机构地区:[1]安徽医科大学第四附属医院呼吸内科,合肥2300222 [2]安徽安科生物工程(集团)股份有限公司,合肥230088
出 处:《安徽医科大学学报》2018年第2期176-181,共6页Acta Universitatis Medicinalis Anhui
基 金:安徽省自然科学基金面上项目(编号:1308085MH141);安徽高校省级自然科学研究项目(编号:KJ2011A173)
摘 要: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 8μg/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PDL1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。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.
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