靶向HER2的CAR-T细胞构建与抗肿瘤活性的体外分析

Construction of HER2-specific CAR-T cells and in vitro analysis of their activity to suppress tumor cell growth

摘要:

CAR-T细胞疗法通过靶向识别肿瘤细胞表面抗原，特异性杀伤肿瘤细胞，近年来已经成为肿瘤免疫疗法的研究热点。通过基因工程方法构建靶向人类表皮生长因子受体2 (HER2) 的CAR慢病毒表达质粒，以磷酸钙沉淀辅助多质粒共转染HEK293T细胞包装，制备CAR慢病毒颗粒lenti-car，感染人外周血单核细胞获得HER2靶向的CAR-T细胞，并分析其对HER2阳性和阴性肿瘤细胞的特异性抑制效果。研究结果表明，构建的CAR-T细胞可被HER-2阳性的肿瘤细胞特异性激活，分泌大量炎症性细胞因子IFN-γ和IL-2。在同样效靶比等处理条件下，构建的HER2靶向CAR-T细胞对HER2阳性的人卵巢癌细胞株SK-OV-3的生长抑制率为 (58.47±1.72)%，显著高于对照组 (P<0.05)；而对HER2阴性的人慢性髓原白血病细胞株K562的生长抑制率为 (11.74±2.37)%，与对照组无显著差异 (P>0.05)。进一步，在K562细胞中转染人HER2表达载体使其成为HER2阳性，则HER2靶向CAR-T细胞对其的生长抑制率上升为 (30.41±7.59)%，较HER2阴性K562具有明显差异 (P<0.05)。研究结果表明，构建的HER2靶向的第二代CAR-T细胞可选择性地抑制高表达HER2蛋白的肿瘤细胞的生长，暗示了其对HER2阳性肿瘤进行细胞免疫治疗的临床应用前景。

Abstract:

CAR-T cell therapy that targets surface antigens to kill tumor cells specifically has recently become another cornerstone in tumor immunotherapy. In this study, a lentiviral expression plasmid of CAR targeting human epidermal growth factor receptor 2 (HER2) was constructed by genetic engineering. The recombinant plasmid was co-transfected with other packaging plasmids into HEK293T cells by calcium phosphate precipitation to generate lenti-car, which are CAR lentiviral particles. HER2-specific CAR-T cells were obtained by transducing human peripheral blood mononuclear cells with lenti-car. Their specific inhibitory effects on HER2-positive and HER2-negative tumor cells were analyzed in vitro. The constructed CAR-T cells were specifically activated by HER2-expressing tumor cells as indicated by secretion of IFN-γ and IL-2. The inhibitory rate on HER2-positive SK-OV-3 cell line was (58.47±1.72)%, significantly higher than that on the mock-treated control group (P<0.05). The inhibitory rate on HER2-negative K562 cell lines was (11.74±2.37)%, which was not significantly different from that on the control group (P>0.05). Furthermore, when we transfected a HER2-expressing vector into K562, the inhibitory rate increased to (30.41±7.59)%, which was higher than that on HER2-negative K562 (P<0.05). Thus, the constructed second-generation HER2-specific CAR-T cells specifically suppressed growth of tumor cells overexpressing HER2 protein, suggesting that HER2-specific CAR-T cells might prove useful for immunotherapy of HER2-positive cancer.