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) Factor VII (FVII), Factor VIIa (FVIIa), or Factor IX (FIX);   b) a half-life enhancing polypeptide (HLEP), and   c) a peptide linker which joins the FVII, FVIIa, or FIX and the half-life enhancing polypeptide;   wherein the HELP is an immunoglobulin without an antigen binding domain, wherein the peptide linker is cleavable by one or more proteases that can activate or cleave FVII, FVIIa, or FIX during coagulation, and wherein the fusion protein has at least one of the following properties, in comparison to the respective fusion protein linked by a non-cleavable linker consisting of 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 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-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 FVII, FVIIa, or FIX when not fused to a half-life enhancing polypeptide. 
     
     
         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 FVII, FVIIa, or FIX when not fused to a half-life enhancing polypeptide. 
     
     
         6 . (canceled) 
     
     
         7 . The fusion protein of  claim 1 , wherein the fusion protein comprises FIX. 
     
     
         8 . (canceled) 
     
     
         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 consisting of the amino acid sequence GGGGGGV (SEQ ID NO: 94) in at least one coagulation-related assays. 
     
     
         11 . The fusion protein of  claim 1 , wherein the inactivation rate of FVII, FVIIa, or FIX after cleavage of the peptide linker which links the FVII, FVIIa, or FIX to the half-life enhancing polypeptide is increased at least 10% as compared to the inactivation rate of FVII, FVIIa, or FIX in the respective fusion protein linked by a non-cleavable linker consisting of the amino acid sequence GGGGGGV (SEQ ID NO: 94). 
     
     
         12 . The fusion protein of  claim 1 , wherein the elimination rate of FVII, FVIIa, or FIX after cleavage of the peptide linker which links the FVII, FVIIa, or FIX to the half-life enhancing polypeptide is increased by at least 10% as compared to the elimination rate of FVII, FVIIa, or FIX in the respective fusion protein linked by a non-cleavable linker consisting of the amino acid sequence GGGGGGV (SEQ ID NO: 94). 
     
     
         13 . The fusion protein of  claim 1 , wherein the peptide linker is cleavable by a proteases that naturally activates FVII, FVIIa, or FIX 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 the FVII, FVIIa, or FIX. 
     
     
         15 . (canceled) 
     
     
         16 . The fusion protein of  claim 1 , wherein the peptide linker is cleavable by FXIa and/or by FVIIa/TF, and wherein the fusion protein comprises FIX. 
     
     
         17 . The fusion protein of  claim 1 , wherein the linker is cleavable by FXIa and/or by FVIIa/TF, and wherein the fusion protein comprises 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 . The plasmid or vector of  claim 20 , wherein the plasmid or vector is an expression vector. 
     
     
         22 . The plasmid or vector of  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) 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, 
 (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 of  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 FVII, FVIIa, or FIX or immunoglobulin, comprises a sequence that is 95% identical to the sequence of a wild-type human FVII, FVIIa, or FIX or a wild-type human immunoglobulin, respectively. 
     
     
         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 . 
     
     
         36 . The fusion protein of  claim 1 , wherein the linker is cleavable by FVII, FVIIa, or FIX during coagulation. 
     
     
         37 . The fusion protein of  claim 1 , wherein the linker comprises a sequence from FVII, FVIIa, or FIX. 
     
     
         38 . The fusion protein of  claim 1 , wherein the linker is capable of being cleaved simultaneously with the activation of FVII, FVIIa, or FIX, or with comparable kinetics, during coagulation. 
     
     
         39 . The fusion protein of  claim 1 , wherein the fusion protein comprises FVIIa.