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 . A fusion protein comprising:
 a) a coagulation factor,   b) a half-life enhancing polypeptide (HLEP), and   c) a peptide linker which joins the coagulation 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 proteases involved in coagulation or activated by coagulation enzymes, and wherein the fusion protein has, in comparison to a respective fusion protein linked by a non-cleavable linker having the amino acid sequence GGGGGGV (SEQ ID NO: 94), at least one of the following properties:   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 of the activated coagulation factor after the peptide linker is proteolytically cleaved in a coagulation-related mode.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The fusion protein of  claim 1 , wherein said fusion protein has a higher in vivo recovery compared to the in vivo recovery of the coagulation factor when it is not fused to a half-life enhancing polylpeptide. 
     
     
         5 . The fusion protein of  claim 1 , wherein said fusion protein has an increased half-life in plasma compared to the half-life in plasma of the coagulation factor when it is not fused to a half-life enhancing polylpeptide. 
     
     
         6 . The fusion protein of  claim 1  wherein the coagulation factor is a vitamin-K dependent coagulation factor. 
     
     
         7 . The fusion protein according of  claim 1  wherein the coagulation factor is FVIIa or FIX. 
     
     
         8 . The fusion protein of  claim 1  wherein the half-life enhancing polypeptide is an immunoglobulin without an antigen binding domain. 
     
     
         9 . The fusion protein of  claim 1  wherein the peptide linker is cleavable by FXIa and/or FVIIa/TF. 
     
     
         10 . The fusion protein of  claim 1 , 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 having the amino acid sequence GGGGGGV (SEQ ID NO: 94) in at least one coagulation-related assay. 
     
     
         11 . The fusion protein of  claim 1 , wherein the inactivation rate of the coagulation factor after cleavage of the peptide linker which links the coagulation factor to the half-life enhancing polypeptide is increased by at least 10% as compared to the inactivation rate of the coagulation factor in the corresponding fusion protein linked by a non-cleavable linker having the amino acid sequence GGGGGGV (SEQ ID NO: 94). 
     
     
         12 . The fusion protein of  claim 1 , wherein the elimination rate of the coagulation factor after cleavage of the peptide linker which links the coagulation factor to the half-life enhancing polypeptide is increased by at least 10% as compared to the elimination rate of the coagulation factor in the corresponding fusion protein linked by a non-cleavable linker having the amino acid sequence GGGGGGV (SEQ ID NO: 94). 
     
     
         13 . The fusion protein of  claim 1 , wherein the linker is cleavable by a protease that naturally activates the coagulation factor in vivo. 
     
     
         14 . The fusion protein of  claim 13 , wherein the kinetics of the linker cleavage by the protease is not delayed by more than a factor of 3 compared to the kinetics of the activation of said coagulation factor. 
     
     
         15 . The fusion protein of  claim 1 , wherein the linker is cleavable by a protease that is naturally activated in vivo by the coagulation factor. 
     
     
         16 . The fusion protein of  claim 1 , wherein the linker is cleavable by FXIa and/or by FVIIa/TF, and wherein the coagulation factor is FIX. 
     
     
         17 . The fusion protein of  claim 1  wherein the linker is cleavable by FXa and/or FVIIa/TF, and wherein the coagulation factor is FVIIa. 
     
     
         18 . The fusion protein of  claim 1 , wherein the linker comprises SEQ ID NO:113 or SEQ ID NO:114. 
     
     
         19 . A polynucleotide encoding the fusion protein of  claim 1 . 
     
     
         20 . A plasmid or vector comprising the polynucleotide of  claim 19 . 
     
     
         21 . A plasmid or vector according to  claim 20 , which is an expression vector. 
     
     
         22 . A plasmid or vector according to  claim 20 , wherein the vector is a transfer vector for use in human gene therapy. 
     
     
         23 . A host cell comprising a polynucleotide according to  claim 19 . 
     
     
         24 . A method of producing a fusion protein of  claim 1 , comprising culturing host cells comprising a polynucleotide encoding the fusion protein under conditions such that the fusion protein is expressed. 
     
     
         25 . A pharmaceutical composition comprising a
 (a) the fusion protein of  claim 1 ,   (b) a polynucleotide encoding said fusion protein, or   (c) a plasmid or vector comprising a polynucleotide encoding said fusion protein.   
     
     
         26 . A method of administering an effective amount of the fusion protein of  claim 1  to a patient in need thereof, comprising
 (a) administering said fusion protein, or 
 (b) administering a composition comprising a polynucleotide encoding said fusion protein via a gene therapy protocol or 
 (c) administering a composition comprising a plasmid or vector comprising a polynucleotide encoding said fusion protein via a gene therapy protocol. 
 
     
     
         27 . The method of  claim 26 , wherein the patient suffers from a blood coagulation disorder. 
     
     
         28 . The method of  claim 27 , wherein the blood coagulation disorder is hemophilia B. 
     
     
         29 . The method of  claim 27 , wherein the blood coagulation disorder is FVII and/or FVIIa deficiency. 
     
     
         30 . The method according to  claim 27 , wherein the blood coagulation disorder is hemophilia A. 
     
     
         31 . The method of  claim 27 , wherein the administration comprises administering via a gene therapy protocol
 (a) a composition comprising a polynucleotide encoding said fusion protein or   (b) a composition comprising a plasmid or vector comprising a polynucleotide encoding said fusion protein.   
     
     
         32 . The method according to  claim 27 , wherein the fusion protein is effective to act in the patient as a procoagulant. 
     
     
         33 . The fusion protein of  claim 1 , wherein the coagulation factor, albumin, or immunoglobulin comprises a sequence that is 95% identical to the sequence of a wild-type human coagulation factor, human serum albumin, or a wild-type human immunoglobulin. 
     
     
         34 . The method of  claim 24 , further comprising recovering the fusion protein from the host cells or from the culture medium. 
     
     
         35 . The method of  claim 27 , wherein the administration comprises administering a composition comprising the fusion protein of  claim 1 .