小鼠抗人PD-1单克隆抗体的制备及其生物学活性评价

Preparation of a mouse anti-human PD-1 monoclonal antibody and identification of its bioactivity

目的制备鼠抗人PD-1单克隆抗体,并对其生物阻断活性进行评价。方法利用重组人源PD-1/PDCD1蛋白作为免疫蛋白,免疫接种Balb/c小鼠,分离并提取小鼠脾淋巴细胞,然后与小鼠骨髓瘤细胞SP2/0细胞融合培养,经过多次克隆化培养筛选出稳定分泌鼠抗人PD-1单抗的杂交瘤细胞株。鉴定单抗Ig G类别及亚类,并分析其特异性和细胞毒性;建立Hep G2细胞和Jurkat细胞共同培养体系,ELISA法检测PD-1单抗对Jurkat细胞分泌抗肿瘤细胞因子的影响,MTT法分析PD-1单抗对Jurkat细胞杀伤Hep G2细胞能力的影响。结果获得了1株新的小鼠抗人PD-1单克隆抗体,命名为2H7。单抗2H7能够特异性识别T细胞表面的PD-1蛋白;高浓度长时间处理对Jurkat细胞有一定毒性作用;低浓度单抗2H7可阻断PD-1/PD-L1信号通路,使Jurkat细胞对Hep G2细胞杀伤能力显著增强(P<0.01),且Jurkat细胞分泌的IL-2和IFN-γ等细胞因子水平显著高于对照组(P<0.01)。结论成功制备了1株具有良好生物阻断活性的小鼠抗人PD-1单克隆抗体2H7,该抗体能够显著提高T细胞对肝癌细胞Hep G2的杀伤能力。

This study aimed to prepare a new mouse anti-human PD-1 monoclonal antibody（m Ab） and identify its blocking bioactivity. The recombinant human PD-1/PDCD1 protein was used as an immunogen to immune Balb/c mice, and then the spleen cells from immunized Balb/c mice were fused with mouse myeloma cells（SP2/0）. Hybridoma cell lines stably secreting anti-PD-1 m Ab were screened out by multiple cloning. Then, the subtype of m Ab Ig G was identified, and its specificity and cytotoxicity were analyzed. Hep G2 cells were co-cultured with Jurkat cells, the effect of anti-PD-1 m Ab on the secretion of anti-tumor cytokines in Jurkat cells was measured by ELISA, and the effect of anti-PD-1 m Ab on the ability of Jurkat cells to kill Hep G2 cells was analyzed by MTT assay. At last, a new mouse anti-human PD-1 m Ab was generated and named 2H7. The m Ab 2H7 could specifically recognize the PD-1 protein expressed on T lymphocytes. High concentration of m Ab 2H7 was toxic to Jurkat cells,while low concentration of m Ab 2H7 could block the PD-1/PD-L1 pathway and significantly enhance the function of Jurkat cells to kill target Hep G2 cells（P〈0.01）. Moreover, the levels of IL-2 and IFN-γ secreted by Jurkat cells in 2H7 treated group were higher than those in control group（P〈0.01）. In conclusion, a new mouse anti-humanPD-1 m Ab 2H7 with good blocking bioactivity is developed successfully, which can significantly improve the function of Jurkat cells to kill target HepG2 cells.