Construction And Bioactivity Verification Of Anti-gastric Cancer HER2 ScFv-CCL19-IL7 Fusion Gene Engineering Protein

Gastric cancer is a common malignant tumor of digestive system,the prognosis is relatively poor,seriously threatening human health.The clinical symptoms of early gastric cancer are atypical,so most patients are advanced when they are diagnosed,and the prognosis of these patients is poor.In addition to surgical treatment,they all need auxiliary internal medicine chemotherapy and other follow-up treatment.The medical treatment of gastric cancer mainly includes chemotherapy,immunotherapy and targeted therapy.However,the effective rate of chemotherapy is limited,and the toxic and side effects are high.Immunotherapy has not yet found a specific target for gastric cancer,so the progress is slow.Targeted therapy has been widely used in breast cancer and lung cancer treatment,especially trastuzumab has been widely recognized in the treatment of breast cancer.In view of the precision of targeted therapy,people have begun to explore the application of targeted therapy in gastric cancer.The purpose of this study was to construct and synthesize fusion genetic engineering protein targeting HER2 protein and to explore its killing effect on HER2 positive gastric cancer cells.Monoclonal antibodies are currently widely used as targeted therapeutic drugs.Antibody dependent cytotoxicity(ADCC)is an important mechanism for monoclonal antibody to exert killing effect,but monoclonal antibody has large molecular weight and is difficult to penetrate tumor tissue.These disadvantages limit the application of monoclonal antibody,while the immune killing of T cells can not be effectively mediated by the lack of receptors bound to monoclonal antibodies on the surface,thus weakening the body's immune response to tumors.Single-chain antibody(sc Fv)contains only variable region fragments of total antibody,which has the characteristics of small immunogenic,small molecular weight and strong penetration,so it is an ideal target vector for tumor therapy.CCL19 is a kind of small molecular protein that can be moved by chemotactic immune cells,which can induce T cells,NK cells and DC cells to play an anti-tumor role.IL 7 plays an important role in the development,differentiation and proliferation of T cells.It can inhibit the growth of many solid tumors by promoting the secretion of IFN-?,TNF-?and other cytokines.In this study,a lentiviral vector against the whole gene sequence of HER2 scFv-CCL19-IL7 was constructed by using human HER2 sc Fv as a target vector,connecting CCL19 and IL 7 mediating T cell activation.HEK293T cell line stably expressing fusion genetic engineering protein was transfected and screened,and the target protein was successfully obtained from its culture medium.After obtaining the target protein,the second part of the experiment verified the HER2 targeting and biological activity of the anti-HER2sc Fv-CCL19-IL7 fusion gene engineering protein.The third part of the experiment is the anti-tumor effect of fusion protein,NCI-N87 gastric cancer cells with high expression of HER2 protein and HER2 negative SGC7901cells were given different doses of fusion protein to observe the killing effect.At the same time,a humanized NOD/SCID mouse model was established,and a mouse model of gastric cancer xenograft was established.The anti-HER2sc Fv-CCL19-IL7 fusion gene engineering protein was treated to observe the tumor volume change and detect IFN-?and IL-2 cytokine secretion levels to evaluated their anti-tumor effects in vivo.Part one Construction and eukaryotic expression of anti-gastric cancer HER2 scFv-CCL19-IL7 fusion gene engineering protein expression vectorObjective:To construct the expression plasmid of anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein and screen out the stable HEK293T cell line which stably expresses the fusion gene engineering protein.Methods:Human CCL19 c DNA sequence(gene ID 6363),target gene size 294bp,human IL7 c DNA sequence(gene ID 9606),target gene size531bp,anti-human HER2 sc Fv gene were obtained from hybridoma cell lines of mouse anti-human HER2 monoclonal antibody prepared in our laboratory,the VH,VL gene was obtained by codon optimization and humanization.According to the form of anti-HER2 sc Fv gene-ligated peptide gene-CCL19gene-ligated peptide gene-IL7 gene,the whole gene sequence was synthesized by Xho?/Hind?restriction site located at the two ends of the whole gene synthesis sequence,and the synthesized gene sequence was subcloned into pc DNA3.1 vector.The anti-HER2 scFv-CCL19-IL7 fusion gene in the HCI-pc DNA3.1 plasmid was ligated into the lentiviral expression vector plasmid by Xho I/Hind III endonuclease digestion,and the target gene fragment size was digested and sequenced.After transfection,the target gene was transfected into HEK293T cells by lentiviral transfection,and the transfected cells were selected by PURO to obtain a HEK293T cell system stably expressing the fusion protein.The highly expressed HEK293T cells were cultured in large quantities,and the fusion protein was extracted.The target protein was purified by nickel column affinity chromatography using His tag.The expression of the fusion protein in the supernatant of HEK293T cells was detected by Western-blot.Result:1.The whole anti-HER2 scFv-CCL19-IL7 gene sequence was succes-sfully synthesized by gene engineering,and subcloned into pc DNA3.1 vector.No gene mutation was found in the target fragment by gene sequencing.2.The lentivirus vector containing the whole gene sequence of anti-HER2 scFv-CCL19-IL7 was successfully constructed,and the target gene sequence was stably transfected into HEK293T cells by lentivirus transfection,and the HEK293T cell line stably expressing anti-HER2sc Fv-CCL19-IL7 fusion gene engineering protein was screened out.3.Westernblot detected expression of anti-HER2 scFv-CCL19-IL7fusion gene engineering protein in HEK293T cell culture supernatants.Part two Targeting verification and biological activity analysis of anti-gastric cancer HER2 scFv-CCL19-IL7 fusion gene engineering proteinObjective:Verification of HER2 targeting and biological activity of anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein.Methods:The expression of HER2 protein in human NCI-N87 gastric cancer cells and SGC7901 gastric cancer cells was detected by flow cytometry,and the anti-HER2 antigen targeting and biological activity of anti-HER2sc Fv-CCL19-IL7 fusion gene engineering protein were analyzed by flow cytometry.The content and biological activity of IL7 in the purified fusion protein was determined by ELISA.The binding constant of anti-HER2sc Fv-CCL19-IL7 fusion gene engineering protein was determined,and it was confirmed that anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein had affinity to HER2 antigen and has biological activity,which lays a foundation for further verification of its antitumor activity in vitro and in vivo.Result:1.Flow cytometry showed that the expression of HER2 protein on the surface of human NCI-N87 gastric cancer cells was high,while the expression of HER2 protein on the surface of SGC7901 gastric cancer cells was low or not.2.The fluorescence intensity after flow cytometry showed that anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein could compete with herceptin and HER2-FITC to bind HER2 antigen on the surface of human gastric cancer NCI-N87 cells.3.The assay of protein binding constant showed that the anti-HER2sc Fv-CCL19-IL7 fusion gene engineering protein had the activity of antibody binding to HER2 antigen,and had a certain affinity.4.IL7 was detected in the HEK293T cell culture medium by ELISA,and the results showed that the IL7 content in the medium increased with the prolongation of the culture time.Part three Experimental study on antitumor effect of anti-gastric cancer HER2 scFv-CCL19-IL7 fusion genetic engineering protein in vitro and in vivoObjective:To investigate the killing effect of anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein on HER2-overexpressed gastric cancer cells in vitro and the anti-tumor effect on HER2-positive transplanted tumor model of gastric cancer.Methods:CCK-8 assay was used to detect the killing effect of PBMCs mediated by HER2 scFv-CCL19-IL7 fusion genetic engineering protein on HER2 overexpression and low expression of gastric cancer cells,and ELISA assay was used to detect the secretion of cytokines IL-2 and IFN-?.Humanized NOD/SCID mouse model was established by injecting human peripheral blood mononuclear cells into tail vein.The expression of human CD3~+T lymphocytes and CD19~+B lymphocytes in peripheral blood of mice was detected by flow cytometry.The animal model of gastric cancer transplantation was established by inoculating human gastric cancer cells with HER2 overexpression in humanized NOD/SCID mice.After tumor formation,the animals were randomly divided into three groups:saline,Herceptin,anti-HER2 scFv-CCL19-IL7 fusion genetic engineering protein,each group give initial load therapy once,maintenance treatment dose 3 times,once a week for a total of 4 times.The tumor volume changes were measured regularly,and the average tumor volume and SD value of the three groups were calculated.The secretion of IFN-?and IL-2 in serum of mice in each group was detected by ELISA.The morphology of tumor specimens was observed by HE staining,and the infiltration of T lymphocytes and DC cells in tumor tissue was observed by immunofluorescence histochemical method.Result:1.The anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein could mediate the killing efficiency of PBMCs against HER2-positive NCI-N87 cells and HER2-negative SGC7901 cells in vitro under different effect-target ratios.With the increase of the effect-target ratio,the killing efficiency of both of them increased.2.Anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein can mediate the activation of immune cells and increase the secretion of IFN-?and IL-2.3.Anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein has significant killing effect on HER2-positive gastric cancer cells in vivo.4.Anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein can chemotactic T cells,DC cells and other lymphocytes into the tumor.Conclusion:1.The full-length gene sequence of anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein was successfully transfected into HEK293T cells by lentivirus transfection,and the anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein was stably expressed.2.Anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein can specifically target HER2 protein on the surface of tumor cells and has antibody antigen binding activity.3.Anti-HER2 scFv-CCL19-IL7 fusion gene engineering protein can mediate the activation of immune cells and increase the secretion of IFN-?and IL-2.4.The humanized NOD/SCID mouse model was successfully established by injecting peripheral blood mononuclear cells into the tail vein of mice,and the tumor model of gastric cancer transplantation was successfully established by subcutaneous injection of tumor cell lines.Anti-HER2 scFv-CCL19-IL7fusion genetic engineering protein can specifically recognize HER2 antigen and mediated the immune system to play an anti-tumor role by chemotactic DC cells and activated T cells.