Expression, Purification, And Preliminary Functional Study Of PD-1-CEGF Multi-targeting Fc Fusion Protein PCP

Monoclonal antibodies (mAbs) have high targeting specificity, safety and clinical efficiency,Till now,10mAbs have been approved by the US Food and Drug Administration (FDA), andwas considered as the first-tier drugs for a variety of malignant tumor therapies. However, tumoris composed of cells of high heterogy, while single-targeting cancer drug only targets a smallportion of cancer cells, so that other cancer cells probably escape, which may be the source ofcancer relapse. As a result, researches of antibody-like protein drugs with small molecular weight,and multi-targeting capability has become a hotspot in this area.Programmed death-1receptor(PD-1) is one of the B7-CD28family. PD-1with its ligandsPD-L1and PD-L2contribute inhibitory signals that modulate T-cell responses. Recently, MoffittCancer Center found that20%~25%patients with non-small-cell lung cancer, melanoma or renalcell carcinoma have complete response or partial response. At present, several clinic trials havebeen carried out. Therefore, PD-1has become an important target for the treatment of cancer.Vascular Endothelial Growth Factor (VEGF) is a promoting angiogenesis factor. As ananti-VEGF antibody, Avastin has been proved to be an effective anti-tumor drug. Thus, VEGFand its receptor have become an important target for the treatment of cancer.In my research, the fusion protein PVP can target PD-1and VEGF at the same time. Mostimportantly, PVP targets T cells to suppress T cell inactivation. PVP will target VEGF in thetumor microenvironment.First, we generated a recombinant vector containing the PD-L2-Flt1D2-Fc expressioncassette, which includes the extracellular of PD-L2, the second structural domain of Flt1D2andFc of IgG4. The expressed protein will target PD-1and VEGF. Then we generated the Ad5tocarry the gene PD-L2-Flt1D2-Fc. We used Ad5to infect293cells to make it express the fusionprotein (named PD-1-VEGF Multi-targeted Fc Fusion Protein, PVP). We purified the proteinwith protein A affinity chromatography. The purified protein was identified with western blotting.Finally, we used interaction analysis system to study the affinity with PD-1and VEGF. Ourresearch shows that PVP has high affinity with PD-1and VEGF. Our research makes afoundation for the following studies in vitro and in vivo.