Study On Inhibitory Effect Of Humanized Monoclonal Antibody E12 Against Human FGF2 With Chemotherapy Drugs On Breast Cancer MCF7 And MDA-MB453 Cell-lines In Vitro

Objective: Make characterization and identification of the humanized FGF2 monoclonal antibody E12;Study the inhibition of proliferation and promotion of apoptosis of humanized FGF2 monoclonal antibody E12 alone and in combination with cisplatin and paclitaxel on breast cancer cell lines MCF-7 and MDA-MB453;Analyze the molecular mechanism of the biological activity of the humanized FGF2 monoclonal antibody E12.Methods: 1.Make statistical analysis of humanization transformation of the humanized FGF2 monoclonal antibody E12.The purity of the monoclonal antibody was analyzed by SDS-PAGE and HPLC,and the titer of the monoclonal antibody was detected by indirect ELISA.The BCA was used to detects the protein concentration of humanized monoclonal antibodies;2.Analysis of the expression of FGF2 and FGFR1 genes in tumor cell lines MDAMB453 and MCF7 by CCLE database.MTT assay was used to detect the sensitivity of tumor cell lines MDA-MB453 and MCF7 to FGF2.CCK8 assay was used to detect the inhibition of proliferation of MDA cell-lines MDA-MB453 and MCF-7 induced by FGF2 monoclonal antibody alone and in combination with chemotherapy drugs cisplatin and paclitaxel;3.Using flow cytometry Annexin V-FITC/PI double staining to detect the pro-apoptotic effects of FGF2 monoclonal antibody and chemotherapeutic drugs cisplatin and paclitaxel on breast cancer cell-lines MDA-MB453 and MCF-7;4.Use the Discovery Studio software to analyze the epitope of the antibody and the biologically active site of FGF2.Explain the molecular mechanism of the biological activity of the FGF2 monoclonal antibody by comparing the overlapping regions of the m Ab epitope and FGF2 bioactive sites.Virtual mutations screened for amino acids that make important contributions to binding and amino acid mutation combinations that may increase antibody affinity.Results: 1.Anti-human FGF2 humanized antibody E12 antibody variable region The ratio of human antibody amino acid sequence was 98.89% for heavy chain and 100% for light chain.Purity identification by SDS-PAGE and HPLC showed that the purity of antibody reached 99.02%.ELISA test showed that the human monoclonal antibody titer was 0.004?g/ml,and BCA assay showed that the concentration of humanized monoclonal antibody was 8.74mg/ml;2.The m RNA expression of FGF2/FGFR1 gene was relatively high in both cells.FGF2 can promote the growth of both tumor cell lines.When the concentration of humanized FGF2 monoclonal antibody was 500 ?g/ml,the inhibition rate of MCF-7 was 32%,and the inhibition rate of MDA-MB453 was 37%.When the cisplatin concentrations were 0.625,2.5,5,10,?g/ml,the inhibitory rate of MCF-7 was increased by 14.5%,10.4%,1.5%,and 0.5%,respectively,in the combination group compared to the cisplatin alone.Increased inhibition rate of MDA-MB453 was 15.5%,14.8%,13.0%,6.8%.When the paclitaxel concentrations were 1.0,0.1,0.01,?g/ml,the inhibitory rate of MCF-7 in the combination relative to paclitaxel alone increased by 14.5%,11.1%,and 9.3%,respectively.When the paclitaxel concentrations were 0.1,0.01,0.001,?g/ml respectively,the inhibitory rates of MDA-MB453 cells increased by 18.6%,19.9%,and 6.8%,respectively,in the combination relative to paclitaxel alone;3.Flow cytometry results showed that compared with the blank control group,MCF-7 cells treated with FGF2 monoclonal antibody,paclitaxel alone,and FGF2 monoclonal antibody+paclitaxel combined group accounted for the Annexin V/PI double positive region.The proportions of MCF-7 cells treated with FGF2 monoclonal antibody,paclitaxel alone,and FGF2 monoclonal antibody + paclitaxel were increased in the Annexin V/PI double positive regions,respectively,in proportions of 6.13%,7.36%,and 12.83%,respectively.The proportions of MCF-7 cells treated with FGF2 monoclonal antibody,cisplatin alone,and FGF2 monoclonal antibody + cisplatin were increased in the Annexin V/PI double positive regions,respectively,in proportions of 6.39%,9.46%,17.76%,respectively.The proportions of MDA-MB453 cells treated with FGF2 monoclonal antibody,paclitaxel alone,and FGF2 monoclonal antibody + paclitaxel were increased in the Annexin V/PI double positive regions,respectively,in proportions of 4.86%,7.1%,14.1%,respectively.The proportions of MDA-MB453 cells treated with FGF2 monoclonal antibody,cisplatin alone,and FGF2 monoclonal antibody + cisplatin were increased in the Annexin V/PI double positive regions,respectively,in proportions of 5.38%,9.06%,19.39%,respectively.4.The successful docking of FGF2 and FGF2 monoclonal antibody E12 homologous modeling structures predicted that L:ASN28,H:VAL38 and other amino acids are the epitopes of antibodies.Crystal structure analysis identified amino acids such as FGF2:HIS16 as the biologically active site of FGF2.Comparison of the two found to have 9 amino acid overlapping sites.The amino acid alanine scan of the antigenantibody binding site identified H:GLY111Y,H:GLY113 and other amino acids as important amino acids for antigen-antibody binding.Subsequent saturation mutations identified L:TYR38>TYRL,ASP114>ASP and other mutation combinations can be used as candidate mutations that increase antibody affinity.