Covalent complex of von willebrand factor and factor viii, compositions, and uses relating thereto

Abstract

The present invention relates to a covalent complex of von Willebrand Factor (VWF) and Factor VIII, wherein the complex is modified such that it has an extended half-life in vivo. The invention further relates to a method of producing the complex, as well as the therapeutic or prophylactic use of the complex for treating or preventing bleeding events.

1 . A complex comprising a von Willebrand factor (VWF) covalently linked to a Factor VIII (FVIII), wherein the complex comprises a half-life extending moiety, and wherein the FVIII is modified by substitution of a naturally occurring amino acid with a cysteine residue or insertion of a cysteine residue which forms a disulphide bridge with a cysteine residue in VWF. 
     
     
         2 . The complex of  claim 1 , wherein the covalent link is not provided by the half-life extending moiety. 
     
     
         3 .- 4 . (canceled) 
     
     
         5 . The complex of  claim 1 , wherein the naturally occurring amino acid in FVIII that is substituted is selected from an amino acid in the FVIII a3 domain. 
     
     
         6 . The complex of  claim 1 , wherein the naturally occurring amino acid in FVIII that is substituted is located within amino acids 1653 to 1660 or within amino acids 1667 to 1674 or within amino acids 1675 to 1688 of the FVIII a3 domain, or the inserted cysteine is introduced into the sequence of amino acids 1653 to 1660 or amino acids 1667 to 1674 or amino acids 1675 to 1688 of the FVIII a3 domain. 
     
     
         7 . The complex of  claim 1 , wherein the naturally occurring amino acid that is substituted in FVIII is in the C-terminal domain. 
     
     
         8 . The complex of  claim 1 , wherein the VWF is modified by substitution of a naturally occurring amino acid with a cysteine residue or insertion of a cysteine residue which forms the disulphide bridge with the substituted cysteine residue in the FVIII or the cysteine residue inserted into the FVIII. 
     
     
         9 . The complex of  claim 8 , wherein the naturally occurring amino acid in VWF is an amino acid in the D′ or D3 domain, or wherein the inserted cysteine residue in the VWF is in the D′ or D3 domain. 
     
     
         10 . The complex of  claim 8 , wherein the inserted cysteine residue in the VWF is introduced in the TIL′ domain, the E′ domain, the D3 domain, the C8-3 domain, the TIL-3 domain or the E-3 domain of VWF, or the naturally occurring amino acid in the VWF is in the TIL′ domain, the E′ domain, the D3 domain, the C8-3 domain, the TIL-3 domain or the E-3 domain of VWF. 
     
     
         11 . The complex of  claim 1 , wherein the VWF comprises a FVIII binding domain. 
     
     
         12 . The complex of  claim 1 , wherein the FVIII is modified to comprise one or more domains of VWF. 
     
     
         13 . The complex of  claim 12 , wherein the FVIII is modified to comprise the C-terminal domain CK of VWF. 
     
     
         14 . The complex of  claim 13 , wherein the FVIII is modified to further comprise any one or more of VWF domains C1 to C6, or variants thereof. 
     
     
         15 . The complex of  claim 12 , wherein the FVIII comprises amino acids 2724 to 2812 of SEQ ID NO: 2 or a variant thereof, provided that cysteine residue 2773 (or equivalent thereof) is preserved in the variant. 
     
     
         16 . The complex of  claim 13 , wherein the C-terminal VWF domain is attached to FVIII by a cleavable linker. 
     
     
         17 . The complex of  claim 16 , wherein the cleavable linker comprises a thrombin cleavage site of FVIII. 
     
     
         18 . The complex of  claim 16 , wherein the linker sequence comprises additional amino acids providing a peptide of sufficient length to permit an intramolecular interaction of FVIII and VWF via the a3 and D′D3 domains, respectively. 
     
     
         19 . The complex of  claim 12 , wherein the FVIII is modified at its C-terminus or at its N-terminus, or within the B-domain of FVIII, or partially or completely replacing the B-domain of FVIII. 
     
     
         20 . The complex of  claim 12 , wherein the FVIII is modified to comprise the D′D3 domain of VWF or a fragment or a variant thereof. 
     
     
         21 . The complex of  claim 20 , wherein the FVIII is modified by replacing its B domain partially or completely by the VWF D′D3 domain or a fragment or a variant thereof. 
     
     
         22 . The complex of  claim 21 , wherein the FVIII comprises within its B domain or instead of its B domain or instead of part of its B domain amino acids 764 to 1241 of SEQ ID NO: 2. 
     
     
         23 . The complex of  claim 19 , wherein the FVIII is expressed as a two-chain molecule with the VWF D′D3 domain representing the N-terminus of the FVIII light chain. 
     
     
         24 . The complex of  claim 19 , wherein an additional linker sequence is introduced between the D′D3 region of VWF and the FVIII light chain domains. 
     
     
         25 . The complex of  claim 1 , wherein the FVIII is a genetically engineered FVIII. 
     
     
         26 . The complex of  claim 25 , wherein the engineered FVIII has a full or partial B-domain deletion, or is a mutated FVIII comprising one or more amino acid substitutions, insertions, deletions or combinations thereof, or is a fusion polypeptide with a half-life extending moiety. 
     
     
         27 . The complex of  claim 1 , wherein the VWF is a half-life extended form of VWF. 
     
     
         28 . The complex of  claim 27 , wherein the half-life extended form of VWF is a genetically engineered fusion protein of VWF with a half-life extending moiety. 
     
     
         29 . The complex of  claim 27 , wherein the half-life extending moiety is selected from albumin or a variant or fragment thereof, an immunoglobulin constant region or a portion or variant thereof, an Fc fragment or variant thereof, a solvated random chain with large hydrodynamic volume, afamin or a variant thereof, alpha-fetoprotein or a variant thereof, Vitamin D binding protein or a variant thereof, transferrin or a variant thereof, a carboxyl-terminal peptide (CTP) of human chorionic gonadotropin-β subunit, and a polypeptide or lipid capable of binding under physiological conditions to albumin or an immunoglobulin constant region. 
     
     
         30 . The complex of  claim 1 , wherein the plasma half-life of VWF is extended by one or more chemical modifications selected from polyethylene glycol (PEGylation), glycosylated PEG, hydroxyl ethyl starch (HESylation), polysialic acids, heparosan polymers, elastin-like polypeptides, and hyaluronic acid. 
     
     
         31 . The complex of  claim 1 , wherein the VWF is expressed as a monomer or a dimer. 
     
     
         32 . The complex of  claim 1 , wherein the VWF forms a multimer. 
     
     
         33 . The complex of  claim 1 , wherein the covalent link is obtained by one or more chemically synthesized cross-linkers. 
     
     
         34 . The complex of  claim 1 , wherein the FVIII and VWF are connected by more than one covalent disulfide bond or peptide or proteinaceous linker. 
     
     
         35 . A method of producing the complex of  claim 1 , comprising co-expressing the FVIII and VWF in a eukaryotic cell line. 
     
     
         36 . A method for treatment of hemophilia A or von Willebrand disease, comprising administering to a subject in need thereof an effective amount of the complex of  claim 1 . 
     
     
         37 . (canceled) 
     
     
         38 . A pharmaceutical composition comprising the complex of  claim 1 . 
     
     
         39 . The complex of  claim 1 , wherein the FVIII is modified by substitution of a naturally occurring amino acid with a cysteine residue. 
     
     
         40 . The complex of  claim 1 , wherein the FVIII is modified by insertion of a cysteine residue. 
     
     
         41 . A method for reducing or preventing bleeding events in a subject suffering from hemophilia A or von Willebrand disease, comprising administering to the subject an effective amount of the complex of  claim 1 . 
     
     
         42 . The method of  claim 41 , wherein the VWF is modified by substitution of a naturally occurring amino acid with a cysteine residue or insertion of a cysteine residue which forms the disulphide bridge with the substituted cysteine residue in the FVIII or the cysteine residue inserted into the FVIII. 
     
     
         43 . The method of  claim 41 , wherein the VWF comprises a FVIII binding domain. 
     
     
         44 . The method of  claim 41 , wherein the FVIII is modified to comprise one or more domains of VWF. 
     
     
         45 . The method of  claim 41 , wherein the FVIII has a full or partial B-domain deletion, or is a mutated FVIII comprising one or more amino acid substitutions, insertions, deletions or combinations thereof, or is a fusion polypeptide with a half-life extending moiety. 
     
     
         46 . The complex of  claim 45 , wherein the half-life extending moiety is selected from albumin or a variant or fragment thereof, an immunoglobulin constant region or a portion or variant thereof, an Fc fragment or variant thereof, a solvated random chain with large hydrodynamic volume, afamin or a variant thereof, alpha-fetoprotein or a variant thereof, Vitamin D binding protein or a variant thereof, transferrin or a variant thereof, a carboxyl-terminal peptide (CTP) of human chorionic gonadotropin-β subunit, and a polypeptide or lipid capable of binding under physiological conditions to albumin or an immunoglobulin constant region. 
     
     
         47 . The method of  claim 41 , wherein the VWF is fused to a half-life extending moiety. 
     
     
         48 . The method of  claim 47 , wherein the half-life extending moiety is selected from albumin or a variant or fragment thereof, an immunoglobulin constant region or a portion or variant thereof, an Fc fragment or variant thereof, a solvated random chain with large hydrodynamic volume, afamin or a variant thereof, alpha-fetoprotein or a variant thereof, Vitamin D binding protein or a variant thereof, transferrin or a variant thereof, a carboxyl-terminal peptide (CTP) of human chorionic gonadotropin-β subunit, and a polypeptide or lipid capable of binding under physiological conditions to albumin or an immunoglobulin constant region. 
     
     
         49 . The method of  claim 41 , wherein the VWF is expressed as a monomer, a dimer, or a multimer.