The Research Of Fc Fusion Protein Binding To VEGF And EGFRs With High Efficiency

Due to the complication of tumor aetiology and the tedious and intersectedintracellular signal transductions of tumor cells, medical treatment of cancer has long beenproved as a forbidden task. Recently, the treatment of tumor has witnessed tremendousdevelopment due to the discover and progress made in monoclonal antibody. monoclonalantibodies（mAb） have the reputation of high targeting specificity, safety and clinicalefficiency, they are now considered as the first-line drugs for a variety of malignant tumortherapy. However, the applicability of monoclonal antibodies is relatively narrow, and thistype of drugs can easily induce drug tolerance. Meanwhile, full-length antibodies areproved to be hard to penetrate solid tumors because of the huge molecular weight.Therefore, antibody-like protein drugs with small molecular weight and multi-target are ofgreat significance in this area.It has been demonstrated that a large amount of vascular endothelial growthfactor（VEGF） would be secreted during the process of carcinogenesis. VEGF could induceAngiogenesis in tumor cells so as to provide necessary nutriment for the vast expansion oftumor. In the meatime, the proliferation and migration of tumor cells are closely connectedwith THE up-regulation of the epidermal growth factor receptor（EGFR） family. As aresult, the EGFR family and the VEGF signal pathway are regarded as the most importanttarget for tumor treatment.In this research, the second domain of FLT1（KDR-D3） was combined with HERINthrough Linker in order to construct an EGFR-VEGFR multi-targeted fusion protein. Thenthe protein was fused with the IgG1Fc fragment undergone Site-directed Mutagenesis. Inthis way, antibody-dependent cell-mediated cytotoxicity（ADCC） has been introduced in tothe protein to enhance its stability and prolong the half-life in vivo. Based on such afoundation, a chimeric cilia type adenovirus carrying FLT1D2-HERIN-FC cassette （EVP3）was utilized to constitute recombinant adenovirus Ad5/35-EVP3. This virus was then usedto transfect the293cells in vitro to produce the fused protein EVP3, which was entitledEVP3. After affinitive layer purification, Western blot experiment and double antibodysandwich method of enzyme linked immunosorbent assay were carried out to qualitativelyand quantitatively study the fusion protein EVP3. Subsequently, the affinity of the fusionprotein EVP3was qualitatively and quantitatively studied through the indirect immunofluorescent assay （IFA） and ForteBio Octet Red96Biomolecular InteractionAnalysis System. The results of all these experiments demonstrated that the adenovirusAd5/35-293cell system can express fusion protein EVP1effectively, and EVP1binds withhigh affinity to EGFR, HER2and VEGFR, their affinity constants are1.95×10^-8mol/L,8.47×10^-9mol/L,5.75×10^-9mol/L. these have laid a reasonable foundation for futureresearch of the anti-tumor effect in vitro and in vivo.