Preparation Of Anti-CD47 Antibodies And Its Treatment Of Human EC9706 Xenograft Tumors In Mice

CD47 also known as integrin-related protein,is a five-time transmembrane protein,belonging to the immunoglobulin superfamily member.CD47 is expressed as the most important "self" recognition molecule on most cell surfaces.In addition,CD47 also plays a key role in immune recognition,especially in innate immune recognition,and is thought to be a marker molecule that regulates innate immunity.CD47 binds to bone marrow-derived innate immune cells,especially the immunoreactive regulatory molecules on the surface of macrophages and the signal regulatory protein alpha(SIRP?),which inhibits macrophage phagocytosis.In recent years,CD47 has also been found in most tumors,is one of the mechanisms of cancer to escape immune attacks.Thus,blocking the binding of CD47 to SIRP? is a promising treatment strategy for recovering antitumor immune responses and thus obtaining effective tumor immunotherapy.Recently,anti-CD47 monoclonal antibodies and SIRP? mimics have been shown to inhibit the binding of CD47 to SIRP? in mouse tumor models to effectively remove leukemia,lymphoma and breast cancer,colon cancer,bladder cancer,melanoma,Small cell lung cancer and other solid tumor cells,significantly inhibit tumor growth and metastasis,and significantly prolong the survival of tumor-bearing mice,with encouraging therapeutic effect.Esophageal cancer in China is a high incidence of cancer,the incidence and mortality rate among the top five.There is no effective treatment.In addition,there is no report for esophageal cancer CD47 research and treatment.Thus,in this study,we prepared a genetically engineered single chain antibody and a Nanobody derived from the camel heavy chain variable region to analyze the blocking effect of these two antibodies on CD47 binding to SIRP? and to explore its Macrophage phagocytosis,to evaluate the therapeutic effect of these small molecule antibodies on esophageal cancer,and to develop a new treatment for esophageal cancer innate immunological test points,to improve the treatment of esophageal cancer to contribute.There are three main part research contents in the thesis:1.Preparation of anti-CD47 nanobodies and single chain antibodiesFirst,the CD47 antigen was prepared and the extracellular domain(ECD)gene of CD47 was amplified by PCR.The ECD gene of CD47 was constructed on the empty vector of p ET30 a.The ECD protein of CD47 was expressed by prokaryotic expression system,and the constructed CD47 antigen was identified by commercial anti-CD47 monoclonal antibody.Secondly,anti-CD47 nanobodies were screened by phage display library technique.After two rounds of affinity washing,the nanoantibody VHH-2B1 with high affinity for CD47-ECD was obtained.And VHH-2B1 was subcloned into p ET22 b vector to induce expression of anti-CD47 antibody.Thirdly,the heavy chain variable region(VH)and light chain of anti-CD47 monoclonal antibody B6H12 were obtained by DNA synthesis method.(B6H12-sc Fv),and purified by Ni ion affinity chromatography.The B6H12 single chain antibody(B6H12-sc Fv)was ligated by short peptide(Gly4Ser)3.The anti-CD47 single chain antibody B6H12-sc Fv was obtained.2.Anti-CD47 antibody in vitro binding activity analysis and phagocytosis of esophageal cancer cellsStudies have shown that anti-CD47 monoclonal antibody in the experimental animal tumor treatment has achieved very good results.The aim of this study was to prepare anti-CD47 small molecule Nanobody and single chain antibody,and to analyze the role of antigen-binding activity and in vitro function.ELISA and Western blot to detect its binding to CD47.At the same time,the binding ability of B6H12-sc Fv to CD47 and SIRP? was analyzed by competitive ELISA and the expression level of CD47 in esophageal cancer cells and the phagocytosis of esophageal cancer cells.First,culture of esophageal cancer EC9706 cells,using cell ELISA and flow cytometry to verify whether the expression of EC9706 cell surface CD47 protein.Second,through flow cytometry and cell ELISA was used to verify the binding of the prepared single chain antibody to esophageal cancer cells.Anti-CD47 blocking monoclonal antibody as a positive control,the same type of Ig G1 as a negative control to verify the ability of single-chain antibody binding.Third,phagocytosis experiments,from the mouse's abdominal cavity removed macrophages cultured for 7 days,macrophages and tumor cells were incubated,adding different concentrations of anti-CD47 single-chain antibody to verify the phagocytosis of macrophages and calculate the phagocytic coefficient.3.Inhibitory effect of anti-CD47 antibody on nude mice bearing human esophageal xenograftsFirstly,EC9706 esophageal cancer cells were immunized with Balb/c nude mice.A total of 18 nude mice were injected and divided into three groups.Each group was treated twice a week for 6 weeks.Secondly,the antibody titers VHH-2B1 and B6H12-sc Fv were prepared.Each mouse was immunized with antibody protein twice a week and the blank control group immunized DPBS.Finally,the volume of the tumor was measured weekly and the number of days of survival was calculated before and after treatment,and the significant difference in tumor volume was statistically analyzed.In this study,the anti-CD47 antibody was tested by in vitro and in vivo experiments.In vitro experiments have shown that anti-CD47 antibodies have binding activity to CD47 antigens.In vivo experiments,an esophageal cancer mouse model was established using anti-CD47 antibody therapy but did not achieve the desired therapeutic effect.The reason may be that the preparation of antibodies for small molecular antibodies,and monoclonal antibodies than the lack of glycosylation of the site,in vivo metabolic time is short,the treatment is not easy to produce results.Second,the prepared antibody has binding activity,but the binding activity is still weak,resulting in the treatment effect is not obvious.In this study,we successfully prepared anti-CD47 antibody,verified its function,for the treatment of esophageal cancer provides a new basis.