METHODS OF PROPHYLACTIC TREATMENT USING RECOMBINANT VWF (rVWF)

Abstract

To provide a method for prophylactic treatment of spontaneous bleeding episodes in a subject with severe von Willebrand Disease (VWD).SOLUTION: The method includes administering to the subject at least one dose of recombinant von Willebrand Factor (rVWF) in a range of at least about 40 IU / kg to about 80 IU / kg twice a week, thereby reducing the frequency and / or duration of spontaneous bleeding episodes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 800,370, filed February 1, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Coagulation disorders, such as von Willebrand disease (VWD), are generally caused by defects in the coagulation cascade. Von Willebrand disease (VWD) refers to a group of disorders caused by a deficiency of von Willebrand factor, which helps platelets aggregate and adhere to blood vessel walls and is required for normal blood clotting.

[0003] Von Willebrand disease (VWD) is the most common inherited bleeding disorder, with an estimated prevalence of 1% (Veyradier A, et al., Medicine (Baltimore). 2016, 95(11):e3038). However, excluding milder forms of the disease, only approximately 1 in 10,000 patients actually require treatment. Current treatment for these coagulation disorders involves replacement therapy using pharmaceutical preparations containing normal clotting factors.

[0004] VWF is a glycoprotein that circulates in plasma as a series of multimers ranging in size from approximately 500 to 20,000 kD. A full-length VWF cDNA has been cloned, and its propolypeptide corresponds to amino acid residues 23 to 764 of the full-length preproVWF (Eikenboom et al. (1995) Haemophilia 1, 77 90 (Non-Patent Document 2)). Multimeric forms of VWF consist of 250 kD polypeptide subunits linked to each other by disulfide bonds. VWF mediates initial platelet adhesion to the subendothelium of injured vascular walls, and larger multimers exhibit enhanced hemostatic activity. Multimerized VWF binds to the platelet surface glycoprotein Gp1bα through interactions with the A1 domain of VWF, promoting platelet adhesion. Other sites on VWF mediate binding to the vessel wall. Thus, VWF forms a bridge between platelets and the vessel wall, essential for platelet adhesion and primary hemostasis under high shear stress conditions. Normally, endothelial cells secrete large polymeric forms of VWF, and lower molecular weight forms of VWF are generated by proteolytic cleavage. The very large multimers are stored in Weibel-Palade bodies of endothelial cells and are released upon stimulation by agonists such as thrombin and histamine.

[0005] Patients with VWD are recommended to be treated with von Willebrand factor (VWF) replacement, due to the need for long-term hemostasis, particularly in response to major surgery and other bleeding episodes. However, prophylactic treatments have not been well described or demonstrated. There remains a need in the art to proactively help patients with such bleeding episodes with prophylactic treatment regimens. The present invention addresses this need by providing a method for the prophylactic treatment of VWD with human rVWF. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Veyradier A,et al.,Medicine(Baltimore).2016,95(11):e3038 [Non-patent document 2] Eikenboom et al (1995) Haemophilia 1,77 90 Summary of the Invention

[0007] The present invention provides a method for prophylactically treating spontaneous bleeding episodes in subjects with severe von Willebrand disease (VWD), comprising administering to the subject at least one dose of recombinant von Willebrand factor (rVWF) in the range of at least about 40 IU / kg to about 80 IU / kg twice weekly, thereby reducing the frequency and / or duration of spontaneous bleeding episodes.

[0008] In some embodiments, at least one dose of rVWF ranges from at least about 50 IU / kg to about 80 IU / kg.

[0009] In some embodiments, the subject has a baseline VWF:ristocetin cofactor activity (VWF:RCo) in the range of 20 IU / dL or less or has been diagnosed with VWD type 1. In certain embodiments, the subject has been diagnosed with VWD type 2A, 2B, or 2M. In certain embodiments, the subject has a VWF:antigen load (VWF:Ag) in the range of 3 IU / dL or more or has been diagnosed with VWD type 3.

[0010] In some embodiments, the subject has received prophylactic treatment with plasma-derived VWF (pdVWF) within the past 12 months prior to the first administration of rVWF.

[0011] In some embodiments, the subject has experienced at least three spontaneous bleeding episodes within the past 12 months.

[0012] In some embodiments of the method, administration of rVWF occurs every 3 to 4 days. In some embodiments, administration occurs on days 1 and 5, 2 and 6, or 3 and 7 of a 7-day period. In certain embodiments, administration occurs at least every 24 hours, 36 hours, 48 ​​hours, 72 hours, or 84 hours. In some embodiments, administration occurs at least every 72 hours.

[0013] In some embodiments, the outlined methods further comprise administering to the subject at least one dose of recombinant factor VIII (rFVIII). In some embodiments, the administration of the at least one dose of rFVIII is administered simultaneously or sequentially with the at least one dose of rVWF.

[0014] In some embodiments, the subject resumes prophylactic treatment after undergoing elective surgery or oral surgery. In some embodiments, if the elective surgery is minor or oral surgery and the subject has a FVIII activity (FVIII:C) of at least 0.4 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery. In certain embodiments, if the elective surgery is major surgery and the subject has a FVIII activity (FVIII:C) of at least 0.8 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery.

[0015] In some embodiments, prophylactic treatment efficacy is indicated by a ≧25% reduction in annualized bleeding rate (ABR) for spontaneous bleeding episodes during rVWF prophylaxis compared to the ABR before treatment.

[0016] In some embodiments, prophylactic treatment efficacy is indicated by a reduction of ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 45%, ≥ 50%, ≥ 55%, ≥ 60%, ≥ 65%, ≥ 70%, ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, or ≥ 95% in the annualized bleeding rate (ABR) for spontaneous bleeding episodes during the rVWF prophylactic administration period compared to the ABR before treatment.

[0017] In some embodiments, the efficacy of prophylactic treatment is measured by examining vWF:RCo and / or FVIII activity in samples obtained from the subject before and after prophylactic treatment with rVWF.

[0018] In some embodiments, the preventive therapeutic effect is measured by examining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity in samples obtained from the subject before and after prophylactic treatment with rVWF.

[0019] In some embodiments, samples for testing FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity are obtained 15 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 28 hours, 32 hours, 48 ​​hours, 72 hours, or 96 hours after prophylactic treatment with rVWF.

[0020] In some embodiments, samples for testing FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity are obtained 25-31 days after prophylactic treatment with rVWF.

[0021] In some embodiments, the prophylactic therapeutic effect is determined after or during a bleeding episode, wherein samples for FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity are obtained after the bleeding episode, and further samples are obtained before rVWF administration, 2 hours after rVWF administration, and every 12 to 24 hours thereafter until the bleeding episode has resolved.

[0022] In some embodiments, the prophylactic therapeutic effect is indicated by an improvement in the activity levels of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability after prophylactic treatment with rVWF compared to the levels before prophylactic treatment with rVWF.

[0023] Also provided herein are methods for prophylactically treating spontaneous bleeding episodes in a subject with severe von Willebrand disease (VWD), comprising administering to the subject a weekly dose of recombinant von Willebrand factor (rVWF) substantially equivalent to a corresponding weekly dose of plasma-derived VWF (pdVWF) previously administered to the subject, thereby reducing the frequency and / or duration of spontaneous bleeding episodes.

[0024] In some embodiments, the weekly dose of rVWF is about 10% less than the corresponding weekly dose of pdVWF. In some embodiments, the weekly dose of rVWF is about 10% more than the corresponding weekly dose of pdVWF.

[0025] In some embodiments, the weekly dose of rVWF is two separate infusions administered on separate days. In some embodiments, the weekly dose of rVWF is three separate infusions administered on separate days. In some embodiments, the weekly dose of rVWF is a single infusion.

[0026] In some embodiments, each individual infusion contains up to 80 IU / kg of rVWF.

[0027] In some embodiments, each individual injection contains 80 IU / kg of rVWF.

[0028] In some embodiments, each individual injection contains 50 IU / kg of rVWF.

[0029] In some embodiments, the subject has a baseline VWF:ristocetin cofactor activity (VWF:RCo) in the range of 20 IU / dL or less or has been diagnosed with VWD type 1. In some embodiments, the subject has been diagnosed with VWD type 2A, 2B, or 2M. In some embodiments, the subject has a VWF:antigen load (VWF:Ag) in the range of 3 IU / dL or more or has been diagnosed with VWD type 3.

[0030] In some embodiments, the subject has been receiving prophylactic pdVWF therapy for at least 12 months.

[0031] In some embodiments, two separate infusions are administered on days 1 and 5, or days 2 and 6, or days 3 and 7 within a seven-day period. In certain embodiments, three separate infusions are administered on days 1, 3, and 6 within a seven-day period. In some embodiments, administration occurs at least every 24 hours, 36 hours, 48 ​​hours, 72 hours, or 84 hours. In certain embodiments, administration occurs at least every 72 hours.

[0032] In some embodiments, the methods provided herein further comprise administering to the subject at least one dose of recombinant factor VIII (rFVIII).

[0033] In some embodiments, the administration of at least one dose of rFVIII is administered simultaneously or sequentially with the weekly dose of rVWF.

[0034] In some embodiments, the subject resumes prophylactic treatment after undergoing elective surgery or oral surgery. In some embodiments, if the elective surgery is minor or oral surgery and the subject has a FVIII activity (FVIII:C) of at least 0.4 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery. In some embodiments, if the elective surgery is major surgery and the subject has a FVIII activity (FVIII:C) of at least 0.8 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery.

[0035] In some embodiments, prophylactic treatment efficacy is indicated by a ≧25% reduction in annualized bleeding rate (ABR) for spontaneous bleeding episodes during rVWF prophylaxis compared to the ABR before treatment.

[0036] In some embodiments, prophylactic treatment efficacy is indicated by a reduction of ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 45%, ≥ 50%, ≥ 55%, ≥ 60%, ≥ 65%, ≥ 70%, ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, or ≥ 95% in the annualized bleeding rate (ABR) for spontaneous bleeding episodes during the rVWF prophylactic administration period compared to the ABR before treatment.

[0037] In some embodiments, the efficacy of prophylactic treatment is measured by examining vWF:RCo and / or FVIII activity in samples obtained from the subject before and after prophylactic treatment with rVWF.

[0038] In some embodiments, the preventive therapeutic effect is measured by examining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity in samples obtained from the subject before and after prophylactic treatment with rVWF.

[0039] In some embodiments, samples for testing FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity are obtained 15 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 28 hours, 32 hours, 48 ​​hours, 72 hours, or 96 hours after prophylactic treatment with rVWF.

[0040] In some embodiments, samples for testing FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity are obtained 25-31 days after prophylactic treatment with rVWF.

[0041] In some embodiments, the prophylactic therapeutic effect is determined after or during a bleeding episode, wherein samples for FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity are obtained after the bleeding episode, and further samples are obtained before rVWF administration, 2 hours after rVWF administration, and every 12 to 24 hours thereafter until the bleeding episode has resolved.

[0042] In some embodiments, the prophylactic therapeutic effect is indicated by an improvement in the activity levels of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability after prophylactic treatment with rVWF compared to the levels before prophylactic treatment with rVWF.

[0043] In some embodiments, the spontaneous bleeding or bleeding episode comprises any one selected from the group consisting of hemarthrosis, epistaxis, muscle bleeding, oral bleeding, and gastrointestinal bleeding.

[0044] In some embodiments of any of the methods described, the subject has not been diagnosed with type 2N VWD or pseudo-VWD.

[0045] Other objects, advantages and embodiments of the present invention will become apparent from the following detailed description. [Brief explanation of the drawings]

[0046] [Figure 1] 1 shows the study design for the international, multicenter, open-label, Phase 3 study described herein (NCT02973087, EudraCT registration number: 2016-001478-14). [Figure 2] A table of study inclusion and exclusion criteria is provided. [Figure 3] 1 shows a table of exemplary dosing schedules for rVWF prophylaxis. [Figure 4] A table of the primary endpoints of the study is shown. [Figure 5A] 1 shows the nucleic acid sequence of human recombinant prepro-VWF. [Figure 5B] 1 shows the nucleic acid sequence of human recombinant prepro-VWF. [Figure 5C] 1 shows the nucleic acid sequence of human recombinant prepro-VWF. [Figure 6A] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6B] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6C]The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6D] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6E] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6F] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6G] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6H] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6I] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 6J] The amino acid sequence of human recombinant prepro-VWF is shown. [Figure 7A] The amino acid sequence of human recombinant mature VWF is shown. [Figure 7B] The amino acid sequence of human recombinant mature VWF is shown. [Figure 7C] The amino acid sequence of human recombinant mature VWF is shown. [Figure 7D] The amino acid sequence of human recombinant mature VWF is shown. [Figure 7E] The amino acid sequence of human recombinant mature VWF is shown. [Figure 7F] The amino acid sequence of human recombinant mature VWF is shown. [Figure 7G] The amino acid sequence of human recombinant mature VWF is shown. DETAILED DESCRIPTION OF THE INVENTION

[0047] Detailed Description of the Invention Introduction For patients with VWD, especially those undergoing major surgery, long-term hemostasis is required, and treatment with von Willebrand factor (VWF) replacement is recommended (Mannucci PM and Franchini M., Haemophilia, 2017, 23(2):182-187; National Institutes of Health. National Heart, Lung, and Blood Institute. The Diagnosis, Evaluation, and Management of von Willebrand Disease. NIH Publication No. 08-5832; December 2007). Plasma-derived VWF therapy contains factor VIII (FVIII), which may accumulate with repeated administration.

[0048] VONVENDI®, approved in the United States, and its equivalent, VEYVONDI®, approved in the EU, each contain a recombinant von Willebrand factor (rVWF) concentrate produced by recombinant DNA technology in a Chinese hamster ovary cell line without the addition of exogenous human or animal-derived proteins (Turecek PL, et al. Hamostaseologie. 2009;29(suppl 1):S32-38; Mannucci PM, et al. Blood. 2013;122(5):648-657; Gill JC, et al. Blood. 2015;126(17):2038-2046; European Medicines Agency. VEYVONDI Summary of Product Characteristics). rVWF contains a complete VWF multimer profile, including extra-large multimers that are typically lacking in plasma-derived VWF (pdVWF) concentrates exposed to the VWF cleavage protein ADAMTS13.

[0049] Most patients with VWD experience mild to moderate mucosal bleeding and posttraumatic or postoperative bleeding, but life-threatening bleeding can occur, especially in patients with severe disease (Nichols et al., Haemophilia, 2008, 14:171-232; Leeebeek FW and Eikenboom JC, N Engl J Med, 2016, 375:2067-80). Reducing the frequency and duration of bleeding episodes may be expected to decrease the need for red blood cell transfusions and reduce the risk of debilitating comorbidities such as arthropathy.

[0050] Patients with severe VWD may benefit from prophylactic rVWF treatment to maintain VWF and FVIII levels to reduce the risk of spontaneous bleeding episodes (BE), such as hemarthrosis, epistaxis, and gastrointestinal bleeding (Abshire TC, Thromb Res 2009, 124(suppl 1):S23-6; Berntorp E, Haemophilia 2008, 14(suppl 5):47-53; Berntorp E and Petrini P, Blood Coagul Fibrinolysis, 2005, 16(suppl 1):S23-6; Berntorp E, Semin Thromb Hemost, 2006, 32:621-5; Abshire et al., Haemophilia, 2013, 19:76-81; Abshire TC, J Thromb Haemost, 2015, 13:1585-9).

[0051] The present invention provides methods for prophylactically treating spontaneous bleeding in patients with severe von Willebrand disease (VWD). The methods include administering recombinant von Willebrand factor (rVWF) to a subject. In some embodiments, the subject is administered a twice-weekly dose of rVWF ranging from about 40 to 80 IU / kg. In some embodiments, the subject is administered a weekly dose of rVWF ranging from about 40 to 80 IU / kg. In some cases, the weekly dose is provided as a single weekly administration. In certain cases, the weekly dose is provided as two separate administrations on different days over a one-week period. In other cases, the weekly dose is provided as three separate administrations on different days over a one-week period.

[0052] The disclosure of PCT Application Publication No. WO2012 / 171031 is incorporated herein by reference in its entirety for all purposes.

[0053] definition Before describing the present invention in detail, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0054] As used herein, "rVWF" refers to recombinant VWF.

[0055] As used herein, "rFVIII" refers to recombinant FVIII.

[0056] The term "recombinant," when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that such cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express naturally occurring genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0057] As used herein, "recombinant VWF" includes VWF obtained by recombinant DNA technology. In certain embodiments, the VWF protein of the present invention can include constructs, such as those prepared as described in WO 1986 / 06096, published October 23, 1986, and U.S. Patent Application No. 07 / 559,509, filed July 23, 1990, in the name of Ginsburg et al., which are incorporated herein by reference for methods of making recombinant VWF. VWF in the present invention can include all possible forms, including monomeric and multimeric forms. It should also be understood that the present invention encompasses various forms of VWF used in combination. For example, VWF of the present invention can include different multimers, different derivatives, and both biologically active and non-biologically active derivatives.

[0058] In the context of the present invention, recombinant VWF includes any member of the VWF family from mammals, such as primates, humans, monkeys, rabbits, pigs, rodents, mice, rats, hamsters, gerbils, dogs, and cats, and biologically active derivatives thereof. Active mutant VWF proteins and variant VWF proteins, such as functional fragments and fusion proteins of VWF proteins, are also included. Furthermore, the VWF of the present invention may further comprise tags that facilitate purification, detection, or both. The VWF described herein may be further modified with therapeutic moieties or moieties suitable for in vitro or in vivo imaging.

[0059] As used herein, "plasma-derived VWF" or "pdVWF" includes all forms of the protein found in blood, including mature VWF obtained from a mammal, that have the property of stabilizing, e.g., binding, in vivo, at least one FVIII molecule.

[0060] The term "high multimeric VWF" or "high molecular weight VWF" refers to a VWF containing at least 10 subunits, or ranging from 12, 14, or 16 subunits to about 20, 22, 24, 26, or more subunits. The term "subunit" refers to a VWF monomer. As is known in the art, it is generally a VWF dimer that polymerizes to form higher multimers (see Turecek et al., Semin. Thromb. Hemost. 2010, 36(5):510-521, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings regarding multimeric analysis of VWF).

[0061] As used herein, the term "factor VIII" or "FVIII" refers to any form of factor VIII molecule having the typical characteristics of blood coagulation factor VIII, whether endogenous to a patient, derived from plasma, or produced using recombinant DNA technology, including any modified form of factor VIII. Factor VIII (FVIII) exists in nature and in therapeutic preparations as a heterogeneous distribution of polypeptides resulting from a single gene product (see, e.g., Andersson et al., Proc. Natl. Acad. Sci. USA, 83:2979-2983 (1986)). Examples of commercially available therapeutic preparations containing factor VIII include those sold under the trade names HEMOFIL M, ADVATE, and RECOMBINATE (available from Baxter Healthcare Corporation, Deerfield, Ill., USA).

[0062] As used herein, "plasma FVIII activity" and "in vivo FVIII activity" are used interchangeably. In vivo FVIII activity, measured using standard assays, can be endogenous FVIII activity, the activity of therapeutically administered FVIII (recombinant or plasma-derived), or both endogenous and administered FVIII activity. Similarly, "plasma FVIII" refers to endogenous FVIII or administered recombinant or plasma-derived FVIII.

[0063] As used herein, "von Willebrand disease" refers to a group of diseases caused by a deficiency of von Willebrand factor, which helps platelets aggregate and adhere to blood vessel walls and is required for normal blood clotting. As further detailed herein, there are several types of von Willebrand disease, including type 1, type 2A, type 2B, type 2M, and type 3.

[0064] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it when found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. VWF is the predominant species present in a preparation and is substantially purified. In some embodiments, the term "purified" means that the nucleic acid or protein appears as essentially one band in an electrophoretic gel. In other embodiments, the nucleic acid or protein is at least 50% pure, more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more pure. In other embodiments, "purifying" or "purifying" refers to the removal of at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogeneous, e.g., 100% pure.

[0065] As used herein, "administering" (and all grammatical equivalents) includes intravenous, intramuscular, subcutaneous, oral, as a suppository, topical contact, intraperitoneal, intralesional, or intranasal administration, or implantation of a sustained-release device, e.g., a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0066] The terms "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" refer to a dose that produces a therapeutic effect in the subject to which it is administered. For example, a therapeutically effective amount of a drug useful for treating hemophilia can be an amount that can prevent or alleviate one or more symptoms associated with hemophilia. The exact dose will vary depending on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0067] As used herein, the terms "patient" and "subject" are used interchangeably and refer to a mammal (preferably a human) having or susceptible to a disease.

[0068] As used herein, the term "about" refers to an approximate range of plus or minus 10% from the stated value. For example, the language "about 20%" encompasses a range of 18-22%.

[0069] As used herein, the term "half-life" refers to the period of time it takes for the amount of a substance to be reduced by half due to degradation (or clearance from a sample or patient).

[0070] I. Recombinant von Willebrand Factor (rVWF) The present invention uses compositions comprising von Willebrand factor (rVWF) for the prophylactic treatment of spontaneous bleeding episodes in subjects with severe VWD. In some embodiments, treatment reduces the severity, incidence (frequency), and / or duration of spontaneous bleeding episodes.

[0071] In certain embodiments, the VWF proteins of the present invention can include constructs, such as those prepared as described in WO 1986 / 06096, published October 23, 1986, in the name of Ginsburg et al., and U.S. Patent Application No. 07 / 559,509, filed July 23, 1990, both of which are incorporated herein by reference for methods of producing recombinant VWF. VWF useful in the present invention includes all possible forms, including monomeric and multimeric forms. One particularly useful form of VWF is a homomultimer of at least two VWFs. The VWF protein can be a biologically active derivative, or, when used solely as a stabilizer of FVIII, the VWF can be in a form that is not biologically active. It should also be understood that the present invention encompasses various forms of VWF used in combination. For example, compositions useful in the present invention can include different multimers, different derivatives, and both biologically active and non-biologically active derivatives.

[0072] In primary hemostasis, VWF acts as a bridge between platelets and specific components of the extracellular matrix, such as collagen. The biological activity of VWF in this process can be measured by various in vitro assays (Turecek et al., Semin. Thromb. Hemost. 28:149-160, 2002). The ristocetin cofactor test is based on the aggregation of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF.

[0073] The degree of platelet aggregation varies depending on the VWF concentration and can be measured, for example, by turbidimetry using an aggregometer (Weiss et al., J. Clin. Invest. 52:2708-2716, 1973; Macfarlane et al., Thromb. Diath. Haemorrh. 34:306-308, 1975). The second method is the collagen binding assay, which is based on ELISA technology (Brown et Bosak, Thromb. Res. 43:303-311, 1986; Favaloro, Thromb. Haemost. 83:127-135, 2000). Microtiter plates are coated with type I or type III collagen. VWF is then bound to the collagen surface and subsequently detected with an enzyme-labeled polyclonal antibody. The final step is a substrate reaction, which can be monitored photometrically with an ELISA reader. As provided herein, the specific ristocetin cofactor activity (VWF:RCo) of the VWF of the present invention is generally expressed in mU / μg of VWF as measured using an in vitro assay.

[0074] An advantage of the rVWF compositions of the present invention over pdVWF is that rVWF exhibits a higher specific activity than pdVWF. In some embodiments, the rVWF of the present invention exhibits a specific activity of at least about 20 mU / μg, 22.5 mU / μg, 25 mU / μg, 27.5 mU / μg, 30 mU / μg, 32.5 mU / μg, 35 mU / μg, 37.5 mU / μg, 40 mU / μg, 42.5 mU / μg, 45 mU / μg, 47.5 mU / μg, 50 mU / μg, 52.5 mU / μg, 55 mU / μg, 57.5 mU / μg, 60 mU / μg, 62.5 mU / μg, 65 mU / μg, 67.5 mU / μg, 70 mU / μg , 72.5mU / μg, 75mU / μg, 77.5mU / μg, 80mU / μg, 82.5mU / μg, 85mU / μg, 87.5mU / μg, 90mU / μg, 92.5mU / μg, 95mU / μg, 97.5mU / μg, 100mU / μg g, 105 mU / μg, 110 mU / μg, 115 mU / μg, 120 mU / μg, 125 mU / μg, 130 mU / μg, 135 mU / μg, 140 mU / μg, 145 mU / μg, 150 mU / μg, or more.

[0075] The rVWF of the present invention is a high multimer containing about 10 to about 40 subunits. In further embodiments, the multimeric rVWF produced using the methods of the present invention contains about 10 to 30, 12 to 28, 14 to 26, 16 to 24, 18 to 22, or 20 to 21 subunits. In further embodiments, rVWF exists in multimers of various sizes, ranging from dimers to multimers of more than 40 subunits (>10 million daltons). The largest multimers provide multiple binding sites capable of interacting with both platelet receptors and subendothelial matrix sites of injury and are the most hemostatically active forms of VWF. While ultra-large rVWF multimers are cleaved over time in the presence of ADAMTS13, the rVWF compositions of the present invention are generally not exposed to ADAMTS13 during production (typically by expression in cell culture) and retain their high multimeric structure.

[0076] In one embodiment, the rVWF composition used in the methods described herein has an oligomeric distribution characterized in that 95% of the rVWF oligomers have between 6 and 20 subunits. In another embodiment, the rVWF composition has an oligomeric distribution characterized in that 95% of the rVWF oligomers have a set of subunits selected from variants 458-641 found in Table 2 of WO 2012 / 171031, which is incorporated herein by reference in its entirety for all purposes.

[0077] In one embodiment, an rVWF composition can be characterized according to the percentage of rVWF molecules present in specific higher-order rVWF multimers or larger multimers. For example, in one embodiment, at least 20% of the rVWF molecules in an rVWF composition used in the methods described herein are present in oligomeric complexes of at least 10 subunits. In another embodiment, at least 20% of the rVWF molecules in an rVWF composition used in the methods described herein are present in oligomeric complexes of at least 12 subunits. In yet other embodiments, an rVWF composition used in the methods provided herein has a minimum percentage (e.g., at least X%) of rVWF molecules present in specific higher-order rVWF multimers or larger multimers (e.g., multimers of at least Y subunits) according to any one of variants 134-457 found in Tables 3-5, which are incorporated herein by reference in their entirety for all purposes.

[0078] In accordance with the above, rVWF compositions (with or without FVIII) administered to a subject generally contain a significant proportion of high molecular weight (HMW) rVWF multimers. In further embodiments, HMW rVWF multimer compositions contain at least 10%-80% rVWF decamers or higher multimers. In further embodiments, the compositions contain about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, or 60-70% decamers or higher multimers. In further embodiments, HMW rVWF multimer compositions contain at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decamers or higher multimers.

[0079] Assessment of the number and proportion of rVWF multimers can be performed using methods known in the art, including, but not limited to, methods using electrophoresis and size-exclusion chromatography to separate VWF multimers by size, as discussed, for example, by Cumming et al. (J Clin Pathol. 1993 May;46(5):470-473, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings related to the assessment of VWF multimers). Such techniques can also include immunoblotting (such as Western blotting), in which a gel is immunoblotted with a radiolabeled antibody against VWF, followed by chemiluminescence detection (see, e.g., Wen et al., (1993), J. Clin. Lab. Anal., 7:317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings related to the assessment of VWF multimers). Additional assays for VWF include VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assays (VWF:CBA), which are often used to diagnose and classify von Willebrand disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings regarding assays for VWF).

[0080] In a further embodiment, the higher-ordered rVWF multimers of the present invention are stable for about 1 to about 90 hours after administration. In another further embodiment, the higher-ordered rVWF multimers are stable for about 5 to 80 hours, 10 to 70 hours, 15 to 60 hours, 20 to 50 hours, 25 to 40 hours, or 30 to 35 hours after administration. In yet a further embodiment, the higher-ordered rVWF multimers are stable for at least 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration. In a specific embodiment, the stability of the rVWF multimers is assessed in vitro.

[0081] In one embodiment, the higher-order rVWF multimers used in the compositions and methods provided herein have a half-life of at least 12 hours after administration. In another embodiment, the higher-order rVWF multimers have a half-life of at least 24 hours after administration. In yet other embodiments, the higher-order rVWF multimers have a half-life selected from variants 642-1045 found in Table 6 of WO2012 / 171031, which is incorporated herein by reference in its entirety for all purposes.

[0082] In certain aspects, the rVWF (recombinant or plasma-derived) used in accordance with the present invention is not modified by any conjugation, post-translational modification, or covalent modification, hi certain embodiments, the rVWF of the present invention is not modified with water-soluble polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, poly-carbohydrate moieties, etc.

[0083] In other aspects, the rVWF (recombinant or plasma-derived) used in accordance with the present invention is modified by conjugation, post-translational modification, or covalent modification, including modification of N- or C-terminal residues and selected side chains, e.g., at free sulfhydryl groups, primary amines, and hydroxyl groups. In one embodiment, a water-soluble polymer is linked to the protein (directly or via a linker) through a lysine group or other primary amine. In one embodiment, the rVWF protein of the present invention can be modified by conjugation of a water-soluble polymer, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, poly-carbohydrate moieties, and the like.

[0084] Water-soluble polymers that can be used to modify rVWF and / or FVIII include linear and branched structures. The conjugated polymers can be directly attached to the coagulation proteins of the present invention or, alternatively, can be attached via a linking moiety. Non-limiting examples of protein conjugation with water-soluble polymers can be found in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337, as well as Abuchowski and Davis, "Enzymes as Drugs," Holcenberg and Roberts, Eds., pp. 367-383, John Wiley & Sons, New York (1981), and Hermanson G., Bioconjugate Techniques, 2nd Ed., Academic Press, Inc. 2008.

[0085] Protein conjugation can be carried out by several techniques well known in the art; see, for example, Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include linkage via a peptide bond between a carboxyl group on either the coagulation protein or the water-soluble polymer moiety and an amine group on the other, or via an ester bond between a carboxyl group on one side and a hydroxyl group on the other. Another contemplated linkage for conjugating the coagulation proteins of the invention to water-soluble polymer compounds is via a Schiff base between a free amino group on the polymer moiety, which reacts with an aldehyde group formed at the non-reducing end of the polymer by periodate oxidation (Jennings and Lugowski, J. Immunol. 1981;127:1011-8; Femandes and Gregonradis, Biochim Biophys Acta. 1997;1341;26-34). The resulting Schiff base can be stabilized by specific reduction with NaCNBH3 to form a secondary amine. An alternative approach is to generate terminal free amino groups on the polymer by preoxidation followed by reductive amination with NH4Cl. Bifunctional reagents can be used to link two amino groups or two hydroxyl groups. For example, amino-containing polymers can be coupled to the amino groups of coagulation proteins using reagents such as BS3 (bis(sulfosuccinimidyl) suberic acid / Pierce, Rockford, Ill.). Furthermore, heterobifunctional crosslinkers such as sulfo-EMCS (N-epsilon-maleimidocaproyloxy) sulfosuccinimide ester / Pierce) can be used to link amine and thiol groups.In other embodiments, aldehyde-reactive groups such as PEG alkoxide and diethyl acetal of bromoacetaldehyde; PEG with DMSO and acetic anhydride; and PEG chloride and phenoxide of 4-hydroxybenzaldehyde; succinimidyl active esters; activated dithiocarbonate PEG; and activated PEG with 2,4,5-trichlorophenyl chloroformate and p-nitrophenyl chloroformate may be used in conjugating coagulation proteins.

[0086] In some embodiments, the rVWF used in the methods of the invention has been matured in vitro using furin. In further embodiments, the furin is recombinant furin.

[0087] In a further aspect, the rVWF used in the methods of the invention is produced by expression in mammalian cell culture using methods known in the art. In a specific embodiment, the mammalian culture comprises CHO cells. In an exemplary embodiment, the rVWF of the invention comprises an rVWF protein isolated from a CHO cell expression system. In a further embodiment, propeptide removal is induced in vitro via exposure of pro-VWF to furin, and in yet another further embodiment, the furin used for propeptide removal is recombinant furin. In yet a further embodiment, fully glycosylated / ABO blood group glycans are absent.

[0088] In yet a further embodiment, the rVWF used in the methods and compositions of the present invention is expressed in a suitable eukaryotic host system. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep, and HepG2; insect cells such as SF9, SF21, S2, and High Five cells; and yeast cells such as Saccharomyces or Schizosaccharomyces cells. In one embodiment, VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, etc., such as human cell lines, hamster cell lines, or mouse cell lines. In a specific embodiment, the cell line is a CHO cell line, a BHK cell line, or a HEK cell line. Typically, mammalian cells, such as CHO cells from a continuous cell line, can be used to express the VWF of the present invention.

[0089] In certain embodiments, the nucleic acid sequence comprising the VWF coding sequence can be a vector. The vector can be delivered by a virus or can be a plasmid. The nucleic acid sequence encoding the protein can be a specific gene or a biologically functional portion thereof. In one embodiment, the protein is at least a biologically active portion of VWF. A wide variety of vectors can be used to express VWF and can be selected from eukaryotic expression vectors. Examples of eukaryotic cell expression vectors include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, and pMET, which use promoters such as AOX1, GAP, GAL1, and AUG1; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, and pBAC, which use promoters such as PH, p10, MT, Ac5, OpIE2, gp64, and polh; and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, and pBPV, which use promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin, as well as vectors derived from viruses such as vaccinia virus, adeno-associated virus, herpes virus, and retrovirus.

[0090] In some embodiments of the invention, the nucleic acid sequence further comprises other sequences suitable for controlling protein expression, such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction sites, polyA sequences, protein processing sequences, selectable markers, and the like, commonly known to those skilled in the art.

[0091] In certain embodiments, the cell culture methods of the present invention may include the use of microcarriers. In some embodiments, cell cultures of the present invention can be carried out in large bioreactors under conditions suitable for achieving a high specific culture surface area per volume to achieve high cell densities and protein expression. One means for achieving such culture conditions is the use of cell culture microcarriers in stirred-tank bioreactors. The concept of cell growth on microcarriers, first described by van Wezel (van Wezel, AL, Nature 216:64-5 (1967)), allows cells to attach to the surface of small solid particles suspended in the growth medium. These methods provide a high surface-to-volume ratio, thereby enabling efficient nutrient utilization. Furthermore, in the case of expression of secreted proteins in eukaryotic cell lines, the increased surface-to-volume ratio allows for high levels of secretion in the culture supernatant and, therefore, high protein yields. Ultimately, these methods facilitate the scale-up of eukaryotic cell expression cultures.

[0092] VWF-expressing cells can be attached to spherical or porous microcarriers during cell culture growth. The microcarriers can be selected from the group consisting of dextran-, collagen-, plastic-, gelatin-, and cellulose-based microcarriers, as well as other microcarriers such as those described by Butler (1988). In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). It is also possible to grow cells to a certain biomass on spherical microcarriers, subculture the cells when they reach the final biomass of the fermentor, and then produce the expressed protein on porous microcarriers, or vice versa. Suitable spherical microcarriers include smooth-surfaced microcarriers such as Cytodex™ 1, Cytodex™ 2, and Cytodex™ 3 (GE Healthcare), and macroporous microcarriers such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1, and Cytoline™ 2 (GE Healthcare).

[0093] In certain embodiments, rVWF is expressed in cells cultured in a cell culture medium that produces high molecular weight rVWF. The terms "cell culture medium," "cell culture medium(s)," and "cell culture supernatant" generally refer to aspects of the cell culture process that are well known in the art. In the context of the present invention, cell culture solutions can include cell culture medium and cell culture supernatant. Cell culture medium is added externally to the cell culture medium to provide nutrients and other components for culturing VWF-expressing cells, optionally with additives. Cell culture supernatant refers to a cell culture medium that contains nutrients and other components from the cell culture medium as well as products released, metabolized, and / or excreted by the cells during culture. In further embodiments, the medium can be animal protein-free and of known composition. Methods for preparing animal protein-free, chemically defined media are known in the art, e.g., US2008 / 0009040 and US2007 / 0212770, both of which are incorporated herein for all purposes, particularly for all teachings related to cell culture media. "Protein-free" and related terms refer to proteins that are exogenous to the cells being cultured or derived from sources other than those cells, and which are naturally shed during growth. In another embodiment, the medium is polypeptide-free. In another embodiment, the medium is serum-free. In another embodiment, the medium is animal protein-free. In another embodiment, the medium is animal component-free. In another embodiment, the medium contains protein, e.g., animal protein derived from serum, such as fetal bovine serum. In another embodiment, the culture has exogenously added recombinant protein. In another embodiment, the protein is derived from a certified pathogen-free animal. The term "chemically defined" as used herein means that the medium does not contain any additives whose composition is not defined, such as, for example, extracts of animal components, organs, glands, plants, or yeast. Thus, each component of a chemically defined medium is precisely specified. In a preferred embodiment, the medium is animal-derived component-free and protein-free.

[0094] In a further embodiment, after purification from mammalian cell culture, rFVIII is reconstituted prior to administration. In another further embodiment, rVWF is treated with furin before or after reconstitution. In a further embodiment, furin is recombinant furin. In another further embodiment, the rVWF of the present invention is not exposed to ADAMTS13, and as a result, extra-large (i.e., containing 10 or more subunits) rVWFs are present in the rVWF compositions of the present invention.

[0095] In certain aspects, the rVWF used in the methods of the present invention is contained in a formulation containing a buffer, a sugar and / or sugar alcohol (including, but not limited to, trehalose and mannitol), a stabilizer (such as glycine), and a surfactant (such as polysorbate 80). In further embodiments, in the case of formulations containing rFVIII, the formulation may further include sodium, histidine, calcium, and glutathione.

[0096] In one embodiment, the formulation containing rVWF is lyophilized prior to administration. Lyophilization is performed using techniques common in the art and should be optimized for the composition being prepared (Tang et al., Pharm Res. 21:191-200 (2004) and Chang et al., Pharm Res. 13:243-9 (1996)).

[0097] The method for preparing a pharmaceutical formulation may include one or more of the following steps: adding a stabilizer to the mixture before lyophilization, as described herein; or adding at least one agent selected from a bulking agent, an osmolality adjusting agent, and a surfactant, each of which is described herein, to the mixture before lyophilization. In one embodiment, the lyophilized formulation comprises at least one or more of a buffer, a bulking agent, and a stabilizer. In this embodiment, the usefulness of a surfactant is evaluated and selected when aggregation during the lyophilization step or during reconstitution is a problem. A suitable buffer is included to maintain a stable pH range of the formulation during lyophilization.

[0098] The standard reconstitution technique for lyophilized material is to add back a volume of purified water or sterile water for injection (WFI) (typically a volume equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the manufacture of pharmaceuticals for parenteral administration [Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)]. Thus, a method for preparing a reconstituted recombinant VWF composition of the present invention is provided, which comprises adding a diluent to the lyophilized recombinant VWF composition.

[0099] The lyophilized material can be reconstituted as an aqueous solution with a variety of aqueous carriers, for example, sterile water for injection, water containing a preservative for multiple doses, or water containing an appropriate amount of surfactant (e.g., aqueous suspensions containing the active compound mixed with excipients suitable for the manufacture of aqueous suspensions). In various embodiments, such additives are suspending agents such as, but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and dispersing or wetting agents are naturally occurring phospholipids such as, but not limited to, lecithin, or condensation products of alkylene oxides with fatty acids, such as, but not limited to, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as, but not limited to, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, such as, but not limited to, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as, but not limited to, polyethylene sorbitan monooleate. In various embodiments, aqueous suspensions also contain one or more preservatives, for example, but not limited to, ethyl, or n-propyl p-hydroxybenzoate.

[0100] In certain embodiments, the compositions of the present invention are liquid formulations for administration with the use of a syringe or other storage container, hi further embodiments, these liquid formulations are produced from lyophilized materials described herein that are reconstituted as an aqueous solution.

[0101] In a further embodiment, the compositions of the present invention further comprise one or more pharmaceutically acceptable carriers. The phrases "pharmaceutically" or "pharmacologically" acceptable refer to molecular entities and compositions that are stable, inhibit proteolytic degradation, including aggregation and cleavage products, and do not produce allergic or other adverse reactions when administered using routes well known in the art, such as those described below. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., including those disclosed above.

[0102] II. Production of recombinant VWF The free, mature recombinant von Willebrand factor (rVWF) of the present invention can be produced recombinantly. Those skilled in the art are aware of useful methods for expressing recombinant proteins in host cells. In some cases, the method involves expressing a nucleic acid sequence encoding rVWF in host cells, such as CHO cells, and culturing the resulting host cells under specific conditions to produce rVWF, preproVWF, proVWF, etc.

[0103] In certain embodiments, the nucleic acid sequence comprising a sequence encoding VWF can be an expression vector. The vector can be delivered by a virus or can be a plasmid. The nucleic acid sequence encoding a protein can be a specific gene or a biologically functional portion thereof. In one embodiment, the protein is at least a biologically active portion of VWF. The nucleic acid sequence can further include other sequences suitable for regulating protein expression, such as a promoter sequence, an enhancer, a TATA box, a transcription initiation site, a polylinker, a restriction site, a polyA sequence, a protein processing sequence, a selection marker, and the like, commonly known to those skilled in the art.

[0104] A wide variety of vectors can be used to express VWF, and can be selected from eukaryotic cell expression vectors. Examples of eukaryotic cell expression vectors include: (i) vectors for yeast expression, such as pAO, pPIC, pYES, and pMET, which use promoters such as AOX1, GAP, GAL1, and AUG1; (ii) vectors for insect cell expression, such as pMT, pAc5, pIB, pMIB, and pBAC, which use promoters such as PH, p10, MT, Ac5, OpIE2, gp64, and polh; and (iii) vectors for mammalian cell expression, such as pSVL, pCMV, pRc / RSV, pcDNA3, and pBPV, which use promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin, as well as vectors derived from viruses such as vaccinia virus, adeno-associated virus, herpes virus, and retrovirus.

[0105] In some aspects, the rVWF used in the methods of the invention is produced by expression in mammalian cell culture using methods known in the art. In certain embodiments, the mammalian culture comprises CHO cells. In further embodiments, rVWF is co-expressed with recombinant factor VIII (rFVIII) in the same culture. In such embodiments, rVWF and rFVIII are co-purified (co-purified) or separately purified using methods known in the art. In other embodiments, rVWF is expressed in a culture that does not contain rFVIII.

[0106] In some embodiments, rVWF is expressed and isolated from a suitable eukaryotic host system. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep, and HepG2; insect cells such as SF9, SF21, S2, and High Five cells; and yeast cells such as Saccharomyces or Schizosaccharomyces cells. In one embodiment, VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, etc., such as in a human cell line, a hamster cell line, or a mouse cell line. In a specific embodiment, the cell line is a CHO cell line, a BHK cell line, or a HEK cell line. Typically, mammalian cells, such as CHO cells from a continuous cell line, can be used to express the VWF of the present invention. In certain cases, the VWF protein is expressed and isolated from a CHO cell expression system.

[0107] VWF can be produced in cell culture systems and according to any cell culture method recognized by those skilled in the art. In some embodiments, cell culture can be carried out in large bioreactors under conditions suitable for achieving a high specific culture surface area per volume to achieve high cell density and protein expression. One means of achieving such culture conditions is the use of cell culture microcarriers in stirred-tank bioreactors. The concept of cell growth on microcarriers was first described by van Wezel (van Wezel, AL, Nature, 1967, 216:64-5) and allows cells to attach to the surface of small solid particles suspended in the growth medium. These methods provide a high surface-to-volume ratio, thereby enabling efficient nutrient utilization. Furthermore, in the case of expression of secreted proteins in eukaryotic cell lines, the increased surface-to-volume ratio allows for high levels of secretion in the culture supernatant and, therefore, high protein yields. Ultimately, these methods facilitate the scale-up of eukaryotic cell expression cultures.

[0108] VWF-expressing cells can be attached to spherical or porous microcarriers during cell culture growth. The microcarriers can be selected from the group consisting of dextran-, collagen-, plastic-, gelatin-, and cellulose-based microcarriers, as well as other microcarriers such as those described by Butler (1988). In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). It is also possible to grow cells to a certain biomass on spherical microcarriers, subculture the cells when they reach the final biomass of the fermentor, and then produce the expressed protein on porous microcarriers, or vice versa. Suitable spherical microcarriers include smooth-surfaced microcarriers such as Cytodex™ 1, Cytodex™ 2, and Cytodex™ 3 (GE Healthcare), and macroporous microcarriers such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1, and Cytoline™ 2 (GE Healthcare).

[0109] In further embodiments, the VWF propeptide is cleaved from non-mature VWF in vitro via exposure of pro-VWF to furin. In some embodiments, the furin used to cleave the propeptide is recombinant furin.

[0110] In certain embodiments, rVWF is expressed in cells cultured in a cell culture medium that produces high molecular weight rVWF. The terms "cell culture medium," "cell culture medium(s)," and "cell culture supernatant" generally refer to aspects of the cell culture process that are well known in the art. In the context of the present invention, cell culture solutions can include cell culture medium and cell culture supernatant. Cell culture medium is added externally to the cell culture medium to provide nutrients and other components for culturing VWF-expressing cells, optionally with additives. Cell culture supernatant refers to a cell culture medium that contains nutrients and other components from the cell culture medium as well as products released, metabolized, and / or excreted by the cells during culture. In further embodiments, the medium can be animal protein-free and of known composition. Methods for preparing animal protein-free, chemically defined media are known in the art, e.g., US2006 / 0094104, US2007 / 0212770, and US2008 / 0009040, all of which are incorporated herein for all purposes, particularly for all teachings related to cell culture media. "Protein-free" and related terms refer to proteins that are exogenous to the cells being cultured or derived from sources other than those cells, and which are naturally shed during growth. In another embodiment, the medium is polypeptide-free. In another embodiment, the medium is serum-free. In another embodiment, the medium is animal protein-free. In another embodiment, the medium is animal component-free. In another embodiment, the medium contains protein, e.g., animal protein derived from serum, such as fetal bovine serum. In another embodiment, the culture has exogenously added recombinant protein. In another embodiment, the protein is derived from a certified pathogen-free animal. The term "chemically defined" as used herein means that the medium does not contain any additives whose composition is not defined, such as, for example, extracts of animal components, organs, glands, plants, or yeast. Thus, each component of a chemically defined medium is precisely specified. In a preferred embodiment, the medium is animal-derived component-free and protein-free.

[0111] In certain embodiments, the culture of cells expressing VWF can be maintained for at least about 7 days, or at least about 14 days, 21 days, 28 days, or at least about 5 weeks, 6 weeks, 7 weeks, or at least about 2 months, or 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, or more. The cell density at which the cell culture is maintained for the production of recombinant VWF protein will vary depending on the culture conditions and medium used for protein expression. One skilled in the art will be able to readily determine the optimal cell density for a cell culture producing VWF. In one embodiment, the culture is maintained at a density of about 0.5 x 10 6 ~4×10 7 In another embodiment, the cell density is maintained at about 1.0 x 10 cells / ml for an extended period of time. 6 ~Approx. 1.0×10 7 In another embodiment, the cell density is maintained at about 1.0 x 10 cells / ml for an extended period of time. 6 ~Approx. 4.0×10 6 In another embodiment, the cell density is maintained at about 1.0 x 10 cells / ml for an extended period of time. 6 ~Approx. 4.0×10 6 In yet another embodiment, the cell density is maintained at about 2.0 x 10 cells / ml for an extended period of time. 6 ~Approx. 4.0×10 6 , or approximately 1.0 × 10 6 ~Approx. 2.5×10 6 , or approximately 1.5 × 10 6 ~Approx. 3.5×10 6 or any other similar range of concentrations for extended periods of time. After a suitable period in cell culture, the rVWF can be isolated from the expression system using methods known in the art.

[0112] In a specific embodiment, the cell density of the continuous cell culture for rVWF production is 2.5×10 6 In another specific embodiment, the cell density is maintained at a concentration of 2.0 x 10 cells / mL or less for an extended period of time. 6 Cells / mL or less, 1.5×10 6Cells / mL or less, 1.0×10 6 Cells / mL or less, 0.5×10 6 In one embodiment, the cell density is maintained at a density of 1.5 x 10 cells / mL or less. 6 cells / mL~2.5×10 6 Maintained in cells / mL.

[0113] In one embodiment of the above cell culture, the cell culture medium comprises a media additive comprising copper. Such cell culture medium is described, for example, in U.S. Patent No. 8,852,888 and U.S. Patent No. 9,409,971, which are incorporated herein by reference in their entirety for all purposes, particularly for all teachings related to cell culture methods and compositions for producing recombinant VWF.

[0114] The polynucleotide and amino acid sequences of prepro-VWF are set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively, and are available under GenBank accession numbers NM_000552 (Homo sapiens von Willebrand factor (VWF) mRNA) and NP_000543, respectively. The amino acid sequence corresponding to the mature VWF protein is set forth in SEQ ID NO:3 (corresponding to amino acids 764 to 2813 of the full-length prepro-VWF amino acid sequence). In some embodiments, the VWF exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the sequence of SEQ ID NO:3. In some embodiments, the rVWF of the present invention exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the sequence of SEQ ID NO: 3. See, e.g., U.S. Patent No. 8,597,910, U.S. Patent Publication No. 2016 / 0129090, and Figures 5A-5C, 6A-6J, and 7A-7G.

[0115] A useful form of rVWF has at least the property of stabilizing, e.g., binding, at least one factor VIII (FVIII) molecule in vivo, and optionally has a pharmacologically acceptable glycosylation pattern. Specific examples include VWF lacking the A2 domain and therefore resistant to proteolysis (Lankhof et al., Thromb. Haemost. 77:1008-1013, 1997), and the VWF fragment from Val449 to Asn730, which contains the glycoprotein 1b-binding domain and collagen and heparin-binding sites (Pietu et al., Biochem. Biophys. Res. Commun. 164:1339-1347, 1989). In one embodiment, the ability of VWF to stabilize at least one FVIII molecule is determined in a VWF-deficient mammal according to methods known in the art.

[0116] The rVWF of the present invention can be produced by any method known in the art. Specific examples are disclosed in WO 86 / 06096, published October 23, 1986, and U.S. Patent Application No. 07 / 559,509, filed July 23, 1990, which are incorporated herein by reference for their description of methods for producing recombinant VWF. Thus, methods are known in the art for (i) generating recombinant DNA by genetic engineering, e.g., reverse transcription of RNA and / or amplification of DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, e.g., electroporation or microinjection, (iii) culturing transformed cells, e.g., continuously or batchwise, (iv) expressing VWF, e.g., constitutively or upon induction, and (v) isolating VWF, e.g., from the culture medium or by recovering transformed cells, thereby (vi) obtaining purified rVWF, e.g., by ion exchange or affinity chromatography. In one embodiment, recombinant VWF is produced in transformed host cells using recombinant DNA techniques well known in the art. For example, the sequence encoding the polypeptide could be excised from DNA using appropriate restriction enzymes. Alternatively, in another embodiment, the DNA molecule is synthesized using chemical synthesis techniques such as the phosphoramidate method. In yet another embodiment, a combination of these techniques is used.

[0117] The present invention also provides a vector encoding a polypeptide of the present invention in a suitable host. The vector comprises a polynucleotide encoding the polypeptide operably linked to an appropriate expression control sequence. Methods for achieving this functional linkage, either before or after the polynucleotide is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation. The resulting vector, carrying the polynucleotide therein, is used to transform a suitable host. This transformation can be carried out using methods well known in the art.

[0118] Any of a large number of available, well-known host cells may be used in the practice of the present invention. The selection of a particular host depends on several factors recognized in the art, such as compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation efficiency, ease of peptide recovery, expression characteristics, biosafety, and cost. These factors must be balanced with the understanding that not all host cells are equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial host cells include, but are not limited to, bacteria, yeast and other fungi, insects, plants, mammalian (including human) cultured cells, or other hosts known in the art.

[0119] The transformed host cells are cultured under conventional fermentation conditions to express the desired compound. Such fermentation conditions are well known in the art. Finally, the polypeptide is purified from the culture medium or the host cells themselves by methods well known in the art.

[0120] Depending on the host cell used to express the compounds of the present invention, carbohydrate (oligosaccharide) groups are optionally attached to sites known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues, and N-linked oligosaccharides are attached to asparagine (Asn) residues, when these residues are part of the sequence Asn-X-Ser / Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids, excluding proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type differ. One sugar commonly found in both N-linked and O-linked oligosaccharides is N-acetylneuraminic acid (called sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides, and due to its negative charge, in one aspect, confers acidic properties to glycosylated compounds. Such site(s) may be incorporated into the linker of the compounds of the invention and are preferably glycosylated by the cell (e.g., in mammalian cells such as CHO, BHK, COS, etc.) during recombinant production of the polypeptide compound. In other aspects, such sites are glycosylated by synthetic or semi-synthetic methods known in the art.

[0121] In some embodiments, sialylation (also referred to as sialylation) can be performed on a column as part of the purification procedures described herein (including ion exchange, cation exchange, size exclusion, and / or immunoaffinity). In some embodiments, sialylation results in increased stability of rVWF compared to non-sialylated rVWF. In some embodiments, sialylation results in increased stability of rVWF in the circulation (e.g., after administration to a subject) compared to non-sialylated rVWF. In some embodiments, the increased stability of salivary secreted rVWF is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to non-sialylated rVWF. In some embodiments, sialylation results in an increased half-life of rVWF compared to non-sialylated rVWF. In some embodiments, sialylation results in an increased half-life of rVWF in the circulation (e.g., after administration to a subject) compared to non-sialylated rVWF. In some embodiments, the increased half-life of sialylated rVWF is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to non-sialylated rVWF. In some embodiments, the increased half-life of sialylated rVWF results in rVWF that is stable in the circulation (e.g., after administration to a subject) for 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours, or more compared to non-sialylated rVWF. In some embodiments, sialylation increases the number of 2,3 sialylation sites and / or 2,6 sialylation sites. In some embodiments, sialylation is increased by the addition of a 2,3 sialyltransferase and / or a 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step. In some embodiments, sialylation is increased by the addition of a 2,3 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step.In some embodiments, 2,3 sialylation is increased by the addition of a 2,3 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step.

[0122] In some embodiments, 2,6 sialylation is increased by the addition of a 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step. In some embodiments, 2,3 sialylation and / or 2,6 sialylation is increased by the addition of a 2,3 sialyltransferase and / or a 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step. In some embodiments, CMP-NANA is chemically or enzymatically modified to transfer the modified sialic acid to a potential-free position. In some embodiments, sialylation is carried out by loading rVWF onto a resin, washing with one or more buffers described herein to remove undesired impurities, applying one or more buffers containing sialyltransferase and CMP-NANA under conditions that allow for additional sialylation, washing with one or more buffers to remove excess sialylation reagent, and eluting the enhanced rVWF (e.g., rVWF with increased sialylation) with one or more buffers. In some embodiments, the sialylation step is carried out as part of a cation exchange, anion exchange, size exclusion, or immunoaffinity purification method, as described herein.

[0123] Alternatively, the compounds can be made by synthetic methods, for example, using solid phase synthesis. Suitable techniques are well known in the art, including those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.), Merrifield (1963), J. Am. Chem. Soc. 85:2149, Davis et al. (1985), Biochem. Intl. 10:394-414, Stewart and Young (1969), Solid Phase Peptide Synthesis, U.S. Patent No. 3,941,763, Finn et al. (1976), The Proteins (3rd ed.) 2:105-253, and Erickson et al. (1976), The Proteins (3rd ed.) 2:257-527. Solid phase synthesis is the preferred technique for producing individual peptides because it is the most cost-effective method for producing small peptides.

[0124] Fragments, variants, and analogs of VWF can be produced according to methods well known in the art. Polypeptide fragments can be prepared using, but not limited to, enzymatic cleavage (e.g., trypsin, chymotrypsin) and also using recombinant means to generate polypeptide fragments having specific amino acid sequences. Polypeptide fragments can be generated that contain protein regions with specific activities, such as the multimerization domain or any other identifiable VWF domain known in the art.

[0125] Methods for producing polypeptide analogs are also well known. Amino acid sequence analogs of polypeptides can be substitution, insertion, addition, or deletion analogs. Deletion analogs, including polypeptide fragments, lack one or more residues of the native protein that are not essential for function or immunogenic activity. Insertion analogs involve additions at non-terminal locations of the polypeptide, such as the addition of an amino acid(s). This analog can include, for example, but not limited to, the insertion of an immunoreactive epitope or simply a single residue. Addition analogs, including polypeptide fragments, include the addition of one or more amino acids at either or both ends of the protein, such as fusion proteins. Combinations of the above analogs are also contemplated.

[0126] Substitution analogs typically replace one wild-type amino acid with another at one or more sites within a protein, and can be designed to adjust one or more properties of a polypeptide without completely losing other functions or properties. In one embodiment, the substitution is a conservative substitution. A "conservative amino acid substitution" is the replacement of an amino acid with an amino acid having a side chain or similar chemical properties. Similar amino acids for conservative substitution include those with acidic side chains (glutamic acid, aspartic acid), basic side chains (arginine, lysine, histidine), polar amide side chains (glutamine, asparagine), hydrophobic aliphatic side chains (leucine, isoleucine, valine, alanine, glycine), aromatic side chains (phenylalanine, tryptophan, tyrosine), small side chains (glycine, alanine, serine, threonine, methionine), or aliphatic hydroxyl side chains (serine, threonine).

[0127] In one embodiment, analogs are substantially homologous or substantially identical to the recombinant VWF from which they are derived. Analogs include those that retain at least some of the biological activity of the wild-type polypeptide, for example, blood clotting activity.

[0128] Contemplated polypeptide variants include, but are not limited to, polypeptides that have been chemically modified by techniques not normally occurring in human proteins, such as ubiquitination, glycosylation (including polysialylation (or polysialylation)), conjugation to a therapeutic or diagnostic agent, labeling, covalent polymer attachment such as PEGylation (derivatization with polyethylene glycol), introduction of a non-hydrolyzable bond, and chemically synthesized insertion or substitution of an amino acid such as ornithine. The variants retain the same or essentially the same binding characteristics as the unmodified molecules of the invention. Such chemical modifications can include direct or indirect (e.g., via a linker) attachment of an agent to the VWF polypeptide. In the case of indirect attachment, it is contemplated that the linker may be hydrolyzable or non-hydrolyzable.

[0129] In one aspect, the preparation of a PEGylated polypeptide analog involves (a) reacting a polypeptide with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions such that the polypeptide conjugated construct is conjugated to one or more PEG groups, and (b) obtaining a reaction product(s). Generally, optimal reaction conditions for the acylation reaction are determined based on known parameters and the desired results. For example, the higher the PEG:protein ratio, the greater the proportion of polyPEGylated products. In some embodiments, the conjugated construct has a single PEG moiety at the N-terminus. Polyethylene glycol (PEG) may be attached to a blood clotting factor, for example, to achieve a longer half-life in vivo. The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG ranges from about 2 kilodaltons ("kD") to about 100 kDa, about 5 kDa to about 50 kDa, or about 5 kDa to about 10 kDa. In certain embodiments, the PEG group is attached to the blood clotting factor by acylation or reductive alkylation via a native or engineered reactive group (e.g., an aldehyde, amino, thiol, or ester group) on the PEG moiety and a reactive group (e.g., an aldehyde, amino, or ester group) on the blood clotting factor, or by any other technique known in the art.

[0130] Methods for preparing polysialylated polypeptides are described in U.S. Patent Publication No. 20060160948, Fernandes et Gregoriadis, Biochim. Biophys. Acta 1341:26-34, 1997, and Saenko et al., Haemophilia 12:42-51, 2006. Briefly, a solution of colominic acid (CA) containing 0.1 M NaIO4 is stirred in the dark at room temperature to oxidize the CA. The activated CA solution is dialyzed against, for example, 0.05 M sodium phosphate buffer at pH 7.2 in the dark, and this solution is added to the rVWF solution and incubated for 18 hours in the dark at room temperature with gentle shaking. Optionally, the free reagent is separated from the rVWF-polysialic acid complex by, for example, ultrafiltration / diafiltration. Conjugation of rVWF with polysialic acid is achieved using glutaraldehyde as a cross-linking agent (Migneault et al., Biotechniques 37:790-796, 2004).

[0131] In another aspect, preproVWF and proVWF polypeptides are also contemplated to provide therapeutic benefit in the formulations of the invention. For example, U.S. Patent No. 7,005,502 describes pharmaceutical preparations containing amounts of proVWF sufficient to induce thrombin generation in vitro. In addition to recombinant, biologically active fragments, variants, or other analogs of naturally occurring mature VWF, the invention contemplates the use of recombinant, biologically active fragments, variants, or analogs of preproVWF (as set forth in SEQ ID NO:2) or proVWF polypeptides (amino acid residues 23-764 of SEQ ID NO:2), or rVWF (as set forth in SEQ ID NO:3), in the formulations described herein.

[0132] Polynucleotides encoding fragments, variants, and analogs can be readily generated by a skilled artisan to encode biologically active fragments, variants, or analogs of naturally occurring molecules that have the same or similar biological activity as the naturally occurring molecules. In various embodiments, these polynucleotides are prepared using PCR, digestion / ligation of DNA encoding the molecules, etc. Thus, one skilled in the art can use any method known in the art, including, but not limited to, site-directed mutagenesis, to generate single-base changes in DNA strands to result in modified codons and missense mutations. As used herein, the phrase "moderately stringent hybridization conditions" refers, for example, to hybridization in 50% formamide at 42°C and washing in 0.1×SSC, 0.1% SDS at 60°C. It will be understood by those skilled in the art that these conditions will vary based on the length and GC nucleotide base content of the sequences to be hybridized. Standard procedures in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

[0133] A. VWF multimers Assessment of the number and proportion of rVWF multimers can be performed using methods known in the art, including, but not limited to, methods using electrophoresis and size-exclusion chromatography to separate VWF multimers by size, as discussed, for example, by Cumming et al. (J. Clin. Pathol., 1993 May;46(5):470-473, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings related to the assessment of VWF multimers). Such techniques can also include immunoblotting (such as Western blotting), in which a gel is immunoblotted with a radiolabeled antibody against VWF, followed by chemiluminescence detection (see, e.g., Wen et al., (1993), J. Clin. Lab. Anal., 7:317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings related to the assessment of VWF multimers). Additional assays for VWF include VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assays (VWF:CBA), which are often used to diagnose and classify von Willebrand disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456; Sadler, JE, Annu Rev Biochem, 1998, 67:395-424; and Turecek et al., Semin Thromb Hemost, 2010, 36:510-521, which are incorporated by reference in their entireties for all purposes, particularly for any teachings regarding assays for VWF). In some embodiments, rVWF obtained using the disclosed methods includes any multimer pattern present in the rVWF loading sample. In some embodiments, the rVWF obtained using the methods of the present application comprises a physiologically occurring multimer pattern and an extra-large VWF multimer pattern.

[0134] b. VWF assay In primary hemostasis, VWF acts as a bridge between platelets and specific components of the extracellular matrix, such as collagen. The biological activity of VWF in this process can be measured by various in vitro assays (Turecek et al., Semin Thromb Hemost, 2010, 36:510-521).

[0135] The VWF:Ristocetin Cofactor (VWF:RCo) assay is based on the aggregation of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF. The degree of platelet aggregation varies with VWF concentration and can be measured, for example, turbidimetrically using an aggregometer (Weiss et al., J. Clin. Invest., 1973, 52:2708-2716; Macfarlane et al., Thromb. Diath. Haemorrh., 1975, 34:306-308). As provided herein, the specific ristocetin cofactor activity (VWF:RCo) of the VWF of the present invention is generally expressed in mU / μg of VWF as measured using an in vitro assay.

[0136] In some embodiments, the rVWF purified according to the methods of the present invention has a rVWF concentration of at least about 20mU / μg, 22.5mU / μg, 25mU / μg, 27.5mU / μg, 30mU / μg, 32.5mU / μg, 35mU / μg, 37.5mU / μg, 40mU / μg, 42.5mU / μg, 45mU / μg, 47.5mU / μg, 50mU / μg, 52.5mU / μg, 55mU / μg, 57.5mU / μg, 60mU / μg, 62.5mU / μg, 65mU / μg, 67.5mU / μg, 70 In some embodiments, the rVWF used in the methods described herein has a specific activity of 20 mU / μg to 150 mU / μg. In some embodiments, the rVWF has a specific activity of 30 mU / μg to 120 mU / μg. In some embodiments, the rVWF has a specific activity of 40 mU / μg to 90 mU / μg. In some embodiments, the rVWF has a specific activity selected from variants 1 to 133 found in Table 3 below.

[0137] Table 3. Exemplary embodiments of the specific activity of rVWF found in the compositions provided herein and used in the methods provided herein. TIFF2025128168000001.tif183157Var.=Transform

[0138] The rVWF of the present invention is a high-order multimer containing about 10 to about 40 subunits. In further embodiments, the multimeric rVWF produced using the methods of the present invention contains about 10 to 30, 12 to 28, 14 to 26, 16 to 24, 18 to 22, or 20 to 21 subunits. In some embodiments, rVWF exists in multimers of various sizes, ranging from dimers to multimers of more than 40 subunits (>10 million daltons). The largest multimers provide multiple binding sites capable of interacting with both platelet receptors and subendothelial matrix sites of injury and are the most hemostatically active forms of VWF. In some embodiments, the rVWF of the present invention comprises ultra-large multimers (ULMs). Generally, higher-order and ultra-large multimers are considered to have the greatest hemostatic efficacy (see, e.g., Turecek, P., Hamostaseologie, (Vol. 37): Supplement 1, pages S15-S25 (2017)). In some embodiments, the rVWF is between 500 kDa and 20,000 kDa. In some embodiments, any desired multimer pattern can be obtained using the described methods. In some embodiments, when anion exchange and / or cation exchange methods are used, the pH, conductivity, and / or counterion concentration of the buffer or gradient buffer in one or more wash step(s) can be manipulated to obtain the desired multimer pattern. In some embodiments, size exclusion chromatography methods can then be used, and recovery criteria can be used to obtain the desired multimer pattern. In some embodiments, the described multimer patterns include ultra-large multimers. In some embodiments, ultra-large multimers are at least 10,000 kDa, at least 11,000 kDa, at least 12,000 kDa, at least 13,000 kDa, at least 14,000 kDa, at least 15,000 kDa, at least 16,000 kDa, at least 17,000 kDa, at least 18,000 kDa, at least 19,000 kDa, or at least 20,000 kDa. In some embodiments, ultra-large multimers are between about 10,000 kDa and 20,000 kDa.In some embodiments, the ultra-large multimers are between about 11,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 12,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 13,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 14,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 15,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 16,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 17,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 18,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 19,000 kDa and 20,000 kDa. In some embodiments, the rVWF obtained using the methods of the present application includes any multimer pattern present in a loading sample of rVWF. In some embodiments, the rVWF obtained using the methods of the present application includes a physiologically occurring multimer pattern and an ultra-large VWF multimer pattern.

[0139] In some embodiments, the rVWF compositions prepared by the purification methods described herein have an rVWF oligomer distribution characterized in that 95% of the oligomers have between 6 and 20 subunits. In some embodiments, the rVWF compositions have an oligomer distribution characterized in that 95% of the rVWF oligomers have a set of subunits selected from variants 458 to 641 found in 4.

[0140] Table 4. Exemplary embodiments of rVWF oligomer distributions found in the compositions provided herein and used in the methods provided herein. TIFF2025128168000002.tif246144Var.=Transform

[0141] In some embodiments, the rVWF compositions prepared by the methods provided herein can be characterized according to the percentage of rVWF molecules present in specific higher-order rVWF multimers or larger multimers. For example, in one embodiment, at least 20% of the rVWF molecules in the rVWF compositions used in the methods described herein are present in oligomeric complexes of at least 10 subunits. In another embodiment, at least 20% of the rVWF molecules in the rVWF compositions used in the methods described herein are present in oligomeric complexes of at least 12 subunits. In yet other embodiments, the rVWF compositions used in the methods provided herein have a minimum percentage (e.g., at least X%) of rVWF molecules present in specific higher-order rVWF multimers or larger multimers (e.g., multimers of at least Y subunits) according to any one of variants 134-457 found in Tables 5-7.

[0142] Table 5. Exemplary embodiments of the proportion of rVWF molecules present in specific higher order rVWF multimers or larger multimers found in the compositions provided herein and used in the methods provided herein. TIFF2025128168000003.tif107134Var.=Transform

[0143] Table 6: Exemplary embodiments of the proportion of rVWF molecules present in specific higher order rVWF multimers or larger multimers found in the compositions provided herein and used in the methods provided herein. TIFF2025128168000004.tif107134Var.=Transform

[0144] Table 7. Exemplary embodiments of the proportion of rVWF molecules present in specific higher order rVWF multimers or larger multimers found in the compositions provided herein and used in the methods provided herein. TIFF2025128168000005.tif105134Var.=Transform

[0145] In accordance with the above, rVWF comprises a significant proportion of high molecular weight (HMW) rVWF multimers. In further embodiments, the HMW rVWF multimer composition comprises at least 10%-80% rVWF decamers or higher multimers. In further embodiments, the composition comprises about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, or 60-70% decamers or higher multimers. In further embodiments, the HMW rVWF multimer composition comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decamers or higher multimers.

[0146] Assessment of the number and proportion of rVWF multimers can be performed using methods known in the art, including, but not limited to, methods using electrophoresis and size-exclusion chromatography to separate rVWF multimers by size, as discussed by, for example, Cumming et al. (J Clin Pathol. 1993 May;46(5):470-473, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings related to the assessment of rVWF multimers). Such techniques can also include immunoblotting (such as Western blotting), in which a gel is immunoblotted with a radiolabeled antibody against VWF, followed by chemiluminescence detection (see, e.g., Wen et al., (1993), J. Clin. Lab. Anal., 7:317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings related to the assessment of rVWF multimers). Additional assays for VWF include VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assays (VWF:CBA), which are often used to diagnose and classify von Willebrand disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456, which is incorporated herein by reference in its entirety for all purposes, particularly for any teachings regarding assays for VWF).

[0147] In some embodiments, the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF:ristocetin cofactor activity (IU rVWF:RCo) of rVWF prepared according to the methods of the invention is 3:1 to 1:5. In a further embodiment, the ratio is 2:1 to 1:4. In another further embodiment, the ratio is 5:2 to 1:4. In a further embodiment, the ratio is 3:2 to 1:3. In another further embodiment, the ratio is about 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:4, 2:5, 3:1, 3:2, 3:4, or 3:5. In a further embodiment, the ratio is 1:1 to 1:2. In still further embodiments, the ratio is 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1. In certain embodiments, the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF:ristocetin cofactor activity (IU rVWF:RCo) in the compositions useful in the methods described herein is selected from variants 1988-2140 found in Table 8.

[0148] Table 8: Exemplary embodiments of the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF:ristocetin cofactor activity (IU rVWF:RCo) in the compositions provided herein and used in the methods provided herein. TIFF2025128168000006.tif231158Var.=Transform

[0149] In a further embodiment, the higher-ordered rVWF multimers of the present invention are stable for about 1 to about 90 hours after administration. In another further embodiment, the higher-ordered rVWF multimers are stable for about 5 to 80 hours, 10 to 70 hours, 15 to 60 hours, 20 to 50 hours, 25 to 40 hours, or 30 to 35 hours after administration. In yet a further embodiment, the higher-ordered rVWF multimers are stable for at least 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours after administration. In a specific embodiment, the stability of the rVWF multimers is assessed in vitro.

[0150] In one embodiment, the higher-order rVWF multimers used in the compositions and methods provided herein have a half-life of at least 12 hours after administration. In another embodiment, the higher-order rVWF multimers have a half-life of at least 24 hours after administration. In yet another embodiment, the higher-order rVWF multimers have a half-life selected from variants 642-1045 found in Table 9.

[0151] Table 9. Exemplary embodiments of the half-lives of higher-order rVWF multimers found in compositions prepared by the methods provided herein. TIFF2025128168000007.tif157144TIFF2025128168000008.tif225144TIFF2025128168000009.tif152144Var.=Var

[0152] In some embodiments, the pro-VWF and / or purified rVWF purified according to the present invention is not modified by any conjugation, post-translational modification, or covalent modification. In certain embodiments, the pro-VWF and / or purified rVWF of the present invention is not modified with a water-soluble polymer, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, poly-carbohydrate moieties, etc.

[0153] In some embodiments, pro-VWF and / or purified rVWF purified according to the present invention are modified by conjugation, post-translational modification, or covalent modification, including modification of N- or C-terminal residues and selected side chains, e.g., at free sulfhydryl groups, primary amines, and hydroxyl groups. In one embodiment, a water-soluble polymer is linked to the protein (directly or via a linker) through a lysine group or other primary amine. In some embodiments, pro-VWF and / or purified rVWF of the present invention can be modified by conjugation of a water-soluble polymer, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, poly-carbohydrate moieties, etc.

[0154] Water-soluble polymers that can be used to modify pro-VWF and / or purified rVWF include linear and branched structures. The conjugated polymers can be directly attached to the coagulation proteins of the present invention or, alternatively, can be attached via a linking moiety. Non-limiting examples of protein conjugation with water-soluble polymers can be found in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337, as well as Abuchowski and Davis, "Enzymes as Drugs," Holcenberg and Roberts, Eds., pp. 367-383, John Wiley & Sons, New York (1981), and Hermanson G., Bioconjugate Techniques, 2nd Ed., Academic Press, Inc. 2008.

[0155] Protein conjugation can be carried out by several techniques well known in the art; see, for example, Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include linkage via a peptide bond between a carboxyl group on either the coagulation protein or the water-soluble polymer moiety and an amine group on the other, or via an ester bond between a carboxyl group on one side and a hydroxyl group on the other. Another contemplated linkage for conjugating the coagulation proteins of the invention to water-soluble polymer compounds is via a Schiff base between a free amino group on the polymer moiety, which reacts with an aldehyde group formed at the non-reducing end of the polymer by periodate oxidation (Jennings and Lugowski, J. Immunol. 1981;127:1011-8; Femandes and Gregonradis, Biochim Biophys Acta. 1997;1341;26-34). The resulting Schiff base can be stabilized by specific reduction with NaCNBH3 to form a secondary amine. An alternative approach is to generate terminal free amino groups on the polymer by preoxidation followed by reductive amination with NH4Cl. Bifunctional reagents can be used to link two amino groups or two hydroxyl groups. For example, amino-containing polymers can be coupled to the amino groups of coagulation proteins using reagents such as BS3 (bis(sulfosuccinimidyl) suberic acid / Pierce, Rockford, Ill.). Furthermore, heterobifunctional crosslinkers such as sulfo-EMCS (N-ε-maleimidocaproyloxy) sulfosuccinimide ester / Pierce) can be used to link, for example, amine and thiol groups.In other embodiments, aldehyde-reactive groups such as PEG alkoxide and diethyl acetal of bromoacetaldehyde; PEG with DMSO and acetic anhydride; and PEG chloride and phenoxide of 4-hydroxybenzaldehyde; succinimidyl active esters; activated dithiocarbonate PEG; and activated PEG with 2,4,5-trichlorophenyl chloroformate and p-nitrophenyl chloroformate may be used in conjugating coagulation proteins.

[0156] Another method for measuring the biological activity of VWF is the collagen binding assay, which is based on ELISA technology (Brown and Bosak, Thromb. Res., 1986, 43:303-311; Favaloro, Thromb. Haemost., 2000, 83:127-135). Microtiter plates are coated with type I or type III collagen. VWF is then bound to the collagen surface and subsequently detected with an enzyme-labeled polyclonal antibody. The final step is a substrate reaction, which can be monitored photometrically in an ELISA reader.

[0157] Von Willebrand factor (VWF:Ag) immunoassays are immunoassays that measure VWF protein concentrations in plasma. They do not provide any information about VWF function. Numerous methods exist for measuring VWF:Ag, including either enzyme-linked immunosorbent assays (ELISAs) or automated latex immunoassays (LIAs). Many laboratories now use fully automated latex immunoassays. Historically, laboratories used a variety of technologies, including the Laurell "Laurell Rocket" electroimmunoassay, but these are rarely used in most laboratories today.

[0158] III. Kit In an additional aspect, the present invention includes a kit containing one or more lyophilized compositions packaged in a manner convenient for administration to a subject. In one embodiment, such a kit includes a pharmaceutical formulation described herein (e.g., a composition comprising a therapeutic protein or peptide) packaged in a container, such as a sealed bottle or container, with a label affixed to the container or contained within the package that describes how to use the compound or composition in practicing a method. In one embodiment, the pharmaceutical formulation is packaged in a container such that the amount of headspace within the container (e.g., the amount of air between the liquid formulation and the top of the container) is minimal. Preferably, the amount of headspace is negligible (e.g., virtually nonexistent). In one embodiment, the kit contains a first container with a therapeutic protein or peptide composition and a second container with a physiologically acceptable composition reconstitution solution. In one aspect, the pharmaceutical formulation is packaged in a unit dosage form. The kit may further include a device suitable for administering the pharmaceutical formulation according to a specific route of administration. Preferably, the kit contains a label that describes the use of the pharmaceutical formulation.

[0159] IV. rVWF for the prophylactic treatment of spontaneous bleeding in patients with severe VWD One advantage of administering rVWF to severely ill VWD subjects for the prophylactic treatment of spontaneous bleeding episodes is that the higher specific activity of rVWF compared to pdVWF allows flexibility in the amount of rVWF administered and the number of times the subject is re-administered. As will be understood and discussed in more detail herein, co-administered FVIII can be recombinant or plasma-derived.

[0160] Single or repeated administration of rVWF will be carried out with the dose and pattern being selected by the treating physician. For disease prevention or treatment, the appropriate dosage will vary depending on the type of disease being treated (e.g., von Willebrand's disease), the severity and course of the disease, whether the drug is being administered for prevention or treatment, previous medical treatments, the patient's medical history and response to the drugs, and the discretion of the treating physician.

[0161] In some embodiments, rVWF is administered at a dose ranging from 40 to 80 IU / kg, e.g., 40 IU / kg, 41 IU / kg, 42 IU / kg, 43 IU / kg, 44 IU / kg, 45 IU / kg, 46 IU / kg, 47 IU / kg, 48 IU / kg, 49 IU / kg, 50 IU / kg, 51 IU / kg, 52 IU / kg, 53 IU / kg, 54 IU / kg, 55 IU / kg, 56 IU / kg, 57 IU / kg, 58 IU / kg, 59 IU / kg, 60 IU / kg, 60 IU / kg, 61 IU / kg, 62 IU / kg, 63 IU / kg, 64 IU / kg, 65 IU / kg, 66 IU / kg, 67 IU / kg, 68 IU / kg, 69 IU / kg kg, 70IU / kg, 71IU / kg, 72IU / kg, 73IU / kg, 74IU / kg, 75IU / kg, 76IU / kg, 77IU / kg, 78I U / kg, 79IU / kg, 80IU / kg, 40~80IU / kg, 45~80IU / kg, 50~80IU / kg, 45~70IU / kg, 45~60I The subject is prophylactically administered 45-55 IU / kg, 45-50 IU / kg, 50-60 IU / kg, 55-60 IU / kg, 60-65 IU / kg, 55-65 IU / kg, 60-70 IU / kg, 65-70 IU / kg, 60-75 IU / kg, 70-80 IU / kg, or 75-80 IU / kg. In some embodiments, the dose is in the range of 40 IU / kg to 60 IU / kg. In some embodiments, the dose is in the range of 45 IU / kg to 55 IU / kg. In some embodiments, the dose is about 40 IU / kg, about 50 IU / kg, or about 60 IU / kg. In some embodiments, the dose is about 50 IU / kg. In some embodiments, the dose is about 80 IU / kg. In some embodiments, rVWF is administered once a week, twice a week, three times a week, four times a week, five times a week, or more times a week. In some embodiments, rVWF is administered twice a week. In some embodiments, rVWF is administered twice a week. In some embodiments, rVWF is administered twice a week at a dose ranging from 40 IU / kg to 60 IU / kg. In some embodiments, rVWF is administered by IV infusion twice a week at a dose ranging from 40 IU / kg to 60 IU / kg.

[0162] In some embodiments, blood samples for measuring vWF:Ag, vWF:RCo, vWF:CB, and FVIII activity levels are taken before administration, 15 minutes, 30 minutes, and 60 minutes, and 3 hours, 6 hours, 12 hours, 24 hours, 28 hours, 32 hours, 48 ​​hours, 72 hours, and 96 hours after drug infusion. In some embodiments, samples, including, for example, blood samples, can be obtained to measure vWF:RCo and FVIII activity. In some embodiments, samples for FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF collagen-binding activity are obtained before prophylactic treatment with rVWF. In some embodiments, samples for assessing the collagen-binding activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF are obtained 15 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 28 hours, 32 hours, 48 ​​hours, 72 hours, or 96 hours after prophylactic treatment with rVWF. In some embodiments, samples for assessing the collagen-binding activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF are obtained 25 to 31 days after prophylactic treatment with rVWF. In some embodiments, samples for FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF collagen-binding activity are obtained after a bleeding episode; in such embodiments, samples are obtained before administration of rVWF, 2 hours after administration, and every 12-24 hours thereafter until the bleeding event has resolved. In some embodiments, activity and time profiles of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF collagen-binding activity are determined based on the samples. In some embodiments, FVIII:C is also measured by a one-stage clotting assay and time profile comparison.

[0163] In some embodiments, the rVWF is in the range of 40-80 IU / kg, e.g., 40 IU / kg, 41 IU / kg, 42 IU / kg, 43 IU / kg, 44 IU / kg, 45 IU / kg, 46 IU / kg, 47 IU / kg, 48 IU / kg, 49 IU / kg, 50 IU / kg, 51 IU / kg, 52 IU / kg, 53 IU / kg, 54 IU / kg, 55 IU / kg, 56 IU / kg, 57 IU / kg, 58 IU / kg, 59 IU / kg, 60 IU / kg, 60 IU / kg, 61 IU / kg, 62 IU / kg, 63 IU / kg, 64 IU / kg, 65 IU / kg, 66 IU / kg, 67 IU / kg, 68 IU / kg, 69 IU / kg, 70 IU / kg, 71 IU / kg, 72 IU / kg, 73 IU / kg, 74 IU / kg, 75 IU / kg, 76 IU / kg, 77 IU / kg, 78 IU / kg, 79 IU / kg, 80 IU / kg, 81 IU / kg, 82 IU / kg, 83 IU / kg, 84 IU / kg, 85 IU / kg, 86 IU / kg, 87 IU / kg, 88 IU / kg, 89 IU / kg, 90 IU / kg, 91 IU / kg, 92 IU / kg, 93 IU / kg, 94 IU / kg, 95 IU / kg, 96 IU / kg, 97 IU / kg, 98 IU / kg, IU / kg, 71IU / kg, 72IU / kg, 73IU / kg, 74IU / kg, 75IU / kg, 76IU / kg, 77IU / kg, 78IU / kg, 7 9IU / kg, 80IU / kg, 40~80IU / kg, 45~80IU / kg, 50~80IU / kg, 45~70IU / kg, 45~60IU / kg, 45 ~55 IU / kg, 45-50 IU / kg, 50-60 IU / kg, 55-60 IU / kg, 60-65 IU / kg, 55-65 IU / kg, 60-70 IU / kg, 65-70 IU / kg, 60-75 IU / kg, 70-80 IU / kg, or 75-80 IU / kg is administered to the subject as the initial (first) dose.In some embodiments, the rVWF is administered in the range of 40-80 IU / kg, e.g., 40 IU / kg, 41 IU / kg, 42 IU / kg, 43 IU / kg, 44 IU / kg, 45 IU / kg, 46 IU / kg, 47 IU / kg, 48 IU / kg, 49 IU / kg, 50 IU / kg, 51 IU / kg, 52 IU / kg, 53 IU / kg, 54 IU / kg, 55 IU / kg, 56 IU / kg, 57 IU / kg, 58 IU / kg, 59 IU / kg, 60 IU / kg, 60 IU / kg, 61 IU / kg, 62 IU / kg, 63 IU / kg, 64 IU / kg, 65 IU / kg, 66 IU / kg, 67 IU / kg, 68 IU / kg, 69 IU / kg, 70IU / kg, 71IU / kg, 72IU / kg, 73IU / kg, 74IU / kg, 75IU / kg, 76IU / kg, 77IU / kg, 78IU / kg , 79IU / kg, 80IU / kg, 40~80IU / kg, 45~80IU / kg, 50~80IU / kg, 45~70IU / kg, 45~60IU / kg , 45-55 IU / kg, 45-50 IU / kg, 50-60 IU / kg, 55-60 IU / kg, 60-65 IU / kg, 55-65 IU / kg, 60-70 IU / kg, 65-70 IU / kg, 60-75 IU / kg, 70-80 IU / kg, or 75-80 IU / kg, is administered to the subject as a second dose.In some embodiments, the rVWF is administered in the range of 40-80 IU / kg, e.g., 40 IU / kg, 41 IU / kg, 42 IU / kg, 43 IU / kg, 44 IU / kg, 45 IU / kg, 46 IU / kg, 47 IU / kg, 48 IU / kg, 49 IU / kg, 50 IU / kg, 51 IU / kg, 52 IU / kg, 53 IU / kg, 54 IU / kg, 55 IU / kg, 56 IU / kg, 57 IU / kg, 58 IU / kg, 59 IU / kg, 60 IU / kg, 60 IU / kg, 61 IU / kg, 62 IU / kg, 63 IU / kg, 64 IU / kg, 65 IU / kg, 66 IU / kg, 67 IU / kg, 68 IU / kg, 69 IU / kg, 70IU / kg, 71IU / kg, 72IU / kg, 73IU / kg, 74IU / kg, 75IU / kg, 76IU / kg, 77IU / kg, 78IU / kg , 79IU / kg, 80IU / kg, 40~80IU / kg, 45~80IU / kg, 50~80IU / kg, 45~70IU / kg, 45~60IU / kg , 45-55 IU / kg, 45-50 IU / kg, 50-60 IU / kg, 55-60 IU / kg, 60-65 IU / kg, 55-65 IU / kg, 60-70 IU / kg, 65-70 IU / kg, 60-75 IU / kg, 70-80 IU / kg, or 75-80 IU / kg, administered to the subject as a third dose.In some embodiments, the rVWF is administered in the range of 40-80 IU / kg, e.g., 40 IU / kg, 41 IU / kg, 42 IU / kg, 43 IU / kg, 44 IU / kg, 45 IU / kg, 46 IU / kg, 47 IU / kg, 48 IU / kg, 49 IU / kg, 50 IU / kg, 51 IU / kg, 52 IU / kg, 53 IU / kg, 54 IU / kg, 55 IU / kg, 56 IU / kg, 57 IU / kg, 58 IU / kg, 59 IU / kg, 60 IU / kg, 60 IU / kg, 61 IU / kg, 62 IU / kg, 63 IU / kg, 64 IU / kg, 65 IU / kg, 66 IU / kg, 67 IU / kg, 68 IU / kg, 69 IU / kg, 70IU / kg, 71IU / kg, 72IU / kg, 73IU / kg, 74IU / kg, 75IU / kg, 76IU / kg, 77IU / kg, 78IU / kg , 79IU / kg, 80IU / kg, 40~80IU / kg, 45~80IU / kg, 50~80IU / kg, 45~70IU / kg, 45~60IU / kg , 45-55 IU / kg, 45-50 IU / kg, 50-60 IU / kg, 55-60 IU / kg, 60-65 IU / kg, 55-65 IU / kg, 60-70 IU / kg, 65-70 IU / kg, 60-75 IU / kg, 70-80 IU / kg, or 75-80 IU / kg, and administered to the subject as subsequent doses.

[0164] The rVWF composition can be contained in a pharmaceutical formulation as described herein. Such formulations can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or intracranially. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intracisternal, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, or intrapulmonary injection and / or surgical implantation at a specific site is also contemplated. Generally, compositions are essentially free of pyrogens and other impurities that could be harmful to the recipient.

[0165] In one embodiment, the formulations of the present invention are administered as an initial bolus, followed by continuous infusion to maintain therapeutic circulating concentrations of the formulation. In another example, the compounds of the present invention are administered as a single dose. Those skilled in the art can easily optimize effective doses and administration regimens, as determined by good medical practice and the clinical condition of each individual patient. The route of administration can be, but is not limited to, intravenous, intraperitoneal, subcutaneous, or intramuscular. The frequency of administration will depend on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation will be determined by those skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, Mack Publishing Co., Easton, Pa. 18042, pages 1435-1712, the disclosure of which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings regarding pharmaceutical formulations, routes of administration, and dosages. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, suitable doses are calculated according to body weight, body surface area, or organ size. Appropriate doses can be confirmed by the use of established assays for determining blood concentrations of administered doses in conjunction with appropriate dose-response data. The final dosing regimen will be determined by the attending physician and will take into account various factors that modify the drug's action, such as the specific activity of the drug, the severity and responsiveness of the patient's injury, the patient's age, condition, weight, sex, and diet, the severity of any infection, the time of administration, and other clinical factors. By way of example, a typical dose of the recombinant VWF of the present invention is approximately 50 IU / kg, which is equivalent to 500 μg / kg. As studies are conducted, more information will emerge regarding appropriate dosages and durations of treatment for various diseases and conditions.

[0166] For administration of a composition to a human or experimental animal, in one embodiment, the composition comprises one or more pharmaceutically acceptable carriers. The phrases "pharmaceutically" or "pharmacologically" acceptable refer to molecular entities and compositions that are stable, inhibit proteolytic degradation, including aggregation and cleavage products, and do not produce allergic or other adverse reactions when administered using routes well known in the art, such as those described below. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., including those disclosed above.

[0167] Pharmaceutical preparations are administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or intracranially. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intracisternal, or infusion techniques. Administration by injection at a specific site, such as intravenously, intradermally, intramuscularly, intramammary, intraperitoneally, intrathecally, retrobulbarly, and / or intrapulmonary, is also contemplated. Generally, compositions are essentially free of pyrogens and other impurities that may be harmful to the recipient.

[0168] Single or repeated administration of rVWF will be carried out with the dose and pattern being selected by the treating physician. For disease prevention or treatment, the appropriate dosage will vary depending on the type of disease being treated (e.g., von Willebrand's disease), the severity and course of the disease, whether the drug is being administered for prevention or treatment, previous medical treatments, the patient's medical history and response to the drugs, and the discretion of the treating physician.

[0169] a. Lyophilized VWF preparation The present invention also provides rVWF formulations for use in the therapeutic methods provided herein. In some embodiments, the rVWF compositions are used to produce pharmaceutical compositions. In some embodiments, the rVWF can be formulated into a lyophilized preparation.

[0170] In some embodiments, formulations containing the VWF polypeptides of the present invention are lyophilized after purification and before administration to a subject. Lyophilization is performed using techniques common in the art and should be optimized for the composition being produced (Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996)).

[0171] In one embodiment, the lyophilization cycle consists of three steps: freezing, primary drying, and secondary drying (AP Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)). In the freezing step, the solution is cooled until ice formation begins. This step also induces crystallization of the bulking agent. The ice is sublimated in the primary drying step by using a vacuum, introducing heat to promote sublimation, and reducing the chamber pressure below the vapor pressure of the ice. Finally, a secondary drying step at low chamber pressure and elevated shelf temperature removes adsorbed or bound water. This process produces a material known as a lyophilized cake. The cake can then be reconstituted with sterile water for injection or a suitable diluent.

[0172] The lyophilization cycle not only determines the final physical state of the excipient, but also influences other parameters such as reconstitution time, appearance, stability, and final moisture content. The structure of a composition in the frozen state progresses through several transitions (e.g., glass transition, wetting, and crystallization) that occur at specific temperatures, and this structure can be used to understand and optimize the lyophilization process. The glass transition temperature (Tg and / or Tg') provides information about the physical state of the solute and can be determined by differential scanning calorimetry (DSC). Tg and Tg' are important parameters that must be considered when designing a lyophilization cycle. For example, Tg' is important for primary drying. Furthermore, in the dried state, the glass transition temperature provides information about the storage temperature of the final product.

[0173] b. Pharmaceutical preparations and additives in general Additives are additives that confer or enhance the stability and delivery of formulations (e.g., proteins). Regardless of the reason for their inclusion, additives are essential components of formulations and therefore must be safe and well tolerated by patients. For protein drugs, the selection of additives is particularly important because they can affect both the efficacy and immunogenicity of the drug. Therefore, protein formulations must be developed with appropriate selection of additives that provide suitable stability, safety, and marketability.

[0174] In one embodiment, the lyophilized formulation is comprised of at least one or more of a buffer, bulking agent, and stabilizer. In this embodiment, the usefulness of a surfactant is evaluated and selected when aggregation during the lyophilization step or reconstitution is an issue. An appropriate buffer is included to maintain the formulation in a stable pH range during lyophilization. A comparison of the additive components intended for liquid and lyophilized protein formulations is provided in Table 10.

[0175] Table 10. Excipient components of lyophilized protein formulations TIFF2025128168000010.tif148147

[0176] A major challenge in developing protein formulations is stabilizing the product against the stresses of manufacturing, transportation, and storage. The role of formulation additives is to provide stabilization against these stresses. Additives are also used to reduce the viscosity of highly concentrated protein formulations, enabling the delivery of these formulations and improving patient convenience. Generally, additives can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stresses. Some additives are used to mitigate the effects of specific stresses or to adjust the individual susceptibility of a particular protein. Other additives have a more general effect on the physical and covalent stability of proteins. The additives described herein are organized by either their chemical type or their functional role in the formulation. A brief description of the mode of stabilization is provided when discussing each type of additive.

[0177] Given the teachings and guidance provided herein, one of skill in the art will know what amounts or ranges of additives can be included in any particular formulation to achieve a biopharmaceutical formulation of the invention that promotes the retention of stability of a biopharmaceutical (e.g., protein). For example, the amount and type of salt to include in a biopharmaceutical formulation of the invention will be selected based on the desired osmolality (e.g., isotonic, hypotonic, or hypertonic) of the final solution and the amounts and osmolality of other components to be included in the formulation.

[0178] As an example, isotonicity can be achieved by including about 5% sorbitol, while about 9% sucrose additive is required to achieve isotonicity. The selection of the amount or concentration range of one or more additives that can be included in the biopharmaceutical formulations of the invention is exemplified above with reference to salts, polyols, and sugars. However, those skilled in the art will understand that the considerations described herein, and further exemplified with reference to specific additives, are equally applicable to all types of additives and combinations thereof, such as salts, amino acids, other tonicity agents, surfactants, stabilizers, bulking agents, cryoprotectants, lyoprotectants, antioxidants, metal ions, chelating agents, and / or preservatives.

[0179] Additionally, where a particular additive is reported in molar concentration, one of skill in the art will recognize that the equivalent percent (%) w / v solution (e.g., (grams of substance in solution sample / mL of solution) x 100%) is also contemplated.

[0180] Of course, those skilled in the art will recognize that the concentrations of the additives described herein are interdependent within a particular formulation. For example, the concentration of bulking agent may be reduced if, for example, the protein is at a high concentration or, for example, the stabilizer is at a high concentration. Furthermore, those skilled in the art will recognize that to maintain the isotonicity of a particular formulation without the bulking agent, the concentration of the stabilizer will be adjusted accordingly (e.g., a "tonicifying" amount of stabilizer is used). Common additives are known in the art and can be found in Powell et al., Compendium of Excipients for Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology, 52:238-311.

[0181] c. Pharmaceutical buffers and buffering agents The stability of pharmacologically active protein formulations is usually observed to be greatest within a narrow pH range. This pH range for optimal stability needs to be identified early during preformulation studies. Several techniques, such as accelerated stability testing and calorimetric screening studies, are useful in this endeavor (Remmele RL Jr., et al., Biochemistry, 38(16):5241-7(1999)). Once the formulation is finalized, the protein must be manufactured and maintained throughout its shelf life. Therefore, buffers are almost always used to control the pH in the formulation.

[0182] The buffering capacity of a buffering species is greatest at a pH equal to its pKa and decreases as the pH increases or decreases from this value. Ninety percent of the buffering capacity is within one pH unit of its pKa. Buffering capacity also increases proportionally with increasing buffer concentration.

[0183] Several factors must be considered when selecting a buffer. First and foremost, the buffer species and its concentration must be defined based on its pKa and the desired formulation pH. Equally important is ensuring that the buffer is compatible with proteins and other formulation excipients and does not catalyze any degradation reactions. A third important aspect to consider is the stinging and irritation that the buffer may induce upon administration. For example, citrate is known to cause stinging upon injection (Laursen T, et al., Basic Clin Pharmacol Toxicol., 98(2):218-21(2006)). The potential for stinging and irritation is higher for drugs administered via the subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains at the site for a relatively longer time than with IV administration, where the formulation is rapidly diluted in the blood upon administration. For formulations administered via direct IV infusion, the total amount of buffer (and any other formulation components) must be monitored. Particular caution must be exercised with potassium ions administered in the form of potassium phosphate buffer, which can induce cardiovascular effects in patients (Hollander-Rodriguez JC, et al., Am. Fam. Physician., 73(2):283-90(2006)).

[0184] Buffers for lyophilized formulations require additional consideration. Some buffers, such as sodium phosphate, can crystallize from the amorphous protein phase during freezing, causing a pH shift. Other common buffers, such as acetate and imidazole, can sublime or evaporate during the lyophilization process, which can shift the formulation pH during lyophilization or after reconstitution.

[0185] The buffer system present in the composition is selected to be physiologically compatible and to maintain the desired pH of the pharmaceutical formulation. In one embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, the pH of the solution can be 2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, 4.0, 4.3, 4.5, 4.7, 5.0, 5.3, 5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7, 9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5, 10.7, 11.0, 11.3, 11.5, 11.7, or 12.0.

[0186] The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level. In one embodiment, the pH buffering concentration is between 0.1 mM and 500 mM (1 M). For example, it is contemplated that the pH buffering agent is at least 0.1 mM, 0.5 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.7 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, or 500 mM.

[0187] Exemplary pH buffering agents used to buffer the formulations described herein include, but are not limited to, organic acids, glycine, histidine, glutamate, succinate, phosphate, acetate, citrate, Tris, HEPES, and amino acids or mixtures of amino acids, including, but not limited to, aspartic acid, histidine, and glycine. In one embodiment of the present invention, the buffering agent is citrate.

[0188] d. Pharmaceutical stabilizers and bulking agents In one embodiment of the pharmaceutical formulation, a stabilizer (or combination of stabilizers) is added to prevent or reduce storage-induced aggregation and chemical degradation. A cloudy or turbid solution upon reconstitution indicates that the protein has precipitated or at least aggregated. The term "stabilizer" refers to an additive that can prevent aggregation or physical degradation, including chemical degradation (e.g., autolysis, deamidation, oxidation, etc.) in aqueous conditions. Contemplated stabilizers include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCl, polyhydroxy compounds (including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methyl pyrollidene, cellulose, and hyaluronic acid), and sodium chloride (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In the present formulations, stabilizers are incorporated at a concentration of about 0.1 mM, 0.5 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.7 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 500 mM, 700 mM, 900 mM, or 1000 mM. In one embodiment of the present invention, mannitol and trehalose are used as stabilizers.

[0189] If desired, the formulation also includes appropriate amounts of bulking agents and osmolality adjusters. Bulking agents include, but are not limited to, mannitol, glycine, sucrose, polymers such as dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. In one embodiment, the bulking agent is mannitol. Bulking agents are incorporated at a concentration of about 0.1 mM, 0.5 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.7 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 500 mM, 700 mM, 900 mM, or 1000 mM.

[0190] e. Pharmaceutical surfactants Proteins have a high tendency to interact with surfaces, making them susceptible to adsorption and denaturation at air-liquid, vial-liquid, and liquid-liquid (silicone oil) interfaces. This degradation pathway has been observed to be inversely dependent on protein concentration, resulting in the formation of soluble and insoluble protein aggregates or loss of protein from solution due to adsorption to surfaces. In addition to adsorption to container surfaces, surface-induced degradation is exacerbated by physical agitation, such as may be experienced during product transportation and handling.

[0191] Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to compete with proteins for interfacial positions. The hydrophobic portion of the surfactant molecule occupies an interfacial position (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically around the detergent's critical micelle concentration), the surface layer of the surfactant molecule acts to prevent protein molecules from adsorbing to the interface, thereby minimizing surface-induced degradation. Surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylate, such as polysorbate 20 and polysorbate 80. The two differ only in the length of the aliphatic chain that confers hydrophobic properties to the molecule: C-12 and C-18, respectively. Thus, polysorbate 80 has higher surface activity and a lower critical micelle concentration than polysorbate 20.

[0192] The detergent solution can also affect the thermodynamic conformational stability of proteins. Again, the effect of a given detergent additive is protein-specific. For example, polysorbate has been shown to decrease the stability of some proteins and increase the stability of others. Detergent destabilization of proteins can be explained in terms of the hydrophobic tails of the detergent molecule, which can associate with partially or completely unfolded protein states through specific binding. These types of interactions are thought to shift the conformational equilibrium toward a more unfolded protein state (e.g., complementing bound polysorbate to increase the exposure of hydrophobic portions of the protein molecule). Alternatively, if the native state of a protein exhibits a certain degree of hydrophobic surface, detergents binding to the native state may stabilize that conformation.

[0193] Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation.In many cases, as raw materials, they contain sufficient amounts of peroxides to cause the oxidation of protein residue side chains, especially methionine.Since oxidative damage may occur from the addition of stabilizers, it is emphasized that the lowest effective concentration of additives should be used in formulations.For surfactants, the effective concentration for a given protein varies depending on the stabilization mechanism.

[0194] Surfactants are also added in an appropriate amount to prevent surface-related aggregation phenomena during freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Accordingly, exemplary surfactants include, but are not limited to, anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and glycodeoxycholic acid sodium salt. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic detergents include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Nonionic detergents include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. Surfactants also include, but are not limited to, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbates 40, 60, 65, and 80, soybean lecithin, and other phospholipids such as dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), and (dioleylphosphatidylglycerol)DOPG; sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Therefore, compositions containing these surfactants individually or as a mixture in different ratios are further provided. In one embodiment of the present invention, the surfactant is TWEEN-80. In the present formulations, the surfactant is incorporated at a concentration of about 0.01 to about 0.5 g / L.In the provided formulations, the surfactant concentration is 0.005 g / L, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1.0 g / L.

[0195] f. Pharmaceutical salts Salts are often added to increase the ionic strength of a formulation and can be important for protein solubility, physical stability, and tonicity. Salts can affect the physical stability of proteins in various ways. Ions can stabilize the native state of a protein by binding to charged residues on the protein surface. Alternatively, salts can stabilize the denatured state by binding to peptide groups along the protein backbone (-CONH-). Salts can also stabilize the native conformation of a protein by shielding repulsive electrostatic interactions between residues within the protein molecule. Salts in protein formulations can also shield attractive electrostatic interactions between protein molecules, which can lead to protein aggregation and insolubility. In the provided formulations, the salt concentration is between 0.1 mM, 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 80 mM, 100 mM, 120 mM, 150 mM, 200 mM, 300 mM, and 500 mM.

[0196] g. Other common additive ingredients: pharmaceutical amino acids Amino acids are versatile agents in protein formulations, serving as buffers, bulking agents, stabilizers, and antioxidants. Thus, in one embodiment, histidine and glutamic acid are used to buffer protein formulations in the pH ranges of 5.5-6.5 and 4.0-5.5, respectively. Because the imidazole group of histidine has a pKa of 6.0, and the carboxyl group of the glutamic acid side chain has a pKa of 4.3, these amino acids are suitable for buffering in their respective pH ranges. Glutamic acid is particularly useful in such cases. Histidine is commonly found in commercial protein formulations, and this amino acid offers an alternative to citrate, a buffer known for its stinging sensation upon injection. Interestingly, histidine has also been reported to have a stabilizing effect against aggregation when used at high concentrations in both liquid and lyophilized formulations (Chen B, et al., Pharm Res., 20(12):1952-60 (2003)). Histidine has also been observed by others to reduce the viscosity of high-protein formulations. However, in the same study, the authors observed increased aggregation and discoloration in histidine-containing formulations during freeze-thaw studies of antibodies in stainless steel containers. Another point of note regarding histidine is that it is subject to photooxidation in the presence of metal ions (Tomita M, et al., Biochemistry, 8(12):5149-60 (1969)). The use of methionine as a formulation antioxidant appears promising and has been observed to be effective against some oxidative stresses (Lam XM, et al., J Pharm ScL, 86(11):1250-5 (1997)).

[0197] In various aspects, formulations are provided that include one or more of the amino acids glycine, proline, serine, arginine, and alanine, which have been shown to stabilize proteins by a selective exclusion mechanism. Glycine is also a commonly used bulking agent in lyophilized formulations. Arginine has been shown to be an effective agent in inhibiting aggregation and is used in both liquid and lyophilized formulations. In the provided formulations, the amino acid concentration is between 0.1 mM, 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 80 mM, 100 mM, 120 mM, 150 mM, 200 mM, 300 mM, and 500 mM. In one embodiment of the present invention, the amino acid is glycine.

[0198] h. Other common additive ingredients: pharmaceutical antioxidants Oxidation of protein residues can arise from several different sources. Beyond the addition of specific antioxidants, preventing oxidative damage to proteins involves careful control of several factors, including atmospheric oxygen, temperature, light exposure, and chemical contamination, throughout the manufacturing process and product storage. Accordingly, the present invention contemplates the use of pharmaceutical antioxidants, including, but not limited to, reducing agents, oxygen / free radical scavengers, or chelating agents. In one aspect, antioxidants in therapeutic protein formulations are water-soluble and maintain activity throughout the product's shelf life. Reducing agents and oxygen / free radical scavengers act by quenching reactive oxygen species in solution. Chelating agents, such as EDTA, work by binding trace metal contaminants that promote free radical formation. For example, EDTA was used in liquid formulations of acidic fibroblast growth factor to inhibit metal ion-catalyzed oxidation of cysteine ​​residues.

[0199] In addition to the effectiveness of various additives in preventing protein oxidation, there is concern that antioxidants themselves may induce other covalent or physical changes in proteins, for example, reducing agents can cause the disruption of intramolecular disulfide bonds, leading to disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote the oxidation of methionine in some proteins and peptides (Akers MJ, and Defelippis MR. Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Sven Frokjaer, Lars Hovgaard, editors. Pharmaceutical Science. Taylor and Francis, UK (1999)); Fransson JR, / . Pharm. Sci. 86(9):4046-1050 (1997); Yin J, et al., Pharm Res., 21(12):2377-83 (2004)). Sodium thiosulfate has been reported to reduce the level of light- and temperature-induced methionine oxidation in rhuMab HER2, although the formation of thiosulfate-protein adducts was also reported in this study (Lam XM, Yang JY, et al., J Pharm Sci. 86(11):1250-5(1997)). The selection of an appropriate antioxidant is based on the specific stress and sensitivity of the protein. Antioxidants contemplated in certain embodiments include, but are not limited to, reducing agents and oxygen / free radical scavengers, EDTA, and sodium thiosulfate.

[0200] i. Other common additive ingredients: pharmaceutical metal ions Generally, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical decomposition reactions in proteins. However, certain metal ions are included in formulations when they are protein cofactors and when they form coordination complexes with protein suspensions (e.g., zinc suspensions of insulin). Recently, magnesium ions (10-120 mM) have been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (WO2004039337).

[0201] Two examples of metal ions conferring stability or increasing activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme®) and factor VIII. In the case of rhDNase, Ca⁺⁺ ions (up to 100 mM) increased the enzyme's stability through specific binding sites (Chen B, et al., Pharm Sci., 88(4):477-82 (1999)). In fact, removing calcium ions from solution with EGTA resulted in increased deamidation and aggregation. However, this effect was observed only with Ca⁺⁺ ions; other divalent cations, Mg⁺⁺, Mn⁺⁺, and Zn⁺⁺, were observed to destabilize rhDNase. A similar effect was observed with factor VIII. Ca+2 and Sr+2 ions stabilized the protein, whereas other ions such as Mg+2, Mn+2, Zn+2, Cu+2, and Fe+2 destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997)). In another study using factor VIII, a significant increase in the rate of aggregation was observed in the presence of Al+3 ions (Derrick TS, et al., / . Pharm. Sci., 93(10):2549-57 (2004)). The authors noted that other additives, such as buffer salts, are often contaminated with Al+3 ions, and emphasized the importance of using additives of appropriate quality in pharmaceutical products.

[0202] j. Other common excipient ingredients: Pharmaceutical preservatives Preservatives are necessary when developing multi-dose parenteral formulations involving two or more extractions from the same container. Their primary function is to inhibit microbial growth and ensure product sterility throughout the formulation's shelf life or usage period. Commonly used preservatives include, but are not limited to, benzyl alcohol, phenol, and m-cresol. Although preservatives have been used for a long time, developing protein formulations that contain preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations (Roy S, et al., J Pharm ScL, 94(2):382-96(2005)).

[0203] To date, most protein drugs have been formulated for single use only. However, where multi-dose formulations are possible, they offer the added benefit of patient convenience and increased marketability. A good example is that of human growth hormone (hGH), where the development of a preserved formulation has led to the commercialization of a more convenient multi-dose injection pen dosage form. At least four such pen devices containing preserved formulations of hGH are currently on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech), and Genotropin (lyophilized dual-chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope® (Eli Lilly) is formulated with m-cresol.

[0204] Several aspects must be considered during formulation development of a preserved dosage form. The effective concentration of the preservative in the formulation must be optimized. This requires testing a given preservative in the dosage form over a range of concentrations that confer antimicrobial efficacy without compromising protein stability. For example, using differential scanning calorimetry (DSC), three preservatives were successfully screened in the development of a liquid formulation of interleukin-1 receptor (type I). The preservatives were ranked in order based on their impact on stability at concentrations commonly used in commercial products (Remmele RL Jr., et al., Pharm Res., 15(2):200-8 (1998)).

[0205] Developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without preservatives and reconstituted immediately with a preservative-containing diluent. This reduces the time the preservative is in contact with the protein, significantly minimizing the associated stability risks. For liquid formulations, the preservative's effectiveness and stability must be maintained throughout the product's shelf life (18-24 months). It is important to note that the preservative's effectiveness must be demonstrated in the final formulation containing the active drug and all excipient components.

[0206] Some preservatives can cause injection site reactions, another factor that must be considered when selecting a preservative. A clinical trial focused on evaluating preservatives and buffers in Norditropin found that formulations containing phenol and benzyl alcohol resulted in lower pain perception compared with formulations containing m-cresol (Kappelgaard AM, Horm Res. 62 Suppl 3:98-103 (2004)). Interestingly, among commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue SC, and Sun DA., Anesth Analg., 100(3):683-6 (2005)). In various aspects, the use of preservatives provides benefits that outweigh any side effects.

[0207] k. Methods for preparing pharmaceutical preparations The present invention further contemplates a method for the preparation of a pharmaceutical formulation.

[0208] The method may further include one or more of the following steps: adding a stabilizer, as described herein, to the mixture prior to lyophilization; adding at least one agent selected from a bulking agent, an osmolality adjusting agent, and a surfactant, each as described herein, to the mixture prior to lyophilization.

[0209] The standard reconstitution technique for lyophilized material is to add back a volume of purified water or sterile water for injection (WFI) (typically a volume equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the manufacture of pharmaceuticals for parenteral administration (Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)). Accordingly, a method for preparing a reconstituted rVWF composition of the present invention is provided, which comprises adding a diluent to the lyophilized rVWF composition.

[0210] The lyophilized material can be reconstituted as an aqueous solution with a variety of aqueous carriers, for example, sterile water for injection, water containing a preservative for multiple doses, or water containing an appropriate amount of surfactant (e.g., aqueous suspensions containing the active compound mixed with excipients suitable for the manufacture of aqueous suspensions). In various embodiments, such additives are suspending agents such as, but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and dispersing or wetting agents are naturally occurring phospholipids such as, but not limited to, lecithin, or condensation products of alkylene oxides with fatty acids, such as, but not limited to, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as, but not limited to, heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, such as, but not limited to, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as, but not limited to, polyethylene sorbitan monooleate. In various embodiments, aqueous suspensions also contain one or more preservatives, for example, but not limited to, ethyl, or n-propyl p-hydroxybenzoate.

[0211] l. Exemplary rVWF Formulations for Administration In some embodiments, the methods provide enhanced formulations that allow for a more potent (higher rVWF concentration and enhanced long-term stability) final product with a smaller therapeutic dose (100 IU / ml to 10,000 IU / ml). In some embodiments, the rVWF concentration in the formulation for administration is about 100 IU / ml to 10,000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 500 IU / ml to 10,000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 1,000 IU / ml to 10,000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 2,000 IU / ml to 10,000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 3,000 IU / ml to 10,000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 4000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 5000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 6000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 7000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 8000 IU / ml to 10000 IU / ml. In some embodiments, the rVWF concentration in the formulation for administration is about 9000 IU / ml to 10000 IU / ml.

[0212] In some embodiments, the dosage formulation contains one or more zwitterionic compounds, including, for example, amino acids such as histidine, glycine, and arginine. In some embodiments, the dosage formulation contains an amphiphilic component having at least one hydrophobic group and one hydrophilic group, such as polysorbate 80, octylpyranoside, a dipeptide, and / or an amphipathic peptide. In some embodiments, the dosage formulation contains a non-reducing sugar or sugar alcohol or disaccharide, such as sorbitol, mannitol, sucrose, or trehalose. In some embodiments, the dosage formulation contains a non-toxic, water-soluble salt, such as sodium chloride, that provides physiological osmolality. In some embodiments, the dosage formulation contains a pH in the range of 6.0 to 8.0. In some embodiments, the dosage formulation contains a pH of about 6.0, about 6.5, about 7, about 7.5, or about 8.0. In some embodiments, the formulation for administration includes one or more divalent cations that stabilize rVWF, such as, for example, Ca2+, Mg2+, Zn2+, Mn2+, and / or combinations thereof. In some embodiments, the formulation for administration includes about 1 mM to about 50 mM glycine, about 1 mM to about 50 mM histidine, about 0 mM to about 300 mM sodium chloride (e.g., less than 300 mM sodium), about 0.01% to about 0.05% polysorbate 20 (or polysorbate 80), and about 0.5% to about 20% (w / w) sucrose, and has a pH of about 7.0 and a physiological osmolality at the time of administration.

[0213] In some embodiments, the formulation for administration can be freeze-dried. In some embodiments, the formulation for administration is stable and can be stored in a liquid state at about 2°C to about 8°C, and about 18°C ​​to about 25°C. In some embodiments, the formulation for administration is stable and can be stored in a liquid state at about 2°C to about 8°C. In some embodiments, the formulation for administration is stable and can be stored in a liquid state at about 18°C ​​to about 25°C.

[0214] V. Administration of rVWF for preventive therapy in patients with severe VWD In some embodiments, the present invention provides for prophylactically treating spontaneous bleeding episodes in subjects with severe von Willebrand disease (VWD), hi some embodiments, the prophylactic treatment comprises administering recombinant von Willebrand factor (rVWF) to the subject to reduce the frequency and / or duration of spontaneous bleeding episodes.

[0215] In some embodiments, spontaneous bleeding episodes include any episode not associated with trauma. In some embodiments, the efficacy of treatment is indicated by a decrease in the number of spontaneous bleeding episodes. In some embodiments, a decrease in the number of spontaneous bleeding episodes is indicated by a decrease in the annualized bleeding rate (ABR). In some embodiments, the ABR before treatment is determined based on the following formula: number of bleeding episodes on days off the treatment regimen / number of days off the treatment regimen. In some embodiments, the ABR before treatment is determined based on the following formula: number of bleeding episodes before prophylactic treatment with rVWF / 12 months. In some embodiments, the prophylactic ABR (ABR after prophylactic treatment) is determined based on the following formula: number of bleeding episodes on days on the treatment regimen / number of days on the treatment regimen.

[0216] In some embodiments, a 25% or greater decrease in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic administration period indicates a prophylactic treatment effect. In some embodiments, a 30% or greater decrease in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 35% or greater decrease in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 40% or greater decrease in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 45% or greater decrease in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 50% or greater decrease in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 55% or greater reduction in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 60% or greater reduction in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 65% or greater reduction in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 70% or greater reduction in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, a 75% or greater reduction in the annualized bleeding rate (ABR) compared to the pre-treatment ABR for spontaneous bleeding episodes during the rVWF prophylactic treatment period indicates a prophylactic treatment effect. In some embodiments, for spontaneous bleeding episodes during the rVWF prophylactic treatment period, a reduction of 80% or more in annualized bleeding rate (ABR) compared to the pre-treatment ABR indicates efficacy of the prophylactic treatment.In some embodiments, a reduction of 85% or more in annualized bleeding rate (ABR) compared to pre-treatment ABR for spontaneous bleeding episodes during rVWF prophylactic treatment indicates efficacy of prophylactic treatment. In some embodiments, a reduction of 90% or more in annualized bleeding rate (ABR) compared to pre-treatment ABR for spontaneous bleeding episodes during rVWF prophylactic treatment indicates efficacy of prophylactic treatment.

[0217] In some embodiments, the efficacy of prophylactic treatment can be measured by measuring vWF:RCo and / or FVIII activity in samples obtained before and after prophylactic treatment with rVWF. In some embodiments, samples for measuring FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen-binding activity are obtained before and after prophylactic treatment with rVWF. In some embodiments, samples for measuring FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF collagen-binding activity are obtained 15 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 28 hours, 32 hours, 48 ​​hours, 72 hours, or 96 hours after prophylactic treatment with rVWF. In some embodiments, samples for FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF collagen-binding activity are obtained 25-31 days after prophylactic treatment with rVWF. In some embodiments, samples for FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF collagen-binding activity are obtained after or during a bleeding episode, in such embodiments, samples are obtained before administration of rVWF, 2 hours after administration, and every 12-24 hours thereafter until the bleeding event has resolved. In some embodiments, the therapeutic effect is determined after or during a bleeding episode. In such embodiments, samples for FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen-binding activity are obtained after a bleeding episode, where additional samples are obtained before administration of rVWF, 2 hours after administration, and every 12 to 24 hours thereafter until the bleeding event has resolved. In some embodiments, activity and time profiles of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and VWF collagen-binding activity are determined based on the samples to monitor the therapeutic effect of rVWF prophylactic treatment. In some embodiments, FVIII:C is also measured by a one-stage clotting assay and time profile comparison to monitor the therapeutic effect of rVWF prophylactic treatment.In some embodiments, the activity levels of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen-binding ability are improved after prophylactic treatment with rVWF compared to levels before prophylactic treatment with rVWF. In some embodiments, the activity levels of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen-binding ability are improved after prophylactic treatment with rVWF compared to levels before prophylactic treatment with rVWF, and this improvement indicates a therapeutic effect. In some embodiments, a prophylactic therapeutic effect is indicated by an improvement in the activity levels of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen-binding ability after prophylactic treatment with rVWF compared to levels before prophylactic treatment with rVWF. In some embodiments, improving the activity level of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability includes changing the activity level such that the activity level approaches a normal level, e.g., the level in a subject without VWD.

[0218] In some aspects, the prophylactic therapeutic effect of rVWF administration is determined after or during a bleeding episode. In some embodiments, the sample for determining FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity is obtained after a bleeding episode. In some embodiments, the sample for determining FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity is obtained during a bleeding episode. In some cases, a sample is obtained from a patient during a bleeding episode, and the sample can be used to determine FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity. In other cases, the sample is obtained from a patient after a bleeding episode. In some embodiments, the sample is obtained before rVWF administration. In some embodiments, the sample is obtained after rVWF administration. In some embodiments, the sample is obtained 2 hours after rVWF administration. In some embodiments, samples are obtained every 12-24 hours until the end of the bleeding episode. In certain embodiments, samples are obtained before rVWF administration and 2 hours after rVWF administration. In some embodiments, samples are obtained before rVWF administration and every 12-24 hours until the end of the bleeding episode. In other embodiments, samples are obtained before rVWF administration, 2 hours after rVWF administration, and every 12-24 hours until the end of the bleeding episode. Thus, samples can be obtained before rVWF administration and before the bleeding episode. In some embodiments, samples are obtained after rVWF administration and before the bleeding episode. In some embodiments, samples are obtained after rVWF administration and before the bleeding episode. In certain embodiments, samples are obtained after rVWF administration and after the end of the bleeding episode.

[0219] In some embodiments, rVWF is administered to a subject in the range of 40-80 IU / kg, e.g., 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 90 IU / kg, 100 IU / kg, 40-100 IU / kg, 40-80 IU / kg, 50-80 IU / kg, 60-80 IU / kg, 70-80 IU / kg, 40-50 IU / kg, 40-60 IU / kg, 40-70 IU / kg, 40-50 IU / kg, 50-60 IU / kg, 60-70 IU / kg, or 70-80 IU / kg. In some embodiments, rVWF is administered at least once a week to prevent spontaneous bleeding episodes. Optionally, the subject receives a single dose of rVWF. Optionally, the subject receives a single infusion of rVWF.

[0220] In some embodiments, rVWF is administered to a subject in the range of 40-80 IU / kg, e.g., 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 90 IU / kg, 100 IU / kg, 40-100 IU / kg, 40-80 IU / kg, 50-80 IU / kg, 60-80 IU / kg, 70-80 IU / kg, 40-50 IU / kg, 40-60 IU / kg, 40-70 IU / kg, 40-50 IU / kg, 50-60 IU / kg, 60-70 IU / kg, or 70-80 IU / kg. In some embodiments, the dose ranges from 40 IU / kg to 60 IU / kg. In some embodiments, the dose ranges from 45 IU / kg to 55 IU / kg. In some embodiments, the dose is about 40 IU / kg, about 50 IU / kg, or about 60 IU / kg. In some embodiments, rVWF is administered at least twice weekly to prevent spontaneous bleeding episodes. In other embodiments, rVWF is administered more than once weekly, e.g., two, three, four, five, or more times, to prevent spontaneous bleeding episodes. Optionally, the subject receives two administrations of rVWF. Optionally, the subject receives two infusions of rVWF. In some embodiments, rVWF is administered twice weekly. In some embodiments, rVWF is administered twice weekly as an infusion dose ranging from 40 IU / kg to 60 IU / kg. In some embodiments, rVWF is administered by IV infusion twice weekly at an infusion dose ranging from 40 IU / kg to 60 IU / kg, with each infusion containing about 40-80 IU / kg of rVWF, e.g., 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 40-80 IU / kg, 50-80 IU / kg, 60-80 IU / kg, 70-80 IU / kg, 40-50 IU / kg, 40-60 IU / kg, 40-70 IU / kg, 40-50 IU / kg, 50-60 IU / kg, 60-70 IU / kg, or 70-80 IU / kg of rVWF. In some embodiments, each injection ranges from 40 IU / kg to 60 IU / kg.In some embodiments, each injection ranges from 45 IU / kg to 55 IU / kg. In some embodiments, each injection is about 40 IU / kg, about 50 IU / kg, or about 60 IU / kg. In some embodiments, the injections can be substantially equivalent in volume. For example, the first injection and the second injection can be substantially equivalent in volume. In some embodiments, the total dose of rVWF administered to a subject is about 40-160 IU / kg, e.g., 40-150 IU / kg, 40-125 IU / kg, 40-100 IU / kg, 40-90 IU / kg, 40-75 IU / kg, 50-150 IU / kg, 50-100 IU / kg, 75-150 IU / kg, 100-125 IU / kg, or 100-160 IU / kg.

[0221] In some embodiments, rVWF is administered to a subject in the range of 40-80 IU / kg, e.g., 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 90 IU / kg, 100 IU / kg, 40-100 IU / kg, 40-80 IU / kg, 50-80 IU / kg, 60-80 IU / kg, 70-80 IU / kg, 40-50 IU / kg, 40-60 IU / kg, 40-70 IU / kg, 40-50 IU / kg, 50-60 IU / kg, 60-70 IU / kg, or 70-80 IU / kg. In some embodiments, rVWF is administered at least twice a week to prevent spontaneous bleeding episodes. In some embodiments, rVWF is administered at least three times a week to prevent spontaneous bleeding episodes. In other embodiments, rVWF is administered three or more times a week to prevent spontaneous bleeding episodes, for example, three, four, five, or more times a week. In some embodiments, the subject receives three administrations of rVWF. In some embodiments, the subject receives three infusions of rVWF. Each infusion may contain rVWF in the range of about 40-80 IU / kg, e.g., 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, 65 IU / kg, 70 IU / kg, 75 IU / kg, 80 IU / kg, 40-80 IU / kg, 50-80 IU / kg, 60-80 IU / kg, 70-80 IU / kg, 40-50 IU / kg, 40-60 IU / kg, 40-70 IU / kg, 40-50 IU / kg, 50-60 IU / kg, 60-70 IU / kg, or 70-80 IU / kg. In some embodiments, the infusions may be substantially equivalent in volume. For example, the first, second, and third infusions may be substantially equivalent in volume. In some embodiments, the total dose of rVWF administered to a subject is about 80-240 IU / kg, e.g., 120-240 IU / kg, 140-240 IU / kg, 140-200 IU / kg, 160-240 IU / kg, 180-240 IU / kg, 200-240 IU / kg, 80-120 IU / kg, 80-160 IU / kg, 80-200 IU / kg, 120-220 IU / kg, or 220-240 IU / kg.In some embodiments, the total dose of rVWF administered to a subject is less than about 160 IU / kg per week, hi some embodiments, the total dose of rVWF administered to a subject is less than about 240 IU / kg per week.

[0222] In some embodiments, rVWF is administered at least once a week, at least twice (2 times) a week, at least three (3) times a week, daily, every other day, every 2-3 days, every 2-4 days, every 2-5 days, etc. Optionally, rVWF is administered for a total of 1, 2, 3, 4, 5, 6, or 7 days over a 7-day period. In some embodiments, rVWF is not administered on consecutive days. In some embodiments, rVWF is administered on consecutive days.

[0223] In some embodiments, rVWF is administered at least every 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, or 96 hours. In some cases, rVWF is administered at least every 60 hours, 72 hours, or 84 hours. In some cases, rVWF is administered at least every 72 hours.

[0224] In some embodiments, recombinant factor VIII (rFVIII) is also administered to subjects with severe VWD to prevent or reduce the frequency and / or duration of spontaneous bleeding episodes. In some cases, the administered treatment includes rVWF and rFVIII. In other cases, the administered treatment does not include rFVIII. In some embodiments, rFVIII is administered to a subject at a dose in the range of about 10-70 IU / kg, e.g., 10-70 IU / kg, 10-60 IU / kg, 10-50 IU / kg, 10-40 IU / kg, 10-30 IU / kg, 10-20 IU / kg, 20-30 IU / kg, 30-40 IU / kg, 40-50 IU / kg, 50-60 IU / kg, or 60-70 IU / kg. In some cases, rFVIII is administered as an initial (first) dose or initial (first) infusion. In some cases, rFVIII is not administered in the initial (first) dose or initial (first) infusion. In some cases, rFVIII is administered as part of the second dose or second infusion. In some cases, rFVIII is not administered as part of the second dose or second infusion. In some cases, rFVIII is administered as part of the third dose or third infusion. In some cases, rFVIII is not administered as part of the third dose or third infusion.

[0225] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes are administered a single infusion of rVWF and rFVIII. In some embodiments, the second administration of rVWF is not administered with FVIII. In some embodiments, the third administration of rVWF is not administered with FVIII.

[0226] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes are administered a single infusion of rVWF and rFVIII. In some embodiments, the second administration of rVWF is administered with FVIII. In some embodiments, the third administration of rVWF is not administered with FVIII.

[0227] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes are administered a single infusion of rVWF and rFVIII. In some embodiments, a second administration of rVWF is administered with FVIII. In some embodiments, a third administration of rVWF is administered with FVIII.

[0228] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes are administered a single infusion of rVWF without rFVIII. In some embodiments, a second administration of rVWF is administered with FVIII. In some embodiments, a third administration of rVWF is administered with FVIII.

[0229] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes are administered a single infusion of rVWF without rFVIII. In some embodiments, a second administration of rVWF is not administered with FVIII. In some embodiments, a third administration of rVWF is administered with FVIII.

[0230] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes are administered a single infusion of rVWF without rFVIII. In some embodiments, a second administration of rVWF is administered with FVIII. In some embodiments, a third administration of rVWF is not administered with FVIII.

[0231] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes receive a first infusion and a second infusion of rVWF, hi some embodiments, the first and / or second infusion of rVWF is administered with FVIII.

[0232] In some embodiments, VWD subjects at risk of experiencing spontaneous bleeding episodes receive a first infusion, a second infusion, and a third infusion of rVWF. In some embodiments, the first, second, and / or third infusion of rVWF are administered with FVIII.

[0233] In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.5:0.8. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.3:1. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.1:0.8. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.5:1. In some embodiments of the method, when rVWF and FVIII are administered together, the ratio of rVWF to FVIII is about 1.1:1.2.

[0234] In some embodiments, about 40 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 45 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 50 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 55 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 60 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 65 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 70 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 75 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 80 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 40 IU / kg to 80 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 50 IU / kg to 80 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 40 IU / kg to 70 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 50 IU / kg to 80 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 40 IU / kg to 60 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding. In some embodiments, about 50 IU / kg to 80 IU / kg of such rVWF is administered for the prevention of spontaneous bleeding.

[0235] In some embodiments, 40-80 IU / kg of such rVWF is administered twice weekly for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50-80 IU / kg of such rVWF is administered twice weekly for the prophylactic treatment of spontaneous bleeding. In some embodiments, 40 IU / kg of such rVWF is administered twice weekly for the prophylactic treatment of spontaneous bleeding. In some embodiments, 45 IU / kg of such rVWF is administered twice weekly for the prophylactic treatment of spontaneous bleeding. In some embodiments, about 50 IU / kg of such rVWF is administered twice weekly for the prophylactic treatment of spontaneous bleeding. In some embodiments, about 55 IU / kg of such rVWF is administered twice weekly for the prophylactic treatment of spontaneous bleeding. In some embodiments, about 60 IU / kg of such rVWF is administered twice weekly for the prophylactic treatment of spontaneous bleeding. In some embodiments, about 65 IU / kg of such rVWF is administered twice weekly for prophylactic treatment of spontaneous bleeding. In some embodiments, about 70 IU / kg of such rVWF is administered twice weekly for prophylactic treatment of spontaneous bleeding. In some embodiments, about 75 IU / kg of such rVWF is administered twice weekly for prophylactic treatment of spontaneous bleeding. In some embodiments, about 80 IU / kg of such rVWF is administered twice weekly for prophylactic treatment of spontaneous bleeding.

[0236] In some embodiments, 40-80 IU / kg of such rVWF is administered three times per week for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50-80 IU / kg of such rVWF is administered three times per week for the prophylactic treatment of spontaneous bleeding. In some embodiments, 40 IU / kg of such rVWF is administered three times per week for the prophylactic treatment of spontaneous bleeding. In some embodiments, 45 IU / kg of such rVWF is administered three times per week for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50 IU / kg of such rVWF is administered three times per week for the prophylactic treatment of spontaneous bleeding. In some embodiments, 55 IU / kg of such rVWF is administered three times per week for the prophylactic treatment of spontaneous bleeding. In some embodiments, 60 IU / kg of such rVWF is administered three times per week for the prophylactic treatment of spontaneous bleeding. In some embodiments, 65 IU / kg of such rVWF is administered three times per week for prophylactic treatment of spontaneous bleeding. In some embodiments, 70 IU / kg of such rVWF is administered three times per week for prophylactic treatment of spontaneous bleeding. In some embodiments, 75 IU / kg of such rVWF is administered three times per week for prophylactic treatment of spontaneous bleeding. In some embodiments, 80 IU / kg of such rVWF is administered three times per week for prophylactic treatment of spontaneous bleeding. In some embodiments, 50 IU / kg of such rVWF is administered at least every 72 hours for prophylactic treatment of spontaneous bleeding.

[0237] In some embodiments, 40-80 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50-80 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 40 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 45 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 55 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 60 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 65 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 70 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 75 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 80 IU / kg of such rVWF is administered every 2-3 days for the prophylactic treatment of spontaneous bleeding.

[0238] In some embodiments, 40-80 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50-80 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 40 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 45 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 55 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 60 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 65 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 70 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 75 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding. In some embodiments, 80 IU / kg of such rVWF is administered every 3-4 days for the prophylactic treatment of spontaneous bleeding.

[0239] In some embodiments, 40 to 80 IU / kg of the rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50 to 80 IU / kg of the rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for the prophylactic treatment of spontaneous bleeding. In some embodiments, 40 IU / kg of the rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for the prophylactic treatment of spontaneous bleeding. In some embodiments, 45 IU / kg of the rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for the prophylactic treatment of spontaneous bleeding. In some embodiments, 50 IU / kg of such rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for prophylactic treatment of spontaneous bleeding. In some embodiments, 55 IU / kg of such rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for prophylactic treatment of spontaneous bleeding. In some embodiments, 60 IU / kg of such rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for prophylactic treatment of spontaneous bleeding. In some embodiments, 65 IU / kg of such rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for prophylactic treatment of spontaneous bleeding. In some embodiments, 70 IU / kg of such rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for prophylactic treatment of spontaneous bleeding. In some embodiments, 75 IU / kg of such rVWF is administered once on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven-day (weekly) period for prophylactic treatment of spontaneous bleeding.In some embodiments, 80 IU / kg of such rVWF is administered once on days 1 and 5, once on days 2 and 6, or once on days 3 and 7 of a 7-day (weekly) period for prophylactic treatment of spontaneous bleeding.

[0240] In some embodiments, the subject receives prophylactic treatment for spontaneous bleeding for at least one month. In some embodiments, the subject receives prophylactic treatment for spontaneous bleeding for at least six months. In some embodiments, the subject receives prophylactic treatment for spontaneous bleeding for at least one year. In some embodiments, the subject receives prophylactic treatment for spontaneous bleeding for at least two years. In some embodiments, the subject receives prophylactic treatment for spontaneous bleeding for at least five years. In some embodiments, the subject receives prophylactic treatment for spontaneous bleeding for at least ten years.

[0241] In some embodiments, subjects with severe VWD are administered a weekly dose of rVWF for the prophylactic treatment of spontaneous bleeding episodes. In some embodiments, a weekly dose of rVWF substantially equivalent to the weekly dose of plasma-derived VWF (pdVWF) is administered to the subject for the prophylaxis of spontaneous bleeding. In some embodiments, a weekly dose of rVWF functionally equivalent to the weekly dose of plasma-derived VWF (pdVWF) is administered to the subject for the prophylaxis of spontaneous bleeding. In some embodiments, a weekly dose of rVWF about 10% less than the weekly dose of plasma-derived VWF (pdVWF) is administered to the subject for the prophylaxis of spontaneous bleeding. In some embodiments, a weekly dose of rVWF about 10% greater than the weekly dose of plasma-derived VWF (pdVWF) is administered to the subject for the prophylaxis of spontaneous bleeding.

[0242] In some embodiments, the weekly dose of rVWF is administered as at least two separate doses or at least two infusions. In some embodiments, the subject is provided with at least two doses per week. In some embodiments, the weekly dose of rVWF is administered as two infusions. In some embodiments, the weekly dose of rVWF is administered as two intravenous infusions. In some cases, the subject is administered two doses of rVWF per week, i.e., the subject is administered rVWF twice per week. In some embodiments, the weekly dose of rVWF is administered as two intravenous infusions. In some embodiments, rVWF is administered to the subject twice per week. In some embodiments, rVWF is administered to the subject every 3 to 4 days. In some embodiments, a first infusion of rVWF is administered during a 7-day period, followed by a second infusion of rVWF four days later. In some embodiments, rVWF is administered once on day 1 and once on day 5 of a 7-day (weekly) period for the prophylactic treatment of spontaneous bleeding. In some embodiments, subjects are administered a weekly dose split into two infusions, with the subject receiving the first infusion on day 1 and the second infusion on day 5 of the week. In some embodiments, rVWF is administered once per week on days 2 and 6 for prophylactic treatment of spontaneous bleeding. In some embodiments, subjects are administered a weekly dose split into two infusions, with the subject receiving the first infusion on day 2 and the second infusion on day 6 of the week. In some embodiments, rVWF is administered once per week on days 3 and 7 for prophylactic treatment of spontaneous bleeding. In some embodiments, subjects are administered a weekly dose split into two infusions, with the subject receiving the first infusion on day 3 and the second infusion on day 7 of the week. In some embodiments, the weekly dose of rVWF is up to 80 IU / kg of rVWF per infusion. In some embodiments, the weekly dose of rVWF is divided into two intravenous infusions of up to 80 IU / kg of rVWF per infusion.

[0243] In some embodiments, the weekly dose of rVWF is administered as at least three separate doses or at least three infusions. In some embodiments, the subject is provided with at least three doses per week. In some embodiments, the weekly dose of rVWF is administered as three intravenous infusions. Optionally, the subject receives three doses of rVWF per week, i.e., the subject receives rVWF three times per week. In some embodiments, rVWF is administered to the subject three times per week. In some embodiments, the weekly dose of rVWF is administered as three intravenous infusions, with each infusion administered on a different day. In some embodiments, rVWF is administered to the subject every two to three days. In some embodiments, during a seven-day period, a first infusion of rVWF is administered, followed two days later by a second infusion of rVWF, and three days later by a third infusion of rVWF. In some embodiments, rVWF is administered once on days 1, 3, and 6 of a seven-day period for prophylactic treatment of spontaneous bleeding. In some embodiments, subjects are administered a weekly dose divided into three infusions, such that the subject receives a first infusion on day 1, a second infusion on day 3, and a third infusion on day 6 of the week. In some embodiments, rVWF is administered once each on days 1, 3, and 6 of the week for prophylactic treatment of spontaneous bleeding. In some embodiments, subjects are administered a weekly dose divided into three infusions, such that the subject receives a first infusion on day 2, a second infusion on day 4, and a third infusion on day 7 of the week. In some embodiments, rVWF is administered once each on days 2, 4, and 7 of the week for prophylactic treatment of spontaneous bleeding. In some embodiments, the weekly dose of rVWF is up to 80 IU / kg of rVWF per infusion. In some embodiments, the weekly dose of rVWF is divided into three intravenous infusions of up to 80 IU / kg of rVWF per infusion.

[0244] In some embodiments, the weekly dose of rVWF is administered as a single dose or a single infusion. In some embodiments, the weekly dose of rVWF is administered as a single infusion. In some embodiments, the weekly dose of rVWF is administered as a single intravenous infusion. In some embodiments, if the subject has previously received a weekly dose of pdVWF, the subject is administered a weekly dose of rVWF. In some embodiments, in such an infusion, the weekly dose of rVWF is up to 80 IU / kg of rVWF.

[0245] In some embodiments, a subject experiences at least a 5% reduction (e.g., a 5%, 7%, 10%, 12%, 14%, 15%, 17%, 20%, 22%, 24%, 25%, 27%, 30%, 32%, 34%, 35%, 37%, 40%, 42%, 44%, 45%, 47%, 50%, or more reduction) in annualized bleeding rate (ABR) for spontaneous bleeding events when receiving a prophylactic treatment described herein. In some embodiments, a subject experiences at least a 25% reduction (e.g., a 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more reduction) in ABR for spontaneous bleeding events when receiving a prophylactic treatment described herein. In some embodiments, a reduction in the number of spontaneous bleeding episodes is indicated by a reduction in the annualized bleeding rate (ABR). In some embodiments, the pre-treatment ABR is determined based on the following formula: number of bleeding episodes on days off the treatment regimen / number of days off the treatment regimen. In some embodiments, the pre-treatment ABR is determined based on the following formula: number of bleeding episodes before prophylactic treatment with rVWF / 12 months. In some embodiments, the prophylactic ABR (ABR after prophylactic treatment) is determined based on the following formula: number of bleeding episodes on the treatment regimen / number of days on the treatment regimen.

[0246] In some embodiments, the subject does not experience any spontaneous bleeding events during prophylactic treatment with rVWF. In some embodiments, the subject experiences 1 to 2 spontaneous bleeding events during prophylactic treatment with rVWF. In some embodiments, the subject experiences 1 to 3 spontaneous bleeding events during prophylactic treatment with rVWF. In some embodiments, the subject experiences 3 to 5 spontaneous bleeding events during prophylactic treatment with rVWF. In some embodiments, the subject experiences 1 to 5 spontaneous bleeding events during prophylactic treatment with rVWF. In some embodiments, the subject experiences 5 or more spontaneous bleeding events during prophylactic treatment with rVWF.

[0247] In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to zero per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to one per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to two per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to three per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to four per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to five per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to five or more per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to one to five per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to three to five per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to 1-3 per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to fewer than 5 per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to fewer than 4 per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to fewer than 3 per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to fewer than 2 per year. In some embodiments, administration of rVWF reduces the number of spontaneous bleeding events to fewer than 1 per year.

[0248] In some embodiments, prophylactic treatment reduces or decreases the severity (e.g., mild, moderate, and severe bleeding episodes), incidence, frequency, or duration of spontaneous bleeding episodes. In some embodiments, subjects administered rVWF experience fewer spontaneous bleeding episodes than control subjects who do not receive prophylactic treatment. In some embodiments, subjects administered rVWF experience fewer spontaneous bleeding episodes throughout the treatment period compared to before treatment began.

[0249] Generally, type 1 VWD is indicated by VWF:RCo<30 IU / dL, VWF:Ag<30 IU / dL, low or normal FVIII levels, and a VWF:RCo / VWF:Ag ratio >0.5-0.7 IU / dL. In some embodiments, a subject diagnosed with type 1 VWD has a VWF:RCo<20 IU / dL. Type 2A VWD is indicated by VWF:RCo<30 IU / dL, VWF:Ag<30-200 IU / dL, low or normal FVIII levels, and a VWF:RCo / VWF:Ag ratio <0.5-0.7 IU / dL. Type 2B VWD is characterized by VWF:RCo<30-200 IU / dL, VWF:Ag<30 IU / dL, low or normal FVIII levels, and a VWF:RCo / VWF:Ag ratio usually <0.5-0.7 IU / dL. Type 2M VWD is characterized by VWF:RCo<30 IU / dL, VWF:Ag<30-200 IU / dL, low or normal FVIII levels, and a VWF:RCo / VWF:Ag ratio <0.5-0.7 IU / dL. Type 2N VWD is indicated by VWF:RCo between 30 and 2000 IU / dL, VWF:Ag between 30 and 200 IU / dL, very low FVIII levels, and a VWF:RCo / VWF:Ag ratio >0.5 to 0.7 IU / dL. Type 3 VWD is indicated by VWF:RCo <3 IU / dL, VWF:Ag <3 IU / dL, very low FVIII levels (<10 IU / dL), and a VWF:RCo / VWF:Ag ratio not applicable. In some embodiments, subjects diagnosed with type 3 VWD have a VWF:Ag <3 IU / dL. Normal is indicated by VWF:RCo between 50 and 200 IU / dL, VWF:Ag between 50 and 200 IU / dL, normal FVIII levels, and a VWF:RCo / VWF:Ag ratio >0.5 to 0.7 IU / dL. In some embodiments, the subject has type 3 VWD. In some embodiments, the subject has severe type 1 VWD. In some embodiments, the subject has severe type 2 VWD. In some embodiments, the subject has severe type 2A VWD. In some embodiments, the subject has severe type 2B VWD. In some embodiments, the subject has severe type 2M VWD. In some embodiments, the subject has severe type 3 VWD.

[0250] In some embodiments, the preventive therapeutic effect is demonstrated by an improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in type 1 VWD patients compared to the levels before prophylactic treatment with rVWF. In some embodiments, the improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag includes a change in the activity levels such that the activity levels approach normal levels, e.g., levels in subjects without type 1 VWD. In some embodiments, the preventive therapeutic effect is demonstrated by an improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in severe type 1 VWD patients compared to the levels before prophylactic treatment with rVWF. In some embodiments, the improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag includes a change in the activity levels such that the activity levels approach normal levels, e.g., levels in subjects without severe type 1 VWD. In some embodiments, the preventive therapeutic effect is demonstrated by an improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in type 2A VWD patients compared to the levels before prophylactic treatment with rVWF. In some embodiments, the improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag includes a change in the activity levels such that the activity levels approach normal levels, e.g., levels in subjects without type 2A VWD. In some embodiments, the preventive therapeutic effect is demonstrated by an improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in severe type 2A VWD patients compared to the levels before prophylactic treatment with rVWF. In some embodiments, the improvement in the activity levels of FVIII, VWF:RCo, and / or VWF:Ag includes a change in the activity levels such that the activity levels approach normal levels, e.g., levels in subjects without severe type 2A VWD. In some embodiments, the preventive treatment effect is demonstrated by improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag after preventive treatment with rVWF compared to levels before preventive treatment with rVWF in type 2M VWD patients.In some embodiments, improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag include changes in activity levels such that the activity levels approach normal levels, e.g., levels in subjects without type 2M VWD. In some embodiments, a prophylactic therapeutic effect is demonstrated by improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in patients with severe type 2M VWD compared to levels before prophylactic treatment with rVWF. In some embodiments, improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag include changes in activity levels such that the activity levels approach normal levels, e.g., levels in subjects without severe type 2M VWD. In some embodiments, a prophylactic therapeutic effect is demonstrated by improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in patients with type 2N VWD compared to levels before prophylactic treatment with rVWF. In some embodiments, improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag include changes in activity levels such that the activity levels approach normal levels, e.g., levels in subjects without type 2N VWD. In some embodiments, a prophylactic therapeutic effect is demonstrated by improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in patients with severe type 2N VWD compared to levels before prophylactic treatment with rVWF. In some embodiments, improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag include changes in activity levels such that the activity levels approach normal levels, e.g., levels in subjects without severe type 2N VWD. In some embodiments, a prophylactic therapeutic effect is demonstrated by improved activity levels of FVIII, VWF:RCo, and / or VWF:Ag after prophylactic treatment with rVWF in patients with type 3 VWD compared to levels before prophylactic treatment with rVWF. In some embodiments, improving the activity levels of FVIII, VWF:RCo, and / or VWF:Ag includes changing the activity levels such that the activity levels approach normal levels, e.g., levels in subjects who do not have type 3 VWD.In some embodiments, the preventive treatment effect is demonstrated by an improvement in FVIII, VWF:RCo, and / or VWF:Ag activity levels after prophylactic treatment with rVWF compared to levels before prophylactic treatment with rVWF in patients with severe type 3 VWD. In some embodiments, an improvement in FVIII, VWF:RCo, and / or VWF:Ag activity levels includes a change in activity levels such that the activity levels approach normal levels, e.g., levels in subjects without severe type 3 VWD. In some embodiments, an improvement in FVIII, VWF:RCo, and / or VWF:Ag activity levels includes a change in activity levels such that the activity levels approach normal levels, e.g., levels in subjects without any type of VWD.

[0251] In some embodiments, the subject has been diagnosed with VWD. In some cases, the diagnosis of VWD is confirmed by genetic testing, multimer analysis, patient history, or a combination thereof.

[0252] In some embodiments, the subject has been treated for at least one spontaneous bleeding event within the past 12 months. In some embodiments, the subject has been treated for one or more spontaneous bleeding events within the past 12 months. In some embodiments, the subject has been treated for at least three spontaneous bleeding events within the past 12 months. In some embodiments, the subject has been treated for three or more spontaneous bleeding events within the past 12 months.

[0253] In some embodiments, the subject is currently receiving on-demand treatment for a spontaneous bleeding event (e.g., episode). In some embodiments, the subject is currently receiving VWF therapy (e.g., rVWF or pdVWF therapy) to treat the spontaneous bleeding event. In some embodiments, the subject is not currently receiving on-demand treatment for a spontaneous bleeding event (e.g., episode). In some embodiments, the subject is not currently receiving VWF therapy (e.g., rVWF or pdVWF therapy) to treat the spontaneous bleeding event.

[0254] In some embodiments, the subject has been treated prophylactically against spontaneous bleeding with administration of pdVWF within the past 12 months. In some embodiments, the subject has been treated prophylactically against spontaneous bleeding with administration of pdVWF for the past 12 months. In some embodiments, the subject has been treated prophylactically against spontaneous bleeding with administration of pdVWF for at least the past 12 months. In some embodiments, the subject has not been treated prophylactically against spontaneous bleeding with administration of pdVWF within the past 12 months. In some embodiments, the subject has not been treated prophylactically against spontaneous bleeding with administration of pdVWF for the past 12 months. In some embodiments, the subject has not been treated prophylactically against spontaneous bleeding with administration of pdVWF for at least the past 12 months.

[0255] In general, minor bleeding is characterized by acute or subacute clinical overt bleeding that does not meet the criteria for major bleeding and that leads to hospitalization for bleeding, physician-ordered medical or surgical treatment for bleeding, or a change in antithrombotic therapy (including study drug) for bleeding (Aristotle clinical definition); any other bleeding (excluding major bleeding and ICH) (RE-LY clinical definition); or overt bleeding that does not meet the criteria for major bleeding but requires medical intervention, unscheduled contact with a physician (in-office or by phone), temporary interruption (i.e., delayed administration) of study drug (pain, or impairment of activities of daily living) (Rocket-AF clinical definition); clinically significant bleeding is defined as a cutaneous hematoma >25 cm2 , defined as spontaneous epistaxis of >5 minutes duration, gross hematuria, spontaneous rectal bleeding, gingival bleeding >5 minutes, any bleeding resulting in hospitalization, any bleeding resulting in transfusion <2U, or any other bleeding deemed significant by the investigator (Petro's clinical definition); and / or CRNM (clinically significant non-major bleeding), defined as acute or subacute clinically overt non-major bleeding resulting in hospitalization for bleeding, physician-directed medical or surgical treatment for bleeding, or change in antithrombotic therapy, and minor bleeding events, defined as acute clinically overt events that did not meet the criteria for either major or CRNM bleeding (Aristotle-J's clinical definition). See, e.g., Wells G, Coyle D, Cameron C, et al. Safety, Effectiveness, and Cost-Effectiveness of New Oral Anticoagulants Compared with Warfarin in Preventing Stroke and Other Cardiovascular Events in Patients with Atrial Fibrillation [Internet]. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health; 2012 Apr 9.3, CLINICAL REVIEW. Available on the World Wide Web at www.ncbi.nlm.nih.gov / books / NBK169813 / .Minor bleeding may include events defined as not meeting criteria for major or clinically important bleeding; minor bleeding from wounds (injection site bleeding, epistaxis, or wound hematoma not requiring decompression); overt bleeding not meeting criteria for major bleeding and associated with one or more of the following: epistaxis lasting ≥ 5 minutes or requiring intervention, ecchymosis or hematoma > 5 cm in greatest dimension, hematuria not associated with urinary catheter-related trauma, GI bleeding not associated with NG tube intubation or placement, wound hematoma or complication, subconjunctival hemorrhage requiring drug discontinuation; minor bleeding in the GI or urinary tract and hematoma at the injection site; and / or overt bleeding not meeting criteria for major bleeding. See, e.g., Sobieraj DM, Coleman CI, Tongbram V, et al. Venous Thromboembolism Prophylaxis in Orthopedic Surgery [Internet]. Rockville, MD: Agency for Healthcare Research and Quality (US); 2012 Mar. (Comparative Effectiveness Reviews, No. 49.) Appendix F, Additional Evidence Tables. Available on the World Wide Web at www.ncbi.nlm.nih.gov / books / NBK92309 / .

[0256] In general, major bleeding is characterized by the International Society on Thrombosis and Haemostasis (ISTH) criteria, which include any life-threatening and / or fatal bleeding; symptomatic bleeding into a critical area or organ and major bleeding divided into intracranial (intracerebral, subdural) and extracranial (GI, non-GI) bleeding (RE-LY clinical definition); symptomatic bleeding into a critical anatomical site (Rocket-AF clinical definition); life-threatening retroperitoneal, intracranial, intraocular, or intraspinal bleeding; or bleeding requiring surgery (Artistotle-J clinical definition). Major bleeding events can include a fall in hemoglobin of at least 20 g / L or transfusion of >2 units of whole blood (blood counts referred to in the life-threatening bleeding definition; RE-LY definition of life-threatening bleeding: one or more of the following criteria: (1) fatal symptomatic intracranial hemorrhage; (2) a fall in hemoglobin level of at least 5.0 g / L; (3) transfusion of at least 4 U of blood or blood counts; (4) associated hypotension requiring the use of intravenous inotropes; or (5) requiring surgical intervention; an episode with a fall in hemoglobin >2 g / dL or transfusion of >2 units of whole blood / red blood cells (ISTH or Rocket-AF clinical definition); and / or an episode of bleeding requiring surgery or transfusion ≥2 U or associated with a fall in hemoglobin ≥2.0 g / L. See, e.g., Wells G, Coyle D, Cameron C, et al. Safety, Effectiveness, and Cost-Effectiveness of New Oral Anticoagulants Compared with Warfarin in Preventing Stroke and Other Cardiovascular Events in Patients with Atrial Fibrillation [Internet]. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health; 2012 Apr 9.3, CLINICAL REVIEW.Available on the World Wide Web at www.ncbi.nlm.nih.gov / books / NBK169813 / . Major bleeding may include clinically overt bleeding with a fall in Hb >20 g / L; clinically overt bleeding resulting in transfusion of >2U of red blood cells or whole blood; fatal retroperitoneal, intracranial, intraocular, or intraspinal bleeding; bleeding requiring discontinuation of treatment or leading to reoperation; fatal retroperitoneal, intracranial, or intraspinal bleeding; bleeding affecting any other vital organ; bleeding leading to reoperation; overt bleeding with a bleeding index >2; major wound-related bleeding (wound hematoma requiring decompression) or major non-wound-related bleeding (gastrointestinal or intracerebral hemorrhage); clinically overt bleeding with a fall in Hb >2 g / dL or requirement for RBC transfusion >2U; intracranial or retroperitoneal bleeding (leading to permanent cessation of anticoagulant therapy). See, e.g., Sobieraj DM, Coleman CI, Tongbram V, et al. Venous Thromboembolism Prophylaxis in Orthopedic Surgery [Internet]. Rockville, MD: Agency for Healthcare Research and Quality (US); 2012 Mar. (Comparative Effectiveness Reviews, No. 49.) Appendix F, Additional Evidence Tables. Available from the World Wide Web at www.ncbi.nlm.nih.gov / books / NBK92309 / .

[0257] The rVWF composition can be contained in a pharmaceutical formulation, as described herein. Such formulations can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or intracranially. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intracisternal, or infusion techniques. In some embodiments, the rVWF is administered intravenously. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, or intrapulmonary injection and / or surgical implantation at a specific site is also contemplated. Generally, the compositions are essentially free of pyrogens and other impurities that may be harmful to the recipient.

[0258] In one embodiment, the formulations of the present invention are administered as an initial bolus, followed by continuous infusion to maintain therapeutic circulating concentrations of the formulation. In another example, the compounds of the present invention are administered as a single dose. Those skilled in the art can easily optimize effective doses and administration regimens, as determined by good medical practice and the clinical condition of each individual patient. The route of administration can be, but is not limited to, intravenous, intraperitoneal, subcutaneous, or intramuscular. The frequency of administration will depend on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation will be determined by those skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, Mack Publishing Co., Easton, Pa. 18042, pages 1435-1712, the disclosure of which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings regarding pharmaceutical formulations, routes of administration, and dosages. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, suitable doses are calculated according to body weight, body surface area, or organ size. Appropriate doses can be confirmed by the use of established assays for determining blood concentrations of administered doses in conjunction with appropriate dose-response data. The final dosing regimen will be determined by the attending physician and will take into account various factors that modify the drug's action, such as the specific activity of the drug, the severity and responsiveness of the patient's injury, the patient's age, condition, weight, sex, and diet, the severity of any infection, the time of administration, and other clinical factors. By way of example, a typical dose of the recombinant VWF of the present invention is approximately 50 IU / kg, which is equivalent to 500 μg / kg. As studies are conducted, more information will emerge regarding appropriate dosages and durations of treatment for various diseases and conditions.

[0259] In carrying out the present invention, unless otherwise indicated, conventional techniques and explanations of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and immunology may be used, which are within the skill of those skilled in the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using labels. Specific examples of suitable techniques can be obtained by reference to the examples herein below. However, it will be appreciated that other equivalent conventional procedures can also be used. Such prior art techniques and explanations can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all published by Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, Highly stabilized New York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub., Highly stabilized New York, NY, and Berg et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., Highly stabilized New York, NY, all of which are incorporated herein by reference in their entireties for all purposes.

[0260] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an antibody" includes a plurality of such antibodies; a reference to "a host cell" includes a reference to one or more host cells and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate basis for using limiting language such as "only," "only," and the like in connection with the recitation of claim elements or for using "negative" limitations.

[0261] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference to describe and disclose the devices, compositions, formulations, and methodologies that are described in the publications and that may be used in connection with the inventions described herein.

[0262] Where a range of values ​​is provided, it is understood that each intervening value (to the tenth of the unit of the lower limit unless the context clearly dictates otherwise) between the upper and lower limit of that range, and any other stated or intervening value within that stated range, is encompassed within the invention. The upper and lower limits of these subranges may independently be included within the subranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. When one or both of the limits is included in the stated range, ranges excluding both of those included limits are also included in the invention.

[0263] In the above description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0264] Although the present invention has been described primarily with reference to specific embodiments, it is also anticipated that other embodiments will become apparent to those skilled in the art upon reading this disclosure, and such embodiments are intended to be encompassed within the methods of the present invention. [Example]

[0265] Example 1: Efficacy and safety of recombinant von Willebrand factor (VWF) prophylaxis in severe von Willebrand disease (VWD): design of a phase 3, prospective, open-label, international, multicenter study Introduction Patients with von Willebrand disease (VWD) have impaired hemostasis due to a quantitative or qualitative deficiency of von Willebrand factor (VWF), a large multimeric plasma glycoprotein with important hemostatic functions that mediate platelet aggregation and stabilize circulating blood coagulation factor VIII (FVIII) (1-3). Most patients with VWD present with mild to moderate mucosal bleeding and posttraumatic or postoperative bleeding, but some patients, particularly those with severe disease, may experience life-threatening bleeding (1, 2).

[0266] Recombinant VWF (rVWF, Vonicog alfa, VEYVONDI™; Baxalta Innovations GmbH, a Takeda company, Vienna, Austria) is produced by recombinant DNA technology in a Chinese hamster ovary cell line without the addition of exogenous human or animal-derived proteins (4). VWF contains the complete VWF multimer profile, including extra-large multimers that are typically lacking in plasma-derived VWF (pdVWF) concentrates exposed to the VWF-cleaving protease ADAMTS13 (5-7).

[0267] Patients with severe VWD may benefit from prophylactic rVWF treatment to maintain VWF and FVIII levels to reduce the risk of spontaneous bleeding episodes (BE), such as hemarthrosis, epistaxis, and gastrointestinal bleeding ( 8 – 13 ).

[0268] Reducing the frequency and duration of BE is thought to reduce the need for red blood cell transfusions and reduce the risk of debilitating comorbidities such as arthropathy ( 8 , 9 ).

[0269] Test Purpose The aim of the study was to investigate the efficacy and safety of long-term prophylactic treatment with rVWF in patients with severe VWD.

[0270] Study design Herein, we describe an international, multicenter, open-label, phase 3 study (NCT02973087, EudraCT registration number: 2016-001478-14) to prospectively evaluate the efficacy, safety, and pharmacokinetics of continuous prophylactic treatment with rVWF in adult patients with severe VWD (baseline VWF:ristocetin cofactor activity [VWF:RCo] <20 IU / dL) who required treatment with pdVWF to manage BE in the year prior to enrollment ( Figure 1 ).

[0271] Patients will be assigned to one or two cohorts depending on the type of VWF regimen they received before enrollment: on-demand treatment (on-demand cohort) and prophylactic treatment with a pdVWF product (switch cohort). Patients in both cohorts will receive prophylactic rVWF treatment for one year.

[0272] patient Patients aged 18 years or older with a diagnosis of severe VWD (baseline VWF:RCo<20IU / dL) requiring VWF treatment to control bleeding (main study eligibility criteria are detailed in Figure 2). The study protocol was approved by the relevant local ethics committee, and all patients provided written informed consent before enrollment.

[0273] Test treatment Patients received rVWF prophylaxis as described below.

[0274] For the on-demand cohort, a standard prophylactic dose of 50±10 IU / kg rVWF:RCo was administered intravenously, with the dose escalable up to 80 IU / kg.

[0275] All patients were initiated on twice-weekly dosing ( Figure 3 , Schedule A).

[0276] For the switch cohort, a weekly dose of rVWF equivalent to ±10% of the previously received weekly dose of pdVWF was divided into two intravenous infusions with a maximum of 80 IU / kg per infusion (Fig. 3, Schedule A). The weekly dose was administered as three infusions according to clinical judgment (Fig. 3, Schedule B), with weekly dosing permitted only if the patient had previously received weekly dosing with pdVWF.

[0277] The rVWF dose could be further individualized using a defined range based on pharmacokinetic data, history of BE, and results from clinical and laboratory evaluations. Patients experiencing BE requiring VWF treatment were considered to have received rVWF to treat the bleeding. Weight-based dose adjustment should be based on the type and severity of BE and appropriate clinical and laboratory measures. Recombinant FVIII (antihemophilic factor [recombinant] [ADVATE; Baxalta US Inc., a Takeda company, Lexington, MA]) was considered to have been administered if an immediate increase in FVIII levels was clinically indicated.

[0278] Patients undergoing elective surgery or dental procedures during the study period were treated with rVWF to manage bleeding during surgery and then resumed their prophylactic rVWF treatment schedule. Before surgery, rVWF was administered without recombinant FVIII if FVIII activity (FVIII:C) levels were at the recommended target levels: ≥0.4 IU / mL for minor or oral surgery and ≥0.8 IU / mL for major surgery.

[0279] Study endpoints and statistical analysis The primary endpoints of the study are described in Figure 4 (Table 3).

[0280] Approximately 22 patients were included in the statistical analysis, with at least five type 3 VWD patients and at least eight patients in each study cohort; no formal subject number calculation was performed.

[0281] The primary efficacy analysis was performed on all patients who received prophylactic rVWF treatment. Spontaneous ABR was estimated using negative binomial regression for each study cohort (on-demand or switch cohort). Comparisons of pre- and on-study spontaneous ABR were based on patients in the switch cohort. A linear mixed-effects model was constructed for the evaluation. ABR ratios were reported with two-sided 95% confidence intervals for each study cohort.

[0282] Test status and conclusions Study enrollment is now closed.

[0283] Results from this prospective phase 3 trial provide data on the efficacy and safety of rVWF for the prevention of spontaneous bleeding in patients with severe VWD.

[0284] The trial duration of prophylactic rVWF treatment was 1 year, and the primary efficacy endpoint was ABR for spontaneous (non-traumatic) bleeding.

[0285] Primary endpoint The annualized bleeding rate (ABR) of spontaneous (non-trauma-related) bleeding episodes during prophylactic treatment with rVWF and participants' ABR history of spontaneous bleeding episodes during on-demand treatment were prospectively recorded (time limit: approximately 1 year).

[0286] Modified secondary endpoints Number of participants who experienced a reduction in annualized bleeding rate (ABR) for spontaneous (non-trauma-related) bleeding episodes during prophylactic treatment compared with their own ABR history during on-demand treatment (time limit: approximately 1 year).

[0287] Number of participants who experienced zero bleeding during prophylactic treatment with recombinant von Willebrand factor (rVWF) (time limit: approximately 1 year).

[0288] Number of injections of recombinant von Willebrand factor (rVWF) and ADVATE per month during prophylactic and on-demand treatment periods (duration: approximately 1 year).

[0289] Number of injections of recombinant von Willebrand factor (rVWF) and ADVATE per year during prophylactic and on-demand treatment periods (duration: approximately 1 year).

[0290] Total weight-adjusted consumption of recombinant von Willebrand factor (rVWF) and ADVATE per month during prophylactic and on-demand treatment periods (duration: approximately 1 year).

[0291] Total weight-adjusted consumption of recombinant von Willebrand factor (rVWF) and ADVATE per year during prophylactic and on-demand treatment periods (duration: approximately 1 year).

[0292] Incidence of thromboembolic events (time period: total study period of approximately 22 months).

[0293] Incidence of severe hypersensitivity reactions (time period: total study period of approximately 22 months).

[0294] Number of participants who developed neutralizing antibodies to recombinant von Willebrand factor (rVWF) and factor VIII (FVIII) (time period: total study period of approximately 22 months).

[0295] Number of participants who developed total binding antibodies to recombinant von Willebrand factor (rVWF) and factor VIII (FVIII) (time period: total study period of approximately 22 months).

[0296] Number of participants who developed antibodies against Chinese hamster ovary (CHO) proteins, mouse immunoglobulin G (IgG), and rFurin (timeline: total study period of approximately 22 months).

[0297] Pharmacokinetics - Incremental recovery (IR) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0298] Pharmacokinetics—Terminal half-life (T1 / 2) (Times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0299] Pharmacokinetics - Mean residence time (MRT) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0300] Pharmacokinetics - Area under the curve / dose (AUC / dose) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0301] Pharmacokinetics - Moment Area Under the Curve / Dose (AUMC / Dose) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0302] Pharmacokinetics - Steady state volume of distribution (Vss) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0303] Pharmacokinetics - Clearance (CL) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0304] Number of recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) infusions per spontaneous bleeding episode (BE) (time limit: total study period up to approximately 22 months).

[0305] Number of infusions of recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) per traumatic bleeding episode (BE) (maximum study duration: approximately 22 months).

[0306] Weight-adjusted recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) consumption per spontaneous bleeding episode (BE) (time period: up to approximately 22 months over the entire study period).

[0307] Weight-adjusted recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) consumption per traumatic bleeding episode (BE) (time period: up to approximately 22 months for the entire study period).

[0308] Evaluation of overall hemostatic effect at the time of bleeding disappearance (maximum duration: approximately 22 months for the entire study period). 4-point scale: excellent, good, fair, none.

[0309] Comparison of actual and predicted blood loss during surgery - when surgery is required (time limit: day 0 (at the completion of surgery)). Assessed by the operating surgeon at the completion of surgery).

[0310] Intraoperative hemostatic effect - if surgery is required (time limit: day 0 (at the completion of surgery)). Score on a scale of excellent, good, fair, or none - assessed by the operating surgeon at the completion of surgery.

[0311] For elective surgery: Global assessment of hemostatic efficacy (time limits: 24 hours after the last perioperative rVWF infusion, and on days 7 and 14) on a scale of excellent, good, fair, or none.

[0312] Daily weight-adjusted doses during and after surgery (time limit: Day 0 (day of surgery) to Day 14 after surgery). Daily weight-adjusted doses of rVWF with or without ADVATE during and after surgery.

[0313] Unmodified secondary endpoints Number of participants who experienced a reduction in annualized bleeding rate (ABR) for spontaneous (non-trauma-related) bleeding episodes during prophylactic treatment compared with their own ABR history during on-demand treatment (time limit: approximately 1 year).

[0314] Number of participants who experienced zero bleeding during prophylactic treatment with recombinant von Willebrand factor (rVWF) (time limit: approximately 1 year).

[0315] Number of injections of recombinant von Willebrand factor (rVWF) and ADVATE per month during on-demand treatment (duration: approximately 1 year).

[0316] Number of injections of recombinant von Willebrand factor (rVWF) and ADVATE per year during on-demand treatment (duration: approximately 1 year).

[0317] Total weight-adjusted consumption of recombinant von Willebrand factor (rVWF) and ADVATE per month of on-demand treatment (duration: approximately 1 year).

[0318] Total weight-adjusted consumption of recombinant von Willebrand factor (rVWF) and ADVATE per year of on-demand treatment (duration: approximately 1 year).

[0319] Incidence of thromboembolic events (time period: total study period of approximately 22 months).

[0320] Incidence of severe hypersensitivity reactions (time period: total study period of approximately 22 months).

[0321] Number of participants who developed neutralizing antibodies to recombinant von Willebrand factor (rVWF) and factor VIII (FVIII) (time period: total study period of approximately 22 months).

[0322] Number of participants who developed total binding antibodies to recombinant von Willebrand factor (rVWF) and factor VIII (FVIII) (time period: total study period of approximately 22 months).

[0323] Number of participants who developed antibodies against Chinese hamster ovary (CHO) proteins, mouse immunoglobulin G (IgG), and rFurin (timeline: total study period of approximately 22 months).

[0324] Pharmacokinetics - Incremental recovery (IR) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0325] Pharmacokinetics—Terminal half-life (T1 / 2) (Times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0326] Pharmacokinetics - Mean residence time (MRT) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0327] Pharmacokinetics - Area under the curve / dose (AUC / dose) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0328] Pharmacokinetics - Moment Area Under the Curve / Dose (AUMC / Dose) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0329] Pharmacokinetics - Steady state volume of distribution (Vss) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0330] Pharmacokinetics - Clearance (CL) (times: 30 minutes pre-infusion; and 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours post-infusion).

[0331] Number of recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) infusions per spontaneous bleeding episode (BE) (time limit: total study period up to approximately 22 months).

[0332] Number of infusions of recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) per traumatic bleeding episode (BE) (maximum study duration: approximately 22 months).

[0333] Weight-adjusted recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) consumption per spontaneous bleeding episode (BE) (time period: up to approximately 22 months over the entire study period).

[0334] Weight-adjusted recombinant von Willebrand factor (rVWF) and ADVATE (rFVIII) consumption per traumatic bleeding episode (BE) (time period: up to approximately 22 months for the entire study period).

[0335] Evaluation of overall hemostatic effect at the time of bleeding disappearance (maximum duration: approximately 22 months for the entire study period). 4-point scale: excellent, good, fair, none.

[0336] References TIFF2025128168000011.tif98156

[0337] While exemplary embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0338] The above examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above modes for carrying out the invention that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0339] All heading and section designations are used for clarity and reference only and should not be considered limiting in any way. For example, one skilled in the art will understand the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0340] All references cited in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0341] Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application should be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0342] Sequence information SEQUENCE LISTING <110> Takeda Pharmaceutical Company Limited <120> METHODS OF PROPHYLACTIC TREATMENT USING RECOMBINANT VWF (rVWF) <150> US 62 / 800,370 <151> 2019-02-01 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 8833 <212> DNA <213> Artificial Sequence <220> <223> prepro-VWF <400> 1 agctcacagc tattgtggtg ggaaagggag ggtggttggt ggatgtcaca gcttgggctt 60 tatctccccc agcagtgggg actccacagc ccctgggcta cataacagca agacagtccg 120 gagctgtagc agacctgatt gagcctttgc agcagctgag agcatggcct agggtgggcg 180 gcaccattgt ccagcagctg agtttcccag ggaccttgga gatagccgca gccctcattt 240 gcagggggaag atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt 300 gccagggacc ctttgtgcag aaagactcg cggcaggtca tccacggccc gatgcagcct 360 tttcggaagt gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg 420 cagttacctc ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca 480 gaatggcaag agagtgagcc tctccgtgta tcttgggggaa ttttttgaca tccatttgtt 540 tgtcaatggt accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg 600 gctgtatcta gaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt 660 ggccaggatc gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa 720 gacctgcggg ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga 780 agggaccttg acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga 840 acagtggtgt gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat 900 gcagaagggc ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg 960 ccaccctctg gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg 1020 tgctgggggg ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca 1080 ggagggaatg gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc 1140 tggtatggag tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat 1200 caatgaaatg tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct 1260 ggatgaaggc ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta 1320 ccctcccggc acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg 1380 gatctgcagc aatgaagaat gtccagggga gtgccttgtc acaggtcaat cacacttcaa 1440 gagctttgac aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga 1500 ttgccaggac cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga 1560 cgctgtgtgc acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa 1620 actgaagcat ggggcaggag ttgccatgga tggccaggac gtccagctcc ccctcctgaa 1680 aggtgacctc cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga 1740 cctgcagatg gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc 1800 cgggaagacc tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac 1860 cccctctggg ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg 1920 ggactgccag gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac 1980 caggttctcc gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg 2040 tgccgtcagc ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga 2100 cggccgcgag tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg 2160 cgtgcgcgtc gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt 2220 gtacctgcag tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga 2280 ggaatgcaat gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga 2340 gaggggggac tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca 2400 gccagaagac atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca 2460 ctgtaccatg agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct 2520 gtctcatcgc agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc 2580 cgctgacaac ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct 2640 ggagtgcatg agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca 2700 tgagaacaga tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc 2760 ccctggagaa acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa 2820 ctgcacagac catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac 2880 cttcgacggg ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta 2940 ctgcggcagt aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc 3000 ctcagtgaaa tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt 3060 tgacggggag gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga 3120 gtctggccgg tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca 3180 cctgagcatc tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg 3240 gaattttgat ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga 3300 ccctgtggac tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt 3360 gcctctggac tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga 3420 ttcctcctgt agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc 3480 cgagccatat ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg 3540 cgcctgcttc tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt 3600 ggtgacctgg aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga 3660 gaacgggtat gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg 3720 tcagcaccct gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg 3780 ccctccaggg aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc 3840 agtgtgtgag gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag 3900 tgaccctgag cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg 3960 ccaggagccg ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct 4020 gtatgtggag gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga 4080 cctggtcttc ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa 4140 ggcctttgtg gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc 4200 cgtggtggag taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc 4260 gtcagagctg cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac 4320 cagcgaggtc ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc 4380 ctcccgcatc accctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt 4440 tgtccgctac gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg 4500 gccccatgcc aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc 4560 cttcgtgctg agcagtgtgg atgagctgga gcaagcaaagg gacgagatcg ttagctacct 4620 ctgtgacctt gcccctgaag cccctcctcc tactctgccc cccgacatgg caaagtcac 4680 tgtgggcccg gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct 4740 ggatgtggcg ttcgtcctgg areatcgga caaaattggt gaagccgact tcaacaggag 4800 caaggagttc atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt 4860 cacggtgctg footctcct acatggtgac tgtggagtac cccttcagcg aggcacagtc 4920 caaaggggac atcctgcagc gggtgcgaga gatccgctac cagggcggca aaggaccaa 4980 cactgggctg gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg 5040 ggagcaggcg cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa 5100 gaggctgcct ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca 5160 ggagctggag aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct 5220 cccccgagag gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat 5280 ccccaccctc tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga 5340 tggctcctcc agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt 5400 catttcaaaa gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag 5460 catcaccacc attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct 5520 tgtggacgtc atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc 5580 tgtgcgatac ttgacttcag aaatgcatgg tgccaggccg ggagcctcaa aggcggtggt 5640 catcctggtc acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc 5700 caacagagtg acagtgttcc ctattggaat tggagatcgc tacgatgcag cccagctacg 5760 gatcttggca ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct 5820 ccctaccatg gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg gatttgttag 5880 gatttgcatg gatgaggatg ggaatgagaa gaggcccggg gacgtctgga ccttgccaga 5940 ccagtgccac accgtgactt gccagccaga tggccagacc ttgctgaaga gtcatcgggt 6000 caactgtgac cgggggctga ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga 6060 agagacctgt ggctgccgct ggacctgccc ctgcgtgtgc acaggcagct ccactcggca 6120 catcgtgacc tttgatgggc agaatttcaa gctgactggc agctgttctt atgtcctatt 6180 tcaaaacaag gagcaggacc tggaggtgat tctccataat ggtgcctgca gccctggagc 6240 aaggcagggc tgcatgaaat ccatcgaggt gaagcacagt gccctctccg tcgagctgca 6300 cagtgacatg gaggtgacgg tgaatgggag actggtctct gttccttacg tgggtgggaa 6360 catggaagtc aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca 6420 catcttcaca ttcactccac aaaacaatga gttccaactg cagctcagcc ccaagacttt 6480 tgcttcaaag acgtatggtc tgtgtgggat ctgtgatgag aacggagcca atgacttcat 6540 gctgagggat ggcacagtca ccacagactg gaaaacactt gttcaggaat ggactgtgca 6600 gcggccaggg cagacgtgcc agcccatcct ggaggagcag tgtcttgtcc ccgacagctc 6660 ccactgccag gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc 6720 cacattctat gccatctgcc agcaggacag ttgccaccag cacaagtgt gtgaggtgat 6780. cgcctcttat gcccacctct gtcggaccaa cggggtctgc gttgactgga ggacacctga 6840 tttctgtgct atgtcatgcc caccatctct ggtctacaac cactgtgagc atggctgtcc ccggcactgt gatggcaacg tgagctcctg tggggaccat ccctccgaag gctgtttctg ccctccagat aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg cattggtgag gatggagtcc agcaccagtt cctggaagcc tgggtcccgg accaccagcc 7080 ctgtcagatc tgcacatgcc tcagcgggcg gaaggtcaac tgcacaacgc agccctgccc cacggccaaa gctcccacgt gtggcctgtg tgaagtagcc cgcctccgcc agaatgcaga ccagtgctgc cccgagtatg agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt 7260 gcctcactgt gaacgtggcc tccagcccac actgaccac cctggcgagt gcagaccca 7320 cttcacctgc gcctgcagga aggaggagtg caaaagagtg tccccaccct cctgcccccc 7380 gcaccgtttg cccacccttc ggaagaccca gtgctgtgat gtgctggt gtgcctgcaa 7440 ctgtgtcaac tccacagtga gctgtcccct tgggtacttg gcctcaactg ccaccaatga 7500 ctgtggctgt accacaacca cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat 7560 ctaccctgtg ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga 7620 ggatgccgtg atgggcctcc gcgtggccca gtgctcccag aagccctgtg aggacagctg 7680 tcggtcgggc ttcacttacg ttctgcatga aggcgagtgc tgtggaaggt gcctgccatc 7740 tgcctgtgag gtggtgactg gctcaccgcg gggggactcc cagtcttcct ggaagagtgt 7800 cggctcccag tgggcctccc cggagaaccc ctgcctcatc aatgagtgtg tccgagtgaa 7860 ggaggaggtc tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg 7920 cccctcgggc tttcagctga gctgtaagac ctcagcgtgc tgcccaagct gtcgctgtga 7980 gcgcatggag gcctgcatgc tcaatggcac tgtcattggg cccgggaaga ctgtgatgat 8040 cgatgtgtgc acgacctgcc gctgcatggt gcaggtgggg gtcatctctg gattcaagct 8100 ggagtgcagg aagaccacct gcaacccctg ccccctgggt tacaaggaag aaaataacac 8160 aggtgaatgt tgtgggagat gttgcctac ggcttgcacc attcagctaa gaggaggaca 8220 gatcatgaca ctgaagcgtg atgagacgct ccaggatggc tgtgatactc acttctgcaa 8280 ggtcaatgag agaggagt acttctggga gaagagggtc acaggctgcc caccctttga 8340 tgaacacaag tgtctggctg agggaggtaa aattatgaaa attccaggca cctgctgtga 8400 cacatgtgag gagcctgagt gcaacgacat cactgccagg ctgcagtatg tcaggtggg 8460 aagctgtaag tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa 8520 agccatgtac tccattgaca tcaacgatgt gcaggaccag tgctcctgct gctctccgac 8580 acggacggag cccatgcagg tggccctgca ctgcaccaat ggctctgttg tgtaccatga 8640 ggttctcaat gccatggagt gcaatgctc ccccaggaag tgcagcaagt gaggctgctg 8700 cagctgcatg gtgcctgct gctgcctgcc ttggctgat ggccaggcca gagtgctgcc 8760 agtcctctgc atgttctgct cttgtgccct tctgagccca cataaaggc tgagctctta 8820 tcttgcaaaa ggc 8833 <210> 2 <211> 2783 <212> PRT <213> Artificial Sequence <220> <223> prepro-VWF <400> 2 Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Leu Ile Leu Pro Gly 1 5 10 15 Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr Ala Arg Cys 20 25 30 Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly Ser Met Tyr 35 40 45 Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly Cys Gln Lys 50 55 60 Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys Arg Val Ser 65 70 75 80 Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu Phe Val Asn 85 90 95 Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro Tyr Ala Ser 100 105 110 Lys Leu Glu Thr Glu Ala Gly Tyr Tyr Lys Leu Ser Gly Glu Ala Tyr 115 120 125 Gly Phe Val Ala Arg Ile Asp Gly Ser Gly Asn Phe Gln Val Leu Leu 130 135 140 Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly Leu Cys Gly Asn Phe Asn 145 150 155 160 Ile Phe Ala Glu Asp Asp Phe Met Thr Gln Glu Gly Thr Leu Thr Ser 165 170 175 Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala Leu Ser Ser Gly Glu Gln 180 185 190 Trp Cys Glu Arg Pro Ser Ser Ser Cys Asn Ile Ser Ser Gly Glu Met 195 200 205 Gln Lys Gly Leu Trp Glu Gln Cys Gln Leu Leu Lys Ser Thr Ser Val 210 215 220 Phe Ala Arg Cys His Pro Leu Val Asp Pro Glu Pro Phe Cys Glu Lys 225 230 235 240 Thr Leu Cys Glu Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu 245 250 255 Leu Glu Tyr Ala Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly 260 265 270 Trp Thr Asp His Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu 275 280 285 Tyr Arg Gln Cys Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His 290 295 300 Ile Asn Glu Met Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro 305 310 315 320 Glu Gly Gln Leu Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys 325 330 335 Pro Cys Val His Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser 340 345 350 Arg Asp Cys Asn Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser 355 360 365 Asn Glu Glu Cys Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe 370 375 380 Lys Ser Phe Asp Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr 385 390 395 400 Leu Leu Ala Arg Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu 405 410 415 Thr Val Gln Cys Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val 420 425 430 Thr Val Arg Leu Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His 435 440 445 Gly Ala Gly Val Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu 450 455 460 Lys Gly Asp Leu Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu 465 470 475 480 Ser Tyr Gly Glu Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu 485 490 495 Leu Val Lys Leu Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys 500 505 510 Gly Asn Tyr Asn Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly 515 520 525 Leu Ala Glu Pro Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His 530 535 540 Gly Asp Cys Gln Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu 545 550 555 560 Asn Pro Arg Met Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr 565 570 575 Ser Pro Thr Phe Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr 580 585 590 Leu Arg Asn Cys Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu 595 600 605 Cys Leu Cys Gly Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg 610 615 620 Val Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly 625 630 635 640 Gln Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser 645 650 655 Leu Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys 660 665 670 Phe Cys Pro Pro Met Asp Glu Arg Gly Asp Cys Val Pro Lys Ala Gln 675 680 685 Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp Ile Phe 690 695 700 Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met His Cys 705 710 715 720 Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val Leu Ser 725 730 735 Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg Pro Pro 740 745 750 Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu Gly Leu 755 760 765 Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met Ser Met 770 775 780 Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg His Glu 785 790 795 800 Asn Arg Cys Glu Arg Cys Pro Cys Phe His Gln Gly Lys Glu Tyr Ala 805 810 815 Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr Cys Val Cys Arg Asp 820 825 830 Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp Ala Thr Cys Ser Thr 835 840 845 Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe 850 855 860 Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp Tyr Cys Gly Ser Asn 865 870 875 880 Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys Gly Cys Ser His Pro 885 890 895 Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val Glu Gly Gly Glu 900 905 910 Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg Pro Met Lys Asp 915 920 925 Glu Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu 930 935 940 Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg His Leu Ser Ile Ser 945 950 955 960 Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly 965 970 975 Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln 980 985 990 Val Glu Glu Asp Pro Val Asp Phe Gly Asn Ser Trp Lys Val Ser Ser 995 1000 1005 Gln Cys Ala Asp Thr Arg Lys Val Pro Leu Asp Ser Ser Pro Ala 1010 1015 1020 Thr Cys His Asn Asn Ile Met Lys Gln Thr Met Val Asp Ser Ser 1025 1030 1035 Cys Arg Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn Lys Leu 1040 1045 1050 Val Asp Pro Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr Cys 1055 1060 1065 Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile 1070 1075 1080 Ala Ala Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr 1085 1090 1095 Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn 1100 1105 1110 Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys 1115 1120 1125 Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala 1130 1135 1140 Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 1145 1150 1155 Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu 1160 1165 1170 Asp Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly 1175 1180 1185 Lys Lys Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile 1190 1195 1200 Cys His Cys Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu 1205 1210 1215 Pro Gly Gly Leu Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro 1220 1225 1230 Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro Pro Leu His Asp 1235 1240 1245 Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly 1250 1255 1260 Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Ala Phe 1265 1270 1275 Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys Trp Val 1280 1285 1290 Arg Val Ala Val Val Glu Tyr His Asp Gly Ser His Ala Tyr Ile 1295 1300 1305 Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala 1310 1315 1320 Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu 1325 1330 1335 Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg 1340 1345 1350 Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met Ala Ser Gln Glu 1355 1360 1365 Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly Leu 1370 1375 1380 Lys Lys Lys Lys Val Ile Val Ile Pro Val Gly Ile Gly Pro His 1385 1390 1395 Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu 1400 1405 1410 Asn Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln 1415 1420 1425 Arg Asp Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala 1430 1435 1440 Pro Pro Pro Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly 1445 1450 1455 Pro Gly Leu Leu Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser 1460 1465 1470 Met Val Leu Asp Val Ala Phe Val Leu Glu Gly Ser Asp Lys Ile 1475 1480 1485 Gly Glu Ala Asp Phe Asn Arg Ser Lys Glu Phe Met Glu Glu Val 1490 1495 1500 Ile Gln Arg Met Asp Val Gly Gln Asp Ser Ile His Val Thr Val 1505 1510 1515 Leu Gln Tyr Ser Tyr Met Val Thr Val Glu Tyr Pro Phe Ser Glu 1520 1525 1530 Ala Gln Ser Lys Gly Asp Ile Leu Gln Arg Val Arg Glu Ile Arg 1535 1540 1545 Tyr Gln Gly Gly Asn Arg Thr Asn Thr Gly Leu Ala Leu Arg Tyr 1550 1555 1560 Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly Asp Arg Glu Gln 1565 1570 1575 Ala Pro Asn Leu Val Tyr Met Val Thr Gly Asn Pro Ala Ser Asp 1580 1585 1590 Glu Ile Lys Arg Leu Pro Gly Asp Ile Gln Val Val Pro Ile Gly 1595 1600 1605 Val Gly Pro Asn Ala Asn Val Gln Glu Leu Glu Arg Ile Gly Trp 1610 1615 1620 Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg 1625 1630 1635 Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys Ser Gly Glu Gly 1640 1645 1650 Leu Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro 1655 1660 1665 Leu Asp Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala 1670 1675 1680 Ser Tyr Phe Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile Ser 1685 1690 1695 Lys Ala Asn Ile Gly Pro Arg Leu Thr Gln Val Ser Val Leu Gln 1700 1705 1710 Tyr Gly Ser Ile Thr Thr Ile Asp Val Pro Trp Asn Val Val Pro 1715 1720 1725 Glu Lys Ala His Leu Leu Ser Leu Val Asp Val Met Gln Arg Glu 1730 1735 1740 Gly Gly Pro Ser Gln Ile Gly Asp Ala Leu Gly Phe Ala Val Arg 1745 1750 1755 Tyr Leu Thr Ser Glu Met His Gly Ala Arg Pro Gly Ala Ser Lys 1760 1765 1770 Ala Val Val Ile Leu Val Thr Asp Val Ser Val Asp Ser Val Asp 1775 1780 1785 Ala Ala Ala Asp Ala Ala Arg Ser Asn Arg Val Thr Val Phe Pro 1790 1795 1800 Ile Gly Ile Gly Asp Arg Tyr Asp Ala Ala Gln Leu Arg Ile Leu 1805 1810 1815 Ala Gly Pro Ala Gly Asp Ser Asn Val Val Lys Leu Gln Arg Ile 1820 1825 1830 Glu Asp Leu Pro Thr Met Val Thr Leu Gly Asn Ser Phe Leu His 1835 1840 1845 Lys Leu Cys Ser Gly Phe Val Arg Ile Cys Met Asp Glu Asp Gly 1850 1855 1860 Asn Glu Lys Arg Pro Gly Asp Val Trp Thr Leu Pro Asp Gln Cys 1865 1870 1875 His Thr Val Thr Cys Gln Pro Asp Gly Gln Thr Leu Leu Lys Ser 1880 1885 1890 His Arg Val Asn Cys Asp Arg Gly Leu Arg Pro Ser Cys Pro Asn 1895 1900 1905 Ser Gln Ser Pro Val Lys Val Glu Glu Thr Cys Gly Cys Arg Trp 1910 1915 1920 Thr Cys Pro Cys Val Cys Thr Gly Ser Ser Thr Arg His Ile Val 1925 1930 1935 Thr Phe Asp Gly Gln Asn Phe Lys Leu Thr Gly Ser Cys Ser Tyr 1940 1945 1950 Val Leu Phe Gln Asn Lys Glu Gln Asp Leu Glu Val Ile Leu His 1955 1960 1965 Asn Gly Ala Cys Ser Pro Gly Ala Arg Gln Gly Cys Met Lys Ser 1970 1975 1980 Ile Glu Val Lys His Ser Ala Leu Ser Val Glu Leu His Ser Asp 1985 1990 1995 Met Glu Val Thr Val Asn Gly Arg Leu Val Ser Val Pro Tyr Val 2000 2005 2010 Gly Gly Asn Met Glu Val Asn Val Tyr Gly Ala Ile Met His Glu 2015 2020 2025 Val Arg Phe Asn His Leu Gly His Ile Phe Thr Phe Thr Pro Gln 2030 2035 2040 Asn Asn Glu Phe Gln Leu Gln Leu Ser Pro Lys Thr Phe Ala Ser 2045 2050 2055 Lys Thr Tyr Gly Leu Cys Gly Ile Cys Asp Glu Asn Gly Ala Asn 2060 2065 2070 Asp Phe Met Leu Arg Asp Gly Thr Val Thr Thr Asp Trp Lys Thr 2075 2080 2085 Leu Val Gln Glu Trp Thr Val Gln Arg Pro Gly Gln Thr Cys Gln 2090 2095 2100 Pro Glu Gln Cys Leu Val Pro Asp Ser Ser His Cys Gln Val Leu 2105 2110 2115 Leu Leu Pro Leu Phe Ala Glu Cys His Lys Val Leu Ala Pro Ala 2120 2125 2130 Thr Phe Tyr Ala Ile Cys Gln Gln Asp Ser Cys His Gln Glu Gln 2135 2140 2145 Val Cys Glu Val Ile Ala Ser Tyr Ala His Leu Cys Arg Thr Asn 2150 2155 2160 Gly Val Cys Val Asp Trp Arg Thr Pro Asp Phe Cys Ala Met Ser 2165 2170 2175 Cys Pro Pro Ser Leu Val Tyr Asn His Cys Glu His Gly Cys Pro 2180 2185 2190 Arg His Cys Asp Gly Asn Val Ser Ser Cys Gly Asp His Pro Ser 2195 2200 2205 Glu Gly Cys Phe Cys Pro Pro Asp Lys Val Met Leu Glu Gly Ser 2210 2215 2220 Cys Val Pro Glu Glu Ala Cys Thr Gln Cys Ile Gly Glu Asp Gly 2225 2230 2235 Val Gln His Gln Phe Leu Glu Ala Trp Val Pro Asp His Gln Pro 2240 2245 2250 Cys Gln Ile Cys Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr 2255 2260 2265 Thr Gln Pro Cys Pro Thr Ala Lys Ala Pro Thr Cys Gly Leu Cys 2270 2275 2280 Glu Val Ala Arg Leu Arg Gln Asn Ala Asp Gln Cys Cys Pro Glu 2285 2290 2295 Tyr Glu Cys Val Cys Asp Pro Val Ser Cys Asp Leu Pro Pro Val 2300 2305 2310 Pro His Cys Glu Arg Gly Leu Gln Pro Thr Leu Thr Asn Pro Gly 2315 2320 2325 Glu Cys Arg Pro Asn Phe Thr Cys Ala Cys Arg Lys Glu Glu Cys 2330 2335 2340 Lys Arg Val Ser Pro Pro Ser Cys Pro Pro His Arg Leu Pro Thr 2345 2350 2355 Leu Arg Lys Thr Gln Cys Cys Asp Glu Tyr Glu Cys Ala Cys Asn 2360 2365 2370 Cys Val Asn Ser Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser 2375 2380 2385 Thr Ala Thr Asn Asp Cys Gly Cys Thr Thr Thr Thr Cys Leu Pro 2390 2395 2400 Asp Lys Val Cys Val His Arg Ser Thr Ile Tyr Pro Val Gly Gln 2405 2410 2415 Phe Trp Glu Glu Gly Cys Asp Val Cys Thr Cys Thr Asp Met Glu 2420 2425 2430 Asp Ala Val Met Gly Leu Arg Val Ala Gln Cys Ser Gln Lys Pro 2435 2440 2445 Cys Glu Asp Ser Cys Arg Ser Gly Phe Thr Tyr Val Leu His Glu 2450 2455 2460 Gly Glu Cys Cys Gly Arg Cys Leu Pro Ser Ala Cys Glu Val Val 2465 2470 2475 Thr Gly Ser Pro Arg Gly Asp Ser Gln Ser Ser Trp Lys Ser Val 2480 2485 2490 Gly Ser Gln Trp Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg 2495 2500 2505 Val Lys Glu Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro 2510 2515 2520 Gln Leu Glu Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys 2525 2530 2535 Lys Thr Ser Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu 2540 2545 2550 Ala Cys Met Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val 2555 2560 2565 Met Ile Asp Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly 2570 2575 2580 Val Ile Ser Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn 2585 2590 2595 Pro Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys 2600 2605 2610 Cys Gly Arg Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly 2615 2620 2625 Gly Gln Ile Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly 2630 2635 2640 Cys Asp Thr His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe 2645 2650 2655 Trp Glu Lys Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys 2660 2665 2670 Cys Leu Ala Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys 2675 2680 2685 Cys Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg 2690 2695 2700 Leu Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val 2705 2710 2715 Asp Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr 2720 2725 2730 Ser Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser 2735 2740 2745 Pro Thr Arg Thr Glu Pro Met Gln His Cys Thr Asn Gly Ser Val 2750 2755 2760 Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys Ser Pro 2765 2770 2775 Arg Lys Cys Ser Lys 2780 <210> 3 <211> 2050 <212> PRT <213> Artificial Sequence <220> <223> mature VWF <400> 3 Ser Leu Ser Cys Arg Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp 1 5 10 15 Asn Leu Arg Ala Glu Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr 20 25 30 Asp Leu Glu Cys Met Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro 35 40 45 Pro Gly Met Val Arg His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys 50 55 60 Pro Cys Phe His Gln Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys 65 70 75 80 Ile Gly Cys Asn Thr Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr 85 90 95 Asp His Val Cys Asp Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr 100 105 110 Leu Thr Phe Asp Gly Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr 115 120 125 Val Leu Val Gln Asp Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile 130 135 140 Leu Val Gly Asn Lys Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys 145 150 155 160 Arg Val Thr Ile Leu Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly 165 170 175 Glu Val Asn Val Lys Arg Pro Met Lys Asp Glu Thr His Phe Glu Val 180 185 190 Val Glu Ser Gly Arg Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser 195 200 205 Val Val Trp Asp Arg His Leu Ser Ile Ser Val Val Leu Lys Gln Thr 210 215 220 Tyr Gln Glu Lys Val Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln 225 230 235 240 Asn Asn Asp Leu Thr Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val 245 250 255 Asp Phe Gly Asn Ser Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg 260 265 270 Lys Val Pro Leu Asp Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met 275 280 285 Lys Gln Thr Met Val Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val 290 295 300 Phe Gln Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 305 310 315 320 Cys Ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys 325 330 335 Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gln His Gly 340 345 350 Lys Val Val Thr Trp Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu 355 360 365 Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn 370 375 380 Ser Cys Ala Pro Ala Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu 385 390 395 400 Ala Cys Pro Val Gln Cys Val Glu Gly Cys His Ala His Cys Pro Pro 405 410 415 Gly Lys Ile Leu Asp Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp 420 425 430 Cys Pro Val Cys Glu Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys 435 440 445 Val Thr Leu Asn Pro Ser Asp Pro Glu His Cys Gln Ile Cys His Cys 450 455 460 Asp Val Val Asn Leu Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu 465 470 475 480 Val Val Pro Pro Thr Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val 485 490 495 Glu Asp Ile Ser Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu 500 505 510 Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala 515 520 525 Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu 530 535 540 Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 545 550 555 560 Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu 565 570 575 Leu Arg Arg Ile Ala Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala 580 585 590 Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys 595 600 605 Ile Asp Arg Pro Glu Ala Ser Arg Ile Thr Leu Leu Leu Met Ala Ser 610 615 620 Gln Glu Pro Gln Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly 625 630 635 640 Leu Light Light Light Light Val Ile Val Ile Pro Val Gly Ile Gly Pro His 645 650 655 Ala Asn Leu Lys Gln Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn 660 665 670 Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp 675 680 685 Glu Ile Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro 690 695 700 Thr Leu Pro Pro Asp Met Ala Gln Val Thr Val Gly Pro Gly Leu Leu 705 710 715 720 Gly Val Ser Thr Leu Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val 725 730 735 Ala Phe Val Leu Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn 740 745 750 Arg Ser Lys Glu Phe Met Glu Glu Val Ile Gln Arg Met Asp Val Gly 755 760 765 Gln Asp Ser Ile His Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr 770 775 780 Val Glu Tyr Pro Phe Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln 785 790 795 800 Arg Val Arg Glu Ile Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr Gly 805 810 815 Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly 820 825 830 Asp Arg Glu Gln Ala Pro Asn Leu Val Tyr Met Val Thr Gly Asn Pro 835 840 845 Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp Ile Gln Val Val Pro 850 855 860 Ile Gly Val Gly Pro Asn Ala Asn Val Gln Glu Leu Glu Arg Ile Gly 865 870 875 880 Trp Pro Asn Ala Pro Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg 885 890 895 Glu Ala Pro Asp Leu Val Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu 900 905 910 Gln Ile Pro Thr Leu Ser Pro Ala Pro Asp Cys Ser Gln Pro Leu Asp 915 920 925 Val Ile Leu Leu Leu Asp Gly Ser Ser Ser Phe Pro Ala Ser Tyr Phe 930 935 940 Asp Glu Met Lys Ser Phe Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile 945 950 955 960 Gly Pro Arg Leu Thr Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr 965 970 975 Thr Ile Asp Val Pro Trp Asn Val Val Pro Glu Lys Ala His Leu Leu 980 985 990 Ser Leu Val Asp Val Met Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly 995 1000 1005 Asp Ala Leu Gly Phe Ala Val Arg Tyr Leu Thr Ser Glu Met His 1010 1015 1020 Gly Ala Arg Pro Gly Ala Ser Lys Ala Val Val Ile Leu Val Thr 1025 1030 1035 Asp Val Ser Val Asp Ser Val Asp Ala Ala Ala Asp Ala Ala Arg 1040 1045 1050 Ser Asn Arg Val Thr Val Phe Pro Ile Gly Ile Gly Asp Arg Tyr 1055 1060 1065 Asp Ala Ala Gln Leu Arg Ile Leu Ala Gly Pro Ala Gly Asp Ser 1070 1075 1080 Asn Val Val Lys Leu Gln Arg Ile Glu Asp Leu Pro Thr Met Val 1085 1090 1095 Thr Leu Gly Asn Ser Phe Leu His Lys Leu Cys Ser Gly Phe Val 1100 1105 1110 Arg Ile Cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp 1115 1120 1125 Val Trp Thr Leu Pro Asp Gln Cys His Thr Val Thr Cys Gln Pro 1130 1135 1140 Asp Gly Gln Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg 1145 1150 1155 Gly Leu Arg Pro Ser Cys Pro Asn Ser Gln Ser Pro Val Lys Val 1160 1165 1170 Glu Glu Thr Cys Gly Cys Arg Trp Thr Cys Pro Cys Val Cys Thr 1175 1180 1185 Gly Ser Ser Thr Arg His Ile Val Thr Phe Asp Gly Gln Asn Phe 1190 1195 1200 Lys Leu Thr Gly Ser Cys Ser Tyr Val Leu Phe Gln Asn Lys Glu 1205 1210 1215 Gln Asp Leu Glu Val Ile Leu His Asn Gly Ala Cys Ser Pro Gly 1220 1225 1230 Ala Arg Gln Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala 1235 1240 1245 Leu Ser Val Glu Leu His Ser Asp Met Glu Val Thr Val Asn Gly 1250 1255 1260 Arg Leu Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu Val Asn 1265 1270 1275 Val Tyr Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly 1280 1285 1290 His Ile Phe Thr Phe Thr Pro Gln Asn Asn Glu Phe Gln Leu Gln 1295 1300 1305 Leu Ser Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly 1310 1315 1320 Ile Cys Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly 1325 1330 1335 Thr Val Thr Thr Asp Trp Lys Thr Leu Val Gln Glu Trp Thr Val 1340 1345 1350 Gln Arg Pro Gly Gln Thr Cys Gln Pro Ile Leu Glu Glu Gln Cys 1355 1360 1365 Leu Val Pro Asp Ser Ser His Cys Gln Val Leu Leu Leu Pro Leu 1370 1375 1380 Phe Ala Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala 1385 1390 1395 Ile Cys Gln Gln Asp Ser Cys His Gln Glu Gln Val Cys Glu Val 1400 1405 1410 Ile Ala Ser Tyr Ala His Leu Cys Arg Thr Asn Gly Val Cys Val 1415 1420 1425 Asp Trp Arg Thr Pro Asp Phe Cys Ala Met Ser Cys Pro Pro Ser 1430 1435 1440 Leu Val Tyr Asn His Cys Glu His Gly Cys Pro Arg His Cys Asp 1445 1450 1455 Gly Asn Val Ser Ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe 1460 1465 1470 Cys Pro Pro Asp Lys Val Met Leu Glu Gly Ser Cys Val Pro Glu 1475 1480 1485 Glu Ala Cys Thr Gln Cys Ile Gly Glu Asp Gly Val Gln His Gln 1490 1495 1500 Phe Leu Glu Ala Trp Val Pro Asp His Gln Pro Cys Gln Ile Cys 1505 1510 1515 Thr Cys Leu Ser Gly Arg Lys Val Asn Cys Thr Thr Gln Pro Cys 1520 1525 1530 Pro Thr Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val Ala Arg 1535 1540 1545 Leu Arg Gln Asn Ala Asp Gln Cys Cys Pro Glu Tyr Glu Cys Val 1550 1555 1560 Cys Asp Pro Val Ser Cys Asp Leu Pro Pro Val Pro His Cys Glu 1565 1570 1575 Arg Gly Leu Gln Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro 1580 1585 1590 Asn Phe Thr Cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val Ser 1595 1600 1605 Pro Pro Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr 1610 1615 1620 Gln Cys Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys Val Asn Ser 1625 1630 1635 Thr Val Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn 1640 1645 1650 Asp Cys Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys Val Cys 1655 1660 1665 Val His Arg Ser Thr Ile Tyr Pro Val Gly Gln Phe Trp Glu Glu 1670 1675 1680 Gly Cys Asp Val Cys Thr Cys Thr Asp Met Glu Asp Ala Val Met 1685 1690 1695 Gly Leu Arg Val Ala Gln Cys Ser Gln Lys Pro Cys Glu Asp Ser 1700 1705 1710 Cys Arg Ser Gly Phe Thr Tyr Val Leu His Glu Gly Glu Cys Cys 1715 1720 1725 Gly Arg Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro 1730 1735 1740 Arg Gly Asp Ser Gln Ser Ser Trp Lys Ser Val Gly Ser Gln Trp 1745 1750 1755 Ala Ser Pro Glu Asn Pro Cys Leu Ile Asn Glu Cys Val Arg Val 1760 1765 1770 Lys Glu Glu Val Phe Ile Gln Gln Arg Asn Val Ser Cys Pro Gln 1775 1780 1785 Leu Glu Val Pro Val Cys Pro Ser Gly Phe Gln Leu Ser Cys Lys 1790 1795 1800 Thr Ser Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala 1805 1810 1815 Cys Met Leu Asn Gly Thr Val Ile Gly Pro Gly Lys Thr Val Met 1820 1825 1830 Ile Asp Val Cys Thr Thr Cys Arg Cys Met Val Gln Val Gly Val 1835 1840 1845 Ile Ser Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro 1850 1855 1860 Cys Pro Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys 1865 1870 1875 Gly Arg Cys Leu Pro Thr Ala Cys Thr Ile Gln Leu Arg Gly Gly 1880 1885 1890 Gln Ile Met Thr Leu Lys Arg Asp Glu Thr Leu Gln Asp Gly Cys 1895 1900 1905 Asp Thr His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp 1910 1915 1920 Glu Lys Arg Val Thr Gly Cys Pro Pro Phe Asp Glu His Lys Cys 1925 1930 1935 Leu Ala Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr Cys Cys 1940 1945 1950 Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg Leu 1955 1960 1965 Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp 1970 1975 1980 Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser 1985 1990 1995 Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro 2000 2005 2010 Thr Arg Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn Gly 2015 2020 2025 Ser Val Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys 2030 2035 2040 Ser Pro Arg Lys Cys Ser Lys 2045 2050

Claims

1. 1. A method for prophylactically treating spontaneous bleeding episodes in a subject with severe von Willebrand disease (VWD), comprising administering to the subject at least one dose of recombinant von Willebrand factor (rVWF) in the range of at least about 40 IU / kg to about 80 IU / kg twice weekly, thereby reducing the frequency and / or duration of spontaneous bleeding episodes.

2. 10. The method of claim 1, wherein at least one dose of rVWF ranges from at least about 50 IU / kg to about 80 IU / kg.

3. 3. The method of claim 1 or 2, wherein the subject has a baseline VWF:ristocetin cofactor activity (VWF:RCo) in the range of less than or equal to 20 IU / dL or has been diagnosed with type 1 VWD.

4. 3. The method of claim 1 or 2, wherein the subject has been diagnosed with type 2A, type 2B, or type 2M VWD.

5. 3. The method of claim 1 or 2, wherein the subject has a VWF:antigen amount (VWF:Ag) in the range of 3 IU / dL or greater or has been diagnosed with type 3 VWD.

6. The method of any one of claims 1 to 5, wherein the subject has received prophylactic treatment with plasma-derived VWF (pdVWF) within the past 12 months prior to the first administration of rVWF.

7. 7. The method of any one of claims 1 to 6, wherein the subject has experienced at least three spontaneous bleeding episodes within the past 12 months.

8. The method of any one of claims 1 to 7, wherein the administration occurs every 3 to 4 days.

9. 9. The method of any one of claims 1 to 8, wherein the administration occurs on days 1 and 5, days 2 and 6, or days 3 and 7 of a seven day period.

10. 10. The method of any one of claims 1 to 9, wherein the administration occurs at least every 24 hours, every 36 hours, every 48 hours, every 72 hours, or every 84 hours.

11. The method of any one of claims 1 to 10, wherein the administration occurs at least every 72 hours.

12. The method of any one of claims 1 to 11, further comprising administering to the subject at least one dose of recombinant factor VIII (rFVIII).

13. 13. The method of claim 12, wherein said administration of at least one dose of rFVIII is administered simultaneously or sequentially with at least one dose of rVWF.

14. 10. The method of any one of the preceding claims, wherein the subject resumes the preventative treatment after undergoing elective surgery or oral surgery.

15. 15. The method of claim 14, wherein if the elective surgery is minor or oral surgery and the subject has a FVIII activity (FVIII:C) of at least 0.4 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery.

16. 15. The method of claim 14, wherein if the elective surgery is major surgery and the subject has a FVIII activity (FVIII:C) of at least 0.8 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery.

17. 17. The method of any one of claims 1 to 16, wherein the prophylactic treatment effect is demonstrated by a ≥ 25% reduction in the annualized bleeding rate (ABR) of spontaneous bleeding episodes during the rVWF prophylaxis period compared to the ABR before treatment.

18. 17. The method of any one of claims 1 to 16, wherein the prophylactic treatment effect is indicated by a reduction of ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 45%, ≥ 50%, ≥ 55%, ≥ 60%, ≥ 65%, ≥ 70%, ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, or ≥ 95% in the annualized bleeding rate (ABR) of spontaneous bleeding episodes during the rVWF prophylactic administration period relative to the ABR before treatment.

19. The method according to any one of claims 1 to 18, wherein the preventive treatment effect is measured by examining the activity of vWF:RCo and / or FVIII in samples obtained from the subject before and after the preventive treatment with rVWF.

20. The method according to any one of claims 1 to 19, wherein the preventive treatment effect is measured by examining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability in samples obtained from the subject before and after the preventive treatment with rVWF.

21. The method of claim 19 or 20, wherein the samples for examining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability are obtained 15 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 28 hours, 32 hours, 48 ​​hours, 72 hours, or 96 hours after prophylactic treatment with rVWF.

22. 22. The method of any one of claims 19 to 21, wherein the sample for determining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability is obtained 25 to 31 days after prophylactic treatment with rVWF.

23. 23. The method of any one of claims 1 to 22, wherein the preventive therapeutic effect is determined after or during a bleeding episode, and wherein samples for examining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability are obtained after the bleeding episode, and further wherein the samples are obtained before rVWF administration, 2 hours after rVWF administration, and every 12 to 24 hours thereafter until the end of the bleeding episode.

24. The method of any one of claims 1 to 23, wherein the prophylactic therapeutic effect is demonstrated by an improvement in the activity levels of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability after prophylactic treatment with rVWF compared to the levels before prophylactic treatment with rVWF.

25. A method for prophylactically treating spontaneous bleeding episodes in a subject with severe von Willebrand disease (VWD), comprising administering to the subject a weekly dose of recombinant von Willebrand factor (rVWF) substantially equivalent to a corresponding weekly dose of plasma-derived VWF (pdVWF) previously administered to the subject, thereby reducing the frequency and / or duration of spontaneous bleeding episodes.

26. 26. The method of claim 25, wherein the weekly dose of rVWF is about 10% less than the corresponding weekly dose of pdVWF.

27. 26. The method of claim 25, wherein the weekly dose of rVWF is about 10% higher than the corresponding weekly dose of pdVWF.

28. The method of any one of claims 25 to 27, wherein the weekly dose of rVWF is two separate injections administered on separate days.

29. The method of any one of claims 25 to 27, wherein the weekly dose of rVWF is three separate injections administered on separate days.

30. The method according to any one of claims 25 to 27, wherein the weekly dose of rVWF is a single injection.

31. The method of any one of claims 28 to 30, wherein each individual injection contains up to 80 IU / kg of rVWF.

32. The method of any one of claims 28 to 31, wherein each individual injection comprises 80 IU / kg of rVWF.

33. The method of any one of claims 28 to 31, wherein each individual injection comprises 50 IU / kg of rVWF.

34. 34. The method of any one of claims 25 to 33, wherein the subject has a baseline VWF:ristocetin cofactor activity (VWF:RCo) in the range of less than or equal to 20 IU / dL or has been diagnosed with type 1 VWD.

35. 35. The method of any one of claims 25 to 34, wherein the subject has been diagnosed with type 2A, type 2B, or type 2M VWD.

36. 36. The method of any one of claims 25 to 35, wherein the subject has a VWF:antigen load (VWF:Ag) in the range of 3 IU / dL or greater or has been diagnosed with type 3 VWD.

37. 37. The method of any one of claims 25 to 36, wherein the subject has been receiving prophylactic treatment for pdVWF for at least 12 months.

38. 38. The method of any one of claims 28 and 31-37, wherein the two separate infusions are administered on days 1 and 5, or days 2 and 6, or days 3 and 7 within a seven day period.

39. 38. The method of any one of claims 27 and 31-37, wherein the three separate infusions are administered on days 1, 3 and 6 within a seven day period.

40. 38. The method of any one of claims 25 to 37, wherein the administration occurs at least every 24 hours, every 36 hours, every 48 hours, every 72 hours, or every 84 hours.

41. 38. The method of any one of claims 25 to 37, wherein the administration occurs at least every 72 hours.

42. 42. The method of any one of claims 25 to 41, further comprising administering to the subject at least one dose of recombinant factor VIII (rFVIII).

43. 43. The method of claim 42, wherein said administration of at least one dose of rFVIII is administered simultaneously or sequentially with a weekly dose of rVWF.

44. 44. The method of any one of claims 25 to 43, wherein the preventative treatment is resumed after the subject has undergone elective surgery or oral surgery.

45. 45. The method of claim 44, wherein if the elective surgery is minor or oral surgery and the subject has a FVIII activity (FVIII:C) of at least 0.4 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery.

46. 45. The method of claim 44, wherein if the elective surgery is major surgery and the subject has a FVIII activity (FVIII:C) of at least 0.8 IU / mL or greater, the subject is administered rVWF without rFVIII prior to surgery.

47. 44. The method of any one of claims 25 to 43, wherein the prophylactic treatment effect is demonstrated by a ≥ 25% reduction in the annualized bleeding rate (ABR) of spontaneous bleeding episodes during the rVWF prophylactic administration period compared to the ABR before treatment.

48. 48. The method of any one of claims 25 to 47, wherein the prophylactic treatment effect is indicated by a reduction of ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 45%, ≥ 50%, ≥ 55%, ≥ 60%, ≥ 65%, ≥ 70%, ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, or ≥ 95% in the annualized bleeding rate (ABR) of spontaneous bleeding episodes during the rVWF prophylactic administration period relative to the ABR before treatment.

49. The method according to any one of claims 25 to 48, wherein the preventive treatment effect is measured by examining the activity of vWF:RCo and / or FVIII in samples obtained from the subject before and after the preventive treatment with rVWF.

50. The method of any one of claims 25 to 49, wherein the preventive treatment effect is measured by examining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability in samples obtained from the subject before and after preventive treatment with rVWF.

51. The method of claim 49 or 50, wherein the samples for examining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability are obtained 15 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 28 hours, 32 hours, 48 ​​hours, 72 hours, or 96 hours after prophylactic treatment with rVWF.

52. 52. The method of any one of claims 49 to 51, wherein the sample for determining the activity of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability is obtained 25 to 31 days after prophylactic treatment with rVWF.

53. 53. The method of any one of claims 25 to 52, wherein the preventive therapeutic effect is determined after or during a bleeding episode, and wherein samples for examining FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding activity are obtained after the bleeding episode, and further wherein the samples are obtained before rVWF administration, 2 hours after rVWF administration, and every 12 to 24 hours thereafter until the end of the bleeding episode.

54. The method of any one of claims 25 to 53, wherein the prophylactic therapeutic effect is demonstrated by an improvement in the activity levels of FVIII, FVIII:C, VWF:RCo, VWF:Ag, and / or VWF collagen binding ability after prophylactic treatment with rVWF compared to the levels before prophylactic treatment with rVWF.

55. 10. The method of any one of the preceding claims, wherein the spontaneous bleeding comprises any one selected from the group consisting of hemarthrosis, epistaxis, muscle bleeding, oral bleeding, and gastrointestinal bleeding.

56. 10. The method of any one of the preceding claims, wherein the subject has not been diagnosed with type 2N VWD or pseudo-VWD.

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