Preparation And Anti-tumor Activity Of Singlechain Variable Fragment Targeting Human Lung Cancer Surface Antigen C-met Protein

Background:So far lung cancer is the leading cause of cancer death worldwide.Lung cancer is divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),of which NSCLC accounts for more than 85% of lung cancer morbidity.NSCLC includes several types of squamous cell carcinoma,lung adenocarcinoma,and large cell lung cancer.The most common one is lung adenocarcinoma.At present,drugs targeting lung cancer are mostly small-molecule inhibitors,and accompanied by drug resistance in tumor cells.Therefore,the study of lung adenocarcinoma is farely challenging.Researches have shown that the cellular-mesenchymal to epithelial transition factor(cmet)protein has tyrosine kinase activity and is over-expressed and abnormally activated on the surface of lung adenocarcinoma cells.It plays a key role in the resistance of tyrosine kinase inhibitors(TKI)and drives the oncogenesis and progression of NSCLC.Therefore,in this study,a single chain variable fragment(sc Fv)specific recognizing the c-met protein was obtained by a yeast surface display technique in the previous researches.The eukaryotic expression vector including sc Fv gene was constructed by molecular cloning technology.And verify the specifity of targeting c-met protein and anti-tumor activity respectively in vitro and in vivo.Objective: To prepare a human single chain antibody targeting the c-met protein and verify its characteristics and anti-tumor activity.Methods: Preparation of single-chain antibodies:The sc Fv gene was inserted into the p Fuse vector to construct an eukaryotic expression plasmid,and the fusion protein fused to the mouse Fc fragment was expressed and designated as met sc Fv.Western blot was used to verify whether met sc Fv was expressed.Met sc Fv was purified through an AKTA protein purification system.And Coomassie blue staining were used to verify whether it was expressed and purified.In vitro verification :Using purified met sc Fv fusion protein,through ELISA,flow cytometry,immunofluorescence assay,cell viability assay,complement dependent cytotoxicity(CDC)and antibody-dependent cell-mediated cytotoxicity(ADCC)experiments to verify its specificity for c-met protein.Anti-tumor activity researches: Fluorescence-marked met sc Fv was intraperitoneally injected into nude mice bearing nude mice and its distribution was observed through in vivo imaging techniques;tumors were isolated and immunostained with met sc Fv.A tumor-bearing nude mouse model was established and met sc Fv was injected with purified met sc Fv regularly and plotted the tumor growth curves.Immunohistochemistry was performed using met sc Fv and human lung adenocarcinoma and hepatocellular carcinoma tissues to verify their affinity to the c-met antigen on the surface of cancer tissue.Results: Preparation of single-chain variable fragment: sequencing testified that the sc Fvp Fuse vector was constructed successfully.Western blot results indicated that met sc Fv was successfully expressed.Coomassie brilliant blue staining results showed that the single chain antibody was purified;In vitro validation: ELISA results suggestted that with increasing sc Fv concentration,the corresponding OD450 nm value showed an upward trend,and compared with the control group,p<0.01,the differences had statistical significance.Immunofluorescence and flow cytometry results showed that the met sc Fv group signal was positive.met sc Fv with recombinant human c-met protein was preincubated,and the positive rate of flow-through result was decreased,suggesting that met sc Fv binds to recombinant human c-met protein,which affects its binding to c-met protein on the surface of A549 cells.Cell viability assay showed that met sc Fv may affect the activity of A549 cells.CDC and ADCC results suggestted that met sc Fv could induce cytotoxicity in vitro and thus kill cells under the conditions of peripheral serum and human lymphocytes involved.Antitumor activity researches: fluorescence-labeled single-chain antibody.The results of in vivo distribution experiments showed that met sc Fv mainly distributed in the abdominal cavity and the tumor growth position in the early stage,about 48 hours,and the overall signal was weak.The fluorescence signal was still visible in the body site.The immunohistochemical results of the tumor showed that the paraffin section was faint yellow and signal positive.The met sc Fv was injected into the nude mice bearing the tumor.The growth rate of the tumor in the experimental group gradual declined after the injection of met sc Fv.Compared with the control group,the difference was statistically significant.Conclusion: Met scFv can specifically recognize c-met protein in vivo and in vitro,and can mediated to kill tumor cells in vitro;In vivo experiments showed that met sc Fv has better anti-tumor activity.The specific recognition of c-met by Met sc Fv laid a solid foundation for the later research of CAR-T cells targeting c-met.