Truncated von Willebrand Factor (VWF) for improving the in vitro stability of coagulation factor VIII

Abstract

The present invention relates to the use of a polypeptide comprising a truncated von Willebrand factor (VWF) for improving the in vitro stability of coagulation factor VIII (FVIII) in a composition, wherein the composition comprises FVIII and the polypeptide, and the molar ratio of the polypeptide to FVIII in the composition is greater than 20.

Description

[Technical Field]

[0001] The present invention relates to a method for improving the in vitro stability of coagulation factor VIII (FVIII), and in particular to the use of a polypeptide comprising a truncated von Willebrand factor (VWF) for improving the in vitro stability of coagulation factor VIII (FVIII). [Background technology]

[0002] Factor VIII (FVIII) is a protein found in plasma that acts as a cofactor in the cascade of reactions that result in blood clotting. A deficiency in the amount of FVIII activity in the blood results in a clotting disorder known as hemophilia A, a genetic disease that primarily affects males. Hemophilia A is currently treated with therapeutic preparations of FVIII derived from human plasma or produced using recombinant DNA technology. Generally, these preparations are administered in response to bleeding episodes (on-demand therapy) or frequently and periodically to prevent uncontrollable bleeding (prophylaxis).

[0003] FVIII has been found to be relatively unstable in therapeutic formulations. In plasma, FVIII is typically complexed with another plasma protein, von Willebrand factor (VWF), which is thought to protect FVIII from premature degradation. Current commercially available FVIII preparations often rely on the use of albumin and / or native VWF to stabilize FVIII during the manufacturing process and storage.

[0004] Several attempts have been described to formulate FVIII without albumin or native VWF (or with relatively low levels of such excipients). For example, Patent Document 1 describes a FVIII formulation that contains, in addition to excipients such as sodium chloride and sucrose, a surfactant and a specific combination of amino acids, specifically arginine and glycine.

[0005] Patent Document 2 also describes a therapeutic FVIII formulation containing 15 to 60 mM sucrose, a maximum of 50 mM NaCl, a maximum of 5 mM calcium chloride, 65 to 400 mM glycine, and a maximum of 50 mM histidine.

[0006] Patent Document 3 discloses a formulation containing 0.01 to 1 mg / ml of surfactant. Other attempts using low or high concentrations of sodium chloride have also been described. Patent Document 4 discloses a formulation having a relatively low concentration of sodium chloride, i.e., 0.5 mM to 15 mM NaCl. Meanwhile, Patent Document 5 teaches the use of a formulation having a relatively high concentration of sodium chloride.

[0007] Further FVIII formulations are disclosed in U.S. Patent No. 5,623,299, ...

[0008] It has been reported that VWF-derived polypeptides, particularly VWF fragments, improve the bioavailability of FVIII in vivo. Patent Document 24 teaches chimeric proteins containing FVIII protein and specific VWF fragments. Such chimeric heterodimers of FVIII and VWF fragments certainly have a constant molar ratio of VWF to FVIII of 1:1.

[0009] Patent Documents 25 and 26 describe VWF fragments and their use in the treatment of hemophilia. It has been found that the bioavailability of FVIII can be significantly improved upon extravascular co-administration with an equivalent molar amount of VWF fragment. Patent Documents 27 and 28 describe truncated VWF polypeptides for the treatment or prevention of hemophilia. Patent Documents 29, 30, and 31 describe modified VWF polypeptides capable of binding to FVIII.

[0010] Patent document 32 discloses fusion polypeptides comprising a specific VWF fragment and an antibody Fc region, and proposes a specific molar ratio of VWF fragment to FVIII of up to 10: 1. Additionally, no in vivo data are presented for the Fc fusion constructs.

[0011] Non-Patent Document 1 found that a VWF fragment containing the D'D3 domain fused to the Fc portion of immunoglobulin G1 was sufficient to stabilize endogenous factor VIII in VWF-deficient mice. Thus, in VWF-deficient mice, the endogenous expression rate of FVIII was increased or the clearance rate of endogenously expressed FVIII was reduced. However, when injected into FVIII-deficient mice, the VWF D'D3-Fc fusion protein showed a significant prolongation of survival, whereas the VWF D'D3-Fc fusion protein did not prolong the survival of co-injected FVIII.

[0012] Patent Document 33 describes a composition comprising a complex of FVIII and one or more VWF peptides, where the VWF peptide contains at least amino acids 764-1035 and 1691-1905 of human VWF (UniProtKB-P04275), but does not contain amino acids 2255-2645 of human VWF. A VWF fragment consisting of amino acids 764-2128 of human VWF ("fragment III") was prepared by digestion of plasma-derived VWF with Staphylococcus aureus V-8 protease. This fragment was conjugated to collagen III and heparin. Fragment III, added at a 5-fold molar excess, stabilized rFVIII in solution, calculated based on the fragment III monomer subunit. Patent Document 33 does not demonstrate the stabilizing effect of a VWF peptide lacking amino acids 1691-1905. WO 02 / 04999 does not disclose any ratio of VWF peptides to FVIII greater than 20.

[0013] There is a continuing need for means and methods to stabilize FVIII preparations in vitro. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 5,565,427 (European Patent No. 0508194) [Patent Document 2] U.S. Patent No. 5,763,401 (European Patent No. 0818204) [Patent Document 3] U.S. Patent No. 5,733,873 (European Patent No. 627924) [Patent Document 4] U.S. Patent No. 4,877,608 (European Patent No. 0315968) [Patent Document 5] U.S. Patent No. 5,605,884 (European Patent No. 0314095) [Patent Document 6] International Publication No. 2010 / 054238 [Patent Document 7] European Patent No. 1712223 [Patent Document 8] International Publication No. 2000 / 48635 [Patent Document 9] International Publication No. 96 / 30041 [Patent Document 10] International Publication No. 96 / 22107 [Patent Document 11] International Publication No. 2011 / 027152 [Patent Document 12] European Patent No. 2361613 [Patent Document 13] European Patent No. 0410207 [Patent Document 14] European Patent No. 0511234 [Patent Document 15] European Patent No. 0638091 [Patent Document 16] European Patent No. 0871476 [Patent Document 17] European Patent No. 0819010 [Patent Document 18] U.S. Patent No. 5,874,408 [Patent Document 19] US Patent No. 2005 / 0256038 [Patent Document 20] US Patent No. 2008 / 0064856 [Patent Document 21] International Publication No. 2005 / 058283 [Patent Document 22] International Publication No. 2012 / 037530 [Patent Document 23] International Publication No. 2014 / 026954 [Patent Document 24] International Publication No. 2013 / 106787 A1 [Patent Document 25] International Publication No. 2014 / 198699 A2 [Patent Document 26] International Publication No. 2013 / 083858 A2 [Patent Document 27] International Publication No. 2018 / 087271 A1 [Patent Document 28] International Publication No. 2016 / 188907 A1 [Patent Document 29] International Publication No. 2016 / 000039 A1 [Patent Document 30] International Publication No. 2017 / 117630 A1 [Patent Document 31] International Publication No. 2017 / 117631 A1 [Patent Document 32] International Publication No. 2011 / 060242 A2 [Patent Document 33] International Publication No. 2015 / 185758 A2 [Non-patent literature]

[0015] [Non-Patent Document 1] Yee et al. (2014) Blood 124(3):445-452 Summary of the Invention [Problem to be solved by the invention]

[0016] The inventors of the present application have surprisingly found that VWF fragments comprising the D'D3 domain of VWF improve the in vitro stability of FVIII at molar ratios of VWF fragment to FVIII greater than 20. [Means for solving the problem]

[0017] Therefore, the present invention relates to the objects defined in the following items [1] to

[80] .

[0018] [1] Use of a polypeptide comprising a truncated von Willebrand factor (VWF) for improving the in vitro stability of coagulation factor VIII (FVIII) in a composition, wherein the composition comprises FVIII and the polypeptide, and the molar ratio of the polypeptide to FVIII in the composition is greater than 20.

[0019] [2] The use according to item [1], wherein the polypeptide improves the storage stability of FVIII.

[0020] [3] The use according to item [1] or [2], wherein the composition does not contain proteases.

[0021] [4] The use according to any one of items [1] to [3], wherein the composition does not contain wild-type VWF.

[0022] [5] The use according to any one of items [1] to [4], wherein the FVIII is either recombinantly produced FVIII or plasma-derived FVIII.

[0023] [6] The use according to any one of items [1] to [5], wherein the molar ratio is at least 50.

[0024] [7] The use according to any one of items [1] to [6], wherein the molar ratio is greater than 50.

[0025] [8] The use according to any one of items [1] to [7], wherein the molar ratio is at least 60.

[0026] [9] The use according to any one of items [1] to [8], wherein the molar ratio is at least 75.

[0027]

[10] The use according to any one of items [1] to [9], wherein the molar ratio is at least 100.

[0028]

[11] The use according to any one of items [1] to

[10] , wherein the molar ratio is at least 200.

[0029]

[12] The use according to any one of items [1] to

[11] , wherein the molar ratio is at least 300.

[0030]

[13] The use according to any one of items [1] to

[12] , wherein the molar ratio is at least 400.

[0031]

[14] The use according to any one of items [1] to

[13] , wherein the molar ratio is at least 500.

[0032]

[15] The use according to any one of items [1] to

[14] , wherein the molar ratio is at least 600.

[0033]

[16] The use according to any one of items [1] to

[15] , wherein the molar ratio is at least 700.

[0034]

[17] The use according to any one of items [1] to

[16] , wherein the molar ratio is less than 10,000.

[0035]

[18] The use according to any one of items [1] to

[17] , wherein the molar ratio is greater than 20 and less than 10,000.

[0036]

[19] The use according to any one of items [1] to

[18] , wherein the molar ratio is from about 25 to about 9,000.

[0037]

[20] The use according to any one of items [1] to

[19] , wherein the molar ratio is about 50 to about 7,500.

[0038]

[21] The use according to any one of items [1] to

[20] , wherein the molar ratio is greater than 50 to about 6,000.

[0039]

[22] The use according to any one of items [1] to

[21] , wherein the molar ratio is about 60 to about 5,000.

[0040]

[23] The use according to any one of items [1] to

[22] , wherein the molar ratio is greater than 75 to about 4,000.

[0041]

[24] The use according to any one of items [1] to

[23] , wherein the molar ratio is about 100 to about 3,000.

[0042]

[25] The use according to any one of items [1] to

[24] , wherein the molar ratio is about 200 to about 2,500.

[0043]

[26] The use according to any one of items [1] to

[25] , wherein the molar ratio is about 300 to about 2,000.

[0044]

[27] The use according to any one of items [1] to

[26] , wherein the molar ratio is about 400 to about 1,750.

[0045]

[28] The use according to any one of items [1] to

[27] , wherein the molar ratio is about 500 to about 1,500.

[0046]

[29] The use according to any one of items [1] to

[28] , wherein the molar ratio is about 600 to about 1,250.

[0047]

[30] The use according to any one of items [1] to

[29] , wherein the molar ratio is about 700 to about 1,000.

[0048]

[31] The use according to any one of items [1] to

[30] , comprising a step of stabilizing FVIII by adding a polypeptide in a molar excess of more than 20 times to FVIII.

[0049]

[32] The use according to item

[31] , wherein the molar excess is at least 25-fold, or at least 50-fold, or more than 50-fold, or at least 60-fold, or at least 75-fold, or at least 100-fold, or at least 200-fold, or at least 300-fold, or at least 400-fold, or at least 500-fold, or at least 600-fold, or at least 700-fold.

[0050]

[33] The use according to item

[31] or

[32] , wherein the molar excess is more than 20 times but less than 10,000 times, or 25 times to 9,000 times, or 50 times to 7,500 times, or 60 times to 5,000 times, or 75 times to 4,000 times, or 100 times to 3,000 times, or 200 times to 2,500 times, or 300 times to 2,000 times, or 400 times to 1,750 times, or 500 times to 1,500 times, or 600 times to 1,250 times, or 700 times to 1,000 times.

[0051]

[34] The use according to any one of items [1] to

[33] , wherein the yield of FVIII upon freeze-drying and reconstitution of a composition comprising FVIII and the polypeptide is greater than the yield of FVIII upon freeze-drying and reconstitution of a control composition lacking the polypeptide.

[0052]

[35] The use according to item

[34] , wherein the freeze-dried composition is reconstituted immediately after freeze-drying.

[0053]

[36] The use according to item

[34] or

[35] , wherein the loss of FVIII activity during the freeze-drying process is less than 13%.

[0054]

[37] The use according to item

[34] or

[35] , wherein the reduction in FVIII activity during the freeze-drying process is 11% or less.

[0055]

[38] The use according to item

[34] or

[35] , wherein the reduction in FVIII activity during the freeze-drying process is 10% or less.

[0056]

[39] The use according to item

[34] or

[35] , wherein the reduction in FVIII activity during the freeze-drying process is 8% or less.

[0057]

[40] The use according to item

[34] or

[35] , wherein the reduction in FVIII activity during the freeze-drying process is 5% or less.

[0058]

[41] The use according to item

[34] or

[35] , wherein the loss of FVIII activity during the freeze-drying process is less than 3%.

[0059]

[42] The use according to item

[34] or

[35] , wherein the reduction in FVIII activity during the freeze-drying process is 2% or less.

[0060]

[43] The use according to any one of items [1] to

[33] , wherein the decrease in FVIII activity of a freeze-dried composition comprising FVIII and a polypeptide during storage at 25°C is less than the decrease in FVIII activity of a freeze-dried control composition lacking the polypeptide.

[0061]

[44] The storage is for a period of 12 months, for the use described in item

[43] .

[0062]

[45] The use according to item

[43] , wherein the decrease in FVIII activity during storage at 25°C is less than 20%, preferably less than 18%, more preferably less than 16%.

[0063]

[46] The storage is for a period of 24 months, for the use described in item

[43] .

[0064]

[47] The use according to item

[43] , wherein the decrease in FVIII activity during storage at 25°C is less than 30%, preferably less than 20%.

[0065]

[48] ​​The use according to any one of items [1] to

[33] , wherein the FVIII activity in a liquid composition comprising the polypeptide and FVIII after storage at 25°C for at least one week is greater than the FVIII activity of a control composition lacking the polypeptide.

[0066]

[49] The use according to item

[48] , wherein the decrease in FVIII activity during storage at 25°C for one week is less than 10%.

[0067]

[50] The use according to item

[48] , wherein the decrease in FVIII activity during storage at 25°C for 4 weeks is less than 20% or less than 15%.

[0068]

[51] The recombinant polypeptide for use according to any one of items [1] to

[50] , wherein the truncated VWF is human truncated VWF.

[0069]

[52] The use according to any one of items [1] to

[51] , wherein the truncated VWF comprises an amino acid sequence having at least 90% sequence identity with amino acids 776 to 805 of SEQ ID NO: 4, preferably an amino acid sequence having at least 90% sequence identity with amino acids 764 to 1242 of SEQ ID NO: 4.

[0070]

[53] The use according to any one of items [1] to

[52] , wherein the truncated VWF lacks amino acids 1243 to 2813 of SEQ ID NO: 4.

[0071]

[54] The use according to any one of items [1] to

[53] , wherein the truncated VWF consists of either (a) amino acids 764 to 1242 of SEQ ID NO: 4, (b) an amino acid sequence having at least 90% sequence identity with amino acids 764 to 1242 of SEQ ID NO: 4, or (c) a fragment of (a) or (b).

[0072]

[55] The polypeptide has a dissociation constant, K, of 1 μM or less. D The use according to any one of items [1] to

[54] , wherein the FVIII binds to FVIII by

[0073]

[56] The polypeptide has a dissociation constant, K, of 1 nM or less. D The use according to any one of items [1] to

[55] , wherein the antibody binds to FVIII by

[0074]

[57] The polypeptide has a dissociation constant, K, of 0.1 nM or less. D The use according to any one of items [1] to

[56] , wherein the antibody binds to FVIII by

[0075]

[58] The use according to any one of items [1] to

[57] , wherein the polypeptide comprises a half-life extending moiety (HLEM).

[0076]

[59] The use according to item

[58] , wherein HLEM is a heterologous amino acid sequence fused to truncated VWF.

[0077]

[60] The use according to item

[59] , wherein the heterologous amino acid sequence comprises or consists of a protein or peptide selected from the group consisting of transferrin and fragments thereof, the C-terminal peptide of human chorionic gonadotropin, an XTEN sequence, homozygous amino acid repeats (HAP), proline-alanine-serine repeats (PAS), albumin and fragments thereof, afamin, alpha-fetoprotein, vitamin D binding protein, polypeptides capable of binding to albumin or immunoglobulin constant regions under physiological conditions, polypeptides capable of binding to fetal Fc receptors (FcRn), in particular immunoglobulin constant regions and parts thereof, preferably the Fc portion of an immunoglobulin, and combinations thereof.

[0078]

[61] The use according to item

[58] , wherein the HLEM is conjugated to a polypeptide comprising a truncated VWF.

[0079]

[62] The use according to item

[61] , wherein the HLEM is conjugated to the C-terminus of the polypeptide comprising the truncated VWF.

[0080]

[63] The use according to items

[61] or

[62] , wherein the HLEM is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acid (PSA), elastin-like polypeptides, heparosan polymers, hyaluronic acid and non-proteinaceous albumin-binding ligands, such as fatty acid chains, and combinations thereof.

[0081]

[64] The use according to item

[58] , wherein the HLEM non-covalently binds to a polypeptide comprising a truncated VWF.

[0082]

[65] The use according to any one of items [1] to

[60] , wherein the polypeptide comprising the truncated VWF does not contain any HLEM conjugated to the polypeptide.

[0083]

[66] The use according to any one of items [1] to

[65] , wherein the polypeptide is a glycoprotein comprising N-glycans, and at least 50%, at least 75%, preferably at least 85% of the N-glycans comprise, on average, at least one sialic acid moiety.

[0084]

[67] The use according to any one of items [1] to

[66] , wherein the polypeptide exists as a dimer or at least has a high proportion of dimers.

[0085]

[68] The use according to item

[67] , wherein at least 50%, or at least 70%, or at least 80%, or at least 90%, or at least 95% of the polypeptides are present as dimers.

[0086]

[69] The use according to item

[67] or

[68] , wherein the dimer is a homodimer, and the two monomers forming the dimer are covalently linked to each other via at least one or more disulfide bridges formed by cysteine ​​residues in the truncated VWF.

[0087]

[70] The use according to item

[69] , wherein the cysteine ​​residues forming one or more disulfide bridges are selected from the group consisting of Cys-1099, Cys-1142, Cys-1222, Cys-1225, Cys-1227 and combinations thereof, preferably Cys-1099 and Cys-1142, wherein the amino acid numbering refers to SEQ ID NO: 4.

[0088]

[71] The use according to any one of items

[67] to

[70] , wherein the affinity of the dimer for FVIII is higher than the affinity of the monomeric polypeptide for FVIII, and the monomeric polypeptide has an amino acid sequence identical to that of the monomeric subunit of the dimeric polypeptide.

[0089]

[72] The use according to any one of items

[67] to

[71] , wherein the dimer:monomer ratio of the polypeptide is at least 1.5, preferably at least 2, more preferably at least 2.5, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20; or the polypeptide is free of monomeric and / or multimeric forms of the polypeptide; or the polypeptide is essentially free of monomeric and / or multimeric forms of the polypeptide.

[0090]

[73] The dimeric polypeptide has a dissociation constant K of less than 1 μM, preferably less than 1 nM, more preferably less than 500 pM, less than 200 pM, less than 100 pM, less than 90 pM, or less than 80 pM. D The use according to any one of items

[67] to

[72] , wherein the FVIII binding affinity is characterized by:

[0091]

[74] K D The use according to item

[73] , wherein the concentration ranges from 0.1 pM to 500 pM, 0.5 pM to 200 pM, 0.75 pM to 100 pM, or most preferably, from 1 pM to 80 pM.

[0092]

[75] The use according to any one of items [1] to

[74] , wherein the polypeptide comprises at least one amino acid substitution compared to the amino acid sequence of wild-type VWF, and the binding affinity of such a modified polypeptide to FVIII is preferably further improved by the introduction of the at least one substitution compared to the binding affinity of a reference polypeptide having the same amino acid sequence except for the modification.

[0093]

[76] The use according to item

[75] , wherein at least one substitution is selected from the group consisting of S764G / S766Y, S764P / S766I, S764P / S766M, S764V / S766Y, S764E / S766Y, S764Y / S766Y, S764L / S766Y, S764P / S766W, S766W / S806A, S766Y / P769K, S766Y / P769N, S766Y / P769R, S764P / S766L and S764E / S766Y / V1083A, and wherein the amino acid numbering refers to the sequence of SEQ ID NO: 4.

[0094]

[77] The use according to item

[76] , wherein at least one substitution is any combination of S764E / S766Y or S764E / S766Y / V1083A.

[0095]

[78] The use according to any one of items [1] to

[77] , wherein the composition is a formulation.

[0096]

[79] The use according to item

[78] , wherein the preparation is suitable for the treatment or prevention of blood clotting disorders.

[0097]

[80] The use according to item

[79] , wherein the blood coagulation disorder is hemophilia A. DETAILED DESCRIPTION OF THE INVENTION

[0098] The present invention relates to the use of a polypeptide comprising a truncated von Willebrand factor (VWF) for improving the in vitro stability of coagulation factor VIII (FVIII) in a composition, wherein the composition comprises FVIII and the polypeptide, and the molar ratio of the polypeptide to FVIII in the composition is greater than 20.

[0099] Polypeptides comprising truncated von Willebrand factor (VWF) are referred to herein as "polypeptides of the invention." Polypeptides of the invention preferably comprise a half-life extending moiety.

[0100] ratio As described in more detail below, the polypeptides of the present invention may be monomers, dimers, or mixtures thereof. Any molar ratio according to the present invention refers to the molar ratio of the monomeric subunits of the polypeptides of the present invention, regardless of whether they are actually present as monomers, dimers, or oligomers. The ratio is formed relative to the molar concentration of co-formulated FVIII. Any ratio of the polypeptides of the present invention to FVIII in this application refers to the amount (moles) of the monomeric subunits contained in the polypeptides of the present invention, which are preferably present as dimers, divided by the amount (moles) of FVIII, unless otherwise indicated. As a non-limiting example, co-formulation of 100 μM of the monomeric polypeptides of the present invention with 1 μM of FVIII means a ratio of 100. This ratio of 100 is obtained when 50 μM of the dimeric polypeptides of the present invention are co-formulated with 1 μM of FVIII.

[0101] The molar ratio of polypeptide of the invention to FVIII is greater than 20, or at least 25, or at least 50, or greater than 50, more preferably the ratio is at least 60, or at least 75, or at least 100, or greater than 100, or at least 200, most preferably at least 300, or at least 400, or at least 500, or at least 600, or at least 700, or at least 800, or at least 900, or at least 1,000, or at least 1,100, or at least 1,200, or at least 1,300, or at least 1,400, or at least 1,500, or at least 1,600, or at least 1,700, or at least 1,800, or at least 1,900, or at least 2,000, or at least 2,500, or at least 3,000, or at least 5,000, or at least 8,000, or up to 10,000. The molar ratio of the polypeptide of the invention to FVIII may, according to particular embodiments, not exceed a ratio of 10,000, a ratio of 5,000, a ratio of 2,500, or a ratio of 2,000.

[0102] The molar ratio of the polypeptide of the present invention to FVIII can range from greater than 20 to 10,000, or greater than 50 to 5,000, or greater than 50 to 4,000, or greater than 50 to 3,000, or greater than 50 to 2,000, or greater than 50 to 1,000. Preferably, the molar ratio of the polypeptide of the present invention to FVIII ranges from 50 to 2,500, or 75 to 2,000, or 100 to 1,500, or 150 to 1,000.

[0103] cutting type VWF The term "von Willebrand factor" (VWF), as used herein, includes naturally occurring (native) VWF, but also includes variants thereof that retain at least the FVIII binding activity of naturally occurring VWF, e.g., sequence variants in which one or more residues have been inserted, deleted, or substituted. FVIII binding activity can be determined by the FVIII binding assay described in Example 2.

[0104] A preferred VWF according to the present invention is human VWF represented by the amino acid sequence shown in SEQ ID NO: 4. The cDNA encoding SEQ ID NO: 4 is shown in SEQ ID NO: 3.

[0105] The gene encoding human native VWF is transcribed into a 9 kb mRNA, which is translated into a 2,813 amino acid prepropolypeptide with a predicted molecular weight of 310,000 Da. The prepropolypeptide contains an N-terminal 22 amino acid signal peptide followed by a 741 amino acid propolypeptide (amino acids 23-763 of SEQ ID NO: 4) and a mature subunit (amino acids 764-2,813 of SEQ ID NO: 4). Cleavage of the 741 amino acid polypeptide from the N-terminus results in mature VWF consisting of 2,050 amino acids. The amino acid sequence of human native VWF prepropolypeptide is set forth in SEQ ID NO: 4. Unless otherwise indicated, the amino acid numbering of VWF residues in this application refers to SEQ ID NO: 4, even if the VWF molecule, particularly truncated VWF, does not contain all of the residues of SEQ ID NO: 4.

[0106] The native VWF propolypeptide contains several domains. Various domain annotations can be found in the literature (see, for example, Zhou et al. (2012) Blood 120(2):449-458). The following domain annotation of the native VWF prepropolypeptide applies to the present application: D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK

[0107] Referring to SEQ ID NO: 4, the D' domain consists of amino acids 764-865, and the D3 domain consists of amino acids 866-1242.

[0108] The term "truncated" refers to the polypeptide in the context of the present invention, which does not contain the entire amino acid sequence of mature VWF (e.g., amino acids 764 to 2813 of SEQ ID NO: 4). Truncated VWF typically does not contain all of amino acids 764 to 2813 of SEQ ID NO: 4, but only a fragment thereof. Truncated VWF may also be referred to as a VWF fragment, or in the plural as VWF fragments.

[0109] Typically, the truncated VWF is capable of binding to factor VIII. Preferably, the truncated VWF is capable of binding to the mature form of human native factor VIII. In another embodiment, the truncated VWF is capable of binding to recombinant FVIII, such as a FVIII described herein, for example, a single-chain FVIII consisting of the amino acid sequence of SEQ ID NO: 5. The binding of truncated VWF to factor FVIII can be determined by the FVIII-VWF binding assay described in Example 2.

[0110] The truncated VWF of the present invention preferably comprises or consists of an amino acid sequence having at least 90% sequence identity to amino acids 776-805 of SEQ ID NO: 4 and is capable of binding to FVIII. In a preferred embodiment, the truncated VWF comprises or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 776-805 of SEQ ID NO: 4 and is capable of binding to FVIII. In one embodiment, the truncated VWF comprises or consists of amino acids 776-805 of SEQ ID NO: 4. Unless otherwise indicated herein, sequence identity is determined relative to the full length of a reference sequence (e.g., amino acids 776-805 of SEQ ID NO: 4).

[0111] The truncated VWF of the present invention preferably comprises or consists of an amino acid sequence having at least 90% sequence identity with amino acids 766 to 864 of SEQ ID NO: 4 and is capable of binding to FVIII. In a preferred embodiment, the truncated VWF comprises or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids 766 to 864 of SEQ ID NO: 4 and is capable of binding to FVIII. In one embodiment, the truncated VWF comprises or consists of amino acids 766 to 864 of SEQ ID NO: 4.

[0112] In another preferred embodiment, the truncated VWF consists of (a) an amino acid sequence having at least 90% sequence identity with amino acids 764-1242 of SEQ ID NO: 4, or (b) a fragment thereof, with the proviso that the truncated VWF is still capable of binding to FVIII. More preferably, the truncated VWF consists of (a) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids 764-1242 of SEQ ID NO: 4, or (b) a fragment thereof, with the proviso that the truncated VWF is still capable of binding to FVIII. In one embodiment, the truncated VWF consists of (a) amino acids 764-1242 of SEQ ID NO: 4, or (b) a fragment thereof, with the proviso that the truncated VWF is still capable of binding to FVIII.

[0113] As described in more detail below, the polypeptides of the invention can be prepared by methods using cells containing nucleic acid encoding a polypeptide comprising a truncated VWF, which nucleic acid is introduced into a suitable host cell by techniques known per se.

[0114] In a preferred embodiment, the nucleic acid encodes, in a host cell, (a) an amino acid sequence having at least 90% sequence identity with amino acids 1 to 1242 of SEQ ID NO: 4, or (b) a fragment thereof, with the proviso that the truncated mature VWF is still capable of binding to FVIII. More preferably, the nucleic acid encodes (a) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with amino acids 1 to 1242 of SEQ ID NO: 4, or (b) a fragment thereof, with the proviso that the truncated VWF is still capable of binding to FVIII. In one embodiment, the nucleic acid encodes (a) amino acids 1 to 1242 of SEQ ID NO: 4, or (b) a fragment thereof, with the proviso that the truncated VWF is still capable of binding to FVIII. In particular, when the polypeptide according to the invention is a dimer, the nucleic acid comprises a sequence encoding also amino acids 1 to 763 of VWF (e.g., SEQ ID NO: 4), even if the truncated VWF does not comprise amino acids 1 to 763 of VWF (e.g., SEQ ID NO: 4) in the polypeptide.

[0115] The truncated VWF of the recombinant polypeptide of the present invention according to a preferred embodiment may not contain the amino acid sequence 1 to 763 of VWF of SEQ ID NO:4.

[0116] According to a further preferred embodiment, the truncated VWF has the following amino acid sequences, respectively with reference to SEQ ID NO: 4: 776-805; 766-805; 764-805; 776-810; 766-810; 764-810; 776-815; 766-815; 764-815; 776~820;766~820;764~820;776~825;766~825;764~825;776~830;766~830;764~830; 776~835;766~835;764~835;776~840;766~840;764~840;776~845;766~845;764~845; 776~850;766~850;764~850;776~855;766~855;764~855;776~860;766~860;764~860; 776~864;766~864;764~864;776~865;766~865;764~865;776~870;766~870;764~870; 776~875;766~875;764~875;776~880;766~880;764~880;776~885;766~885;764~885; 776~890;766~890;764~890;776~895;766~895;764~895;776~900;766~900;764~900; 776~905;766~905;764~905;776~910;766~910;764~910;776~915;766~915;764~915; 776~920;766~920;764~920;776~925;766~925;764~925;776~930;766~930;764~930; 776~935;766~935;764~935;776~940;766~940;764~940;776~945;766~945;764~945; 776~950;766~950;764~950;776~955;766~955;764~955;776~960;766~960;764~960; 776~965;766~965;764~965;776~970;766~970;764~970;776~975;766~975;764~975; 776~980;766~980;764~980;776~985;766~985;764~985;776~990;766~990;764~990; 776~995;766~995;764~995;776~1000;766~1000;764~1000;776~1005;766~1005;764~1005; 776~1010;766~1010;764~1010;776~1015;766~1015;764~1015;776~1020;766~1020;764~1020; 776~1025;766~1025;764~1025;776~1030;766~1030;764~1030;776~1035;766~1035;764~1035; 776~1040;766~1040;764~1040;776~1045;766~1045;764~1045;776~1050;766~1050;764~1050; 776~1055;766~1055;764~1055;776~1060;766~1060;764~1060;776~1065;766~1065;764~1065; 776~1070;766~1070;764~1070;776~1075;766~1075;764~1075;776~1080;766~1080;764~1080; 776~1085;766~1085;764~1085;776~1090;766~1090;764~1090;776~1095;766~1095;764~1095; 776~1100;766~1100;764~1100;776~1105;766~1105;764~1105;776~1110;766~1110;764~1110; 776~1115;766~1115;764~1115;776~1120;766~1120;764~1120;776~1125;766~1125;764~1125; 776~1130;766~1130;764~1130;776~1135;766~1135;764~1135;776~1140;766~1140;764~1140; 776~1145;766~1145;764~1145;776~1150;766~1150;764~1150;776~1155;766~1155;764~1155; 776~1160;766~1160;764~1160;776~1165;766~1165;764~1165;776~1170;766~1170;764~1170; 776~1175;766~1175;764~1175;776~1180;766~1180;764~1180;776~1185;766~1185;764~1185; 776~1190;766~1190;764~1190;776~1195;766~1195;764~1195;776~1200;766~1200;764~1200; 776~1205;766~1205;764~1205;776~1210;766~1210;764~1210;776~1215;766~1215;764~1215; 776~1220;766~1220;764~1220;776~1225;766~1225;764~1225;776~1230;766~1230;764~1230; 776~1235;766~1235;764~1235;776~1240;766~1240;764~1240;776~1242;766~1242;764~1242; 764~1464;764~1250;764~1041;764~828;764~865;764~1045;764~1035;764~1128;764~1198; 764~1268;764~1261;764~1264;764~1459;764~1463;764~1464;764~1683;764~1873;764~1482; Including or consisting of one of 764-1479; 764-1672; and 764-1874.

[0117] In certain embodiments, the truncated VWF has an internal deletion compared to mature wild-type VWF. For example, the A1, A2, A3, D4, C1, C2, C3, C4, C5, C6, CK domains, or a combination thereof, may be deleted, while the D' and / or D3 domains are retained. According to further embodiments, the truncated VWF lacks one or more of domains A1, A2, A3, D4, C1, C2, C3, C4, C5, C6, or CK. According to further embodiments, the truncated VWF lacks amino acids 1243 to 2813 of SEQ ID NO: 4, i.e., domains A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK.

[0118] In a further embodiment, the truncated VWF does not contain the binding sites for platelet glycoprotein Ibα (GPIbα), collagen, and / or integrin αIIbβIII (the RGDS sequence in the C1 domain). In another embodiment, the truncated VWF does not contain the cleavage site for ADAMTS13 (Tyr1605-Met1606), which is located in the central A2 domain of VWF. In yet another embodiment, the truncated VWF does not contain the binding site for GPIbα, and / or does not contain the binding site(s) for collagen, and / or does not contain the binding site for integrin αIIbβIII, and / or it does not contain the cleavage site for ADAMTS13 (Tyr1605-Met1606), which is located in the central A2 domain of VWF. In a preferred embodiment, the truncated VWF does not contain amino acids 1691 to 1905 of SEQ ID NO: 4. In another preferred embodiment, the truncated VWF does not contain amino acids 1691 to 1905 of the amino acid sequence deposited as UniProtKB-P04275. In another preferred embodiment, the truncated VWF does not contain amino acids 1691 to 1905 of human VWF.

[0119] In one embodiment, the polypeptide has a low affinity for platelets, the low affinity being less than a dissociation constant K for binding of the polypeptide to GPIbα. D >1 μM, preferentially K D Characterized by >10 μM.

[0120] In another embodiment, the polypeptide does not comprise VWF domains A1 and / or A3 or a portion thereof and has low or essentially no affinity for type I and type III collagen, wherein low or essentially no affinity means that the polypeptide has a dissociation constant K for binding to type I and type III collagen. D >1 μM, preferentially K D >10 μM. However, the polypeptide may contain one or more copies of a peptide having, preferably consisting of, amino acids 1238 to 1268 of SEQ ID NO: 4 fused at the N- or C-terminus to the polypeptide.

[0121] In other embodiments, the truncated VWF comprises or consists of an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity to one of the amino acid sequences listed in the previous paragraph, provided that the truncated VWF is capable of binding to FVIII.

[0122] A polypeptide of the present invention is referred to as a "dimer" in the present invention when two monomers of the polypeptide of the present invention are covalently linked. Preferably, the covalent bond is located in the VWF cleavage portion of the polypeptide of the present invention. Preferably, the two monomer subunits are covalently linked via at least one disulfide bridge, for example, by one, two, three, or four disulfide bridges. The cysteine ​​residues forming the at least one disulfide bridge are preferably located in the VWF cleavage portion of the polypeptide of the present invention. In one embodiment, these cysteine ​​residues are Cys-1099, Cys-1142, Cys-1222, Cys-1225, or Cys-1227, or a combination thereof. Preferably, the dimeric polypeptide of the present invention does not contain any additional covalent bonds linking the monomers in addition to the covalent bond located in the cleaved VWF portion of the polypeptide, particularly does not contain any additional covalent bonds located in the HLEM or HLEP portion of the polypeptide. However, according to alternative embodiments, the dimeric polypeptides of the present invention may comprise a covalent bond located in the HLEM or HLEP portion of the polypeptide that links the monomers.

[0123] The dimer is preferably a homodimer, whereby each monomer preferably comprises a HLEM or HLEP as disclosed herein. When the polypeptide of the present invention is a dimer, the truncated VWF preferably comprises or consists of two polypeptides each having an amino acid sequence having at least 90% sequence identity with amino acids 764 to 1099, 764 to 1142, 764 to 1222, 764 to 1225, 764 to 1227 or 764 to 1242 of SEQ ID NO: 4, and is capable of binding to FVIII. In a preferred embodiment, the truncated VWF comprises or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 764-1099, 764-1142, 764-1222, 764-1225, 764-1227, or 764-1242 of SEQ ID NO: 4, and is capable of binding to FVIII. Most preferably, the truncated VWF comprises or consists of amino acids 764-1099, 764-1142, 764-1222, 764-1225, 764-1227, or 764-1242 of SEQ ID NO: 4.

[0124] The truncated VWF may be any one of the VWF fragments disclosed in WO 2013 / 093760 A2, the disclosures of which are incorporated herein by reference.

[0125] According to a further preferred embodiment, the truncated VWF disclosed above may contain at least one of the amino acid substitutions disclosed in Patent Document 29. Such modified versions of truncated VWF contain at least one amino acid substitution in the D' domain compared to the amino acid sequence of the D' domain of wild-type VWF according to SEQ ID NO: 4. The amino acid sequence of the modified versions of truncated VWF may have one or more amino acid substitutions compared to the respective wild-type sequence. The amino acid sequence of the D' domain of the modified truncated VWF preferably has one or two amino acid substitutions compared to the D' domain of SEQ ID NO: 4. It is preferred to replace S at position 764 of SEQ ID NO: 4, which corresponds to position 1 of SEQ ID NO: 2, with an amino acid selected from the group consisting of G, P, V, E, Y, A, and L. It is also preferred to replace S at position 766 of SEQ ID NO: 4, which corresponds to position 3 of SEQ ID NO: 2, with an amino acid selected from the group consisting of Y, I, M, V, F, H, R, and W. Preferred substitution combinations include S764G / S766Y, S764P / S766I, S764P / S766M, S764V / S766Y, S764E / S766Y, S764Y / S766Y, S764L / S766Y, S764P / S766W, S766W / S806A, S766Y / P769K, S766Y / P769N, S766Y / P769R and S764P / S766L, and refer to the sequence SEQ ID NO: 4. The introduction of substitutions may further improve the binding affinity of the polypeptides of the invention to FVIII compared to the binding affinity of a reference polypeptide having the same amino acid sequence except for the modifications. Substitutions within truncated VWF may contribute to improving the half-life of co-administered FVIII or the stability of co-formulated FVIII.

[0126] Half-life extending moiety (HLEM) In addition to the truncated VWF, the polypeptide of the present invention may, in certain preferred embodiments, further comprise a half-life extending moiety. The half-life extending moiety may be a heterologous amino acid sequence fused to the truncated VWF. Alternatively, the half-life extending moiety may be chemically conjugated to the polypeptide comprising the truncated VWF by a covalent bond that may be different from a peptide bond.

[0127] In a specific embodiment of the present invention, the half-life of the polypeptide of the present invention is extended by chemical modification, for example, by the attachment of a half-life extending moiety, such as polyethylene glycol (PEGylation), glycosylated PEG, hydroxyethyl starch (hydroxyethylation), polysialic acid, elastin-like polypeptide, heparosan polymer, or hyaluronic acid. In another embodiment, the polypeptide of the present invention is conjugated to a HLEM, such as albumin, via a chemical linker. The principle of this conjugation technology is described in an exemplary manner by Conjuchem LLC (see, for example, U.S. Patent No. 7,256,253).

[0128] In another embodiment, the half-life extending moiety is a half-life enhancing polypeptide (HLEP). Preferably, the HLEP is albumin or a fragment thereof. The N-terminus of albumin may be fused to the C-terminus of the truncated VWF. Alternatively, the C-terminus of albumin may be fused to the N-terminus of the truncated VWF. One or more HLEPs may be fused to the N- or C-terminal portions of VWF, provided that they do not interfere with or abolish the ability of the truncated VWF to bind to FVIII.

[0129] The recombinant polypeptide preferably further comprises a covalent bond located between the truncated VWF and the HLEM, or a linker sequence located between the truncated VWF and the HLEM.

[0130] The linker sequence may be a peptide linker consisting of one or more amino acids, particularly 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 (e.g., 1, 2, or 3) amino acids, which may be equal to or different from each other. Preferably, the linker sequence is not present at the corresponding position in wild-type VWF. Preferred amino acids present in the linker sequence include Gly and Ser. The linker sequence should be non-immunogenic. A preferred linker may consist of alternating glycine and serine residues. Suitable linkers are described, for example, in WO 2007 / 090584.

[0131] In another embodiment of the present invention, the peptide linker between the truncated VWF portion and the HLEP consists of a peptide sequence that acts as a natural interdomain linker or sequence in human proteins. Preferably, such peptide sequences are located near the protein surface in their natural environment and are accessible to the immune system, thus allowing for natural tolerance to this sequence to be assumed. Examples are given in WO 2007 / 090584. Cleavable linker sequences are described, for example, in WO 2013 / 120939 A1.

[0132] In a preferred embodiment of the recombinant polypeptide, the linker between the truncated VWF and the HLEP is a glycine / serine peptide linker having or consisting of amino acid sequence 480 to 510 of SEQ ID NO:2.

[0133] In one embodiment, the polypeptide has the following structure: tVWF-L1-H [Formula 1], where tVWF is a truncated VWF, L1 is a chemical bond or linker sequence, and H is a HLEM, particularly a HLEP.

[0134] L1 can be a chemical bond or a linker sequence consisting of one or more amino acids, e.g., 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 (e.g., 1, 2, or 3) amino acids, which can be equal to or different from each other. Typically, the linker sequence is not present at the corresponding position in wild-type VWF. Examples of suitable amino acids present in L1 include Gly and Ser. The linker must be non-immunogenic and can be a non-cleavable or cleavable linker. A non-cleavable linker can be composed of alternating glycine and serine residues, as exemplified in WO 2007 / 090584 A1. In another embodiment of the present invention, the peptide linker between the truncated VWF moiety and the albumin moiety consists of a peptide sequence that acts as a natural interdomain linker or sequence in human proteins. Preferably, such peptide sequences are located near the protein surface in their natural environment and are accessible to the immune system, thus allowing for natural tolerance to the sequence to be assumed. Examples are given in WO 2007 / 090584. Cleavable linker sequences are described, for example, in WO 2013 / 120939 A1.

[0135] Preferred HLEP sequences are described below. Similarly, fusions to the exact "N-terminal amino acid" or "C-terminal amino acid" of each HLEP, or to the "N-terminal portion" or "C-terminal portion" of each HLEP, including N-terminal deletion of one or more amino acids of the HLEP, are encompassed by the present invention. A polypeptide may contain two or more HLEP sequences, for example, two or three HLEP sequences. These multiple HLEP sequences may be fused in tandem, for example, as consecutive repeats, to the C-terminal portion of VWF.

[0136] Half-life enhancing polypeptide (HLEP) Preferably, the half-life extending moiety is a half-life extending polypeptide (HLEP). More preferably, the HLEP is selected from the group consisting of albumin, a member of the albumin family or a fragment thereof, solvated random chains with large hydrodynamic volumes (e.g., XTEN (Schellenberger et al. 2009; Nature Biotechnol. 27:1186-1190), homoamino acid repeats (HAP) or proline-alanine-serine repeats (PAS), afamin, alpha-fetoprotein, vitamin D binding protein, transferrin or a variant or fragment thereof, the carboxy-terminal peptide of the human chorionic gonadotropin β subunit (CTP), a polypeptide capable of binding to fetal Fc receptor (FcRn), and in particular, immunoglobulin constants. The HLEP may be selected from the group consisting of a constant region and a portion thereof, such as an Fc fragment, a polypeptide or lipid capable of binding under physiological conditions to albumin, a member of the albumin family or a fragment thereof, or an immunoglobulin constant region or a portion thereof. The immunoglobulin constant region or a portion thereof is preferably an Fc fragment of immunoglobulin G1, an Fc fragment of immunoglobulin G2, or an Fc fragment of immunoglobulin A. The HLEP may additionally or alternatively comprise one or more copies of a peptide having, preferably consisting of, amino acids 1238 to 1268 of SEQ ID NO: 4. This peptide preferably contains multiple O-glycosylated amino acids.

[0137] A half-life enhancing polypeptide, as used herein, can be a full-length half-life enhancing protein as described herein or one or more fragments thereof that are capable of stabilizing or extending the therapeutic or biological activity of a coagulation factor, and in particular, of increasing the in vivo half-life of the polypeptides of the invention. Such fragments can be 10 or more amino acids in length, or can comprise at least about 15, at least about 20, at least about 25, at least about 30, at least about 50, at least about 100 or more contiguous amino acids from the HLEP sequence, or can comprise part or all of a particular domain of each of the HLEPs, so long as the HLEP fragment provides at least a 25% extension of functional half-life compared to the respective polypeptide without the HLEP.

[0138] The HLEP portion of the polypeptide of the invention may be a variant of wild-type HLEP. The term "variant" includes conservative or non-conservative insertions, deletions and substitutions, where such changes do not substantially alter the FVIII binding activity of the truncated VWF.

[0139] In particular, the proposed truncated VWF-HLEP fusion constructs of the present invention may include naturally occurring polymorphic variants of HLEP and fragments of HLEP. The HLEP may be derived from any vertebrate, particularly any mammal, such as human, monkey, bovine, ovine, or porcine. Non-mammalian HLEPs include, but are not limited to, chicken and salmon.

[0140] According to certain embodiments of the present disclosure, the HLEM, and in particular the HLEP, portions of the recombinant polypeptides of the present invention may be identified by the alternative term "FP." Preferably, the term "FP" refers to human albumin.

[0141] According to a particularly preferred embodiment, the recombinant polypeptide is a fusion protein. A fusion protein, in the context of the present invention, is a protein produced by the in-frame ligation of at least two DNA sequences encoding a truncated VWF and an HLEP. Those skilled in the art will understand that translation of the fusion protein DNA sequence results in a single protein sequence. In a further preferred embodiment, in-frame insertion of a DNA sequence encoding a peptide linker results in a fusion protein comprising a truncated VWF, a suitable linker, and an HLEP.

[0142] According to some embodiments, the co-formulated FVIII does not include any of the HLEM or HLEP structures described herein. According to certain other embodiments, the co-formulated FVIII may include at least one of the HLEM or HLEP structures described herein.

[0143] Albumin as HLEP The terms "human serum albumin" (HSA) and "human albumin" (HA) are used interchangeably in this application. The terms "albumin" and "serum albumin" are broad and encompass human serum albumin (and fragments and variants thereof) as well as albumins (and fragments and variants thereof) from other species.

[0144] As used herein, "albumin" collectively refers to an albumin polypeptide or amino acid sequence, or an albumin fragment or variant having one or more functional properties (e.g., biological functions) of albumin. In particular, "albumin" refers to human albumin or a fragment thereof, particularly the mature form of human albumin set forth in SEQ ID NO: 6 herein or albumin from other vertebrates or a fragment thereof, or an analog or variant of these molecules or fragments thereof.

[0145] According to certain embodiments of the present disclosure, the alternative term "FP" is used to identify HLEPs, and in particular to define albumin as an HLEP.

[0146] In particular, the proposed polypeptides of the present invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin. Generally speaking, albumin fragments or variants are at least 10, preferably at least 40, and most preferably more than 70 amino acids in length.

[0147] A preferred embodiment of the present invention comprises albumin variants used as HLEPs of the polypeptides of the present invention that have enhanced binding to the FcRn receptor, which may result in a longer plasma half-life of the truncated VWF-albumin variant fusion protein compared to truncated VWF fusions with wild-type albumin.

[0148] The albumin portion of a polypeptide of the invention may comprise at least one subdomain or domain of HA or a conservative modification thereof.

[0149] Immunoglobulin as HLEP The immunoglobulin G (IgG) constant region (Fc) is known in the art to enhance the half-life of therapeutic proteins (Dumont JA et al. 2006. BioDrugs 20:151-160). The IgG constant region of the heavy chain consists of three domains (CH1-CH3) and a hinge region. The immunoglobulin sequence can be derived from any mammal or from the subclasses IgG1, IgG2, IgG3, or IgG4, respectively. IgG and IgG fragments without antigen-binding domains can also be used as HLEPs. The therapeutic polypeptide moiety is preferably linked to the IgG or IgG fragment via the hinge region of the antibody or a peptide linker, which may also be cleavable. Several patents and patent applications describe fusing therapeutic proteins to immunoglobulin constant regions to enhance their in vivo half-life. U.S. Patent No. 2004 / 0087778 and WO 2005 / 001025 describe fusion proteins of the Fc domain, or at least a portion of the immunoglobulin constant region, with biologically active peptides, which improve the half-life of peptides that would otherwise be rapidly cleared in vivo. Fc-IFN-β fusion proteins were described that achieve enhanced biological activity, prolonged circulating half-life, and high solubility (WO 2006 / 000448 A2). Fc-EPO proteins with extended serum half-lives and improved in vivo potency have been disclosed (WO 2005 / 063808 A1), as well as Fc fusions with G-CSF (WO 2003 / 076567 A2), glucagon-like peptide 1 (WO 2005 / 000892 A2), coagulation factors (WO 2004 / 101740 A2), and interleukin-10 (U.S. Pat. No. 6,403,077), all of which possessed half-life enhancing properties.

[0150] Various HLEPs that can be used in accordance with the present invention are described in detail in WO 2013 / 120939 A1.

[0151] Sialylation of N-glycans and polypeptides of the invention The polypeptides of the present invention preferably comprise N-glycans, with at least 50%, preferably at least 75%, more preferably at least 85%, and even more preferably at least 90% of the N-glycans comprising, on average, at least one sialic acid moiety. In preferred embodiments, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the N-glycans comprising, on average, at least one sialic acid moiety.

[0152] When mammalian cells are used to express the polypeptides of the present invention, they often contain α-2,3-linked sialic acid groups. However, the polypeptides of the present invention may also contain N-glycans with α-2,6-linked sialic acid groups. In certain embodiments, at least 50% of the sialic acid groups on the N-glycans of a glycoprotein are α-2,6-linked sialic acid groups. Generally, terminal sialic acid groups can be linked to galactose groups via α-2,3 or α-2,6 linkages. The N-glycans of the polypeptides of the present invention may contain more α-2,6-linked sialic acid groups than α-2,3-linked sialic acid groups. At least 60%, at least 70%, at least 80%, or at least 90% of the sialic acid groups on the N-glycans may be α-2,6-linked sialic acid groups. These embodiments can be achieved by using human-derived cell lines to express the polypeptides of the present invention, or by coexpressing, for example, human α-2,6 sialyltransferase in mammalian cells.

[0153] Suitable methods for producing such glycoproteins are described, for example, in WO 2016 / 188905. Thus, a method for producing a glycoprotein comprising N-glycans with improved sialylation is described therein, the method comprising (i) providing cells comprising a nucleic acid encoding a polypeptide comprising a truncated von Willebrand factor (VWF), and (ii) culturing the cells at a temperature below 36.0° C. Additionally, a method for producing a dimer of a glycoprotein comprising a truncated von Willebrand factor (VWF), or for improving the dimerization of the glycoprotein, is described, the method comprising (i) providing cells comprising a nucleic acid encoding the amino acid sequence of the glycoprotein, and (ii) culturing the cells at a temperature below 36.0° C. Further described herein is a method for producing a glycoprotein comprising an N-glycan with improved sialylation, comprising the steps of (i) providing a cell comprising a nucleic acid encoding a polypeptide comprising a truncated von Willebrand factor (VWF) and a recombinant nucleic acid encoding an α-2,6 sialyltransferase, and (ii) culturing the cell under conditions allowing expression of the glycoprotein and the α-2,6 sialyltransferase.

[0154] In one embodiment, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the N-glycans of a polypeptide of the invention comprise at least one sialic acid group.

[0155] In another embodiment, less than 50%, or less than 25%, or less than 15%, or less than 12%, or less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or even less than 1% of the N-glycans of the polypeptides of the invention are asialo N-glycans, i.e., they are N-glycans lacking sialic acid groups.

[0156] Other embodiments of the invention include a truncated von Willebrand factor (VWF), wherein the truncated VWF is capable of binding to factor VIII (FVIII), and wherein the glycoprotein comprises N-glycans, and wherein less than 50%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 29%, preferably less than 28%, preferably less than 27%, preferably less than 26%, preferably less than 25%, preferably less than 24%, preferably less than 23%, preferably less than 22% of the N-glycans are N-glycans. , preferably less than 21%, preferably less than 20%, preferably less than 19%, preferably less than 18%, preferably less than 17%, preferably less than 16%, preferably less than 15%, preferably less than 14%, preferably less than 13%, preferably less than 12%, preferably less than 11%, preferably less than 10%, preferably less than 9%, preferably less than 8%, preferably less than 7%, preferably less than 6%, preferably less than 5% contain on average two or more terminal and non-sialylated galactose residues.

[0157] Yet another embodiment of the present invention includes a truncated von Willebrand factor (VWF), wherein the truncated VWF is capable of binding to factor VIII (FVIII), and wherein the truncated VWF comprises N-glycans, wherein less than 20%, preferably less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1% of the N-glycans contain an average of three or more terminal and non-sialylated galactose residues.

[0158] The above embodiments can be combined with each other. Any indication of the percentage or degree of sialylation of N-glycans above should be understood as an average percentage or degree, i.e., they refer to a population of molecules, not a single molecule. It is clear that the glycosylation or sialylation of individual glycoprotein molecules in a population of glycoproteins will show some heterogeneity.

[0159] dimer The polypeptides of the present invention have a high proportion of dimers. Thus, the polypeptides of the present invention preferably exist as dimers. In one embodiment, at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% of the polypeptides exist as dimers. In another embodiment, the dimer:monomer ratio of the polypeptides of the present invention is at least 1.5, preferably at least 2, more preferably at least 3 or at least 5. Most preferably, essentially all of the polypeptides of the present invention exist as dimers. In a further preferred embodiment, the polypeptides of the present invention do not contain multimeric forms. The use of dimers is preferred because dimers have improved affinity for factor VIII compared to monomers. The dimer content and dimer-to-monomer ratio of the polypeptides of the present invention can be determined as described in Example 2.

[0160] In one embodiment, the affinity of the polypeptide of the present invention for factor VIII is higher than the affinity of human native VWF for the same factor VIII molecule. The factor VIII affinity of the polypeptide may refer to human native, plasma-derived, or recombinant factor VIII with a truncated or deleted B domain, particularly a recombinant factor VIII molecule, preferably a factor VIII molecule characterized by SEQ ID NO: 5.

[0161] It has been found that the preparation of peptides of the present invention with a high proportion of dimers reliably improves the affinity for factor VIII.Instead of or in combination with an increased proportion of dimers, polypeptides according to the present invention with mutations in the factor VIII binding domain that reliably improve the affinity for factor VIII are also preferred embodiments of the present invention.Suitable mutations are disclosed, for example, in WO 2013 / 120939 A1.

[0162] Preparation of Polypeptides Nucleic acids encoding the polypeptides of the present invention can be prepared according to methods known in the art. Based on the cDNA sequence of the prepro-form of human native VWF (SEQ ID NO: 3), recombinant DNA encoding the truncated VWF constructs or polypeptides of the present invention described above can be designed and produced.

[0163] Even if the polypeptide secreted by the host cell does not contain amino acids 1 to 763 of the prepro-form of human VWF, it is preferred that the nucleic acid (e.g., DNA) encoding the intracellular precursor of the polypeptide comprises a nucleotide sequence encoding amino acids 23 to 763 of SEQ ID NO: 4, or preferably an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 1 to 763. Most preferably, the nucleic acid (e.g., DNA) encoding the intracellular precursor of the polypeptide comprises a nucleotide sequence encoding amino acids 23 to 763 of SEQ ID NO: 4 or amino acids 1 to 763 of SEQ ID NO: 4.

[0164] Constructs containing the entire open reading frame DNA inserted in the correct orientation into an expression plasmid can be used for protein expression. Typical expression vectors contain a promoter that directs the synthesis of large amounts of mRNA corresponding to the inserted nucleic acid in cells harboring the plasmid. They may also contain an origin of replication sequence that allows autonomous replication in the host organism and sequences that improve the efficiency with which the synthesized mRNA is translated. Suitable long-term vectors can be maintained as free-replicating vectors, for example, by using viral regulatory elements (e.g., the OriP sequence from the Epstein-Barr virus genome). Cell lines that integrate the vector into their genomic DNA can also be generated, and in this way, the gene product is continuously produced.

[0165] Typically, the provided cells are obtained by introducing nucleic acid encoding the polypeptide of the invention into a mammalian host cell.

[0166] Any host cell capable of cell culture and expression of glycoproteins may be utilized in accordance with the present invention. In certain embodiments, the host cell is a mammalian cell. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include the BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblastoma (PER.C6 (CruCell, Leiden, The Netherlands)); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243 251, 1980); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY. Acad. Sci., 383:44-68, 1982); MRC5 cells; PS4 cells; human amniotic cells (CAP); and a human stem cell tumor line (HepG2). Preferably, the cell line is a rodent cell line, in particular a hamster cell line, such as CHO or BHK, or a human cell line.

[0167] Suitable methods for introducing nucleic acids sufficient to achieve expression of a glycoprotein of interest into mammalian host cells are known in the art. See, for example, Gething et al., Nature, 293:620-625, 1981; Mantei et al., Nature, 281:40-46, 1979; Levinson et al., EP 117, 060; and EP 117, 058. For mammalian cells, common methods for introducing genetic material into mammalian cells include the calcium phosphate precipitation method of Graham and van der Erb (Virology, 52:456-457, 1978) or the Lipofectamine™ (Gibco BRL) method of Hawley-Nelson (Focus 15:73, 1993). A general aspect of mammalian cell host system transformation is described by Axel in U.S. Pat. No. 4,399,216. For various techniques for introducing genetic material into mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537, 1990 and Mansour et al., Nature, 336:348-352, 1988.

[0168] The cells are cultured under conditions that allow expression of the polypeptide, which can be recovered and purified using methods known to those skilled in the art.

[0169] Factor VIII As used herein, the terms "factor VIII" or "FVIII" refer to a molecule having at least a portion of the clotting activity of human native factor VIII. Human FVIII consists of 2351 amino acids (including the signal peptide) and 2332 amino acids (excluding the signal peptide). "Human native FVIII" is a human plasma-derived FVIII molecule having the full-length sequence set forth in SEQ ID NO: 7 (amino acids 1-2332). The detailed domain structure A1-a1-A2-a2-B-a3-A3-C1-C2 has corresponding amino acid residues (see SEQ ID NO: 7): A1 (1-336), a1 (337-372), A2 (373-710), a2 (711-740), B (741-1648), a3 (1649-1689), A3 (1690-2020), C1 (2021-2173) and C2 (2174-2332).

[0170] The clotting activity of the FVIII molecule can be determined using a one-stage clotting assay (e.g., as described by Lee et al., Thrombosis Research 30, 511-519 (1983)) or a chromogenic substrate assay (e.g., the coamatic FVIII test kit, Chromogenix-Instrumentation Laboratory SpA V., le Monza 338-20128 Milano, Italy). Further details of these activity assays are described below.

[0171] Preferably, the FVIII molecules used according to the invention have a specific molar activity of at least 10% of that of human native FVIII. The term "specific molar activity" refers to the clotting activity per mole of FVIII, e.g., expressed in "IU / mole" FVIII, or more conveniently, in "IU / pmole" FVIII.

[0172] In a preferred embodiment, the FVIII molecule is a non-naturally occurring FVIII molecule. Preferably, the non-naturally occurring FVIII molecule is recombinantly produced. In another embodiment, the FVIII molecule is produced by cell culture. In another preferred embodiment, the non-naturally occurring FVIII molecule has a glycosylation pattern that differs from that of plasma-derived FVIII. In yet another embodiment, the FVIII molecule is selected from the group consisting of: (i) a B-domain deleted or truncated FVIII molecule, (ii) a single-chain FVIII molecule, (iii) a recombinantly produced two-chain FVIII molecule, (iv) a FVIII molecule with a protecting group or half-life extending moiety, (v) a fusion protein comprising a FVIII amino acid sequence fused to a heterologous amino acid sequence, and (vi) a combination thereof.

[0173] In another preferred embodiment, the FVIII molecule is a plasma-derived FVIII molecule.

[0174] The terms "factor VIII" and "FVIII" are used interchangeably herein. A "factor VIII composition" in the sense of the present invention includes a composition comprising FVIII and FVIIIa. FVIIIa may be present in small amounts, typically about 1-2% of FVIIIa based on the total amount of FVIII protein in the composition. Proteolytically cleaved FVIII may be present in small to moderate amounts, typically about 1-50% of FVIII protein based on the total amount of FVIII protein in the composition. "FVIII" includes natural allelic variants of FVIII, each of which may be present and occur depending on the individual. FVIII may be plasma-derived or recombinantly produced using well-known production and purification methods. The degree and location of glycosylation, tyrosine sulfation, and other post-translational modifications may vary depending on the host cell chosen and its growth conditions.

[0175] The term FVIII includes FVIII analogs. The term "FVIII analog," as used herein, refers to a FVIII molecule (full-length or B-domain truncated / deleted) in which one or more amino acids are substituted or deleted compared to SEQ ID NO: 7, or the corresponding portion of SEQ ID NO: 7 in a B-domain truncated / deleted FVIII molecule. FVIII analogs do not occur in nature but are obtained by human manipulation.

[0176] The factor VIII molecules contained in the compositions of the invention may also be B-domain truncated / deleted FVIII molecules, where the remaining domains correspond to the sequences set forth in amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 7. Other forms of B-domain deleted FVIII molecules have partial deletions in their a3 domain, resulting in single-chain FVIII molecules.

[0177] These FVIII molecules are therefore recombinant molecules, preferably of mammalian origin, produced in transformed host cells, although the remaining domains of B-domain deleted FVIII (i.e., the three A domains, the two C domains, and the a1, a2, and a3 regions) may differ slightly, e.g., by about 1%, 2%, 3%, 4%, or 5%, from the respective amino acid sequences set forth in SEQ ID NO: 7 (amino acids 1-740 and 1649-2332).

[0178] The FVIII molecules contained in the compositions of the present invention can be two-chain or single-chain FVIII molecules. Alternatively, the FVIII molecules contained in the compositions of the present invention can be biologically active fragments of FVIII, i.e., FVIII in which one or more domains other than the B domain have been deleted or truncated, but the deleted / truncated FVIII molecule retains the ability to support clot formation. FVIII activity can be assessed in vitro using techniques well known in the art. Preferred tests for determining FVIII activity according to the present invention are chromogenic substrate assays or one-stage clotting assays (see below). Amino acid modifications (substitutions, deletions, etc.) can be introduced into the remaining domains, for example, to modify the binding ability of factor VIII to various other components, such as von Willebrand factor (VWF), low-density lipoprotein receptor-related protein (LPR), various receptors, other coagulation factors, cell surfaces, etc., or to introduce and / or eliminate glycosylation sites, etc. Other mutations that do not abolish FVIII activity may also be applied to FVIII molecules / analogues for use in the compositions of the present invention.

[0179] FVIII analogs also include FVIII molecules in which one or more of the amino acid residues of the polypeptides of the present invention have been deleted or substituted with other amino acid residues, and / or additional amino acid residues have been added to the FVIII polypeptides of the present invention.

[0180] Moreover, the Factor VIII molecule / analog may contain other modifications, for example, in the truncated B domain and / or in one or more of the other domains of the molecule ("FVIII derivative") These other modifications may be in the form of various molecules conjugated to the Factor VIII molecule, for example, polymeric compounds, peptide compounds, fatty acid-derived compounds, etc.

[0181] The term FVIII includes FVIII molecules having a protecting group or half-life extending moiety. The term "protecting group" / "half-life extending moiety" refers to one or more chemical groups attached to one or more amino acid moiety chain functional groups, e.g., -SH, -OH, -COOH, -CONH2, -NH2, or one or more N- and / or O-glycan structures, which, when conjugated to such proteins / peptides, can improve the in vivo circulating half-life of many therapeutic proteins / peptides. Examples of protecting groups / half-life extending moieties include biosynthetic fatty acids and their derivatives, hydroxyalkyl starch (HAS) such as hydroxyethyl starch (HES), poly(Gly), and the like. x -Ser y ) n Examples of suitable polymers include homo-amino acid polymers (HAP), hyaluronic acid (HA), heparosan polymers (HEP), phosphorylcholine-based polymers (PC polymers), Fleximer® polymers (Mersana Therapeutics, MA, USA), dextran, polysialic acid (PSA), polyethylene glycol (PEG), Fc domains, transferrin, albumin, elastin-like peptides, XTEN® polymers (Amunix, CA, USA), albumin-binding peptides, von Willebrand factor fragments (vWF fragments), carboxy-terminal peptides (CTP peptides, Prolor Biotech, IL), and any combination thereof (see, e.g., McCormick, C.L., A.B. Lowe, and N. Ayres, Water-Soluble Polymers, in Encyclopedia of Polymer Science and Technology, 2002, John Wiley & Sons, Inc.). The method of derivatization is not critical and will be apparent from the above.

[0182] The term FVIII includes glycoPEGylated FVIII. In the present context, the term "glycoPEGylated FVIII" is intended to designate a FVIII factor molecule (including full-length FVIII and B-domain truncated / deleted FVIII) in which one or more PEG group(s) are attached to the FVIII polypeptide via the polysaccharide side chain(s) (glycan(s)) of the polypeptide.

[0183] FVIII molecules that can be used according to the present invention include fusion proteins containing a FVIII amino acid sequence fused to a heterologous amino acid sequence, preferably a half-life extending amino acid sequence. Preferred fusion proteins are Fc fusion proteins and albumin fusion proteins. The term "Fc fusion protein" as used herein is meant to encompass FVIII fused to an Fc domain, which may be derived from any antibody isotype. IgG Fc domains are often preferred due to the relatively long circulating half-life of IgG antibodies. Furthermore, Fc domains can be modified to modulate specific effector functions, such as complement fixation and / or binding to specific Fc receptors. Fusion with the Fc domain of FVIII, which has the ability to bind to the FcRn receptor, generally results in an extended circulating half-life of the fusion protein compared to the half-life of wild-type FVIII. Therefore, the FVIII molecule for use in the present invention may also be a derivative of a FVIII analog, such as a fusion protein of a FVIII analog, a PEGylated or glycoPEGylated FVIII analog, or a FVIII analog conjugated to a heparosan polymer. The term "albumin fusion protein" as used herein is meant to include FVIII fused to an albumin amino acid sequence or a fragment or derivative thereof. The heterologous amino acid sequence may be fused to the N- or C-terminus of FVIII or inserted internally into the FVIII amino acid sequence. The heterologous amino acid sequence may be any "half-life extending polypeptide" described in WO 2008 / 077616 A1, the disclosure of which is incorporated herein by reference.

[0184] Examples of FVIII molecules for use in the compositions of the invention include those described, for example, in WO 2010 / 045568, WO 2009 / 062100, WO 2010 / 014708, WO 2008 / 082669, WO 2007 / 126808, U.S. Patent No. 2010 / 0173831, U.S. Patent No. 2010 / 0173830, U.S. Patent No. 2010 / 0168391, U.S. Patent No. 2010 / 0113365, U.S. Patent No. 2010 / 0113364, WO 2010 / 0113366, WO 2010 / 0113368, WO 2010 / 0126808, WO 2010 / 0173831, WO 2010 / 0173830, WO 2010 / 0168391, WO 2010 / 0113365, WO 2010 / 0113364, WO 2010 / 0113366, WO 2010 / 0113368, WO 2010 / 0113368, WO 2010 / 0113369, WO 2010 / 0126808 ... and WO 2004 / 067566.

[0185] Examples of FVIII molecules that can be used in the compositions of the invention include the active ingredients of Advate®, Helixate®, Kogenate®, Xyntha®, Adynovate®, Kovaltrii®, Nuic®, NovoEight®, and Eloctate®, as well as the FVIII molecules described in WO 2008 / 135501 and WO 2009 / 007451 and the construct designated "dBN(64-53)" in WO 2004 / 067566. This construct has the amino acid sequence set forth in SEQ ID NO:5.

[0186] The concentration of factor FVIII in the composition of the present invention is typically in the range of 10 to 10,000 IU / mL. In various embodiments, the concentration of FVIII molecules in the compositions of the invention is within the range of 10 to 8,000 IU / mL, or 10 to 5,000 IU / mL, or 20 to 3,000 IU / mL, or 50 to 1,500 IU / mL, or 3,000 IU / mL, or 2,500 IU / mL, or 2,000 IU / mL, or 1,500 IU / mL, or 1,200 IU / mL, 1,000 IU / mL, or 800 IU / mL, or 600 IU / mL, or 500 IU / mL, or 400 IU / mL, or 300 IU / mL, or 250 IU / mL, or 200 IU / mL, or 150 IU / mL, or 100 IU / mL.

[0187] The "International Unit" or "IU" is a unit of measurement for the blood clotting activity (potency) of FVIII as measured by an FVIII activity assay, e.g., a one-stage clotting assay or a chromogenic substrate FVIII activity assay, using a standard calibrated against an international reference preparation calibrated in "IU." One-stage clotting assays are known in the art, such as those described in N Lee, Martin L et al., An Effect of Predilution on Potency Assays of FVIII Concentrates, Thrombosis Research (Pergamon Press Ltd.) 30, 511-519 (1983). Principle of the one-stage assay: The test is performed as a modified version of the activated partial thromboplastin time (aPTT) assay: incubation of plasma with phospholipids and surfactants results in activation of factors of the intrinsic coagulation system. Addition of calcium ions triggers the clotting cascade. The time to the formation of a measurable fibrin clot is determined. The assay is performed in the presence of factor FVIII-deficient plasma. The clotting ability of the depleted plasma is restored by the FVIII clotting factor contained in the sample being tested. The reduction in clotting time is proportional to the amount of FVIII factor present in the sample. The activity of FVIII clotting factor is quantified by direct comparison in international units to a standard preparation of known FVIII activity.

[0188] Another standard assay is the chromogenic substrate assay. Chromogenic substrate assays are commercially available, for example, the coamatic FVIII test kit (Chromogenix-Instrumentation Laboratory SpA, V.le Monza 338-20128 Milano, Italy). The principle of the chromogenic substrate assay is that in the presence of calcium and phospholipids, factor X is activated to factor Xa by factor IXa. This reaction is stimulated by factor VIIIa as a cofactor. FVIIIa is formed from FVIII in the sample to be measured by a small amount of thrombin in the reaction mixture. The optimal concentration of Ca 2+When using phospholipids and factor IXa and an excess of factor X, activation of factor X is proportional to the potency of factor VIII. Activated factor X releases the chromophore pNA from the chromogenic substrate S-2765. Thus, the release of pNA measured at 405 nm is proportional to the amount of FXa formed and, therefore, to the factor VIII activity in the sample.

[0189] Freeze-drying or lyophilization, unless otherwise indicated by the context in which it appears, shall be used to refer to a drying process that converts a solution of substances (i.e., active pharmaceutical ingredients and various formulation additives or "excipients") into a solid. A typical freeze-drying process consists of three stages: "freezing," "primary drying," and "secondary drying." In the freezing stage, almost all of the contained water is converted to ice and the solute is converted to a solid (crystalline or amorphous). In the primary drying stage, ice is removed from the product by direct sublimation, achieved by maintaining a favorable pressure gradient between water molecules (ice) and the surrounding atmosphere. In the secondary drying stage, residual moisture is removed from the product by desorption.

[0190] When concentrations (w / v) are given for freeze-dried compositions, these refer to the volume immediately before freeze-drying.

[0191] Unless otherwise stated, percentage terms represent weight / weight percentages and temperatures are on the Celsius scale.

[0192] FVIII stability When co-formulated into a composition, the polypeptides of the invention inhibit or reduce the loss of FVIII activity in the composition, such that the loss of FVIII activity of the FVIII formulation over time or during a particular process step is less than the FVIII activity of a control formulation lacking the polypeptides of the invention.

[0193] The term "control formulation" or "control composition," as used herein, refers to a formulation having the same components in the same amounts, except that it lacks the polypeptide of the invention. The reduction in FVIII activity of (i) a composition comprising a polypeptide of the invention and FVIII and (ii) a control composition is measured (i) after storage for the same period under the same conditions and / or (ii) after subjecting both compositions to the same process steps, which may include freeze-drying and, optionally, reconstitution.

[0194] In one embodiment, the polypeptides of the present invention, including truncated VWF, improve the stability of FVIII in liquid compositions. During storage in liquid form at 25°C for one week, the decrease in FVIII activity in a composition comprising FVIII and a polypeptide of the present invention is preferably less than 10%, more preferably less than 9%, and most preferably less than 8%. During storage in liquid form at 25°C for two weeks, the decrease in FVIII activity is preferably less than 15%. During storage in liquid form at 25°C for three weeks, the decrease in FVIII activity is preferably less than 17%. During storage in liquid form at 25°C for four weeks, the decrease in FVIII activity is preferably less than 20%. During storage in liquid form at 25°C for six weeks, the decrease in FVIII activity is preferably less than 30%. The decrease in FVIII activity can be determined as described in the Examples of the present application.

[0195] In another embodiment, the polypeptides of the present invention, including truncated VWF, improve the stability of FVIII during freeze-drying, and the loss of FVIII activity upon freeze-drying of a composition comprising FVIII and a polypeptide of the present invention is preferably less than 15%, more preferably less than 10%, and most preferably less than 5%.

[0196] In another embodiment, the polypeptides of the present invention, including truncated VWF, improve the stability of FVIII in lyophilized compositions. During storage in lyophilized form at 25° C. for 12 months, the FVIII activity of the composition comprising FVIII and the polypeptide of the present invention preferably decreases by less than 16%. During storage in lyophilized form at 25° C. for 18 months, the FVIII activity of the composition comprising FVIII and the polypeptide of the present invention preferably decreases by less than 25%. During storage in lyophilized form at 25° C. for 24 months, the FVIII activity of the composition comprising FVIII and the polypeptide of the present invention preferably decreases by less than 30%.

[0197] Pharmaceutical Composition The polypeptides comprising the truncated VWF described herein may preferably be used to improve the in vitro stability of coagulation factor VIII (FVIII) in a composition comprising said FVIII and said polypeptide, which composition is preferably a formulation.

[0198] The term "formulation," as used herein, refers to a pharmaceutical formulation. The terms "pharmaceutical formulation" and "therapeutic formulation" are used interchangeably herein, unless otherwise indicated.

[0199] The preparation is preferably suitable for the treatment or prevention of a blood clotting disorder, in particular hemophilia A.

[0200] Therapeutic formulations of the polypeptides of the present invention can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the polypeptides of the present invention having the desired degree of purity with, optionally, pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic surfactants, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosages and concentrations employed.

[0201] Buffering agents serve to maintain a pH in a range that approximates physiologically acceptable conditions. They may typically be present at concentrations ranging from about 2 mM to about 100 mM. Suitable buffering agents include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumaric acid buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-fumarate mixtures, etc.), and the like. Examples of suitable buffers include disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can also be used.

[0202] Preservatives can be added to retard microbial growth, typically in amounts ranging from 0.2% to 1% (w / v). Suitable preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0203] Osmolality adjusting agents, which may act as "stabilizers," can be added to ensure pharmaceutically acceptable osmolality, preferably isotonicity, of the liquid composition and include inorganic salts, such as sodium chloride, and polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0204] Stabilizers refer to a broad category of excipients whose function can range from bulking agents to additives that help solubilize the therapeutic agent or prevent denaturation or adhesion to the container wall. Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids, e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threoninine, etc.; organic sugars or sugar alcohols, including cyclitols, e.g., inositol, e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, etc.; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, e.g., glutathione; The stabilizer may be thione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides, such as xylose, mannose, fructose, and glucose; disaccharides, such as lactose, maltose, and sucrose; and trisaccharides, such as raffinose; and polysaccharides, such as dextran. The stabilizer may be present in a range of 0.1 to 10,000 parts by weight per part by weight of the polypeptide of the invention. Non-ionic surfactants or detergents (also known as "wetting agents") may be added to aid in solubilizing the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, allowing the formulation to be subjected to stressful shear surfaces without denaturing the protein. Suitable non-ionic surfactants include pluronic polyols (such as poloxamer 184, 188, etc.) and polyoxyethylene sorbitan monoethers (such as polysorbate 20 and 80, etc.). The non-ionic surfactant may be present in a range of about 0.01 mg / ml to about 1.0 mg / ml, or in a range of about 0.05 mg / ml to about 0.2 mg / ml.

[0205] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0206] In one embodiment, the pharmaceutical compositions described herein comprise an aqueous composition of truncated von Willebrand factor (VWF), the pharmaceutical composition comprising: a.FVIII molecule; b. 40-195 mM sodium salt; c.Histidine; d. at least 1 mM calcium salt; and e. surfactants wherein [His]≧180 mM−20*[Ca 2+ ], wherein [Ca 2+ [His] is the concentration of calcium ions in the aqueous composition in millimoles per liter, and [His] is the concentration of histidine in the aqueous composition in millimoles per liter, provided that [His]>0; the osmolality of the composition is 600 mOsmol / L or less. The concentration of sodium salt in the aqueous composition may be 45-95 mM. The concentration of histidine [His] may be 5-200 mM. The composition may contain calcium ions [Ca] at a concentration of 5-100 mM. 2+The composition may have a pH of 5 to 9. The calcium salt of the composition may preferably be calcium chloride or sodium chloride. The composition may further comprise a sugar. The sugar may preferably be sucrose. The sugar concentration may be 1 to 20% (w / w). The surfactant concentration may be 0.001 to 0.2% (v / v). The surfactant may be a non-naturally occurring surfactant. The composition may further comprise at least one amino acid other than histidine. The at least one amino acid other than histidine may be selected from the group consisting of arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, methionine, phenylalanine, leucine, isoleucine, and combinations thereof. The composition may further comprise at least one antioxidant, wherein the at least one antioxidant may be selected from the group consisting of reduced glutathione, methionine, cysteine, sodium sulfite, vitamin A, vitamin E, ascorbic acid, sodium ascorbate, and combinations thereof. The concentration of the at least one antioxidant may vary from 0.05 to 100 mM.

[0207] In one embodiment, the pharmaceutical composition described herein contains, in addition to truncated von Willebrand factor (VWF), sodium chloride, sucrose, L-arginine, calcium ions, a surfactant, and citric acid. Preferably, the composition further contains factor FVIII. In a preferred embodiment, the pharmaceutical composition of the present invention contains 10-30 mg / mL sodium chloride, 3-8 mg / mL sucrose, 3-8 mg / mL L-Arg×2H2O, 0.1-0.5 mg / mL CaCl2×2H2O, 0.5-2 mg / mL poloxamer 188, and 0.5-2 mg / mL Na citrate×2H2O. In certain preferred embodiments, the pharmaceutical composition of the invention comprises 18 mg / mL sodium chloride, 5.4 mg / mL sucrose, 5.4 mg / mL L-Arg×2H2O, 0.3 mg / mL CaCl2×2H2O, 1.2 mg / mL poloxamer 188, and 1.2 mg / mL Na citrate×2H2O.

[0208] In a further embodiment, the pharmaceutical compositions described herein comprise FVIII factor in addition to truncated von Willebrand factor (VWF), wherein the composition comprises L-histidine, NaCl, CaCl, sucrose, polysorbate 80.

[0209] In a further embodiment, the pharmaceutical compositions described herein comprise FVIII factor in addition to truncated von Willebrand factor (VWF), wherein the composition comprises 20 mM L-histidine, 280 mM NaCl, 3.4 mM CaCl, 0.6% w / v sucrose, 0.02% v / v polysorbate 80 at pH 7.

[0210] The formulations of the present invention may also contain an additional therapeutic agent in addition to the polypeptide of the present invention, or FVIII and the polypeptide of the present invention.

[0211] The nucleotide and amino acid sequences shown in the sequence listing are summarized in Table 1.

[0212] [Table 1]

[0213] Specific embodiments of the present invention will now be described with reference to the following examples, which are intended for purposes of illustration only and are not intended to limit the scope of the general concepts described above. [Example]

[0214] Chromogenic FVIII:C assay The chromogenic FVIII:C assay was performed using the Coamatic FVIII test kit (Chromogenix-Instrumentation Laboratory SpA V., le Monza 338-20128 Milano, Italy).

[0215] Assay principle: In the presence of calcium and phospholipids, factor X is activated to factor Xa by factor IXa. This reaction is stimulated by factor VIIIa as a cofactor. FVIIIa is formed from FVIII in the sample to be measured by a small amount of thrombin in the reaction mixture. The optimal concentration of Ca 2+ When using phospholipids and factor IXA and excess factor X, activation of factor X is proportional to the potency of factor VIII. Activated factor X releases the chromophore pNA from the chromogenic substrate S-2765. Thus, the release of pNA, measured at 405 nm, is proportional to the amount of FXa formed and, therefore, to the factor VIII activity in the sample. The assay was configured to run on either the Behring Coagulation Timer (BCT) or the Behring Coagulation System (BCS), both from Siemens Healthcare Diagnostics GmbH, Ludwig-Erhard-Strasse 12, 65760 Eschborn, Germany.

[0216] Example 1 Generation of D'D3 albumin fusion protein (D'D3-FP) An expression cassette for D'D3-FP, consisting of a cDNA encoding VWF amino acids 1-1242, a glycine / serine linker, and a cDNA encoding human albumin, was prepared by custom gene synthesis (Eurofins Genomics, Ebersberg, Germany). The expression cassette was excised from the supplied cloning vector up to the flanking restriction sites (EcoRI, NotI) and inserted into a linearized pIRESneo3 vector (BD Biosciences, Franklin Lakes, NJ, USA) with EcoRI and NotI. The resulting expression plasmid contained nucleotide sequences encoding the VWF polypeptide, D', and D3 (VWF amino acids 1-1242 of SEQ ID NO: 4) fused to the albumin coding sequence via a short linker coding sequence under the control of a CMV promoter. The nucleotide sequence of the coding sequence is presented as SEQ ID NO: 1, and the amino acid sequence of mature D'D3-FP is presented as SEQ ID NO: 2.

[0217] Expression plasmids were propagated in XL 10 Gold (Agilent Technologies) and purified using standard protocols (Qiagen, Hilden, Germany).

[0218] CHO K1 cells were transfected using Lipofectamine 2000 Reagent (Invitrogen) and grown in serum-free medium (CD-CHO, Invitrogen) in the presence of 500–1000 μg / ml of geneticin. An expression plasmid (pFu-797) encoding PACE / Furin, described in WO 2007 / 144173, was cotransfected to maximize propeptide cleavage. Single-cell derived clones were expanded and selected according to their D'D3-FP expression yield, as quantified by an albumin-specific enzyme immunoassay (see below). The cell line finally selected for D'D3-FP fermentation was designated T2050-CL3.

[0219] The production of D'D3-FP was carried out in a bioreactor using a perfusion fermentation process. The fermentation process for the production of D'D3-containing polypeptides was initiated by thawing the T2050-CL3 cell line, followed by cell growth in shake flasks. Finally, the perfusion fermentation process was carried out using a Sartorius BioStat B-DCU5L bioreactor and a BioStat STR 50L disposable bioreactor. A 10L or 200L BioSep (Applikon) was used as a cell retention device, respectively. The cell culture medium was either PowerCHO3 (Lonza BESP1204) with 8mM L-glutamine and 1µM CuSO4 or ProCHO5 (Lonza BESP1072) with 10mM L-glutamine and 1µM CuSO4.

[0220] Seed trains in shake flasks were carried out at 37°C, 7.5% CO2, and a shaking speed of 160 rpm.

[0221] 2.5 x 10 cells in a 5 L bioreactor 5 The target VCD was seeded at 1000 cells / mL. Cells were cultured in PowerCHO3 with 8 mM L-glutamine and 1 μM CuSO4 at a temperature of +37.0 °C, pH 7.00, and 30% oxygen saturation. A temperature shift to +34.0 °C (evaluation range +31 °C to +35 °C) was performed after the initial harvest from the bioreactor run at +37 °C. pH was controlled by sparging with CO2 as acid and NaHCO3 as base. The overlay airflow rate was set at 0.5 L / min. A ring sparger was used as the sparging unit. The agitation rate was 150 rpm in down-pull mode using a double-pitch impeller.

[0222] 3.0 x 10 in a 50 L bioreactor 5Cells were seeded at 1000 cells / mL of target VCD. Cells were cultured in ProCHO5 medium with 10 mM L-glutamine and 1 μM CuSO4 at a temperature of +37.0°C, pH 6.90, and 30% oxygen saturation. A temperature shift to +34.0°C was performed after the first one or two harvests. pH adjustment was as described above, and the overlay airflow rate was set at 2 L / min. A microsparger was used as the sparging unit. The agitation rate was 90 rpm in down-pull mode with a double-pitch impeller blade.

[0223] VCD in the bioreactor is ≥ 1.0 × 10 6 Once the cell count reached 1.0 cells / mL, perfusion was initiated. The perfusion rate was set to 1.0 vol / vol / day. The BioSep was operated in backflush mode with a power input of 7 (30) W, a run time of 5 (10) min, and a 10-second backflush (numbers in parentheses refer to the 50 L bioreactor). Perfusate and bleed were collected into bags filtered through an in-line filter at +2 to +8°C for 48 h. VCD was adjusted according to active bleeding using a turbidity probe and glucose consumption as a parameter with a target of 2 g / L glucose. Harvested material and bleed were filtered through an in-line filter, and the collection system, consisting of a disposable filter and disposable bag, was changed every 2 days.

[0224] To prepare the material for freeze-drying and the stability studies described below, the D'D3 albumin fusion protein harvest was purified by affinity and size-exclusion chromatography. Briefly, the cell-free harvest from the bioreactor was concentrated 30-fold using a TFF system (e.g., Pall's Centramate 500S) with a 30 kDa membrane (e.g., Pall's Centramate OS030T12). NaCl and EDTA were added to the concentrate to a final concentration of 0.75 M NaCl and 5 mM EDTA, and the concentrate was loaded overnight onto a CaptureSelect human albumin column (Life Technologies) pre-equilibrated with 20 mM Tris buffer, pH 7.4. After washing the column with equilibration buffer, D'D3-FP was eluted with elution buffer (20 mM Tris, 2 M MgCl2, pH 7.4). The eluate was then concentrated 10-fold and dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.4 using a 30 kDa cut-off Ultra centrifugal filter (e.g., Amicon UFC903024). To separate the D'D3-FP dimer from the monomer portion, this material was loaded onto a Superdex 200 pg column (GE Healthcare, code: 17-1069-01) pre-equilibrated with 50 mM Tris, 150 mM NaCl, pH 7.4, and the peak fraction containing the D'D3-FP dimer was pooled. The area under the curve of the dimer and monomer peak fractions was used to calculate the ratio of dimer to monomer. The dimer preparation of D'D3-FP was used in the experiments in the following examples.

[0225] rVIII single chain The examples were carried out using a FVIII molecule (the dBN(64-53) construct described in WO 2004 / 067566), which is referred to below as "rVIII single chain".

[0226] Example 2: Determination of FVIII affinity for VWF fragment dimers and monomers The VWF fragment (1-1242) albumin fusion (D'D3-FP) containing the short linker sequence described in Example 1 was expressed in a bioreactor; after purification as described above and separation of monomers and dimers, the affinity of FVIII for such formulations was evaluated by surface plasmon resonance on a Biacore instrument (T2000, GE Healthcare).

[0227] Anti-albumin antibody (MA1-20124, Thermo Scientific) was covalently coupled to the activated CM3 chip via its N-terminus using NHS (N-hydroxysuccinimide) and EDC (ethanolamine hydrochloride), both of which are included in the GE Healthcare Amine Coupling Kit (BR1000-50). For immobilization, 3 μg / mL of antibody was diluted in sodium acetate buffer (10 mM, pH 5.0), and the antibody solution was flowed over the chip at a flow rate of 10 μL / min for 7 minutes. After the immobilization procedure, unbound dextran filaments were saturated by flowing ethanolamine solution (1 M, pH 8.3) over the chip for 5 minutes (at a flow rate of 10 μL / min). The purpose of flow cell saturation was to minimize nonspecific binding of the analyte to the chip. A reference flow cell was set up by saturating an empty flow cell with ethanolamine using the same procedure as above.

[0228] Dimeric and monomeric D'D3-FP proteins were immobilized on covalently bound anti-albumin antibodies by flowing D'D3-FP protein (5 μg / mL) over the chip for 3 minutes (flow rate 10 μL / min).

[0229] To generate FVIII binding curves, each D'D3-FP protein formulation was diluted to concentrations of 0.25 nM, 0.5 nM, 1 nM, 3 nM, and 4 nM in running buffer (HBS-P+: 0.1 M HEPES, 1.5 M NaCl, and 0.5% v / v surfactant P20, pH 7.4; product code BR100671, GE Healthcare). Single-cycle kinetics was performed, and each diluted sample was run over the chip at increasing concentrations for 2 minutes (flow rate 30 μL / min), followed by a 10-minute dissociation time in running buffer HBS-P+. All measurements were performed in duplicate. The temperature during the measurement procedure was adjusted to +25°C.

[0230] Binding parameters were calculated using BiaEvaluation software. The curve fitting method was based on the Langmuir equation. The input data for the calculation was the molar mass of the FVIII analyte (rVIII single chain), and other parameters were approximately maximum. RU and slope were automatically extracted from the fitted association and dissociation curves. The output of the BiaEvaluation software are the association and dissociation rate constants from which affinity constants were calculated. The results are shown in Table 2.

[0231] [Table 2]

[0232] The dimeric D'D3-FP was synthesized from the D'D3-FP monomer (K D = 30 nM) compared to FVIII. D = 34 pM), both of which result from the fast association and slow dissociation of the rVIII single strand.

[0233] Example 3 Preparation of FVIII+rD'D3-FP formulations and evaluation of FVIII:C recovery (i.e., activity by chromogenic assay) during storage in liquid state The purified rVIII single chain was formulated in the desired buffer and excipient composition (i.e., 20 mM L-histidine, 280 mM NaCl, 3.4 mM CaCl, 0.6% w / v sucrose, 0.02% v / v polysorbate 80, pH 7) by preparative size-exclusion chromatography (Superdex 200 pg; GE Healthcare, Ref. No. 17-1043). Purified rD'D3-FP was formulated in the same buffer and excipient matrix by dialysis. Both components were mixed to achieve the desired FVIII activity and rD'D3-FP concentration as described in Table 3. Prior to storage, the formulation was sterile filtered (Millex GV disposable filter unit; Millipore, Ref. No. SLGV013SL) to avoid potential degradation induced by microbial contamination.

[0234] The various compositions were then investigated for FVIII:C recovery at 25°C over a 6 week period.

[0235] FVIII:C recovery (yield) was calculated as the percentage of the amount of FVIII:C in each formulation after storage divided by the amount of FVIII:C in the solution before storage.

[0236] [Table 3]

[0237] Example 4: Preparation of FVIII+rD'D3-FP formulations and evaluation of FVIII:C recovery (i.e., activity by chromogenic assay) upon freeze-drying The purified rVIII single chain was formulated in a buffer and excipient composition (20 mM L-histidine, 280 mM NaCl, 3.4 mM CaCl, 0.6% w / v sucrose, 0.02% v / v polysorbate 80, pH 7) by preparative size exclusion chromatography (Superdex 200 pg). Purified rD'D3-FP was formulated in the same buffer and excipient matrix. Both components were mixed to achieve the desired FVIII activity and rD'D3-FP concentration as described in Table 4. The formulation was then aliquoted (2.5 mL) and freeze-dried. The various compositions were then investigated for FVIII:C loss during freeze-drying.

[0238] The % loss of FVIII:C was calculated by multiplying the amount of FVIII:C in the reconstituted lyophilisate by 100, dividing by the amount of FVIII:C in the solution before freeze-drying, and subtracting the result from 100. FVIII:C loss (%) = 100 - (100 x FVIII:C after lyophilization / FVIII:C before lyophilization)

[0239] [Table 4]

[0240] Example 5: Preparation of FVIII+rD'D3-FP formulations and evaluation of FVIII:C recovery (i.e., activity by chromogenic assay) in freeze-dried samples upon long-term storage at elevated temperatures Purified rVIII single chain was formulated in a buffer and excipient composition (20 mM L-histidine, 280 mM NaCl, 3.4 mM CaCl, 0.6% w / v sucrose, 0.02% v / v polysorbate 80, pH 7) by preparative size exclusion chromatography (Superdex 200 pg). Purified rD'D3-FP was formulated in the same buffer and excipient matrix. Both components were mixed to achieve the desired FVIII activity and rD'D3-FP concentration as described in Table 5. The formulation was then dispensed into vials (2.5 mL) and freeze-dried. Various compositions were then investigated for FVIII:C recovery after freeze-drying and storage at +25°C for up to 24 months.

[0241] FVIII:C recovery (stability) was calculated as the percentage of FVIII:C still measurable after storage. FVIII:C determined after freeze-drying and before storage was used as the basis for the calculation.

[0242] [Table 5]

[0243] Molar ratio Any molar ratio according to the present invention refers to the ratio of the molar concentrations of the D'D3 (monomeric) subunits comprising the polypeptide used in the present invention, regardless of whether they are actually present as monomers or dimers, and FVIII. More specifically, any ratio of rD'D3-FP to FVIII refers in this application, unless otherwise indicated, to the amount or concentration (moles or mol / L) of rD'D3-FP in a solution or freeze-dried product divided by the amount or concentration (moles or mol / L) of FVIII in a solution or freeze-dried product.

[0244] Typically, the concentration of FVIII is measured by an activity test (FVIII:C) and is given in IU / mL. The concentration of rD'D3-FP (a recombinant fusion protein of albumin and the D'D3 domain of VWF) is typically given in mg / mL. These values ​​can be converted to molarity as described below.

[0245] The molecular weight of the monomeric subunit of rD'D3-FP (including glycosylation) used for calculation is 127,000 Da. The molecular weight of FVIII (rVIII single chain) (including glycosylation) used for calculation is 180,000 Da, and its specific activity used for calculation is 11,000 IU / mg.

[0246] [Table 6]

Claims

1. 1. Use of a polypeptide comprising a truncated von Willebrand factor (VWF) to improve the in vitro stability of coagulation factor VIII (FVIII) in a composition, wherein the composition comprises the FVIII and the polypeptide, the molar ratio of the polypeptide to the FVIII in the composition is at least 200, the truncated VWF comprises an amino acid sequence having at least 90% sequence identity with amino acids 764 to 1242 of SEQ ID NO: 4, and the composition does not comprise wild-type VWF.

2. The use according to claim 1, wherein the polypeptide improves the storage stability of FVIII.

3. 3. The use according to claim 1 or 2, wherein the FVIII is either recombinantly produced FVIII or plasma-derived FVIII.

4. The use described in any one of claims 1 to 3, comprising a step of stabilizing FVIII by adding at least a 200-fold molar excess of the polypeptide to FVIII.

5. The use according to any one of claims 1 to 4, wherein the yield of FVIII upon freeze-drying and reconstitution of a composition comprising FVIII and the polypeptide is greater than the yield of FVIII upon freeze-drying and reconstitution of a control composition lacking the polypeptide.

6. The use according to claim 5, wherein the freeze-dried composition is reconstituted immediately after freeze-drying.

7. The use according to any one of claims 1 to 4, wherein a freeze-dried composition comprising FVIII and a polypeptide exhibits less loss of FVIII activity during storage at 25°C than a freeze-dried control composition lacking the polypeptide.

8. 8. The use according to claim 7, wherein the storage is for a period of 12 months.

9. The use according to any one of claims 1 to 4, wherein the FVIII activity in a liquid composition comprising the polypeptide and FVIII after storage at 25°C for one week is greater than the FVIII activity of a control composition lacking the polypeptide.

10. The use according to any one of claims 1 to 9, wherein the polypeptide binds to FVIII with a dissociation constant KD of 1 nM or less.

11. 11. The use according to any one of claims 1 to 10, wherein the polypeptide has low or essentially no affinity for platelets via GPIbα, said low or essentially no affinity being characterized by a dissociation constant KD > 1 μM, preferentially KD > 10 μM, for the binding of the polypeptide to GPIbα.

12. 12. The use according to any one of claims 1 to 11, wherein the polypeptide does not comprise VWF domains A1 and / or A3 or parts thereof and has low or essentially no affinity for type I and type III collagen, said low or essentially no affinity being characterized by a dissociation constant KD > 1 μM, preferentially KD > 10 μM, for binding of the polypeptide to type I and type III collagen.

13. The use according to any one of claims 1 to 12, wherein the polypeptide is a fusion protein comprising a truncated VWF and a half-life extending polypeptide.

14. 14. The use according to claim 13, wherein the half-life extending polypeptide is albumin or a fragment thereof.

15. The use according to any one of claims 1 to 14, wherein the composition is a formulation.

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