺癌细胞MDA-MB-231的杀伤率只有(13.5±4.7)%.在NOD/SCID小鼠体内,CAR-T细胞治疗组乳腺癌移植瘤的生长速度减慢,实验结束时H1-2 CAR-T细胞治疗组肿瘤质量平均为(0.7±0.1)g,未转染T细胞治疗组为(1.2±0.2)g,PBS组为(1.2±0.2)g.H1-2 CAR-T治疗组与未转染T细胞治疗组相比较有明显差异(P<0.05),而未转染T细胞治疗组与PBS治疗组相比差异无统计学意义(P>0.05).结论 靶向HER2的H1-2 CAR-T细胞能特异性杀伤HER2高表达的肿瘤细胞,为CAR-T靶向治疗HER2+的肿瘤提供了研究基础.

abstractsObjective To construct the third generation chimeric antigen receptor based on a novel humanized anti-HER2 H1-2 scFv, and to investigate the specific cytotoxicity of H1-2 CAR modified T lymphocytes ( CAR-T ) against HER2+ tumor cells. Method The expression cassette of the third generation CAR gene and anti-HER2 H1-2 scFv were constructed and cloned into lentivirus transfer plasmid, and then the third generation H1-2 CAR was transduced into human T lymphocytes using lentivirus. Enzyme linked immunosorbent assay was used to detect the expression of cytokines IL2,and LDH release assay was used to detect the cytotoxic effect of the H1-2 CAR-T. Finally, NOD/SCID mice and HER2+ breast cancer cell line SKBR3 were used to detect the anti-tumor effect of H1-2 CAR-T in vivo. Results The third generation H1-2 CAR was successfully constructed. H1-2 CAR-T secreted high dose of IL2 after confrontationwith HER2+ breast cancer cells. In vitro, the cytolytic rate of H1-2 CAR-T on high expression HER2+ tumor cells was significantly higher than that in low expression HER2 or non-expression HER2 tumor cells. At the efficacy to target ratio of 20, the cytolytic rate of H1-2 CAR-T against breast cancer cell SK-BR-3 could reach (90. 1±2. 8)%, while the cytolytic rate of H1-2 CAR-T against HER2- breast cancer cell MDA-MB-231 was only ( 13. 5 ± 4. 7)%. In the mouse xenograft tumor model, H1-2 CAR-T cells inhibited breast cancer growth in vivo. At the end of the experiments, the average tumor weight in the H1-2 CAR-T cell treatment group was (0. 7±0. 1) g, the non-transfected T cell therapeutic group was (1. 2±0. 2) g, and the PBS group was (1. 2±0. 2) g. There was significant difference between the H1-2 CAR-T therapeutic group and the non-transfected T cell therapeutic group (P<0. 05). However, there was no significant difference between the non-transfected T cell therapeutic group and the PBS treatment group ( P>0. 05) . Conclusion The HER2-sepcific H1-2 CAR-T cells specifically kill HER2 positive cells, and further studies on CAR-T cells for the treatment of HER2+cancers are useful.