Novel recombinant bi-functional fusion protein and preparation and application therefor

Abstract

The present invention provides a recombinant fusion protein containing a first extracellular Ig-like domain of a signal-regulator protein alpha (SIRPα), linked to an Fc fragment of a human IgG1. The present invention also provides a polynucleotide encoding the recombinant fusion protein, an expression vector containing the polynucleotide, a method for producing the recombinant protein and a method for treating a disease caused by over expression of CD47.

What is claimed is: 
     
         1 . A recombinant bi-functional fusion protein, comprising an extracellular Ig-like domain of a signal-regulator protein (SIRP) of SIRPα (SIRPαD1), linked to an Fc region of an immunoglobulin comprising both CH2 and CH3 regions of the immunoglobulin, wherein:
 the SIRPαD1 comprises a mutation, wherein:
 the mutation corresponds to N to A at position 89 in full length SIRPα set forth in SEQ ID NO.: 2; and 
 
 the recombinant bi-functional fusion protein can bind to CD47 and Fcγ Receptor, thereby blocking the binding of CD47 to the SIRP on surfaces of macrophages and stimulating phagocytosis of tumor cells by the macrophages. 
 
     
     
         2 . The recombinant bi-functional fusion protein of  claim 1 , wherein the Fc fragment is an Fc fragment of IgG1. 
     
     
         3 . The recombinant bi-functional fusion protein of  claim 2 , wherein the IgG1 is a human IgG1. 
     
     
         4 . The recombinant bi-functional fusion protein of  claim 3 , wherein the Fc region comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 6 from residue 143 to residue 374. 
     
     
         5 . The recombinant bi-functional fusion protein of  claim 3 , wherein the Fc region comprises an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 6 from residue 143 to residue 374. 
     
     
         6 . A homodimer of the recombinant bi-functional fusion protein of  claim 1 , wherein the homodimer comprises two fusion proteins linked by one or more disulfide bonds. 
     
     
         7 . The recombinant bi-functional fusion protein of  claim 1 , comprising an amino acid sequence having at least 90%, identity to the amino acid sequence set forth in SEQ ID NO.: 6. 
     
     
         8 . The recombinant bi-functional fusion protein of  claim 1 , comprising an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO.: 6. 
     
     
         9 . A polynucleotide encoding the recombinant bi-functional fusion protein of  claim 1 . 
     
     
         10 . A polynucleotide encoding the recombinant bi-functional fusion protein of  claim 8 . 
     
     
         11 . An expression vector comprising the polynucleotide of  claim 9 . 
     
     
         12 . A host cell comprising and expressing the polynucleotide of  claim 9 . 
     
     
         13 . A host cell comprising the expression vector of  claim 11 . 
     
     
         14 . A pharmaceutical composition, comprising an effective amount of the recombinant bi-functional fusion protein of  claim 1  for treating a disease caused by over expression of Cluster of Differentiation 47 (CD47), and at least one pharmaceutically-acceptable excipient, wherein the recombinant bi-functional fusion protein can bind to CD47 and Fcγ Receptor, thereby blocking the binding of CD47 to the SIRP on the cell surface of macrophages and stimulating phagocytosis of tumor cells by macrophages. 
     
     
         15 . A method for treating a disease caused by over-expression of CD47, comprising administering to a patient or a subject a therapeutically effective amount of the pharmaceutical composition of  claim 14 . 
     
     
         16 . The method of  claim 15 , wherein the disease is selected from the group consisting of acute myelocytic leukemia (AML), chronic myelocytic leukemia (C1v1L), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, and renal cell carcinoma. 
     
     
         17 . The method of  claim 15 , wherein the disease is selected from the group consisting of Crohn's disease, allergic asthma and rheumatoid arthritis. 
     
     
         18 . A method of preparing a recombinant bi-functional fusion protein, comprising an extracellular Ig-like domain of a signal-regulator protein (SIRP) of SIRPα (SIRPαD1), linked to an Fc region of an immunoglobulin comprising both CH2 and CH3 regions of the immunoglobulin, wherein:
 the SIRPαD1 comprises a mutation, wherein:
 the mutation corresponds to N to A at position 89 in full length SIRPα set forth in SEQ ID NO.: 2; and 
 
 the recombinant bi-functional fusion protein can bind to CD47 and Fcγ Receptor, thereby blocking the binding of CD47 to the SIRP on surfaces of macrophages and stimulating phagocytosis of tumor cells by the macrophages; 
 
       said method comprising cultivating host cells of  claim 13  to express the protein and purifying the protein. 
     
     
         19 . A host cell comprising an expression vector comprising the polynucleotide of  claim 8 ; wherein said host cell expresses the polynucleotide. 
     
     
         20 . A method of preparing a recombinant bi-functional fusion protein, comprising an extracellular Ig-like domain of a signal-regulator protein (SIRP) of SIRPα (SIRPαD1), linked to an Fc region of an immunoglobulin comprising both CH2 and CH3 regions of the immunoglobulin, wherein:
 the SIRPαD1 comprises a mutation, wherein:
 the mutation corresponds to N to A at position 89 in full length SIRPα set forth in SEQ ID NO.: 2; and 
 
 the recombinant bi-functional fusion protein can bind to CD47 and Fcγ Receptor, thereby blocking the binding of CD47 to the SIRP on surfaces of macrophages and stimulating phagocytosis of tumor cells by the macrophages; 
 
       said method comprising cultivating host cells of  claim 19  to express the protein and purifying the protein.