Preparation And Biological Function Analysis Of An Anti-human CD47 Monoclonal Antibody

Invasion of immune surveillance is one of the most contributing factor in cancer progression while cancer immunotherapy has been advanced as a powerful strategy for treating malignancies.Cluster of differentiation 47(CD47)is an Ig G superfamily membe and functions critically in mediating phagocytosis of cancer cells by macrophages,promoting tumor survival,progression and migration.Strategies blocking the interaction between CD47 and its ligand SIRPα have been validated to restore and strengthen the phagocytosis ability of macrophages,which elicits the potential to treat malignant tumors.Thus,developing anti-CD47 antibodies with blocking the function of anti-phagocytosis elicited by CD47 is a novel approach in cancer immnunotherapy.In this study,using prokaryotic proteisn expression,we expressed and purified the extracellular domain(ECD)of recombinant human CD47 protein as well as characterization of the purified CD47 using Western Blot and enzyme-linked immunosorbent assay(ELISA)and found that CD47 protein could be recognized by commercial anti-CD47 antibody B6H12.With this recombinant protein,one anti-CD47 monoclonal antibody named 13G1 was generated using conventional hybridoma techniques.Epitope of 13G1 which was mainly in the 79-91 amino acid sequence was identified using Western Blot.Besides,the antibody was characterized by flow cytometry and immunofluorescent analysis using different human cancer cell lines including cervical cancer Hela,lung cancer A549,Burkitt lymphoma Raji and gastric cancer MKN-45 and results showed that could be recognized by 13G1.Meanwhile,we used quantitative and qualitative monitoring the phagocytosis rate of macrophages to investigate the phagocytosis of tumor cells by macrophages and found that 13G1 possess the function of promoting macrophage phagocytosis of tumor cells.Furthermore,we amplified the CDR(complementarity-determining regions)sequences of heavy and light chains of the antibody and constructed the human-mouse chimeric antibody to verify its binding efficacy of the sequence using ELISA and the results showed that the chimeric antibody has a good binding activity to CD47 antigen,paving the way for the development of humanized antibodies.Taken together,our study prepared a new anti-CD47 monoclonal antibody which promotes the phagocytosis of cancer cells by macrophages,which will facilitate the development of anti-CD47-based diagnosis and treatment and provide a leading drug.