Proteolytically cleavable fusion proteins with high molar specific activity

Abstract

The invention relates to therapeutic fusion proteins in which a coagulation factor is fused to a half-life enhancing polypeptide, and in which both are connected by a linker peptide that is proteolytically cleavable. The cleavage of such linkers liberates the coagulation factor from activity-compromising steric hindrance caused by the half-life enhancing polypeptide and thereby allows the generation of fusion proteins may show relatively high molar specific activity when tested in coagulation-related assays. Furthermore, the fact that the linker is cleavable can enhance the rates of inactivation and/or elimination after proteolytic cleavage of the peptide linker compared to the rates measured for corresponding therapeutic fusion proteins linked by the non-cleavable linker having the amino acid sequence GGGGGGV.

1 - 33 . (canceled) 
     
     
         34 . A method of administering an effective amount of a coagulation factor fusion protein to a patient in need thereof, wherein the coagulation factor fusion protein comprises:
 a) a von Willebrand Factor;   b) a half-life enhancing polypeptide (HLEP), and   c) a peptide linker which joins the von Willebrand Factor and the half-life enhancing polypeptide;   wherein the HLEP is an immunoglobulin without an antigen binding domain,   wherein the peptide linker is cleavable by one or more proteases involved in coagulation or activated by coagulation enzymes, and   wherein the peptide linker is cleaved during a coagulatory event; and   wherein the patient suffers from a blood coagulation disorder.   
     
     
         35 . The method of  claim 34 , wherein said fusion protein has at least one of the following properties, in comparison to the respective therapeutic fusion protein linked by a non-cleavable linker having the amino acid sequence GGGGGGV (SEQ ID NO: 94):
 i) an increased molar specific activity in at least one coagulation-related assay,   ii) an increased inactivation rate of the activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulation-related mode, and   iii) an increased elimination rate after the peptide linker is proteolytically cleaved in a coagulation-mode.   
     
     
         36 . The method of  claim 34 , wherein said fusion protein has a higher in vivo recovery compared to the in vivo recovery of a von Willebrand Factor when not fused to a half-life enhancing polypeptide. 
     
     
         37 . The method of  claim 34 , wherein said fusion protein has an increased half-life in plasma compared to the half-life in plasma of a von Willebrand Factor when not fused to a half-life enhancing polypeptide. 
     
     
         38 . The method of  claim 34 , wherein the molar specific activity of the fusion protein is increased at least 25% compared to that of the respective fusion protein linked by a non-cleavable linker consisting of the amino acid sequence GGGGGGV (SEQ ID NO: 94) in at least one coagulation-related assays. 
     
     
         39 . The method of  claim 34 , wherein the inactivation rate of the von Willebrand Factor after cleavage of the peptide linker which links the von Willebrand Factor to the half-life enhancing polypeptide is increased at least 10% as compared to the inactivation rate of a von Willebrand Factor in a respective fusion protein linked by a non-cleavable linker consisting of the amino acid sequence GGGGGGV (SEQ ID NO: 94). 
     
     
         40 . The method of  claim 34 , wherein the elimination rate of the von Willebrand Factor after cleavage of the peptide linker which links the von Willebrand Factor to the half-life enhancing polypeptide is increased by at least 10% as compared to the elimination rate of a von Willebrand Factor in a respective fusion protein linked by a non-cleavable linker consisting of the amino acid sequence GGGGGGV (SEQ ID NO: 94). 
     
     
         41 . The method of  claim 34 , wherein the peptide linker is cleavable by a protease that naturally activates FVII or FVIII in vivo. 
     
     
         42 . The method of  claim 34 , wherein the kinetics of linker cleavage by the protease is not delayed by more than a factor of 3 compared to the activation kinetics of the coagulation factor. 
     
     
         43 . The method of  claim 34 , wherein the linker is cleaved by thrombin during coagulation. 
     
     
         44 . The method of  claim 34 , wherein the blood coagulation disorder is hemophilia A. 
     
     
         45 . The method of  claim 34 , wherein the peptide linker is cleavable by a protease that is activated directly or indirectly by a coagulation factor that is activated during a coagulation event. 
     
     
         46 . A method of administering an effective amount of a coagulation factor fusion protein to a patient in need thereof, comprising:
 (a) administering a composition comprising said coagulation factor fusion protein,   (b) administering a composition comprising a polynucleotide encoding said coagulation factor fusion protein via a gene therapy protocol, or   (c) administering a composition comprising a plasmid or vector comprising a polynucleotide encoding said coagulation factor fusion protein via a gene therapy protocol,   wherein the coagulation factor fusion protein comprises:   a) a von Willebrand Factor;   b) a half-life enhancing polypeptide (HLEP), and   c) a peptide linker which joins the von Willebrand Factor and the half-life enhancing polypeptide;   wherein the HLEP is an immunoglobulin without an antigen binding domain,   wherein the peptide linker is cleavable by one or more proteases involved in coagulation or activated by coagulation enzymes, and   wherein the peptide linker is cleaved during coagulation; and   wherein the patient suffers from a blood coagulation disorder.   
     
     
         47 . The method of  claim 46 , wherein the administration comprises administering via a gene therapy protocol
 (a) a composition comprising a polynucleotide encoding said coagulation factor fusion protein or   (b) a composition comprising a plasmid or vector comprising a polynucleotide encoding said coagulation factor fusion protein.   
     
     
         48 . The method of  claim 46 , comprising administering a composition comprising said fusion protein. 
     
     
         49 . The method of  claim 34 , wherein the von Willebrand Factor or the immunoglobulin comprises a sequence that is 95% identical to the sequence of a wild-type human von Willebrand Factor or a wild-type human immunoglobulin, respectively. 
     
     
         50 . The method of  claim 34 , wherein the von Willebrand Factor or the immunoglobulin comprises a sequence that is identical to the sequence of a wild-type human von Willebrand Factor or a wild-type human immunoglobulin respectively.