Construction And Characterization A New Fusion Protein Pro-SIRP?

CD47,a cell membrane glycoprotein belonging to the immunoglobulin superfamily,is widely expressed on the surface of tissue and cell,such as red blood cells,platelets,lymphocytes,hepatocyte,cancer cell and so on.The physiological function of CD47 is diverse.It participates in the interaction of cells and extracellular matrix,regulates a series of cell functions including cell apoptosis,proliferation and migration,and participates in the tumor immunological escape.Drugs targeting CD47 have been used in the treatment of leukemia,non-hodgkin's lymphoma,myeloma and other solid tumors during recent years.Signal regulatory protein-?(SIRP?),a membrane protein belonging to the immunoglobulin superfamily,is a typical inhibitory receptor of SIRP family.It is selectively expressed on the surface of myeloid cells such as macrophages,dendritic cells,and nerve cells.CD47 is a ligand for SIRP?.CD47-SIRP? signaling pathway plays an important role in a variety of mechanisms,especially,the adjustment to the phagorytosis of macrophagocyte.When CD47 interacts with SIRP?,a “don't eat me” signal makes the tumor cells escape from the phagocytosis of macrophages.However,low-level expression of CD47 can be found on the surface of blood cells.CD47-SIRP? signaling pathway adjusts the phagorytosis of red blood cells,platelets and lymphocytes by macrophagocyte.Therefore,anti-CD47 antibody drugs cause certain hematotoxicity,which influences its application.In this study,we constructed tumor-specific freed blocking peptide constituted high-affinity fusion protein pro-SIRP? and investigated its in vitro biologic activity.First,by screening phage-displayed random peptide library,we obtained short peptides which could bind active area of signal regulatory protein(SIRP?-cv1).This short peptide can block the interaction between CD47 and SIRP?-cv1,thus reducing the nonspecific binding and debase antigen sink effect.And then the short peptide was fused to amino terminal of the fusion protein SIRP?-cv1 via a linker containing enzyme digestion site of urokinase(uPA),yielding the fusion protein pro-SIRP?.In tumor microenvironment,pro-SIRP? can be selectively activated by urokinase-type plasminogen activator(uPA)and can competitively bind CD47 and effectively inhibit tumor development.The competitive ELISA assay indicated the blocking peptide of pro-SIRP? had the sealing effect.As a result of the existence of the blocking peptide,the binding capacity ofpro-SIRP? to CD47 had a significant decrease.But uPA is rich in tumor microenvironment,which could hydrolyze the linker of pro-SIRP? that made the blocking peptide dissociate and expose SIRP?-cv1,which recovered the binding activity to CD47.Therefore,pro-SIRP? could have specific effects on tumor cells,decreases non-specific binding with normal blood cells,and reduce haematotoxicity and the possibility of antigen sink effect.Our in vitro study showed that,compared with EGFR-IgG1 antibody,pro-SIRP? in combination with EGFR-IgG1 antibody could significantly enhance the phagocytosis of macrophage to A431 cells.The phagocytosis potency and excellent target selectivity of pro-SIRP? were evidenced by our data,suggesting the potential use of the novel protein in tumor targeting therapy.