Selective Thrombosis In Tumor Vessels Induced By Fusion Proteins Ttf-eg3287 Targeting Neuropilin-1

Selective thrombosis in tumor vessels and induction of subsequent tumor infarction is a promising anti-tumor strategy.tTF(truncated tissue factor) is only the extracellular domain of tissue factor.When tTF is targeted to the tumor vasculature by the ligand of tTF fusion protein and contacts with the cell membrane closely,it could induce thrombosis in tumor vasculature and cut off nutrition and oxygen supplies of tumor,which results in necrosis of the tumor and produces anti-tumor effects. Neuropilin-1(NRP-1) is a 120-130 kDa single spanning transmembrane glycoprotein, was found over expressed on endothelial and some tumor cells.Neuropilin-1 is a non-tyrosine kinase receptor of vascular endothelial growth factor-165(VEGFI65).The over-expression of Neuropilin-1 is correlated with tumor angiogenesis and progression,so NRP-1 could be the target for anti-cancer strategy.EG3287 is a specific binding peptide to NRP-1 on endothelial cells,which developed by Haiyan Jia et al.EG3287 is fragment of VEGF,which remains binding activity to NRP-1,but no signaling.In order to develop a new agent for selective thrombosis of tumor blood vessel,we used EG3287 as a targeting carrier to generated the fusion protein of tTF.The fused genes of tTF with EG3287,were constructed by PCR,and then were inserted into vector pET32b(+),expressed efficiently in E.coli BL21(DE3) under optimization condition.The fusion proteins were expressed as inclusion bodies under IPTG induction,and purified through Nickel-affinity chromatography column.The tTF activity of fusion proteins was analyzed by clotting assay and Fâ…©-activation assay.The results indicated that both of them could activiate Fâ…©and cause blood coagulation.Immunofluorescence experiments showed that ECV304 cells treated with tTF-EG3287 were found to have RBITC red fluorescent signals,but cells treated with the equimolar tTF were found to have.Red fluorescent signals were found on the membrane of cells and distributed in each cell in tTF-EG3287 group,but not found on the the membrane of cells treated with tTF.The results revealed that the fusion protein retained its ability of binging the receptor expressing on the cell surface.In the S180(mouse sarcoma)-bearing mouse models,the tumor growth inhibitions of tTF- EG3287 was 64.6%.In histological studies,the selective localization of the fusion proteins was observed.Blood vessels in the tumors in the treated group were thrombosed and occluded,and tumor cells around the occluded vessels appeared damaged.No thrombosis in blood vessels and cell damage,or other side effects were observed in normal tissues.In summary,the fused genes tTF-EG3287 were constructed by gene engineering technology,and fusion protein were expressed efficiently in E.coli.Fusion proteins tTF-EG3287 could selectively induce thrombosis in tumor vessels and limit the growth of tumor.All these lay the foundation for supplying one new vascular targeting agent for cancer therapeutics.