Development Of Novel Bifunctional VEGFR Fusion Protein For Cancer Therapy

To grow beyond minimal size,tumors must generate a new vascular supply for purposes of gas exchange,cell nutrition and waste disposal.They do so by secreting angiogenic cytokines that induce the formation of new blood vessels.Otherwise,tumor can not grow more than 2 mm~3.Vascular endothelial growth factor(VEGF) is likely one of the most important cytokines in tumor growth and become the most favorable target for cancer therapy.In vascular endothelial growth factor(VEGF) family,VEGF-A and VEGF-B can promote angiogenesis,while VEGF-C and VEGF-D are related to lymphoangiogenesis. Previous study indicates that blocking signal transduction pathway of VEGF/VEGFR using soluble VEGFR fusion proteins is an effective anti-tumor therapeutics. VEGFR1-Fc fusion protein can effectively block angiogenesis and then inhibit tumor growth and metastasis by competitively binding with VEGF-A and VEGF-B. VEGFR3-Fc fusion protein blocked lymphoangiogenesis and tumor metastasis through lymphatics by inhibiting the function of VEGF-C and VEGF-D.In the present study we have constructed a novel bifunctional fusion proteins consisting of the binding domain of VEGFR-1 and VEGFR-3.This fusion protein was expressed in mammalian cell expression system and shown to bind with high affinity to not only VEGF-A and VEGF-B,but also to VEGF-C and VEGF-D,which might be effective in blocking VEGF-VEGFR signal transduction pathway.The bifunctional fusion protein was demonstrated to be able to inhibit the proliferation of vascular endothelium in tumors,ablating angiogenesis and lymphoangiogenesis,reducing vascular permeable, shortening gas and nutrition exchange,and blocking the tumor growth and metastasis.To construct the fusion proteins,total RNA was extracted from peripheral blood mononuclear cells from healthy volunteers.The signal peptide region and Ig1-3 region of VEGFR1 was amplified by RT-PCR.The signal peptide region and Ig1-3 region of VEGFR3 was artificially synthesized.The Ig1-3 region of VEGFR1 was genetically fused in frame to the 5' end of human antibody Fc gene,generating the VEGFR1Fc fusion gene.Likewise,the IgI-3 region of VEGFR3 was fused in frame to the 5' terminus of Fc gene,generating the VEGFR3Fc fusion gene.The Ig1-2 region of VEGFR3 was fused to the 5' terminus of the human gammal chain constant region gene,resulting in the VEGFRIg-H gene,while the Ig2-3 region of VEGFR1 was fused to the 5' terminus of the human kappa chain constant region gene,yielding the VEGFRIg-L gene.In addition, the Ig1-2 region of VEGFR3 was fused in frame to the 5' terminus of the Ig2-3 region of VEGFR1,yielding the VEGFR31 gene.The VEGFR31 gene was then fused to the 5'terminus of Fc gene,generating the VEGFRFc gene.The VEGFR1Fc,VEGFR3Fc, VEGFRIg-H,VEGFRIg-L,and VEGFRFC fusion genes were cloned into the pDR1 vector,yielding the expression vectos pDR(VEGFR1Fc),pDR(VEGFR3Fc), pDR(VEGFRIg-H),pDR(VEGFRIg-L) and pDR(VEGFRFc),respectively.For the expression of bifunctional fusion protein VEGFRIg,the expression vectors pDR(VEGFRIg-H) and pDR(VEGFRIg-L) were cotransfected into CHO cells.The expression vectors pDR(VEGFR1Fc),pDR(VEGFR3Fc) and pDR(VEGFRFc) were transfected into CHO cells to express the VEGFR1Fc,VEGFR3Fc or VEGFRFc fusion protein,respectively.The cell clones producing the highest amount of target proteins were selected and grown in serum-free medium.The expressed proteins in the supernatant were purified by Protein A affinity chromatography and verified by dotblot and SDS-PAGE.The binding affinity of the fusion proteins were analyzed by ELISA and blocking assay,and the affinity constants of them were measured by SPR.The binding of VEGFRIg to VEGF-A was similar to that of VEGFR1Fc.Likewise,VEGFRIg possessed binding affinity with VEGF-C comparably to that of VEGFR3Fc.The inhibition effect of the fusion proteins on the human unbilical vein endothelial cell proliferation and migration induced by VEGF-A and VEGF-C were analyzed using 3[H]thymidine assay and scraping repair assay.The results showed that VEGFRIg could effectively inhibit the cell proliferation and migration induced by VEGF-A and VEGF-C.In chicken embryo CAM assay,gelatin sponge was used as slow-release carrier to observe the function of VEGFR fusion proteins.The results showed that VEGFR1Fc and VEGFRIg significantly inhibited the pro-angiogenic function of VEGF compared with the negative control.In the tumor-bearing mouse model,hepatocellular carcinoma cells LM3 was inoculated to the right flank of nude mice.Mice were randomized into 5 different treatment groups.The results showed that VEGFRIg significantly inhibited the primary tumor growth and metastasis.The results suggested that the fusion protein VEGFRIg could effectively block the angiogenesis and lymphoangiogensis induced by the VEGF-A and VEGF-C in vivo,thus inhibiting the tumor growth and metastasis.Our results indicated that the antitumor activity of VEGFR-Ig in tumor-bearing mice was much more potent than the administration of VEGFR1-Fc,VEGFR3-Fc and VEGFR1-Fc in combination with VEGFR3-Fc,which may be attributable to the markedly increased plasma half-life of VEGFR-Ig.Therefore,to further enhance its antitumor activity,we next constructed a mutant derived from VEGFR-Ig by generating three amino acid mutations on this bifunctional fusion protein denoted VEGFR-IgM.The fusion protein mutant gene was generated by Overlapping PCR using the pDR(VEGFRIg-L) plasmid as template and then ligated into plasmid vector pDR1, denoted as pDR(VEGFRIg-LM).This plasmid was cotransfected with pDR(VEGFRIg-H) into CHO cells and the stable clones highly expressing the VEGFRIgM fusion protein were selected.The expressed proteins in the supernatant were purified by Protein A affinity chromatography and verified by SDS-PAGE.The isoelectric point(pI) of the VEGFRIgM was found to be lower than that of the parental fusion protein VEGFRIg by IEF.The biological functions of the VEGFRIgM fusion protein were analyzed using ELISA,SPR,3[H]thymidine assay,scraping repair assay,chicken embryo CAM assay, pharmacokinetic assay and tumor-bearing mouse model as described above.VEGFRIgM showed significantly longer half-life compared with the parental bifunctional protein VEGFR-Ig.Our study indicated that VEGFR-IgM possessed markedly enhanced antitumor effect than did VEGFR-Ig,suggesting that it had the potential to be developed the approach for an effective antitumor agent.In summary,the VEGFRIgM fusion protein displays better pharmacokinetic properties and is able to effectively block angiogenesis and lymphoangiogensis induced by the VEGF-A and VEGF-C in vitro and in vivo,and then inhibited primary tumor growth and reduced metastasis.This novel approach for generating of effective antitumor fusion proteins may provide a new strategy for treatment of cancer.