Construction, expression, purification, and characterization of a dual-targeting PD-1/VEGF-A fusion protein (P-V)

• Published in: Protein expression and purification 2015-05, Vol.109, p.1-6
• Main Authors: Qian, Niliang, Gao, Liucun, Dong, Lihou, Liu, Hongchuan, Fu, Jie, Meng, Dan, Gao, Xin, Zhang, Jing, Gao, Yucai, Song, Haifeng
• Format: Article
• Language: English
• Subjects: 293E cells; Anti-tumor; Blotting, Western; Dual targeting; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fusion protein; Genetic Vectors - metabolism; Human Umbilical Vein Endothelial Cells - metabolism; Humans; PD-1; Polymerase Chain Reaction; Programmed Cell Death 1 Receptor - isolation & purification; Protein Engineering - methods; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Vascular Endothelial Growth Factor A - isolation & purification; VEGF-A
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.01.004

•A novel and effective fusion protein.•Dual-targeting PD-1/VEGF-A.•Using the eukaryotic expression vector and system. Targeting programmed death-1 (PD-1) is regarded as a novel and promising means for the treatment of many types of solid tumor. In the tumor microenvironment (TME), VEGF expression is dramatically up-regulated, and compounds that neutralize VEGF or block the interaction of VEGF with its receptors exhibit potent antitumor activity, and blocking PD-1 might promote T cell infiltration into TME and significantly enhance local immune activation. Thus, we fused domain II and domain III of kinase-insert domain receptor (KDR), the receptor of VEGF-A, to the Fc side of an anti-PD-1 monoclonal antibody with a (Gly4Ser)3 linker to generate a dual targeting fusion protein. The recombinant plasmid was successfully constructed and the fusion protein was expressed in 293E cells. Protein purification was performed in a single step by using protein A affinity chromatography. The molecular weight of the fusion protein was approximately 220kDa, and the yield was approximately 2.97g/L. Specific binding of recombinant protein to PD-1 and VEGF was detected by enzyme-linked immunosorbent assay (ELISA) analysis. Half maximal effective concentration (EC50) values were 0.561nM for PD-1 and 0.682nM for VEGF-A; accordingly, half maximal inhibitory concentration (IC50) values were 0.914nM and 0.583nM, respectively. Proliferation inhibition assays indicated that the fusion protein could inhibit the growth of human umbilical vein endothelial cells effectively. Taken together, the results indicate that this novel fusion protein can simultaneously target PD-1 and VEGF and may be beneficial for combining anti-angiogenesis with immunotherapeutic approaches for the treatment of patients with cancer.