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

WANG Guanying; LI Bin; LI Ziwei; XING Haiyan; CHEN Jianhong

doi:10.13431/j.cnki.immunol.j.20170063

Abstract：This study aimed to prepare a new mouse anti-human PD-1 monoclonal antibody (mAb) 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 mAb were screened out by muhiple cloning.Then,the subtype of mAb IgG was identified,and its specificity and cytotoxicity were analyzed.HepG2 cells were co-cultured with Jurkat cells,the effect of anti-PD-1 mAb on the secretion of anti-tumor cytokines in Jurkat cells was measured by ELISA,and the effect of anti-PD-1 mAb on the ability of Jurkat cells to kill HepG2 cells was analyzed by MTT assay.At last,a new mouse anti-human PD-1 mAb was generated and named 2H7.The mAb 2H7 could specifically recognize the PD-1 protein expressed on T lymphocytes.High concentration of mAb 2H7 was toxic to Jurkat cells,while low concentration of mAb 2H7 could block the PD-1/PD-L1 pathway and significantly enhance the function of Jurkat cells to kill target HepG2 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-human PD-1 mAb 2H7 with good blocking bioactivity is developed successfully,which can significantly improve the function of Jurkat cells to kill target HepG2 cells.