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目的:研制特异性鼠抗人PD-L1单克隆抗体(mAb)并鉴定其生物学特性。方法:以高表达人PD-L1分子的基因转染细胞L929/PD-L1为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-L1细胞为抗体筛选细胞,L929/mock细胞为对照细胞,经选择性克隆化培养及流式细胞术(FCM)分析,筛选稳定和持久分泌鼠抗人PD-L1 mAb的杂交瘤细胞株;采用Ig亚型快速定性试纸法、Western blot、间接免疫荧光法和竞争结合抑制实验等方法对mAb进行生物学特性鉴定;继而体外实验分析mAb对T细胞增殖的影响。结果:成功获得3株鼠抗人PD-L1mAb,分别命名为11G8,2G5和5C3;对其生物学功能的研究结果显示,3株mAb均能识别活化T细胞表面PD-L1分子,和在体外显著促进T细胞的增殖。结论:获得的3株鼠抗人PD-L1功能性mAb,通过阻断PD-1/PD-L1抑制途径能够有效增强T细胞体外增殖,这为进一步研究PD-1/PD-L1信号通路提供了物质基础。
Objective: To develop a specific mouse anti-human PD-L1 monoclonal antibody (mAb) and identify its biological characteristics. METHODS: BALB / c mice were routinely immunized with L929 / PD-L1 gene transfected cells, which were highly expressed in human PD-L1 molecule. B7 cells were fused with B lymphocyte hybridoma. For antibody screening cells, L929 / mock cells were control cells, and the hybridoma cell lines which stably and durably secreted mouse anti-human PD-L1 mAb were screened by selective clonal culture and flow cytometry (FCM) analysis; Rapid qualitative test paper method, Western blot, indirect immunofluorescence and competition binding inhibition experiments on the biological characteristics of mAb; then in vitro experiments to analyze the impact of mAb on T cell proliferation. RESULTS: Three murine anti-human PD-L1 mAbs were successfully obtained and named 11G8, 2G5 and 5C3, respectively. The biological function of the three mAbs showed that all three mAbs could recognize PD-L1 on the surface of activated T cells and in vitro Significantly promote T cell proliferation. CONCLUSION: The three murine anti-human PD-L1 functional mAbs can effectively enhance the proliferation of T cells by blocking the PD-1 / PD-L1 pathway, which is of great value in further researching PD-1 / PD-L1 signaling pathway The material basis.