肿瘤特异性嵌合抗原受体EGFRvⅢ-CAR的构建及体外活性分析

嵌合抗原受体T细胞免疫疗法（chimeric antigen receptor-T, CAR-T），是通过体外激活和扩增肿瘤特异或非特异性杀伤细胞达到抗肿瘤效果，在肿瘤免疫治疗方面具有良好的应用前景。本研究构建靶向EGFRⅢ(epidermal growth factor receptor variant III)的嵌合抗原受体（CAR）的重组慢病毒表达载体，利用慢病毒感染并筛选能够稳定表达该嵌合抗原受体的Jurkat细胞系。通过EGFRvⅢ分子刺激、与U87MG细胞共培养的方式检测细胞系的活化状况。结果显示，成功构建了pCDH-EGFRvⅢscFv-CAR-copGFP-T2A-puro慢病毒表达重组质粒，并筛选出可稳定表达EGFRⅢ-CAR的Jurkat 细胞系。CCK-8 法检测显示，EGFRvⅢ分子刺激12 h的Jurkat-CAR细胞增殖率约是对照组的1.36倍（P<0.05）；ELISA法检测显示，与U87MG细胞共孵育后，细胞上清中IL-2的浓度约是单独培养分泌在上清中IL-2的1.625倍（P < 0.01）。以上结果表明，稳定表达CAR的jurkat细胞，可以靶向性识别EGFRvⅢ分子及EGFRvⅢ阳性的靶细胞，并引起IL-2细胞因子释放，为后续临床细胞免疫治疗提供了理论基础。

Construction of the Anti-EGFRvⅢ Chimeric Antigen Receptor(CAR) and Analysis of Its Activity in vitro

The Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) plays its anti-tumor role by activating and amplifying tumor-specific or non-specific killer cells in vitro. It has a good prospect in tumor immunotherapy. In this study, a recombinant lentiviral vector encoding the anti EGFRⅢ chimeric antigen receptor (CAR) was constructed. Then a stable CAR-modified Jurkat cell line was generated using the lentivirus. When stimulated with EGFRvⅢ or U87MG, the cell line was activated, suggesting that a recombinant plasmid pCDH-EGFRvⅢ-scFv-CAR-copGFP-T2A-puro was successfully constructed and an EGFRⅢ-CAR-expressing Jurkat cell line was successfully generated. After EGFRvⅢ stimulation for 12 h, the CCK 8 assay showed that the proliferation rate of CAR-Jurkat cells was about 1.36 times faster than the control group (P<0.05). After co-incubation with U87MG cells, the ELISA assay showed that the concentration of IL-2 in the cell supernatant was 1.625 times more than the control group (P <0.01). The results above showed that the EGFRvⅢ-CAR expressing Jurkat cell line specifically targets the EGFRvⅢ molecule and EGFRvⅢ-positive cells and releases IL-2 cytokine, which provides a theoretical basis for the subsequent clinical application of the cell-based immunotherapy.