Clotting factor-Fc chimeric proteins to treat hemophilia

Abstract

The invention relates to a chimeric protein comprising at least one clotting factor and at least a portion of an immunoglobulin constant region. The invention relates to a method of treating a hemostatic disorder comprising administering a therapeutically effective amount of a chimeric protein wherein the chimeric protein comprises at least one clotting factor and at least a portion of an immunoglobulin constant region.

1 . A chimeric protein comprising at least a portion of an immunoglobulin constant region, at least one clotting factor, and optionally at least one linker.  
     
     
         2 . The chimeric protein of  claim 1 , wherein said immunoglobulin is an IgG.  
     
     
         3 . The chimeric protein of  claim 2 , wherein the portion of an immunoglobulin constant region is an Fc fragment.  
     
     
         4 . The chimeric protein of  claim 2 , wherein the portion of an immunoglobulin constant region is an FcRn binding partner.  
     
     
         5 . The chimeric protein of  claim 2 , wherein the Fc fragment comprises an amino acid sequence having at least 80% identity with the sequence set forth in SEQ ID NO:3.  
     
     
         6 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , wherein the clotting factor is factor VII or VIIa.  
     
     
         7 . The chimeric protein of  claim 6 , wherein the clotting factor is an analog of factor VII or factor VIIa.  
     
     
         8 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , wherein the clotting factor is factor VIII, or factor VIIIa, factor V, factor Va, factor IX, factor X, factor XI, factor XII, factor XIII or von Willebrand's factor.  
     
     
         9 . A pharmaceutical composition comprising the chimeric protein of  claim 6 , and pharmaceutically acceptable excipient.  
     
     
         10 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , wherein said chimeric protein is a dimer.  
     
     
         11 . The chimeric protein of  claim 10 , wherein the dimer is a homodimer.  
     
     
         12 . The chimeric protein of  claim 10 , wherein the chimeric protein is a dimer comprising a first chain and a second chain, wherein said first chain comprises an Fc fragment of an immunoglobulin and Factor VII or Factor VIIa and said second chain comprises an Fc fragment of an immunoglobulin without factor VII or factor VIIa.  
     
     
         13 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , comprising the formula  
         X-L-F  
       wherein F is an Fc fragment of an immunoglobulin and L is a linker or a direct bond, and X is factor VII, or factor VIIa.  
     
     
         14 . The chimeric protein of  claim 13 , wherein F comprises an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO:2.  
     
     
         15 . The chimeric protein of  claim 13 , wherein L is a linker comprising about 1 to about 20 amino acids.  
     
     
         16 . The chimeric protein of  claim 13 , wherein L is a linker comprising about 1 to about 10 amino acids.  
     
     
         17 . The chimeric protein of  claim 13 , wherein L is a linker comprising about 1 to about 5 amino acids.  
     
     
         18 . The chimeric protein of  claim 13 , wherein L is a linker comprising the sequence -(Gly) n - (SEQ ID NO: 30), wherein n is an integer of about 1 to about 10.  
     
     
         19 . The chimeric protein of  claim 13 , wherein L is a linker comprising the sequence -(GGS) n - (SEQ ID NO: 5) or (GGGGS) n  (SEQ ID NO: 31), wherein n is an integer of about 1 to about 10.  
     
     
         20 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , wherein the protein comprises SEQ ID NO:1.  
     
     
         21 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , wherein the chimeric protein promotes hemostasis.  
     
     
         22 . A method of treating a hemostatic disorder comprising administering a therapeutically effective amount of at least one chimeric protein having clotting activity to a subject, wherein said chimeric protein comprises at least a portion of an immunoglobulin and a clotting factor, and allowing the chimeric protein to treat the hemostatic disorder.  
     
     
         23 . The method of  claim 22 , wherein the hemostatic disorder is hemophilia A or B.  
     
     
         24 . The method of  claim 22 , wherein the portion of an immunoglobulin constant region is an Fc fragment.  
     
     
         25 . The method of  claim 22 , wherein the clotting factor is factor VII or factor VIIa.  
     
     
         26 . The method of  claim 24 , wherein said portion of an immunoglobulin constant region is an FcRn binding partner.  
     
     
         27 . The method of  claim 22 ,  24 , or  26 , wherein the chimeric protein is administered intravenously, subcutaneously, intra-muscularly, or via a mucosal surface.  
     
     
         28 . The method of  claim 27 , wherein the chimeric protein is administered orally, nasally, rectally, vaginally, or via buccal administration.  
     
     
         29 . The method of  claim 27 , wherein the chimeric protein is administered via a pulmonary route.  
     
     
         30 . The method of  claim 22 , wherein the chimeric protein is administered non-parenterally.  
     
     
         31 . The method of  claim 22 , wherein the chimeric protein is administered at a dose of 0.1-1000 μg/kg.  
     
     
         32 . The method of  claim 22 , wherein the chimeric protein is administered daily.  
     
     
         33 . A method of making a chimeric protein having clotting activity comprising at least a portion of an immunoglobulin constant region and a clotting factor, said method comprising: 
 a) transfecting a cell with a DNA construct comprising a first DNA sequence encoding at least a portion of an immunoglobulin constant region operatively linked to a second DNA sequence encoding a clotting factor and optionally a third DNA sequence encoding an endoprotease;    b) culturing said cell under conditions such that the chimeric protein is expressed; and    c) isolating said chimeric protein from said cell.    
     
     
         34 . The method of  claim 33 , wherein the culture conditions include vitamin K.  
     
     
         35 . The method of  claim 34 , wherein the endoprotease is PACE.  
     
     
         36 . The method of  claim 33 , wherein the cell is a eukaryotic cell.  
     
     
         37 . The method of  claim 36 , wherein the cell is a CHO cell or a BHK cell.  
     
     
         38 . The method of  claim 33  further comprising transfecting a cell with a second DNA construct comprising a DNA sequence encoding at least a portion of an immunoglobulin constant region without said second sequence encoding a clotting factor.  
     
     
         39 . The method of  claim 33  or  38 , wherein said portion of an immunoglobulin constant region is an Fc fragment.  
     
     
         40 . The method of  claim 33  or  38 , wherein said portion of an immunoglobulin constant region is an IgG Fc fragment.  
     
     
         41 . The method of  claim 33  or  38 , wherein said portion of an immunoglobulin constant region is FcRn binding partner.  
     
     
         42 . The method of  claim 33  or  38 , wherein the clotting factor is factor VII or factor VIIa.  
     
     
         43 . A nucleic acid molecule comprising a nucleic acid sequence encoding at least one clotting factor, and at least a portion of an immunoglobulin constant region.  
     
     
         44 . A vector comprising the nucleic acid molecule of  claim 43 .  
     
     
         45 . The nucleic acid molecule of  claim 43 , wherein said portion of an immunoglobulin constant region is an Fc fragment.  
     
     
         46 . The nucleic acid molecule of  claim 43 , wherein said portion of an immunoglobulin constant region is an FcRn binding partner.  
     
     
         47 . The vector of  claim 43 , wherein said portion of an immunoglobulin constant region is a portion of an IgG.  
     
     
         48 . The vector of  claim 43 , wherein the clotting factor is factor VII or factor VIIa.  
     
     
         49 . A host cell comprising the vector of  claim 44 .  
     
     
         50 . The host cell of  claim 49 , wherein said host cell is a CHO cell or a BHK cell.  
     
     
         51 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , comprising a protein encoded by the DNA sequence of SEQ ID NO:1.  
     
     
         52 . The chimeric protein of  claim 1 ,  2 ,  3 , or  4 , comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         53 . The vector of  claim 48 , comprising the DNA sequence of SEQ ID NO:2.  
     
     
         54 . The vector of  claim 48 , comprising the DNA sequence of SEQ ID NO:3.  
     
     
         55 . A method of treating a hemostatic disorder comprising administering to a subject a therapeutically effective amount of a first agent to treat a hemostatic disorder, wherein said first agent comprises at least one chimeric protein having clotting activity, and wherein said chimeric protein comprises at least a portion of an immunoglobulin and a clotting factor, in combination with a second agent, wherein said second agent is any agent known to have clotting factor activity and allowing the first and second agent to treat the hemostatic disorder.  
     
     
         56 . The method of  claim 55  wherein said second agent is factor VIII, factor IX, factor XI, factor XII, fibrinogen, prothrombin, factor V, factor VII, factor X, factor XIII or von Willebrand factor.  
     
     
         57 . The chimeric protein of  claim 10 , wherein the chimeric protein is a dimer comprising a first chain and a second chain, wherein said first chain comprises an Fc fragment of an immunoglobulin and Factor VIII and said second chain comprises an Fc fragment of an immunoglobulin without factor VIII.  
     
     
         58 . The chimeric protein of  claim 10 , wherein the chimeric protein is a dimer comprising a first chain and a second chain, wherein said first chain comprises an Fc fragment of an immunoglobulin and Factor IX and said second chain comprises an Fc fragment of an immunoglobulin without factor IX.