Chimeric factor viii polypeptides and uses thereof

Abstract

The present invention provides a VWF fragment comprising the D′ domain and D3 domain of VWF, a chimeric protein comprising the VWF fragment and a heterologous moiety, or a chimeric protein comprising the VWF fragment and a FVIII protein and methods of using the same. A polypeptide chain comprising a VWF fragment of the invention binds to or is associated with a polypeptide chain comprising a FVIII protein and the polypeptide chain comprising the VWF fragment can prevent or inhibit binding of endogenous VWF to the FVIII protein. By preventing or inhibiting binding of endogenous VWF to the FVIII, which is a half-life limiting factor for FVIII, the VWF fragment can induce extension of half-life of the FVIII protein. The invention also includes nucleotides, vectors, host cells, methods of using the VWF fragment, or the chimeric proteins.

1 - 147 . (canceled) 
     
     
         148 . A chimeric protein comprising a first polypeptide and a second polypeptide,
 wherein the first polypeptide comprises:
 (a) a Factor VIII (“FVIII”) protein comprising amino acid residues 1 to 740 of SEQ ID NO: 16 and a B-domain deletion; and 
 (b) a first immunoglobulin constant region, 
   wherein the second polypeptide comprises:
 (a) a von Willebrand Factor (VWF) fragment comprising a D′ and a D3 domain of VWF, wherein the VWF fragment comprises an alanine substitution for cysteine at residues corresponding to residue 1099 and residue 1142 of SEQ ID NO: 2; 
 (b) a second immunoglobulin constant region; and 
 (c) a cleavable linker located between the VWF fragment and the second immunoglobulin constant region, 
   wherein the first polypeptide and the second polypeptide are linked by a disulfide bond between the first and second immunoglobulin constant regions or the portions thereof.   
     
     
         149 . A polynucleotide encoding a chimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises:
 (a) a Factor VIII (“FVIII”) protein comprising amino acid residues 1 to 740 of SEQ ID NO: 16 and a B-domain deletion; and   (b) a first immunoglobulin constant region,   
       wherein the second polypeptide comprises:
 (a) a von Willebrand Factor (VWF) fragment comprising a D′ and a D3 domain of VWF, wherein the VWF fragment comprises an alanine substitution for cysteine at residues corresponding to residue 1099 and residue 1142 of SEQ ID NO: 2; 
 (b) a second immunoglobulin constant region; and 
 (c) a cleavable linker located between the VWF fragment and the second immunoglobulin constant region, 
 
       wherein the first polypeptide and the second polypeptide are linked by a disulfide bond between the first and second immunoglobulin constant regions or the portions thereof. 
     
     
         150 . A host cell comprising a polynucleotide encoding a chimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises:
 (a) a Factor VIII (“FVIII”) protein comprising amino acid residues 1 to 740 of SEQ ID NO: 16 and a B-domain deletion; and   (b) a first immunoglobulin constant region,   
       wherein the second polypeptide comprises:
 (a) a von Willebrand Factor (VWF) fragment comprising a D′ and a D3 domain of VWF, wherein the VWF fragment comprises an alanine substitution for cysteine at residues corresponding to residue 1099 and residue 1142 of SEQ ID NO: 2; 
 (b) a second immunoglobulin constant region; and 
 (c) a cleavable linker located between the VWF fragment and the second immunoglobulin constant region, 
 
       wherein the first polypeptide and the second polypeptide are linked by a disulfide bond between the first and second immunoglobulin constant regions or the portions thereof. 
     
     
         151 . A method of treating hemophilia A in a subject in need thereof comprising administering an effective amount of a chimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises:
 (a) a Factor VIII (“FVIII”) protein comprising amino acid residues 1 to 740 of SEQ ID NO: 16 and a B-domain deletion; and   (b) a first immunoglobulin constant region,   
       wherein the second polypeptide comprises:
 (a) a von Willebrand Factor (VWF) fragment comprising a D′ and a D3 domain of VWF, wherein the VWF fragment comprises an alanine substitution for cysteine at residues corresponding to residue 1099 and residue 1142 of SEQ ID NO: 2; 
 (b) a second immunoglobulin constant region; and 
 (c) a cleavable linker located between the VWF fragment and the second immunoglobulin constant region, 
 
       wherein the first polypeptide and the second polypeptide are linked by a disulfide bond between the first and second immunoglobulin constant regions or the portions thereof. 
     
     
         152 . The method of  claim 151 , wherein the chimeric protein is administered intravenously. 
     
     
         153 . A method of treating von Willebrand's disease (VWD) in a subject in need thereof comprising administering an effective amount of a chimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises:
 (a) a Factor VIII (“FVIII”) protein comprising amino acid residues 1 to 740 of SEQ ID NO: 16 and a B-domain deletion; and   (b) a first immunoglobulin constant region,   
       wherein the second polypeptide comprises:
 (a) a von Willebrand Factor (VWF) fragment comprising a D′ and a D3 domain of VWF, wherein the VWF fragment comprises an alanine substitution for cysteine at residues corresponding to residue 1099 and residue 1142 of SEQ ID NO: 2; 
 (b) a second immunoglobulin constant region; and 
 (c) a cleavable linker located between the VWF fragment and the second immunoglobulin constant region, 
 
       wherein the first polypeptide and the second polypeptide are linked by a disulfide bond between the first and second immunoglobulin constant regions or the portions thereof. 
     
     
         154 . The method of  claim 153 , wherein the chimeric protein is administered intravenously. 
     
     
         155 . The method of  claim 153 , wherein the VWD is Type 2N VWD.