全人源化PD-L1单抗功能活性及其对人肺腺癌HCC827细胞毒性作用的研究

目的 初步探索自行研制的全人源化PD-L1单抗的功能活性及其对人肺腺癌HCC827细胞毒性作用的影响.方法 利用十二烷基硫酸钠聚丙烯凝胶电泳(SDS-PAGE)鉴定阿特朱单抗及自研PD-L1单抗的纯度;以阿特朱单抗为阳性对照,酶联免疫吸附测定法(ELISA)检测该自研PD-L1单抗的结合活性和竞争性阻断活性;细胞阻断试验分别测定两抗体的细胞阻断活性;流式细胞术(FACS)测定人肺腺癌 HCC827细胞表面PD-L1的表达;检测两抗体的抗体依赖的细胞介导的细胞毒作用(ADCC)效应.结果 SDS-PAGE鉴定出高纯度阿特朱单抗及自研PD-L1单抗;ELISA法检测结果显示自研PD-L1单抗、阿特朱单抗均可与人PD-L1结合并且阻断PD-L1与PD-1结合;自研PD-L1单抗及阿特朱单抗均可以阻断两细胞表面分子PD-L1与PD-1的结合,其EC50分别为0. 208 5,0. 154 8 μg/ml,在一定浓度上两者差异无统计学意义;FACS结果显示人肺腺癌HCC827细胞高表达PD-L1分子;细胞毒性实验结果表明当效应细胞与靶细胞比值一定时,自研PD-L1单抗与阿特朱单抗对人肺腺癌HCC827细胞都有强的毒性作用.结论 该自研全人源化PD-L1单抗有一定的功能活性,可通过阻断细胞表面的PD-L1与PD-1的结合而减弱T细胞受体信号,并可通过ADCC效应发挥一定的抗肿瘤作用.

Functional activity of humanized PD-L1 monoclonal antibody and its toxic effects on human lung adenocarcinoma cell line HCC827

Objective To explore the function and activity of human humanized PD-L1 monoclonal antibody and its toxicity to human lung adenocarcinoma cell line HCC827. Methods Sodium dodecyl sulfate polypropylene gel electrophoresis (SDS-PAGE) was used to determine the purity of Atezolizumab and PD-L1 monoclonal antibodies. The binding and competitive blocking activity of the self-developed PD-L1 monoclonal antibody were determined by enzyme linked immunosorbent assay (ELISA) with Atezolizumab as a positive control. Cell blocking activity of two antibody was measured by cell interruption test. Flow cytometry (FACS) was used to detect the expression of PD-L1 on the surface of human lung adenocarcinoma cell line HCC827, antibody dependent cell-mediated cytotoxicity(ADCC) effects were detected in two of antibodies. Results High purity Atezolizumab and PD-L1 monoclonal antibodies were identified by SDS-PAGE. Cell blocking activity of two antibody was measured by cell interruption test. ELISA showed that both PD-L1 monoclonal antibodies and Atezolizumab could bind to human PD-L1 and block the binding of PD-L1 to PD-1. Both PD-L1 monoclonal antibodies and Atezolizumab could block the binding of two cell surface molecules PD-L1 with PD-1, EC50 were 0. 208 5 μg/ml and 0. 154 8 μg/ml, respectively, there was no significant differences in certain concentration. FACS results showed that high expression of PD-L1 in human lung adenocarcinoma HCC827 cells. Cytotoxicity experiments showed that when the ratio between effector cells and target cells was constant, monoclonal antibodies against PD-L1 and Atezolizumab have strong toxic effects on human lung adenocarcinoma cell line HCC827. Conclusion The research of humanized monoclonal antibody PD-L1 has functional activity, T cell receptor signaling can be attenuated by blocking the combination of PD-L1 and PD-1 on the cell surface, and may exert a certain antitumor effect through the ADCC effect.