Methods of treating blood disorders

Abstract

Provided herein are antibodies, compositions, and methods of use thereof. The disclosed antibodies bind to von Willebrand factor in blood plasma of a subject. Such antibodies are useful for methods of treatment, such methods of treating one or more blood disorders characterized by excessive bleeding. Also provided herein are methods of treating blood disorders by administering a combination of an anti-VWF antibody and von Willebrand Factor.

Description

METHODS OF TREATING BLOOD DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Application Nos. 63 / 760,613, filed February 19, 2025; 63 / 881,170, filed September 12, 2025; and 63 / 955,870, filed January 7, 2026, each of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Von Willebrand factor (VWF) plays a central role in primary hemostasis by mediating platelet adhesion and aggregation at sites of vascular injury. Therapeutic antibodies that bind VWF can modulate this activity and are therefore useful in preventing or reducing excessive bleeding in subjects at risk. However, the hemostatic system is highly sensitive to changes in VWF function, and even modest alterations in VWF activity can significantly affect bleeding outcomes. As a result, the clinical performance of anti-VWF antibodies is strongly dependent on the dosing strategy used.

[0003] Determining an appropriate dose of an anti-VWF antibody presents substantial challenges. Underdosing may result in insufficient modulation of VWF activity, leaving the subject inadequately protected and at continued risk of excessive or uncontrolled bleeding. In contrast, overdosing may excessively inhibit normal hemostatic function, increasing the risk of prolonged bleeding, delayed wound healing, or bleeding at sites unrelated to the original risk event. These risks may be further compounded by inter-subject variability in VWF levels, antibody pharmacokinetics, and clinical context. Accordingly, careful dosing strategies are critical to achieving a balance between effective prevention of excessive bleeding and maintenance of safe, functional hemostasis.BRIEF SUMMARY

[0004] The present disclosure provides a method of treating a bleeding disorder in a subject in need thereof, comprising administering to the subject a flat dose of a pharmaceutical composition comprising an antibody or antigen binding site thereof that specifically binds to anepitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region (“anti-VWF antibody), wherein the flat dose is about 10 mg to about 300 mg of the anti-VWF antibody.

[0005] In some aspects, the flat dose is about 20 mg to 100 mg of the anti-VWF antibody. In some aspects, the flat dose is about 20 mg of the anti-VWF antibody. In some aspects, the flat dose is about 30 mg of the anti-VWF antibody. In some aspects, the flat dose is about 50 mg of the anti-VWF antibody. In some aspects, the flat dose is about 100 mg of the anti-VWF antibody. In some aspects, the flat dose is about 150 mg of the anti-VWf antibody.

[0006] The present disclosure also provides a method of treating a bleeding disorder in a subject in need thereof, comprising administering to the subject a weight-based dose of a pharmaceutical composition comprising an antibody or antigen binding portion thereof that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region (“anti- VWF antibody), wherein the weight-based dose is about 0.1 mg / kg to about 6 mg / kg of the anti-VWF antibody.

[0007] In some aspects, the bleeding disorder comprises von Willebrand disease (VWD), Heydes syndrome, hemophilia A, disorders of platelet function, connective tissue disorders, or any combination thereof. In some aspects, the bleeding disorder comprises Von Willebrand disease type 1, type 2 A, type 2B, type 2M, type 2N, or type 3. In some aspects, the bleeding disorder comprises congenital Von Willebrand disease (cVWD) or acquired Von Willebrand disease (a VWD).

[0008] In some aspects, the bleeding disorder in the subject comprises symptoms of epistaxis, cutaneous bleeding, bleeding from minor wounds, oral-cavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, central nervous system (CNS) bleeding, or any combination thereof.

[0009] In some aspects of the disclosure, the pharmaceutical composition is administered subcutaneously.

[0010] In some aspects of the disclosure, the pharmaceutical composition is administered repeatedly at a regular interval. In some aspects, the regular interval is about once weekly. In some aspects, the regular interval is about once every two weeks. In some aspects, the regular interval is about once every three weeks. In some aspects, the regular interval is about once every four weeks. In some aspects, the regular interval is about once a month. In some aspects, the regular interval is about once every two months.

[0011] The present disclosure also provides a method of treating one or more symptoms of Von Willebrand disease (VWD) in a subject in need thereof, comprising administering to the subject a dose of a pharmaceutical composition comprising an antibody or an antigen-binding site that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region, wherein the one more symptoms comprise epistaxis, cutaneous bleeding, bleeding from minor wounds, oral-cavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, central nervous system (CNS) bleeding, or any combination thereof.

[0012] The present disclosure also provides a method of accumulating a von Willebrand Factor (VWF) protein in blood plasma, accumulating a FVIII protein in blood plasma, and / or increasing blood plasma half-life of a VWF protein in a subject in need thereof, comprising administering to the subject a dose of a pharmaceutical composition comprising an antibody or an antigen-binding site thereof that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region.

[0013] In some aspects of the disclosure, the anti-VWF antibody comprises a variable heavy chain (VH) region having an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 7 and a variable light chain (VL) region having an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 10.

[0014] In some aspects, the anti-VWF antibody comprises a VH comprising a complementarity determining region (CDR) Hl having the amino acid sequence set forth in SEQ ID NO: 2, a CDR H2 having the amino acid sequence set forth in SEQ ID NO: 3, and a CDR H3 having the amino acid sequence set forth in SEQ ID NO: 8; and a VL comprising a CDR LI having the amino acid sequence set forth in SEQ ID NO: 11, a CDR L2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR L3 having the amino acid sequence set forth in SEQ ID NO: 9.

[0015] In some aspects, the anti-VWF antibody comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10.

[0016] In some aspects, the anti-VWF antibody comprises a first heavy chain (HC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13, a second HC comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4, and a light chain (LC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 14.

[0017] In some aspects, the anti-VWF antibody comprises a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14. In some aspects, the second HC does not comprise a variable heavy chain (VH) region. In some aspects, the second HC chain does not comprise a VH region and also does not comprise a CHI domain. In some aspects, the second HC consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0018] In some aspects, the methods of the present disclosure further comprise administering to the subject a second pharmaceutical composition comprising a mature VWF protein or a functional fragment thereof or a therapeutic agent that induces the release of VWF from the vascular endothelium.

[0019] The present disclosure also provides a method of treating a bleeding disorder in a subject in need thereof, the method comprising administering to the patient (a) a first pharmaceutical composition comprising an anti-VWF antibody targeted against von Willebrand factor (VWF), wherein binding of the anti-VWF antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or a functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium.

[0020] The present disclosure also provides a method of treating an episode of unwanted or excessive bleeding in a subject in need thereof, the method comprising administering to the subject (a) a first pharmaceutical composition comprising an anti-VWF antibody targeted against von Willebrand factor (VWF), wherein binding of the anti-VWF antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceuticalcomposition comprising (i) a mature VWF protein or a functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium.

[0021] In some aspects, the agent that induces the release of VWF from the vascular endothelium is desmopressin (DDAVP).

[0022] In some aspects, the anti-VWF antibody is administered prior to administration of the second pharmaceutical composition. In some aspects, the anti-VWF antibody is administered after administration of the second pharmaceutical composition.

[0023] In some aspects, the methods of the disclosure comprise prophylactically administering to the patient the anti-VWF antibody.

[0024] In some aspects, the methods of the disclosure comprise (a) prophylactically administering to the patient the anti-VWF antibody and (b) in response to a minor bleeding event, a major bleeding event, or the blood plasma level of VWF in the subject dropping below 100 lU / dL, administering to the subject the second pharmaceutical composition. In some aspects, the second composition further comprises FVIII.

[0025] In some aspects, the bleeding disorder comprises von Willebrand disease (VWD), Heydes syndrome, hemophilia A, disorders of platelet function, connective tissue disorders, or any combination thereof.

[0026] In some aspects, the unwanted or excessive bleeding episode is associated with von Willebrand disease (VWD), Heydes syndrome, hemophilia A, disorders of platelet function, connective tissue disorders, or any combination thereof.

[0027] In some aspects of the disclosure, the mature VWF protein comprises a polypeptide comprising (i) an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, recombinant human VWF (rhVWF), or plasma-derived VWF (pdVWF) or (ii) a functional fragment thereof.

[0028] In some aspects, the anti-VWF antibody comprises a variable heavy chain (VH) region comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 7 and a variable light chain (VL) region comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 10.

[0029] In some aspects, the anti-VWF antibody comprises a VH comprising a complementarity determining region (CDR) Hl having the amino acid sequence set forth in SEQ ID NO: 2, a CDR H2 having the amino acid sequence set forth in SEQ ID NO: 3, and a CDR H3 having the amino acid sequence set forth in SEQ ID NO: 8; and a VL comprising a CDR LI having the amino acid sequence set forth in SEQ ID NO: 11, a CDR L2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR L3 having the amino acid sequence set forth in SEQ ID NO: 9.

[0030] In some aspects, the anti-VWF antibody comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10.

[0031] In some aspects, the anti-VWF antibody comprises a first heavy chain (HC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13, a second HC comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4, and a light chain (LC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 14.

[0032] In some aspects, the anti-VWF antibody comprises a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14. In some aspects, the second HC does not comprise a variable heavy chain (VH) region. In some aspects, the second HC chain does not comprise a VH region and also does not comprise a CHI domain. In some aspects, the second HC consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0033] The present disclosure also provides a method of treating a bleeding disorder in a subject in need thereof comprising subcutaneously administering to the subject a flat dose of 20 mg of an anti-VWF antibody, wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising a VH-CHl-hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising a hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising a VL-CLpolypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14, wherein, the CHI domain of the first HC is operably linked to the CL region of the LC via a disulfide bridge, and wherein the hinge region of the first HC and the hinge region of the second HC are operablylinked to one another by a disulfide bridge.

[0034] The present disclosure also provides a method of treating a bleeding disorder in a subject in need thereof comprising subcutaneously administering to the subject a flat dose of 50 mg of an anti-VWF antibody, wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising a VH-CHl-hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising a hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising a VL-CL polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14, wherein, the CHI domain of the first HC is operably linked to the CL region of the LC via a disulfide bridge, and wherein the hinge region of the first HC and the hinge region of the second HC are operablylinked to one another by a disulfide bridge.ASPECTS

[0035] Aspect 1. A method of treating von Willebrand disease (VWD) in a subject, the method comprising a step of administering to the subject a pharmaceutical composition comprising a monospecific antibody comprising an antigen-binding site that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region, wherein binding of the antibody to the VWF protein in blood plasma results in accumulation of the VWF protein in blood plasma as compared to a blood plasma level of the VWF protein in the absence of the antibody, at a dose from about 0.1 mg / kg to about 6 mg / kg of the monospecific antibody.

[0036] Aspect 2. The method of Aspect 1, wherein the antibody comprises a variable heavy chain (VH) region having an amino acid sequence with at least 80% identity to SEQ ID NO: 7 a variable light chain (VL) region having an amino acid sequence with at least 80% identity to SEQ ID NO: 10.

[0037] Aspect 3. The method of Aspect 1 or 2, wherein the antibody comprises a VH comprising a complementarity determining region (CDR) Hl having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 2, a CDR H2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 3, and a CDR H3 having anamino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 8; and a VL comprising therein a CDR LI having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 11, a CDR L2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 12, and a CDR L3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0038] Aspect 4. The method of Aspect 1, wherein the antibody comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10.

[0039] Aspect 5. The method of any one of Aspects 1-4, wherein the antibody is a monovalent antibody.

[0040] Aspect 6. The method of any one of Aspects 1-5, wherein the Fc region is a wild-type Fc region.

[0041] Aspect 7. The method of any one of Aspects 1-5, wherein the Fc region is a modified Fe region.

[0042] Aspect 8. The method of any one of Aspects 1-7, wherein the antibody is a human IgG4 antibody comprising a first heavy chain and a second heavy chain.

[0043] Aspect 9. The method of Aspect 8, wherein the first heavy chain comprises a S228P / T366W7F234A / L235A / delta-K447 mutation and the second heavy chain comprises a S228P / T366S / L368A / Y407V / F234A / L235A / delta-K447 mutation.

[0044] Aspect 10. The method of any one of Aspects 1-9, wherein the antibody binds to one or more amino acid residues of the VWF CK domain selected from D2726, R2730, K2735, E2744, D2746, K2757, D2763, and T2789.

[0045] Aspect 11. The method of any one of Aspects 1-9, wherein the antibody binds to at least the following amino acid residues of the VWF CK domain: R2730, K2735, E2744, and D2746.

[0046] Aspect 12. The method of any one of Aspects 1-9, wherein the antibody binds to at least the following amino acid residues of the VWF CK domain: K2735, D2746, and K2757.

[0047] Aspect 13. The method of any one of Aspects 1-9, wherein the antibody binds to at least the following amino acid residues of the VWF CK domain: R2730, D2746, and K2757.

[0048] Aspect 14. The method of any one of Aspects 1-9, wherein the antibody binds to at least amino acid residue K2735 of the VWF CK domain.

[0049] Aspect 15. The method of any one of Aspects 1-9, wherein the antibody binds to at least one of the following amino acid residues of VWF CK domain: T2728, R2730, L2731,Q2732, Y2733, V2734, K2735, E2744, D2746, K2757, M2759, Y2760, and 12762 of SEQ ID NO: 1.

[0050] Aspect 16. The method of any one of Aspects 1-15, wherein the pharmaceutical composition comprises a complex comprising a VWF protein and the monospecific antibody.

[0051] Aspect 17. The method of any one of Aspects 1-16, wherein the pharmaceutical composition comprises further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0052] Aspect 18. The method of any one of Aspects 1-17, wherein the subject suffers from excessive bleeding.

[0053] Aspect 19. The method of any one of Aspects 1-17, wherein the subject suffers from one or more symptoms of VWD selected from the group consisting of epistaxis, cutaneous bleeding, bleeding from minor wounds, oral-cavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, and central nervous system (CNS) bleeding.

[0054] Aspect 20. A method of treating one or more symptoms of VWF selected from the group consisting of epistaxis, cutaneous bleeding, bleeding from minor wounds, oral-cavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, and central nervous system (CNS) bleeding, comprising administering to a subject in need thereof the pharmaceutical composition of any one of Aspects 1-17 at a dose from about 0.3 mg / kg to about 5 mg / kg of the monospecific antibody.

[0055] Aspect 21. A method of accumulating a VWF protein in blood plasma, accumulating a FVIII protein in blood plasma, and / or increasing blood plasma half-life of a VWF protein in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of Aspects 1-17 at a dose from about 0.3 mg / kg to about 5 mg / kg of the monospecific antibody.

[0056] Aspect 22. The method of any one of Aspects 1-21, wherein the subject is a human.

[0057] Aspect 23. The method of any one of Aspects 1-22, wherein the step of administering comprises systemic administration.

[0058] Aspect 24. The method of any one of Aspects 1-23, wherein the step of administering is by injection, optionally subcutaneously or intravenously.

[0059] Aspect 25. The method of any one of Aspects 1-24, wherein the pharmaceutical composition is administered as a single dose.

[0060] Aspect 26. The method of any one of Aspects 1-24, wherein the pharmaceutical composition is administered at a regular interval.

[0061] Aspect 27. The method of Aspect 26, wherein the regular interval is at least about one week (Q1W), about two weeks (Q2W), about three weeks (Q3W), about one month, or about two months.

[0062] Aspect 28. The method of Aspect 26, wherein the regular interval is at least about one week.

[0063] Aspect 29. A method of treating a blood disorder in a patient in need thereof, the method comprising administering to the patient (a) a first pharmaceutical composition comprising a monospecific antibody targeted against von Willebrand factor (VWF), wherein binding of the antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium.

[0064] Aspect 30. The method of Aspect 29, wherein the agent that induces the release of VWF from the vascular endothelium is desmopressin (DDAVP).

[0065] Aspect 31. The method of Aspect 29, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or sequentially.

[0066] Aspect 32. The method of Aspect 29, wherein the first pharmaceutical composition is administered prior to administration of the second pharmaceutical composition.

[0067] Aspect 33. The method of Aspect 29, wherein the first pharmaceutical composition is administered after administration of the second pharmaceutical composition.

[0068] Aspect 34. The method of Aspect 29, comprising prophylactically administering to the patient the first pharmaceutical composition.

[0069] Aspect 35. The method of Aspect 29, comprising (a) prophylactically administering to the patient the first pharmaceutical composition and (b) in response to a minor bleeding event, administering to the patient the second pharmaceutical composition.

[0070] Aspect 36. The method of Aspect 29, comprising (a) prophylactically administering to the patient the first pharmaceutical composition and (b) in response to a major bleeding event, administering to the patient the second pharmaceutical composition.

[0071] Aspect 37. The method of Aspect 29, comprising (a) prophylactically administering to the patient the first pharmaceutical composition and (b) in response to the blood plasma level of VWF of the patient dropping below 100 lU / dL, administering to the patient the second pharmaceutical composition.

[0072] Aspect 38. The method of Aspect 29, comprising (a) prophylactically administering to the patient the first pharmaceutical composition and (b) in response to the blood plasma level of VWF of the patient dropping below 50 lU / dL or 30 lU / dL, administering to the patient the second pharmaceutical composition.

[0073] Aspect 39. The method of Aspect 29, comprising prophylactically administering to the patient the second pharmaceutical composition.

[0074] Aspect 40. The method of Aspect 29, comprising prophylactically administering to the patient (a) the first pharmaceutical composition and (b) the second pharmaceutical composition.

[0075] Aspect 41. The method of Aspect 29, wherein the mature VWF protein has a polypeptide sequence according to the amino acid sequence of SEQ ID NO: 16.

[0076] Aspect 42. The method of Aspect 29, wherein the mature VWF is a recombinant human VWF (rhVWF).

[0077] Aspect 43. The method of Aspect 29, wherein the mature VWF protein is plasma-derived VWF (pdVWF).

[0078] Aspect 44. The method of Aspect 43, wherein the pdVWF is derived from pooled human plasma.

[0079] Aspect 45. The method of Aspect 43, wherein the second composition further comprises FVIII.

[0080] Aspect 46. The method of Aspect 43, wherein the second composition does not comprise FVIII.

[0081] Aspect 47. The method of any of Aspects 41-16, wherein the monospecific antibody extends half-life of the mature VWF or the functional fragment thereof of the second pharmaceutical composition.

[0082] Aspect 48. The method of Aspect 47, wherein the monospecific antibody at least partially preserves activity of the mature VWF of the second pharmaceutical composition.

[0083] Aspect 49. The method of Aspect 30, wherein the monospecific antibody extends half-life of circulating VWF the release of which is induced by the DDAVP.

[0084] Aspect 50. The method of Aspect 49, wherein the monospecific antibody at least partially preserves activity of the circulating VWF the release of which is induced by the DDAVP.

[0085] Aspect 51. A method of treating an episode of unwanted or excessive bleeding in a patient in need thereof, the method comprising administering to the patient (a) a first pharmaceutical composition comprising a monospecific antibody targeted against von Willebrand factor (VWF), wherein binding of the antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium.

[0086] Aspect 52. The method of Aspect 51, wherein the agent that induces the release of VWF from the vascular endothelium is desmopressin (DDAVP).

[0087] Aspect 53. The method of Aspect 51, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or sequentially.

[0088] Aspect 54. The method of Aspect 51, wherein the first pharmaceutical composition is administered prior to administration of the second pharmaceutical composition.

[0089] Aspect 55. The method of Aspect 51, wherein the first pharmaceutical composition is administered after administration of the second pharmaceutical composition.

[0090] Aspect 56. The method of Aspect 51, comprising prophylactically administering to the patient the first pharmaceutical composition.

[0091] Aspect 57. The method of Aspect 51, comprising (a) prophylactically administering to the patient the first pharmaceutical composition and (b) in response to a minor bleeding event, administering to the patient the second pharmaceutical composition.

[0092] Aspect 58. The method of Aspect 51, comprising (a) prophylactically administering to the patient the first pharmaceutical composition and (b) in response to a major bleeding event, administering to the patient the second pharmaceutical composition.

[0093] Aspect 59. The method of Aspect 51, comprising prophylactically administering to the patient the second pharmaceutical composition.

[0094] Aspect 60. The method of Aspect 51, comprising prophylactically administering to the patient (a) the first pharmaceutical composition and (b) the second pharmaceutical composition.

[0095] Aspect 61. The method of Aspect 51, wherein the mature VWF protein has a polypeptide sequence according to the amino acid sequence of SEQ ID NO: 16.

[0096] Aspect 62. The method of Aspect 51, wherein the mature VWF is a recombinant human VWF (rhVWF).

[0097] Aspect 63. The method of Aspect 51, wherein the mature VWF protein is plasma-derived VWF (pdVWF).

[0098] Aspect 64. The method of Aspect 63, wherein the pdVWF is derived from pooled human plasma.

[0099] Aspect 65. The method of Aspect 63, wherein the second composition further comprises FVIII.

[0100] Aspect 66. The method of Aspect 63, wherein the second composition does not comprise FVIII.

[0101] Aspect 67. The method of any of Aspects 51-66, wherein the monospecific antibody extends half-life of the mature VWF of the second pharmaceutical composition.

[0102] Aspect 68. The method of Aspect 67, wherein the monospecific antibody at least partially preserves activity of the mature VWF of the second pharmaceutical composition.

[0103] Aspect 69. The method of Aspect 52, wherein the monospecific antibody extends half-life of circulating VWF the release of which is induced by the DDAVP.

[0104] Aspect 70. The method of Aspect 69, wherein the monospecific antibody at least partially preserves activity of the circulating VWF the release of which is induced by the DDAVP.

[0105] Aspect 71. The method of any one of Aspects 29-50 or 51-70, wherein the monospecific antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0106] Aspect 72. The method of any one of Aspects 29-50 or 51-70, wherein the monospecific antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0107] Aspect 73. The method of any one of Aspects 29-50 or 51-72, wherein the monospecific antibody has (a) (i) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (ii) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (iii) a heavy chain CDR1 (CDR H3) according to the amino acidsequence of SEQ ID NO: 8; and (b) (i) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (ii) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (iii) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0108] Aspect 74. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) that is at least 80% identical to the amino acid sequence of SEQ ID NO: 7, provided that the antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0109] Aspect 75. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) that is at least 85% identical to the amino acid sequence of SEQ ID NO: 7, provided that the antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0110] Aspect 76. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, provided that the antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0111] Aspect 77. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7, provided that the antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0112] Aspect 78. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) that is at least 97% identical to the amino acid sequence of SEQ ID NO: 7, provided that the antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2(CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0113] Aspect 79. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) that is at least 98% identical to the amino acid sequence of SEQ ID NO: 7, provided that the antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0114] Aspect 80. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7, provided that the antibody has (a) a heavy chain CDR1 (CDR Hl) according to the amino acid sequence of SEQ ID NO: 2, (b) a heavy chain CDR2 (CDR H2) according to the amino acid sequence of SEQ ID NO: 3, and (c) a heavy chain CDR1 (CDR H3) according to the amino acid sequence of SEQ ID NO: 8.

[0115] Aspect 81. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a heavy chain variable region (VH) according to the amino acid sequence of SEQ ID NO: 7.

[0116] Aspect 82. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) that is at least 80% identical to the amino acid sequence of SEQ ID NO: 10, provided that the antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0117] Aspect 83. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) that is at least 85% identical to the amino acid sequence of SEQ ID NO: 10, provided that the antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0118] Aspect 84. The method of any one of Aspect 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10, provided that the antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2(CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0119] Aspect 85. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10, provided that the antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0120] Aspect 86. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) that is at least 97% identical to the amino acid sequence of SEQ ID NO: 10, provided that the antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0121] Aspect 87. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) that is at least 98% identical to the amino acid sequence of SEQ ID NO: 10, provided that the antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0122] Aspect 88. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) that is at least 99% identical to the amino acid sequence of SEQ ID NO: 10, provided that the antibody has (a) a light chain CDR1 (CDR LI) according to the amino acid sequence of SEQ ID NO: 11, (b) a light chain CDR2 (CDR L2) according to the amino acid sequence of SEQ ID NO: 12, and (c) a light chain CDR1 (CDR L3) according to the amino acid sequence of SEQ ID NO: 9.

[0123] Aspect 89. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody has a light chain variable region (VL) according to the amino acid sequence of SEQ ID NO: 10.

[0124] Aspect 90. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody comprises a light chain according to the amino acid sequence of SEQ ID

[0125] Aspect 91. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody comprises a first heavy chain according to the amino acid sequence of SEQ ID NO: 4.

[0126] Aspect 92. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody comprises a second heavy chain according to the amino acid sequence of SEQ ID NO: 13.

[0127] Aspect 93. The method of any one of Aspects 29-50 or 51-73, wherein the monospecific antibody comprises (a) a light chain according to the amino acid sequence of SEQ ID NO: 14, (b) a first heavy chain according to the amino acid sequence of SEQ ID NO: 4, and (c) a second heavy chain according to the amino acid sequence of SEQ ID NO: 13.

[0128] Aspect 94. The method of any one of Aspects 29-50 or 71-93, wherein the blood disorder is selected from a von Willebrand disease (VWD), hemophilia A, disorders of platelet function, and connective tissue disorders.

[0129] Aspect 95. The method of Aspect 94, wherein the blood disorder is VWD.

[0130] Aspect 96. The method of Aspect 95, wherein the VWD is congenital VWD (cVWD) or acquired (a VWD).

[0131] Aspect 97. The method of Aspect 95, wherein the VWD is type 1 VWD.

[0132] Aspect 98. The method of Aspect 95, wherein the VWD is type 2a VWD, type 2M VWD, or type 2N VWD.

[0133] Aspect 99. The method of Aspect 95, wherein the VWD is type 3 VWD.

[0134] Aspect 100. The method of Aspect 94, wherein the bleeding disorder is hemophilia A.

[0135] Aspect 101. The method of any one of Aspects 81-100, wherein the unwanted or excessive bleeding episode is associated with von Willebrand disease (VWD), hemophilia A, disorders of platelet function, and connective tissue disorders.

[0136] Aspect 102. A method of treating a bleeding disorder in a subject population in need thereof, comprising subcutaneously administering to the subject population a dose of a pharmaceutical composition comprising a monospecific antibody comprising an antigen-binding site that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region; wherein the dose comprises a flat dose of about 10 mg to about 300 mg of the monospecific antibody.

[0137] Aspect 103. The method of Aspect 102, wherein the antibody comprises a variable heavy chain (VH) region having an amino acid sequence with at least 80% identity to SEQ IDNO: 7 a variable light chain (VL) region having an amino acid sequence with at least 80% identity to SEQ ID NO: 10.

[0138] Aspect 104. The method of Aspects 102 or 103, wherein the antibody comprises a VH comprising a complementarity determining region (CDR) Hl having the amino acid sequence set forth in SEQ ID NO: 2, a CDR H2 having the amino acid sequence set forth in SEQ ID NO: 3, and a CDR H3 having the amino acid sequence set forth in SEQ ID NO: 8; and a VL comprising therein a CDR LI having the amino acid sequence set forth in SEQ ID NO: 11, a CDR L2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR L3 having the amino acid sequence set forth in SEQ ID NO: 9.

[0139] Aspect 105. The method of Aspect 102, wherein the antibody comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10.

[0140] Aspect 106. The method of Aspect 102, wherein the antibody comprises a first heavy chain (HC) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 4, a second HC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 13, and a light chain (LC) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 14.

[0141] Aspect 107. The method of Aspect 106, wherein the antibody comprises a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 4, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0142] Aspect 108. The method of any one of Aspects 102 to 107, wherein the flat dose is about 20 mg.

[0143] Aspect 109. The method of any one of Aspects 102 to 107, wherein the flat dose is about 50 mg.

[0144] Aspect 110. The method of any one of Aspects 102 to 109, wherein the composition is administered repeatedly at regular intervals.

[0145] Aspect 111. The method of Aspect 110, wherein the composition is administered once weekly.

[0146] Aspect 112. The method of Aspect 110, wherein the composition is administered about once every two weeks.

[0147] Aspect 113. The method of Aspect 110, wherein the composition is administered about once every three weeks.

[0148] Aspect 114. The method of Aspect 110, wherein the composition is administered about once every four weeks.

[0149] Aspect 115. The method of Aspect 110, wherein the composition is administered about once a month.

[0150] Aspect 116. The method of Aspect 110, wherein the composition is administered or about once every two months.

[0151] Aspect 117. The method of any one of Aspects 102-116, wherein the blood plasma level of Von Willebrand Factor (VWF) in the subject population increases after the administration compared to the blood plasma level of VWF in the absence of the administration.

[0152] Aspect 118. The method of Aspect 117, wherein the the plasma level of Von Willebrand Factor (VWF) in the subject increases 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater as compared to a blood plasma level of VWF in the absence of the administration.

[0153] Aspect 119. The method of Aspects 117 or 118, wherein the multimer distribution of Von Willebrand Factor (VWF) (e.g. low, medium and / or high molecular weight VWF fraction) in the blood plasma of the subject population remains unchanged after the administration.

[0154] Aspect 120. The method of any one of Aspects 102 to 116, where the plasma level of Von Willebrand Factor (VWF) activity in the subject population increases after the administration compared to the blood plasma level of VWF activity in the absence of the administration.

[0155] Aspect 121. The method of Aspect 120, wherein the blood plasma level of Von Willebrand Factor (VWF) activity in the subject population increases 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater as compared to blood plasma level of VWF activity in the absence of the administration.

[0156] Aspect 122. The method of any one of Aspects 102 to 116, where the plasma level of FVIII activity in the subject population increases after the administration compared to the blood plasma level of FVIII activity in the absence of the administration.

[0157] Aspect 123. The method of Aspect 122, wherein the blood plasma level of FVIII activity in the subject population increases 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater as compared to blood plasma level of FVIII activity in the absence of the administration.

[0158] Aspect 124. The method of any one of Aspects 102 to 116, wherein the blood plasma level of the monospecific antibody in the subject population increases after the administration compared to the blood plasma level of the monospecific antibody in the absence of the administration.

[0159] Aspect 125. The method of Aspect 124, wherein the blood plasma level of the antibody in the subject population remains increased for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 20 days, or at least 21 days after the administration, compared to the blood plasma level of the antibody in the absence of the administration.

[0160] Aspect 126. The method of any one of Aspects 102 to 125, wherein the bleeding disorder is selected from von Willebrand disease (VWD), Heydes syndrome, hemophilia A, disorders of platelet function, connective tissue disorders, or any combination thereof.

[0161] Aspect 127. The method of Aspect 119, wherein the bleeding disorder is Von Willebrand disease type 1, type 2A, type 2B, type 2M, type 2N, or type 3.

[0162] Aspect 128. The method of Aspect 127, wherein the bleeding disorder is congenital Von Willebrand disease (cVWD) or acquired Von Willebrand disease (aVWD).

[0163] Aspect 129. The method of any one of Aspects 102 to 128, wherein the bleeding disorder comprises symptoms of epistaxis, cutaneous bleeding, bleeding from minor wounds, oral-cavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, central nervous system (CNS) bleeding, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0164] FIG. 1 depicts the binding of FVIII to VWF from citrated healthy human plasma in presence of monovalent (one-arm) antibody AB001 or a blocking anti-VWF polyclonal control antibody.

[0165] FIG. 2 depicts the proteolytic processing of VWF multimers by ADAMTS13 in citrated human plasma. Processing of VWF was induced by addition of ristocetin in presence of monovalent (one-arm) antibody AB001 or isotype control antibody. The distribution of VWF into low, medium, and high molecular weight fractions is shown as percent of total VWF for each timepoint and condition. The change in distribution of each VWF fraction at end (2 hours) relative to start (0 hours) of incubation with ristocetin is given in parenthesis. Isotype control antibody is identical to AB001, except that the Fab is replaced with a non-binding Fab.

[0166] FIG. 3A-3B depict VWF:platelet gplb interactions. FIG. 3A depicts RCo activity of VWF in citrated human plasma with monovalent (one-arm) antibody AB001 or a control antibody (400 nM). FIG. 3B depicts the binding of human plasma-derived VWF to recombinant GPlba extracellular domain in presence of AB001 or a control antibody (400 nM). Isotype control antibody is identical to AB001, except that the Fab is replaced with a non-binding Fab. Anti-VWF-Al domain antibody blocks gplba binding. Anti-gplba antibody blocks VWF binding.

[0167] FIG. 4A-4B depict VWF:collagen III interactions. FIG. 4A depicts collagen-binding activity of VWF in citrated human plasma with monovalent (one-arm) antibody AB001 or a control antibody. FIG. 4B depicts binding of human plasma-derived VWF to human collagen III in presence of AB001 or a control antibody. Isotype control antibody is identical to AB001, except that the Fab is replaced with a non-binding Fab. Anti-VWF-A3 domain antibody blocks collagen binding.

[0168] FIG. 5A-5C show predicted human PK profiles for single-dose administration of AB001. Predicted PK Profiles of AB001 from baseline with doses of 20 mg (FIG. 5A) and 300 mg (FIG. 5B) in a typical 70 kg, with residual VWF baseline of 2, 4, 8, 12 and 16 nM. FIG. 5C shows linear scale concentration time profiles in humans administered the starting (20 mg) and maximum (300 mg; upper curve) single dose.

[0169] FIGS. 6A-6B show predicted accumulation of VWF:Ag from baseline in a typical 70 kg individual with residual baseline of 2, 4, 8, 12 and 16 nM (5, 10, 20, 30 and 40 ILJ / dL), using ratio scaling from cynomolgus monkey to human setting. In FIG. 6A, the projected accumulation of VWF:Ag (nM) following a dose of 20 mg is presented for modeling patients with baseline residual VWF ranging from 2 nM (5 lU / dL) to 16 nM (40 ILJ / dL). In FIG. 6B, the projected accumulation of VWF:Ag (nM) following the predicted maximum dose of 300 mg is presented for modeling patients with baseline residual VWF ranging from 2 nM (5 lU / dL) to 16 nM (40lU / dL). Maximum levels predicted by both ratio and allometric scaling to the human setting are presented.

[0170] FIG. 7 shows PK profiles of AB001 in cynomolgus monkeys after a single subcutaneous (SC) administration of 30 mg / kg at day 1. Vertical and horizontal dashed lines indicate time of AB001 dosing and lower limit of quantification, respectively.

[0171] FIGs. 8A-8B show plasma VWF profiles after AB001, rhVWF, and vehicle (buffer) dosing as indicated. FIG. 8A shows Group 1 with intravenous (IV) administration of 400 lU / kg rhVWF at day -7, SC administration of 30 mg / kg AB001 at day 1, and IV administration of vehicle (buffer) at day 6. FIG. 8B shows Group 2 with IV administration of vehicle (buffer) at day -7, SC administration of 30 mg / kg AB001 at day 1, and IV administration of 400 lU / kg rhVWF at day 6. Mean values are shown (n=2 (M+F)) for each group.

[0172] FIG. 9 shows plasma profiles of VWF after rhVWF administration (400 lU / kg) in absence of AB001 (Group 1; animal 710M and 730F) or following AB001 administration (Group 2; animal 714M and 73 IF). X-axis is time (hrs) after rhVWF dosing. Elimination half-lives of VWF were estimated for each animal by non-linear fit of each profile to a mono-exponential function as represented by the dashed lines.

[0173] FIG. 10 shows a connected scatter plot representing pharmacokinetics of AB001 measured by mean SEM (nM) in cohorts Al and A2.

[0174] FIGs. 11A-11C show pharmacodynamics of AB001. FIG. 11A shows a connected scatter plot representing plasma VWF antigen levels measured by mean fold changes in cohorts Al and A2. FIG. 11B shows a connected scatter plot representing plasma VWF activity levels measured by mean fold changes in cohorts Al and A2. FIG. 11C shows a connected scatter plot representing plasma FVIII activity levels measured by mean fold changes in cohorts Al and A2. In cohort A2 (50 mg), last sample was collected at day 15 at the time of data cut.

[0175] FIGs. 12A-12C show thrombin generation (TG) and activated partial thromboplastin time (APTT) measured in cohort Al treated with AB001. FIG. 12A shows a connected scatter plot representing thrombin generation levels measured by mean (SEM) peak thrombin (nM) in cohort Al. FIG. 12B shows a connected scatter plot representing APTT levels measured by mean (SEM) activated partial thromboplastin in cohort Al. FIG. 12C shows a connected scatter plot representing multimer distribution measured in cohort Al. Abbreviations: APTT, activated partial thromboplastin time; HWM, high-molecular weight multimers; IMW, intermediate-molecular weight multimers; LMW, low-molecular weight multimers; SEM, standard error of the mean; and TG, thrombin generation.DETAILED DESCRIPTION

[0176] Disclosed herein, in certain aspects, are antibodies that specifically bind to von Willebrand Factor (VWF). Also disclosed herein are pharmaceutical compositions containing the disclosed antibody and one or more pharmaceutically acceptable carriers, diluents or excipients. The disclosure also provides methods of using the disclosed antibodies or compositions containing the same for the treatment of a blood disorder characterized by excessive bleeding (e.g., von Willebrand disease (VWD), Hey des syndrome, hemophilia A, disorders of platelet function, and connective tissue disorders). A subject with a blood disorder characterized by excessive bleeding is treated in accord with the methods disclosed herein by administering an antibody of the disclosure or a composition containing the same (e.g., a pharmaceutical composition) to the subject by any acceptable route (e.g., subcutaneously).

[0177] Also disclosed herein, in certain aspects, are methods for the treatment of blood disorders in a patient in need thereof, comprising administering to the patient (a) a first pharmaceutical composition comprising a monospecific antibody targeted against von Willebrand factor (VWF), wherein binding of the antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium. In some aspects, the mature VWF is a recombinant human von Willebrand Factor (rhVWF). In some aspects, the mature VWF is plasma-derived human von Willebrand Factor (pdVWF). In some aspects, the agent that induces the release of VWF from the vascular endothelium is desmopressin (DDAVP).Definitions

[0178] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. Generally, nomenclatures utilized in connection with, and techniques of, immunology, oncology, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0179] As used herein, singular forms “a,” “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, e.g., reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some aspects includes multiple antibodies, and so forth.

[0180] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, e.g., reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth.

[0181] As used herein, the phrase “at least [X]% identity” refers to any percent identity value from (and including) X up to (and including) 100% identity, where X can be any integer from 1-99%. For example, the phrase “at least 80% identity” means any percent identity from (and including) 80% identity up to (and including) 100% identity, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. For example, in some aspects, “at least 80% identity” in reference to a particular sequence also includes at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity, at least 98% identity, at least 99% identity, and 100% identity.

[0182] “About” a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0183] As used herein, the term “accumulation” refers to an increase in a level (e.g., concentration) of a particular protein, such as, e.g., VWF or FVIII, in a blood compartment (e.g., blood plasma) of a subject following administration of an antibody of the disclosure or a composition (e.g., pharmaceutical composition) containing the same to the subject. Various factors may influence the rate of accumulation of a protein in a blood compartment of a subject, including but not limited to, the half-life of the target protein and / or the antibody in the blood compartment, the rate of sequestration of the protein by other tissues, clearance of the protein from the body, among others. According to the present disclosure, accumulation of a target protein (e.g, VWF or FVIII) may result in a blood plasma level of the protein that is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater as compared to a blood plasma level of the protein in the absence of the antibody. Alternatively or additionally, accumulation of the target protein may results in a blood plasma level of the protein that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, 5,000%, 5,500%, 6,000%, 6,500%, 7,000%, 7,500%, 8,000%, 8,500%, 9,000%, 9,500%, 10,000%, or greater as compared to a blood plasma level of the protein in the absence of the antibody. In some aspects, accumulation of a protein (e.g., VWF) results directly from specific binding of an antibody of the disclosure to the protein. In some aspects, accumulation of a protein (e.g., FVIII) results indirectly from binding of the protein (e.g., FVIII) to another protein (e.g., VWF) bound by an antibody of the disclosure.

[0184] As used herein “antibody” refers to a naturally occurring or non-naturally occurring protein that binds an antigen. An antibody often comprises a variable domain and a constant domain in each of a heavy chain and a light chain. Accordingly, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the portion of the antibody that binds to the antigen, sometimes referred to as the “antigen receptor.” Within each variable domain are three complementarity-determining regions (CDR), which form loops in the VH and VL and contact the surface of the antigen. “Antibody” includes, but is not limited to monoclonal antibodies, monospecific antibodies, monovalent antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, grafted antibodies, antibody fragments, and in vitro-generated antibodies having the antigen-binding activity. The term also includes antibody conjugates having advantageous properties as compared to an unconjugated antibody (e.g., antibodies conjugated to a half-life extending moiety, e.g., a fatty acid)).

[0185] As used herein, “complementarity-determining regions,” “CDRs,” and “hypervariable regions” refer to the parts of the variable domains in antibodies that determine the antibodies’ binding specificities to their specific antigen. A single variable region of an antibody polypeptide will typically comprise three CDRs, usually designated CDR1, CDR2, and CDR3. More particularly, a heavy chain variable region may contain CDRs designated Hl, H2, and H3; likewise, light chain variable region may contain CDRs LI, L2, and L3. Multiple methods may be used to define a CDR. The current art utilizes various numbering schemes with different definitions of CDR lengths and positions. For example, IMGT numbering scheme is astandardized numbering system based on alignments of sequences from a complete reference gene database including the whole immunoglobulin superfamily. The Kabat numbering scheme is based on sequence alignment and uses "variability parameter" of a given amino acid position (the number of different amino acids at a given position divided by the frequency of the most occurring amino acid at that position) to predict CDRs. The Chothia numbering scheme, on the other hand, is a structure-based numbering scheme where antibody crystal structures are aligned as define the loop structures as CDRs. The Martin numbering scheme focuses on the structure alignment of different framework regions of unconventional lengths. Honneger's numbering scheme (AHo's) is based on structural alignments of the 3D structure of the variable regions and uses structurally conserved Ca positions to deduce framework and CDR lengths. One of skill in the art will note that the definition of a CDR will vary based on the method used. Accordingly, CDR sequences of a given heavy or light chain variable region may vary depending on the numbering system used. Any method of defining a CDR is contemplated with the sequences disclosed herein. Unless stated otherwise, the CDRs disclosed herein are defined according to the IMGT numbering scheme.

[0186] The terms “antigen-binding portion of an antibody,” “antigen-binding fragment,” “antigen-binding domain,” “antibody fragment” are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to the antigen. Nonlimiting examples of antibody fragments included within such terms include, but are not limited to, e.g., a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains. Also included are “one-arm” antibodies comprising a single heavy chain, a truncated heavy chain lacking a Fab region, and a single light chain.

[0187] The term “functional fragment” in the context of a mature VWF protein refers to a fragment of a mature VWF protein that retains functional capabilities of VWF. For example, in some aspects, a functional fragment of a mature VWF protein retains the ability to bind to Factor VIII, platelet gplb receptor, heparin, collagen, gpIIa / IIIb, or any combination thereof. In some aspects, a functional fragment of a mature VWF protein retains the ability to form homodimers with VWF monomers. In some aspects, a functional fragment of a mature VWF protein retains the ability to be cleaved by ADAMTS13.

[0188] As used herein, the phrase “blood disorder characterized by excessive bleeding” refers to one or more disorders in a subject (e.g., a human) that confers a susceptibility to bleed to the subject (e.g., bleeding diathesis). Such a disorder is, in some aspects, due to a condition associated with atypical and slow blood clotting. A “blood disorder characterized by excessivebleeding” may be genetic or acquired in origin. Platelet activation, adhesion, and / or aggregation (e.g., primary hemostasis) is, in some aspects, impaired in a subject with said disorder. In some aspects, function or expression of blood proteins that mediate or facilitate the production of insoluble cross-linked fibrin proteins during blood coagulation (e.g., secondary hemostasis) is impaired in a subject with the disorder. Non-limiting examples of a blood disorder characterized by excessive bleeding include von Willebrand disease (VWD), hemophilia A, disorders of platelet function, and connective tissue disorders. In some aspects, the VWD is congenital VWD (cVWD) or acquired VWD (a VWD). In some aspects, the aVWD includes Heydes syndrome. In some aspects, the VWD is type 1, type 2 A, type 2B, type 2M, type 2N, or type 3.

[0189] As used herein, the term “complex” refers to a molecular entity formed by two or more physically associated molecules (e.g., proteins), such as, e.g., a VWF protein and an antibody described herein. The two or more molecules, in some aspects, are associated by any physical means, including covalent bonds, ionic bonds, hydrogen bonds, and van der Waal forces. In some aspects, a physical association between the two or more molecules is reversible. In some aspects, a physical association between the two or more molecules is irreversible.

[0190] The term “effective amount” as used herein, refers to that amount of an antibody, or an antigen-binding portion thereof as described herein, that is sufficient to induce a disclosed effect, e.g., to effect treatment (e.g., accumulation of a VWF and / or FVIII protein in blood plasma), prognosis, or diagnosis of a disease (e.g., a blood disorder characterized by excessive bleeding), as described herein, when administered to a subject. Therapeutically effective amounts of antibodies provided herein, when used alone or in combination, will vary depending upon the relative activity of the disclosed antibodies and combinations (e.g., in treating, reducing, or ameliorating a disease or disorder described herein) and depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration, and the like, which, in some cases, are readily determined by one of ordinary skill in the art.

[0191] As used herein, the term “monospecific” refers to an antibody that displays a preferential affinity for one particular epitope (e.g., an epitope contained in a CK domain of the VWF protein, such as, e.g., a human, NHP, murine, or canine VWF protein). Within the context of the present disclosure, a monospecific antibody is, in some aspects, a monovalent antibody, a bivalent antibody, or a trivalent antibody. In some aspects of the disclosure, a monospecific antibody is a monovalent antibody.

[0192] As used herein, the term “monovalent” refers to a property of an antibody or antigenbinding fragment thereof in which the antibody has one antigen-binding site to a target epitope of a particular protein. For example, an monovalent antibody of the disclosure binds to an epitope (e.g., an epitope in a CK domain) of a VWF protein with a single antigen-binding site. Within the context of the present disclosure, a monovalent antibody is, in some aspects, a one-armed antibody (e.g., having a heavy chain, a truncated heavy chain lacking a Fab region, and a single light chain).

[0193] As used herein, the terms “one-armed antibody” or “single-armed antibody” refer to a type of monovalent antibody fragment containing an antibody heavy chain, a truncated heavy chain lacking a Fab region, and a single light chain.

[0194] “Percent identity” and “% identity” refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar; 4(1): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA. 1988 Apr; 85(8):2444-8; Pearson, Methods Enzymol. 1990; 183:63-98), gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep 1; 25(17): 3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11; 12(1 Pt l):387-95).

[0195] The term “polypeptide” refers to a chain of amino acids (e.g., naturally occurring or non-naturally occurring amino acids). In some aspects, the polypeptide comprises amino acids that are naturally occurring. In some aspects, the polypeptide comprises amino acids that are non-naturally-occurring (cystine, desmosine, isodesmosine, hydroxyproline, hydroxylysine, gammacarboxyglutamate, phosphoserine, phosphothreonine, phosphotyrosine, N-acetyl lysine, methyllysine, inositol, L-homoalanine, L-2-aminobutyric acid, L-azidohomoalanine, betaphenylalanine, norleucine, 3 -fluorotyrosine, 4-fluorophenylalanine, L-3,4-dyhydroxyphenylalanine, pNCSF, L-(7-hydroxycoumarin-4-yl)ethylglycine, 2,3-dihydroxypropyl cysteine, arylglycine, PrDiAzK, (S)-N-(2-Benzoylphenyl)-l-(2-flurobenzyl)-pyrolidine-2-carboxamide, 4-Phenanthracen-9-yl-l-phenylalanine, BN tryptophan analog, p-acetylphenylalanine, L-azidohomoalanine, among others). The polypeptides are not limited to a specific length of the product (e.g., polypeptides can comprise at least 2 amino acids linkedtogether by way of peptide bonds). Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms are used interchangeably herein unless specifically indicated otherwise. This term also encompasses chains of peptides with post-expression modifications, e.g., glycosylation, acetylation, phosphorylation, and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In some aspects, a polypeptide is an entire protein, or a fragment thereof.

[0196] The terms “preferentially binds” or “specifically binds” mean that the antibodies or fragments thereof bind to an epitope with greater affinity than it binds unrelated amino acid sequences, and, if cross-reactive to other polypeptides containing the epitope, are not toxic at the levels at which they are formulated for administration to human use. In some aspects, such affinity is at least 1.5-fold greater, 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 58-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 94-fold greater, or at least 1000-fold greater than the affinity of the antibody or fragment thereof for unrelated amino acid sequences.

[0197] As used herein, “VWF:Ag” refers to von Willebrand factor antigen. In some aspects, VWF:Ag is used a surrogate for predicting VWF activity.

[0198] The terms “recipient,” “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some aspects, the mammal is a human. In some aspects, the subject is a human. None of the terms require the supervision of a medical professional.

[0199] As used herein, “subject population” or “population of subjects” refers to one or more individuals to whom a treatment, composition, method, or use described herein is administered, applied, or otherwise directed. The subject population may comprise a single subject or a plurality of subjects, including but not limited to human subjects, veterinary subjects, or both, depending on the context. In some aspects, the subject population consists of at least one human subject in need of, suspected of needing, or receiving the treatment. In some aspects, the subject population comprises two or more, three or more, four or more, five or more subjects. In someaspects, the subject population can share one or more common characteristics, such as a disease state, genetic marker, physiological condition, demographic attribute, or treatment history.

[0200] The term “specific” refers to a situation in which an antibody will preferentially bind to molecules other than the antigen containing the epitope recognized by the antibody. The term is also applicable where e.g., an antigen-binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the antibody or antigen-binding fragment thereof carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope.

[0201] The term “therapeutically effective amount” generally refers to an amount of a disclosed antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. In some aspects, a composition described herein is administered to a subject in an amount that is effective for producing some desired therapeutic effect by inhibiting a disease or disorder as described herein at a reasonable benefit / risk ratio applicable to any medical treatment. A therapeutically effective amount is an amount that achieves at least partially a desired therapeutic or prophylactic effect in an organ or tissue. The amount of an antibody necessary to bring about prevention and / or therapeutic treatment of a disease or disorder is not fixed per se. In some aspects, the amount of the antibody administered varies with the type of disease, extensiveness of the disease, and size of the mammal suffering from the disease or disorder. When used in conjunction with therapeutic methods involving administration of a therapeutic agent after the subject presents symptoms of a disease or disorder, the term “therapeutically effective” means that, after treatment, one or more signs or symptoms of the disease or disorder is ameliorated or eliminated.

[0202] In some aspects, an effective response of the present disclosure is achieved when the subject experiences partial or total alleviation or reduction of signs or symptoms of illness and, in the case of the treatment of a disease (e.g., a blood disorder characterized by excessive bleeding), specifically includes, without limitation, amelioration of symptoms, prolongation of progression, cure, remission, prolongation of survival, or other objective responses. In some aspects, the expected progression-free survival times are measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors.Prolonging survival includes without limitation times of at least 1 month (mo.), about at least 2 mos., about at least 3 mos., about at least 4 mos., about at least 6 mos., about at least 1 year, about at least 2 years, about at least 3 years, etc. Overall survival is also measured, e.g., in months to years. Alternatively, an effective response, in some aspects, is that a subject’ssymptoms remain static. Further indications of treatment of indications are described in more detail below.

[0203] In some aspects, administration of a therapeutic agent in a prophylactic method occurs prior to the manifestation of symptoms of an undesired disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. Thus, when used in conjunction with prophylactic methods, the term “therapeutically effective” means that, after treatment, a smaller number of subjects (on average) develop the undesired disease or disorder or progress in severity of symptoms.

[0204] As used herein, the terms “treatment,” “treating,” and the like, in some cases, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or is therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, includes treatment of a disease or disorder (e.g., blood disorder characterized by excessive bleeding) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which is predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that is associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. The term treating includes to any indicia of success in the treatment or amelioration or prevention of a disease or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the agents of the present disclosure to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases. The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. A subject is “treated” for a disease or disorder if, after receiving a therapeutic amount of an antibody of the present disclosure, the patient shows observable and / or measurable change in a parameter or symptom of the disease or disorder.Regulation of Hemostasis

[0205] Blood circulation provides cells and tissues with necessary substances such as nutrients and oxygen and disposes of metabolic waste products from these cells. The blood vasculature establishes a physical barrier that ensures the containment of blood, regulation of its pressure throughout the organism, and tissue-directed delivery of nutrients and waste products. Disruption or damage to this barrier can result in potentially life-threatening blood loss. Blood plasma contains numerous soluble proteins that act in a concerted cascade of enzymatic reactions to produce a blood clot that can readily “plug” the damaged blood vessel and prevent or reduce further blood loss. This plug is stabilized by the proteolytic cleavage of fibrinogen into fibrin, which forms a mesh that ensheathes and reinforces the clot. Formation and degradation of blood clots are part of a process called hemostasis, which is regulated by a number of soluble and membrane-tethered pro-coagulant and anti-coagulant proteins. A balance between coagulation and fibrinolysis (i.e., degradation of insoluble fibrin proteins) controls the stability of the blood clot.

[0206] There are two central pathways that trigger blood clotting, namely the tissue factor (or extrinsic) pathway and the contact (or intrinsic) pathway. These clotting cascades proceed by sequential activation of zymogens (i.e., inactive protein precursors) by proteolytic cleavage. Each zymogen, when activated, acts as a serine protease that activates downstream zymogens to drive clot formation. Protein cofactors also act in the blood clotting cascade, generally circulating as inactive pro-cofactors in blood plasma until activated by limited proteolysis.

[0207] The extrinsic pathway is initiated when a cell-surface complex of an integral membrane protein, tissue factor (TF), and Factor Vila (TF:VIIa) activates Factor IX (FIX) and Factor X (FX) by limited proteolysis, thereby producing activated FIX (FIXa) and activated FX (FXa). The contact pathway initiates when Factor Xlla (FXII), prekallikrein (PK), and kininogen (HK) assemble on a surface or polymer. Assembly results in reciprocal activation of FXII to FXIIa by kallikrein (KK), and PK to KK by FXIIa. FXIIa then activates Factor XI (FXI) to FXIa, which then enzymatically converts FIX to FIXa. Activated FVIII (FVIIIa) combines with FIXa to form an Xase (FVIIIa:FIXa) complex responsible for the generation of FXa together with the initiating TF:FVIIa complex. Extrinsic and intrinsic pathways ultimately converge on the production of FXa, which leads to the activation of thrombin from prothrombin, thrombin-mediated cleavage of fibrinogen to fibrin, and activation, adhesion, and aggregation of platelets at the site of the clot.

[0208] VWF plays a critical role in platelet adhesion, activation, and aggregation during blood coagulation. Increased local shear stress near sites of vascular injury results in VWF binding to collagen in the sub-endothelial membrane, which in turn induces platelets to attach to tethered VWF by way of surface glycoprotein to initiate formation of the platelet plug. Under normal physiological conditions, the interaction between VWF and platelets occurs strictly in the context of vascular damage, since VWF does not interact with circulating platelets in the absence of injury. The Al and C4 domains of VWF are essential for binding with platelet glycoproteins, such as, e.g., the platelet gplb and gpllbllla receptors, respectively. Because VWF and FVIII circulate as a complex in plasma under physiological conditions, accumulation of VWF at the site of vascular injury may increase the local concentration of FVIII. Similarly, reduced levels of VWF, as occurs, e.g., in VWD, may lead to reduced levels of FVIII.

[0209] Regulation of hemostasis is also critical in preventing excessive blood clotting, which can lead to the formation of thrombi that can dislodge from their site of formation and cause a potentially life-threatening embolism. Endothelial cells of intact blood vessels regulate clotting by way of thrombomodulin, an integral membrane protein, which reduces clotting by converting thrombin to an anti-coagulant form of the enzyme. At the same time, the thrombomodulinthrombin complex can enzymatically activate the thrombin-activatable fibrinolysis inhibitor (TAFI) protein, a carboxypeptidase enzyme that reduces fibrinolysis by removing C-terminal lysine residues exposed on fibrin that are critical for the conversion of plasminogen to plasmin. TAFFs ability to attenuate fibrinolysis is highly dependent on its concentration, rate of activation, and blood plasma half-life.

[0210] Another factor important for the control of hemostasis is antithrombin (AT or AT3), a serine protease inhibitor (also known as serpin) that serves to regulate coagulation by inhibiting multiple proteases in the coagulation cascade, including FIXa, FXa, and thrombin. Its ability to inhibit coagulation factors is generally strongly enhanced by binding to heparin. By inhibiting proteins in the coagulation cascade, AT regulates the blood clotting process to prevent excessive clotting and thrombosis. In humans, AT deficiency is associated with an increased risk for thrombotic disease and pulmonary embolism.Anti-von Willebrand Factor Antibodies

[0211] The mature VWF polypeptide is a large (2,050 amino acid residues) blood glycoprotein that functions in hemostasis and platelet adhesion. VWF is generally localized to the blood plasma, vascular endothelium, megakaryocytes, and subendothelial connective tissue, andexists in multimers of various sizes ranging from about 500 to 20,000 kDa. VWF multimer size typically scales with its role in hemostasis, i.e., larger VWF multimers are more effective at promoting clotting. Different domains of VWF are involved different functions. VWF contains the following domains, from N-terminus to C-terminus: D1-D2-D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK. The D1-D2 domains, corresponding to the VWF propeptide, are proteolytically cleaved during processing of VWF into mature VWF. The D’-D3 domain mediates multimerization of VWF dimers and also binds to Factor VIII. The Al domain binds to platelet gplb receptor, heparin, and collagen. The A2 domain is cleaved by ADAMTS13, resulting in generation of smaller VWF multimers. The A3 domain binds to collagen. The C4 domain binds to platelet to gpIIa / IIIb. The CK domain is important for tail-to-tail homodimerization of VWF monomers. Binding of VWF to other proteins, such as, e.g., FVIII, results in the stabilization of the binding partner and increases its blood plasma half-life. For example, in the absence of VWF, FVIII has a half-life of about 1-2 hours, but extends to 8-12 hours when bound by VWF. In addition to acting as a carrier of FVIII, VWF promotes clotting by facilitating platelet adhesion, activation, and aggregation. Deficiency or dysfunction of VWF is associated with a tendency to bleed and occurs in a number of blood disorders characterized by excessive bleeding, including, e.g., VWD and Hey des syndrome.

[0212] Provided herein, in certain aspects, are antibodies that specifically bind to VWF proteins. In some aspects, the VWF protein is a human VWF protein. In some aspects, the human VWF protein has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some aspects, the human VWF protein has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some aspects, the human VWF protein has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some aspects, the human VWF protein has an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1. In some aspects, the human VWF protein has an amino acid sequence of SEQ ID NO: 1. MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYL LAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLY LETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEG TLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARC HPLVDPEPFVALCEKTLCECAGGLECACP ALLEY ARTCAQEGMVLYGWTDHSACSPVC PAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSG KRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLA RDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGN QGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLT SPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPG RCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKA QCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSL SCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDG LKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGE VNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNF DGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVD SSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKV VTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHA HCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCE ACQEPGGLVVPPTD APVSPTTL YVEDISEPPLHDF YC SRLLDLVFLLDGS SRLSEAEFEVL KAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQV ASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVG IGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMAQV TVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSI HVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQ GDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDF ETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAK AFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALG FAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDA AQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDV WTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVC TGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHS ALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEF QLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPI LEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRT NGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVML EGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPT CGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCAC RKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSG FTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRVKEE VFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTVMIDV CTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQI MTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPGTCC DTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCS PTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK(SEQ ID NO: 1; UniProtNo. P04275-1).Antibody Sequences

[0213] In some aspects, the antibody comprises a complementarity determining region (CDR) Hl having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 2, a CDR H2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 3, and a CDR H3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 8. In some aspects, the antibody comprises a CDR LI having 100% sequence identity to an amino acid sequence of SEQ ID NO: 11, a CDR L2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 12, and a CDR L3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0214] In some aspects, the antibody comprises a VH having an amino acid sequence with at least 80% identity to SEQ ID NO: 7. In some aspects, the antibody comprises a VL having an amino acid sequence with at least 80% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 85% identity to SEQ ID NO: 7. In some aspects, the antibody comprises a VL having an amino acid sequence with at least 85% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 90% identity to SEQ ID NO: 7. In some aspects, the antibody comprises a VL having an amino acid sequence with at least 90% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 95% identity to SEQ ID NO: 7. In some aspects, the antibody comprises a VL having an amino acid sequence with at least 95% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 97% identity to SEQ ID NO: 7. In some aspects, the antibody comprises a VL having an amino acid sequence with at least 97% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 98% identity to SEQ ID NO: 7. In some aspects, the antibody comprises a VL having anamino acid sequence with at least 98% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 99% identity to SEQ ID NO: 7. In some aspects, the antibody comprises a VL having an amino acid sequence with at least 99% identity to SEQ ID NO: 8. In some aspects, the antibody comprises a VH having an amino acid sequence 100% identical with the amino acid sequence of SEQ ID NO: 7. In some aspects, the antibody comprises a VL having an amino acid sequence 100% identical with the amino acid sequence of SEQ ID NO: 10.

[0215] In some aspects, the antibody comprises: (a) a CDR Hl having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 2, a CDR H2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 3, and a CDR H3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 8; and (b) a CDR LI having 100% sequence identity to an amino acid sequence of SEQ ID NO: 11, a CDR L2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 12, and a CDR L3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 9. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 80% identity to SEQ ID NO: 7 a VL having an amino acid sequence with at least 80% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 85% identity to SEQ ID NO: 7 and a VL having an amino acid sequence with at least 85% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 90% identity to SEQ ID NO: 7 a VL having an amino acid sequence with at least 90% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 95% identity to SEQ ID NO: 7 and a VL having an amino acid sequence with at least 95% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 97% identity to SEQ ID NO: 7 and a VL having an amino acid sequence with at least 97% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence with at least 99% identity to SEQ ID NO: 7 and a VL having an amino acid sequence with at least 99% identity to SEQ ID NO: 10. In some aspects, the antibody comprises a VH having an amino acid sequence 100% identical with the amino acid sequence of SEQ ID NO: 7 and a VL having an amino acid sequence 100% identical with the amino acid sequence of SEQ ID NO: 10.

[0216] In some aspects, the antibody comprises a VH having an amino acid sequence with at least 80% identity to SEQ ID NO: 7 and comprising therein a CDR Hl having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 2, a CDR H2 having anamino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 3, and a CDR H3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 8. In some aspects, the antibody comprises a VL having an amino acid sequence with at least 80% identity to SEQ ID NO: 10 and comprising therein a CDR LI having 100% sequence identity to an amino acid sequence of SEQ ID NO: 11, a CDR L2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO. 12, and a CDR L3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 9. In some aspects, the antibody comprises: (a) a VH having an amino acid sequence with at least 80% identity to SEQ ID NO: 7 and comprising therein a CDR Hl having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 2, a CDR H2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 3, and a CDR H3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 8; and (b) a VL having an amino acid sequence with at least 80% identity to SEQ ID NO: 10 and comprising therein a CDR LI having 100% sequence identity to an amino acid sequence of SEQ ID NO: 11, a CDR L2 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 12, and a CDRL3 having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 9.Heavy Chain

[0217] Antibody AB001 is a one-arm antibody comprising a first polypeptide chain comprising a first heavy chain (HC) comprising, from N-terminus to C-terminus: a VH, a first heavy chain constant (CHI) region operably linked (e.g., via a peptide bond) to a first hinge domain at its C-terminus, a first CH2 region operably linked at its N-terminus (e.g., via a peptide bond) to the C-terminus of the first hinge domain, and a first CH3 region operably linked at its N-terminus (e.g., via a peptide bond) to the C-terminus of the first CH2 region. In some aspects, the first HC region comprises a first CHl-hinge-CH2-CH3 polypeptide comprising an amino acid sequence of SEQ ID NO: 5 or a variant thereof having at least 80% sequence identity to SEQ ID NO: 5.ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 5).

[0218] In some aspects, the first HC region comprises an amino acid sequence of SEQ ID NO: 15 or a variant thereof having at least 80% sequence identity to SEQ ID NO: 15.QVQLVQSGAEVKKPGASVKVSCKVSGSTFSNYAIHWVRQAPGKGLEWMGGIVPLSGTA IYAQKFQGRVTMTEDTSTDTAYMELSSLKSEDTAVYYCARDQGESSGWYAIYEGMDV WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQ S SGL YSLS S VVTVP S S SLGTKT YTCNVDHKP SNTKVDKRV(SEQ ID NO: 15).

[0219] In some aspects, the first HC region comprises an amino acid sequence according to the amino acid sequence of SEQ ID NO: 13 or a variant thereof having at least 80% identity to SEQ ID NO: 13.QVQLVQSGAEVKKPGASVKVSCKVSGSTFSNYAIHWVRQAPGKGLEWMGGIVPLSGTA IYAQKFQGRVTMTEDTSTDTAYMELSSLKSEDTAVYYCARDQGESSGWYAIYEGMDV WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL VSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 13).Light Chain

[0220] In some aspects, the first polypeptide chain of the one-arm antibody is operably linked (e.g., via a disulfide bridge) to second polypeptide chain comprising a light chain (LC). In some aspects, the light chain region is a polypeptide comprising, from N-terminus to C-terminus: a VL region operably linked at its C-terminus to the N-terminus of a light chain constant (CL) region. In some aspects, the CL region is a kappa chain. In some aspects, the CL region comprises an amino acid sequence of SEQ ID NO: 6 or variant thereof having at least 80% sequence identity to SEQ ID NO: 6.RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 6).

[0221] In some aspects, the LC region comprises an amino acid sequence according to the amino acid sequence of SEQ ID NO: 14 or a variant thereof having at least 80% identity to SEQ ID NO: 14.DIQMTQSPSSVSASVGDRVTITCRASQGIGTYLAWYQQKPGKAPKLLIYGASTRATGVPS RFSGSGSGTDFTLTISSLQPEDFANYYCQQSYSKVTFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLS SP VTKSFNRGEC(SEQ ID NO: 14).Second Heavy Chain

[0222] The one-arm antibody of the disclosure further comprises a third polypeptide chain comprising a second HC region, wherein the second HC region does not include a VH region. In some aspects, the second HC region comprises a second hinge domain, a second CH2 region operably linked at its N-terminus (e.g., via a peptide bond) to the C-terminus of the second hinge domain, and a second CH3 region operably linked at its N-terminus (e.g., via a peptide bond) to the C-terminus of the second CH2 region. In some aspects, the second HC region comprises an amino acid sequence of SEQ ID NO: 4 or is a variant thereof having at least 80% sequence identity to SEQ ID NO: 4.ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 4).Full-Length Antibody

[0223] In some aspects of the disclosure, the CHI domain of the first HC region is operably linked to the CL region via a disulfide bridge. In some aspects, the first HC region and the second HC region are operably linked to one another by a disulfide bridge between the first hinge region and the second hinge region.

[0224] Accordingly, disclosed herein, in some aspects, are antibodies comprising an antigen binding domain that specifically binds to the CK domain of a VWF protein, the antigen binding domain comprising: (1) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 or avariant thereof having at least 80% sequence identity thereto, the VH domain being operably linked (e.g., via a peptide bond) at its C-terminus to the N-terminus of a CHl-hinge-CH2-CH3 polypeptide of SEQ ID NO: 5 or a variant thereof having at least 80% sequence identity thereto; and (2) a VL domain comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof having at least 80% sequence identity thereto operably linked (e.g., via a peptide bond) at its C-terminus to the N-terminus of a CL region having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 6 or a variant thereof having at least 80% sequence identity thereto; wherein the antibody further comprises a polypeptide of SEQ ID NO: 4 or a variant thereof having at least 80% sequence identity thereto operably linked (e.g., via a disulfide bridge) to the hinge region of the CHl-hinge-CH2-CH3 polypeptide.

[0225] Also disclosed herein, in some aspects, are antibodies comprising an antigen binding domain that specifically binds to the CK domain of a VWF protein, the antibody comprising: (1) a heavy chain variable region (VH) domain according to the amino acid sequence of SEQ ID NO: 7, the VH domain being operably linked (e.g., via a peptide bond) at its C-terminus to the N-terminus of a CHl-hinge-CH2-CH3 polypeptide of SEQ ID NO: 5 or a variant thereof having at least 80% sequence identity thereto; (2) a light chain variable region (VL) domain according to the amino acid sequence of SEQ ID NO: 10, the VL domain being operably linked (e.g., via a peptide bond) at its C-terminus to the N-terminus of a CL region having an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 6 or a variant thereof having at least 80% sequence identity thereto; and (3) a polypeptide of SEQ ID NO: 4 or a variant thereof having at least 80% sequence identity thereto operably linked (e.g., via a disulfide bridge) to the hinge region of the CHl-hinge-CH2-CH3 polypeptide of SEQ ID NO: 5.

[0226] In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 13. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 15. In some aspects, the antibody comprises a second HC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 4. In some aspects, the antibody comprises a LC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 14. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 13 or 15, a second HC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 4, and a LC comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 14.

[0227] In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 13. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 15. In some aspects, the antibody comprises a second HC comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4. In some aspects, the antibody comprises a LC comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 14. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 13 or 15, a second HC comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4, and a LC comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 14.

[0228] In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 15. In some aspects, the antibody comprises a second HC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 4. In some aspects, the antibody comprises a LC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 14. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13 or 15, a second HC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 4, and a LC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 14.

[0229] In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 13. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 15. In some aspects, the antibody comprises a second HC comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 4. In some aspects, the antibody comprises a LC comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 14. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 13 or 15, a second HC comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 4, and a LC comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 14.

[0230] In some aspects, the antibody comprises a first HC comprising an amino acid sequence having 100% identity to SEQ ID NO: 13. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having 100% identity to SEQ ID NO: 15. In someaspects, the antibody comprises a second HC comprising an amino acid sequence having 100% identity to SEQ ID NO: 4. In some aspects, the antibody comprises a LC comprising an amino acid sequence having 100% identity to SEQ ID NO: 14. In some aspects, the antibody comprises a first HC comprising an amino acid sequence having 100% identity to SEQ ID NO: 13 or 15, a second HC comprising an amino acid sequence having 100% identity to SEQ ID NO: 4, and a LC comprising an amino acid sequence having at 100% identity to SEQ ID NO: 14.

[0231] In some aspects, the antibody comprises a first HC comprising an amino acid sequence having 100% identity to SEQ ID NO: 13, a second HC comprising an amino acid sequence having 100% identity to SEQ ID NO: 4, and a LC comprising an amino acid sequence having 100% identity to SEQ ID NO: 14.

[0232] In some aspects, the antibody comprises a first HC comprising a VH-CHl-hinge-CH2-CH3 polypeptide comprising an amino acid sequence having 100% identity to SEQ ID NO: 13, a second HC comprising a hinge-CH2-CH3 polypeptide comprising an amino acid sequence having 100% identity to SEQ ID NO: 4, and a LC comprising a VL-CL polypeptide comprising an amino acid sequence having 100% identity to SEQ ID NO: 14, wherein, the CHI domain of the first HC is operably linked to the CL region of the LC via a disulfide bridge, and wherein the hinge region of the first HC and the hinge region of the second HC are operably linked to one another by a disulfide bridge.

[0233] In some aspects, the antibody is AB001. The amino acid sequences of antibody AB001 are described in Table 1.Table 1: AB001 Amino Acid Sequence

[0234] CDR sequences are defined using the ImMunoGeneTics (IMGT) scheme except for the LC CDR2 which is defined using the Kabat scheme.Pharmaceutical Compositions

[0235] Disclosed herein, in certain aspects, are pharmaceutical compositions comprising an antibody as disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions are useful for in vitro or in vivo analysis or, in the case of pharmaceutical compositions, for administration to a subject in vivo or ex vivo for treating a subject with the disclosed antibodies.

[0236] In some aspects, the carrier, diluent, or excipient is a stabilizer, buffer, surfactant, filler, solvent, tonicity or osmolarity adjusting agent, antioxidant, adjuvant, and antimicrobial agent or other suitable materials known to those skilled in the art. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.

[0237] Pharmaceutical formulations comprising an antibody identified by the methods described herein are prepared for storage by mixing the protein having the desired degree of purity with optional physiologically acceptable carrier, diluent, or excipient (see, e.g., Remington ’s Pharmaceutical Sciences, 16thedition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions in some aspects. Acceptable carrier, diluent, or excipient are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

[0238] In some aspects, the pharmaceutical composition comprises the antibody at a concentration of between 1-200 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 1 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 2 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 3 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 4 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 5 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 6 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 7 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 8 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 9 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 10 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 20 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 30 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 40 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 50 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 60 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 70 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 80 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 90 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 100 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 110 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 120 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 130 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 140 mg / mL. In some aspects, the pharmaceutical composition comprises the antibody at a concentration of 150 mg / mL. In some aspects, the pharmaceutical composition is in a volume of no greater than 1.0 mL (e.g., 1.0 mL, 0.9 mL, 0.8 mL, 0.7 mL, mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, or less). In some aspects, the pharmaceutical composition is stable as a liquid solution at room temperature.

[0239] Acceptable carriers are physiologically acceptable to the administered subject and retain the therapeutic properties of the compounds with / in which it is administered. Acceptable carriers and their formulations are and generally described in, e.g., Remington ’s Pharmaceutical Sciences, supra. One exemplary carrier is physiological saline. The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compounds from the administration site of one organ, or portion of the body, to another organ, or portion of the body, or in an in vitro assay system. Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to a subject to whom it is administered. Nor should an acceptable carrier alter the specific activity of the subject compounds.

[0240] In some aspects, a pharmaceutical composition disclosed herein further comprises an acceptable additive to improve the stability of the compounds in composition and / or to control the release rate of the composition. Acceptable additives do not alter the specific activity of the subject compounds. Exemplary acceptable additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. Acceptable additives are combined with acceptable carriers and / or excipients such as dextrose, in some aspects. Alternatively, exemplary acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase stability of the peptide and decrease gelling of the solution. In some aspects, the surfactant is added to the composition in an amount of 0.01% to 5% of the solution. Addition of such acceptable additives increases the stability and half-life of the composition in storage.

[0241] In one aspect, a pharmaceutical composition disclosed herein contains an isotonic buffer such as a phosphate, acetate, or TRIS buffer in combination with a tonicity agent such as a polyol, sorbitol, sucrose or sodium chloride, which tonicifies and stabilizes. In some aspects, a tonicity agent is present in the composition in an amount of about 5%.

[0242] In another aspect, a pharmaceutical composition disclosed herein includes a surfactant such as to prevent aggregation and for stabilization at 0.01 to 0.02% w / v.

[0243] In another aspect, the pH of a pharmaceutical composition disclosed herein ranges from 4.5-6.5 or 4.5-5.5.

[0244] In some aspects, a pharmaceutical composition disclosed herein also contains more than one active compound as necessary for the indication being treated, such as those withcomplementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0245] In some aspects, active ingredients are entrapped in microcapsule prepared, e.g., by coacervation techniques or by interfacial polymerization, e.g., hydroxy methylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington ’s Pharmaceutical Sciences, supra.

[0246] Suspensions and crystal forms of antibodies are also contemplated herein; methods to make suspensions and crystal forms are known to one of skill in the art.

[0247] In some aspects, a pharmaceutical composition disclosed herein is sterile. In some aspects, a pharmaceutical composition disclosed herein is sterilized by conventional, well known sterilization techniques. For example, sterilization is readily accomplished by filtration through sterile filtration membranes. In some aspects, the resulting solutions is packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

[0248] Freeze-drying is employed to stabilize polypeptides for long-term storage, such as when a polypeptide is relatively unstable in liquid compositions, in some aspects.

[0249] In some aspects, excipients such as, e.g., polyols (including mannitol, sorbitol, and glycerol), sugars (including glucose and sucrose), and amino acids (including alanine, glycine, and glutamic acid) act as stabilizers for freeze-dried products. Polyols and sugars are also used to protect polypeptides from freezing and drying-induced damage and to enhance the stability during storage in the dried state in some aspects. Sugars are, in some aspects, effective in both the freeze-drying process and during storage. Other classes of molecules, including mono- and disaccharides and polymers, such as PVP, have also been reported as stabilizers of lyophilized products.

[0250] For injection, in some aspects, a pharmaceutical composition disclosed herein is a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried, or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the compositions optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers, and combinations of these.

[0251] Sustained-release preparations are prepared, in some aspects. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (see, e.g., U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylenevinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3 -hydroxybutyric acid. While polymers such as ethyl ene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. In some aspects, while encapsulated antibodies remain in the body for a long time, they denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies devised for stabilization are, in some cases, dependent on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— bond formation through thio-disulfide interchange, stabilization is achieved, in some cases, by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0252] In some aspects, a pharmaceutical composition disclosed herein is designed to be short-acting, fast-releasing, long-acting, or sustained-releasing as described herein. In some aspects, a pharmaceutical composition disclosed herein is formulated for controlled release or for slow release.

[0253] The pharmaceutical composition is administered, e.g., by injection, including, but not limited to, subcutaneous, intravitreal, intradermal, intravenous, intra-arterial, intraperitoneal, intracerebrospinal, or intramuscular injection. In some aspects, the composition is administered by subcutaneous injection. Excipients and carriers for use in formulation of compositions for each type of injection are contemplated herein. The following descriptions are by example only and are not meant to limit the scope of the compositions. Compositions for injection include, but are not limited to, aqueous solutions (where water soluble) or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In some aspects, the carrier is a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g.,glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity is maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Antibacterial and antifungal agents include, e.g., parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride are included in the composition in some aspects. In some aspects, the resulting solutions are packaged for use as is, or lyophilized; the lyophilized preparation is later combined with a sterile solution prior to administration, in some aspects. For intravenous injection or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, e.g., isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, and Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives are included as needed, in some aspects.

[0254] Sterile injectable solutions are prepared by incorporating an active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization, in some aspects. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0255] Compositions are conventionally administered intravenously in some aspects, such as by injection of a unit dose. For injection, in some aspects, an active ingredient is in the form of a parenterally acceptable aqueous solution which is substantially pyrogen-free and has suitable pH, isotonicity, and stability. In some aspects, one prepares suitable solutions using, e.g., isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives are included, as required, in some aspects. Additionally, compositions are administered via aerosolization in some aspects.

[0256] For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, or emulsion) in association with a pharmaceutically acceptable, parenteral vehicle. Examples of such vehicles are water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate are also used. In some aspects, liposomes are used as carriers. The vehicle contains minoramounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies are typically formulated in such vehicles at concentrations of about 1 mg / mL to 10 mg / mL.

[0257] In one aspect, a pharmaceutical composition disclosed herein is lyophilized, e.g., to increase shelf-life in storage. When the compositions are considered for use in medicaments or any of the methods provided herein, in some aspects, it is contemplated that the composition are substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human subject. Testing compositions for pyrogens and preparing compositions substantially free of pyrogens are well understood to one or ordinary skill of the art and are accomplished using commercially available kits in some aspects.

[0258] In some aspects, acceptable carriers contain a compound that stabilizes, increases, or delays absorption or clearance. Such compounds include, e.g., carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, e.g., aluminum monostearate and gelatin. In some aspects, detergents also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. To protect from digestion the compound, in some aspects, is complexed with a composition to render it resistant to acidic and enzymatic hydrolysis, or the compound is, in some aspects, complexed in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are known in the art.Methods of Treatment

[0259] Disclosed herein, in certain aspects, are methods of treating a blood disorder characterized by excessive bleeding in a subject population in need thereof, comprising administering to the subject an antibody or pharmaceutical composition containing the antibody disclosed herein. In some aspects, the disclosure provides a use of an antibody as described herein for the manufacture of a medicament. In some aspects, the disclosure provides a use of an antibody as described herein for the treatment of a blood disorder characterized by excessive bleeding.

[0260] Blood disorders characterized by excessive bleeding arise, in part, due to dysregulation of hemostasis, which can occur when proteins involved in the clotting signaling cascades are disrupted. For example, von Willebrand disease (VWD) is an inherited bleeding disorder that results from deficiency in VWF. Such deficiency can arise from low levels of VWF(i.e., quantitative effect) or from presence of dysfunctional VWF (i.e., qualitative effect). It is one of the most common inherited blood clotting disorders in humans.

[0261] Other blood disorders include Hemophilia A, Heydes syndrome, abnormal uterine bleeding (AUB), Alpha-2-antiplasmin (A2AP / SERPINF2) deficiency, platelet disorders (such as, but not limited to, Glanzmann' s thrombasthenia, Bernard-Soulier syndrome, Chromosome 22qll.2 deletion syndromes, platelet-type VWF (PT-VWD), gray platelet syndrome, May-Hegglin anomaly, Epstein syndrome, Fechter syndrome, Sebastian syndrome, Wiskott-Aldrich syndrome, Chediak- Higashi syndrome, Hermansky-Pudlak syndrome, and storage pool / secretion disorder), connective tissue disorders (e.g., hereditary hemorrhagic telangiectasia (HHT) / Osler-Weber-Rendu (OWR) disease), and congenital antithrombin (AT) deficiency.

[0262] Hereditary forms of VWD include type 1, type 2, type 3, and platelet-type VWD. In addition to inherited pathology, VWD exists in acquired forms. Generally, VWD is characterized by tendency to bleed, easy bruising, nosebleeds, bleeding gums, heavy menstrual bleeding, and blood loss during childbirth. Recently, another diagnostic category of “low VWF” has emerged, corresponding to individuals with VWF levels that are below normal but insufficient to meet diagnostic criteria for VWD. VWF deficiency in VWD presents primarily in organs with small vessels, including skin, gastrointestinal tract, uterus, and colon.

[0263] As mentioned above, hereditary VWD occurs in multiple forms, including type 1, type 2, type 3, and platelet-type. Type 1 VWD is the predominant form (40-80% of all cases), which presents as VWF levels that are less than 50% of levels in healthy subjects. Type 1 VWD patients can be asymptomatic and lead a normal life without complications. Therefore, although VWD occurs in about 1% of the population, clinically significant cases are observed in about 1 / 10,000 cases. Type 2 VWD corresponds to 15-50% of all cases and generally presents as a bleeding tendency that varies between patients. Type 2 VWD can be further classified into subtypes based on the presence and behavior of VWF multimers, including Types 2A, 2B, 2M, and 2N. Type 2A is characterized by a defect in the ability to form large VWF multimers. Type 2B is characterized by a gain of function in which the binding between VWF and platelet gplb receptor is strongly enhanced, resulting in abnormal platelet binding and rapid clearance of platelet- VWF multimer complexes. This leads to reduced or absent levels of large VWF multimers. Type 2M VWD results from reduced binding between VWF and gplb platelet receptor, resulting in reduced platelet adhesion and aggregation. Type 2N VWD corresponds to a deficiency in binding between VWF and FVIII, resulting in reduced blood plasma levels of F VIII similar to those seen in hemophilia A. Type 3 VWD is characterized by homozygosity for thedefective VWF gene and features complete absence of VWF in plasma, resulting in the most severe form of VWD. Type 3 VWD is also associated with very low blood plasma levels of FVIII. Platelet-type VWD presents with qualitatively and quantitatively normal VWF protein, but features a defect in the platelet gplb receptor that results in increased affinity to VWF. This increased affinity to VWF produces abnormally large platelet aggregates that are cleared from circulation along with VWF multimers. The pathogenesis of acquired VWD is variable. It can be associated with the presence of autoantibodies to VWF, resulting in rapid clearance of VWF -autoantibody complexes from the circulation, or interference with its function. Acquired VWD can also occur with adsorption of VWF by tumor cells and non-immunologic mechanisms of destruction. Current treatments for VWD include use of desmopressin, tranexamic acid (TXA), and concentrates of plasma-derived VWF and / or FVIII or recombinant VWF. However, these therapies suffer from several shortcomings, including but not limited to short half-lives not suitable for bleeding prophylaxis, increased thrombotic and cardiovascular risk, gastrointestinal side effects, limited efficacy in major bleeds, high cost (e.g., VWF / FVIII supplementation), inconvenient intravenous administration (e.g., VWF and FVIII supplementation), and / or risk of blood-transmitted diseases. Therefore, there is an unmet need for the treatment of VWF deficiency that cannot be remedied by currently available therapies.

[0264] Hemophilia A is another disease characterized by disrupted hemostasis resulting from a genetic deficiency in functional FVIII. Such a deficiency can arise from low levels of normal FVIII protein and / or the presence of a dysfunctional variant of FVIII. Hemophilia A presents with internal or external bleeding episodes, with bleeding severity corresponding to the degree of FVIII deficiency. Patients with hemophilia A have an elevated risk for excessive bleeding from common injuries and often exhibit spontaneous bleeding without cause. Sites of bleeding may include skin, joints, muscles, digestive tract, and brain. Various mutations in FVIII can result in a positive diagnosis, with the severity of disease resulting from differences in amount of functional FVIII protein. Patients with less than 1% of normal levels of active FVIII exhibit severe hemophilia, those with 1-5% exhibit moderate hemophilia, and those with 5-40% have mild hemophilia.

[0265] Hey des syndrome is a syndrome featuring abnormal hemostasis from acquired VWD and gastrointestinal bleeding from angiodysplasia, in the presence of aortic stenosis. Patients with aortic valve stenosis have a narrowed valve, resulting in increased sheer stress on blood passing through it. The increase in shear stress causes platelet and VWF activation, further resulting in the activation of ADAMTS13 and degradation of VWF. Accordingly, the reduced levels of VWFin the blood increase the propensity to bleed. Surgical correction of aortic stenosis results in resolution of the acquired VWD in most cases, but not all patients are suitable for surgery.

[0266] Abnormal uterine bleeding (AUB) is a disorder featuring vaginal bleeding from the uterus that is frequent, long-lasting, heavier than normal, and / or irregular, which may lead to anemia and reduced quality of life. AUB affects as many as 25% of women and may be associated with structural and non-structural causes. Examples of structural causes for AUB include leiomyoma (fibroids), adenomyosis (endometrial invasion of myometrium), polyps, and cancer. Non-structural causes for AUB may be medical (e.g., hypothyroidism) or may result from a disorder of hemostasis (accounting for about 10% of all AUB cases). Due to its heterogenous etiology, treatment of AUB is complicated, but generally involves use of hormonal therapies, non-steroidal anti-inflammatory drugs (NSAIDS), and TXA. A recent UK survey of AUB patients (Cox et al., BMC Health Serv Res, 13 :491 (2013)) has reported a low rate of satisfaction with treatment outcomes after one year, highlighting an unmet need for improved therapies to treat AUB.

[0267] Alpha-2-antiplasmin (A2AP / SERPINF2) deficiency is yet another blood disorder characterized by excessive bleeding. A2AP is a serine protease inhibitor that functions in inactivating plasmin and thereby inhibits fibrinolysis. A2AP deficiency is an especially rare disorder with a prevalence of less than 1 in 1,000,000 that occurs in a homozygous or heterozygous form. In the homozygous form, A2AP deficiency presents with severe bleeding characterized by spontaneous hemorrhage and hemarthrosis. The heterozygous form has variable presentation that can be associated with bleeding post-surgery / injury, and spontaneous musculoskeletal bleeds. No effective, long-lasting prophylactic treatments exist for the treatment of A2AP deficiency.

[0268] Disrupted hemostasis can also result from disorders of platelet function. As discussed above, platelet activation, adhesion, and aggregation are critical steps in clot formation and stabilization. Abnormal levels and / or functioning of platelets can result in excessive bleeding (e.g., epistaxis, nosebleeds, gum bleeding, menorrhagia, and prolonged bleeding after cuts or surgery). Platelet disorders can be heritable or acquired. Examples of congenital disorders of platelet function include but are not limited to Glanzmann’s thrombasthenia, Bernard-Soulier syndrome, Chromosome 22qll.2 deletion syndromes, platelet-type VWF (PT-VWD), gray platelet syndrome, May -Hegglin anomaly, Epstein syndrome, Fechter syndrome, Sebastian syndrome, Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, Hermansky-Pudlak syndrome, and storage pool / secretion disorder Non-limiting examples of acquired disorders ofplatelet function include use of certain medications (e.g., aspirin, dipyridamole, clopidogrel, and ibuprofen), liver disease, uremia, and myeloproliferative disorders (e.g., essentialthromb ocy themi a) .

[0269] Abnormal bleeding can also be hallmarks of certain heritable connective tissue disorders, resulting from abnormal vasculature with increased vulnerability to injury and abnormal interactions with platelets and coagulation factors. Examples of such disorders include hereditary hemorrhagic telangiectasia (HHT) / Osler-Weber-Rendu (OWR) disease, which is characterized by formation of anteriovenous malformations (AVM) in internal organs, and telangiectasia on mucocutaneous surfaces including skin, gastrointestinal mucosa, and upper respiratory tract. HHT features an autosomal dominant pattern of inheritance and has a prevalence of 1 / 5000. The majority of HHT patients (80%) have an identifiable causative mutation in ENG, ACVRL1, or MADH4 genes, all of which are involved in TGF-p / BMP signaling pathway required for smooth muscle differentiation and vascular modeling. Mutations in each gene are distinctly associated with particular clinical subtypes of HHT. Specifically, HHT Type 1, accounting for about 60% of all HHT cases, is associated with mutations in ENG (chromosome 9q34.11) and presents with AVM in lungs and brain. HHT Type 2, accounting for about 38% of all HHT cases, is associated with mutations in ACVRL1 (chromosome 12ql3.13) and presents with AVM in liver, lungs, and spine. The third subtype, HHT with juvenile polyposis coli, is associated with mutations in MADH4 (chromosome 18q21.2), presents with AVM and gastrointestinal polyps, and accounts for about 2% of all HHT cases. Among HHT patients, clinical presentation is highly variable and is associated with an increased risk for cerebral abscess, migraine, ischemic / embolic stroke, heart failure, colon cancer, and bleeding complications such as, e.g., epistaxis, gastrointestinal bleeding, hemorrhagic stroke secondary to CNS malformation, pulmonary hemorrhage, increased risk of mortality in pregnancy associated with pulmonary hemorrhage and CNS bleeds, and iron deficiency. Available treatment options for HHT include invasive techniques such as surgery cryotherapy, and ablation, treatment with TXA, hormonal therapy (e.g., estrogen, tamoxifen, or danazol), or an anti-angiogenic therapy (e.g., thalidomide and bevacizumab).

[0270] Deficiency in anti-coagulation factors can also result in abnormal hemostasis. Loss of regulation of these factors results in an increased tendency to form a thrombus. For example, congenital deficiency in AT is an autosomal dominant disease state characterized by increased propensity for clotting, resulting in a 5-50 fold increase in the risk of thrombosis. Congenital AT deficiency is reported to occur with a prevalence of 1 :500 to 1 : 5000. Two subtypes of AT havebeen identified, Type 1 (quantitative defect) and Type 2 (qualitative defect). Type 1 AT deficiency is associated with a decrease in AT activity and blood concentration and can be further divided into subgroup la, featuring normal affinity for heparin, and subgroup lb which features reduced affinity for heparin. Type 1 AT deficiency are predominately due to point mutations, deletions, or insertions within the AT gene. Type 2 AT deficiency is associated with normal AT levels, but reduced AT activity and is further divided into 3 subgroups, namely subgroup 2a, featuring decreased thrombin inactivation, FXa inactivation, and heparin affinity, subgroup 2b, featuring decreased thrombin inactivation and normal heparin affinity, and subgroup 2c, featuring normal thrombin inactivation and Xa inactivation and decreased heparin affinity. AT deficiency can also be acquired by one of three ways: (1) increased AT excretion, e.g., in renal failure associated with proteinuria nephrotic syndrome or in bums; (2) decreased AT production, e.g., as observed in liver dysfunction; or (3) accelerated consumption of AT, e.g., as a result of injury or invasive surgical procedures. Current therapies for congenital AT deficiency rely on anti coagulation therapies, however, these treatments are associated with an increased risk of spontaneous hemorrhage. Other therapies include use of low molecular weight heparin with addition of AT concentrate to overcome heparin resistance. However, AT concentrate is administered as an intravenous infusion, making it suitable only for short-term treatments (e.g., peri-operative bridging).

[0271] In some aspects, a blood disorder characterized by excessive bleeding is selected from a von Willebrand disease (VWD), hemophilia A, disorders of platelet function, and connective tissue disorders. In some aspects, a blood disorder characterized by excessive bleeding is VWD. In some aspects, the VWD is congenital VWD (cVWD). In some aspects, the VWD is acquired VWD (a VWD). In some aspects, the aVWD includes Heydes syndrome. In some aspects, the VWD is type 1 VWD. In some aspects, the VWD is type 2 A VWD. In some aspects, the VWD is type 2B VWD. In some aspects, the VWD is type 2M VWD. In some aspects, the VWD is type 2N VWD. In some aspects, the VWD is type 3 VWD. In some aspects, a blood disorder characterized by excessive bleeding is hemophilia A. In some aspects, a blood disorder characterized by excessive bleeding is a disorder of platelet function. In some aspects, a disorder of platelet function is Glanzmann’s thrombasthenia. In some aspects, a disorder of platelet function is Bernard-Soulier syndrome. In some aspects, a disorder of platelet function is a chromosome 22ql 1.2 deletion syndrome. In some aspects, a disorder of platelet function is platelet type VWD. In some aspects, a disorder of platelet function is gray platelet syndrome. In some aspects, a disorder of platelet function is May-Hegglin anomaly. In some aspects, adisorder of platelet function is Epstein syndrome. In some aspects, a disorder of platelet function is Fechter syndrome. In some aspects, a disorder of platelet function is Sebastian syndrome. In some aspects, a disorder of platelet function is Wiskott-Aldrich syndrome. In some aspects, a disorder of platelet function is Chediak-Higashi syndrome. In some aspects, a disorder of platelet function is Hermansky-Pudlak syndrome. In some aspects, a disorder of platelet function is a storage pool / secretion disorder. In some aspects, a disorder of platelet function is an acquired disorder of platelet function resulting from use of certain medications (e.g., aspirin, dipyridamole, clopidogrel, and ibuprofen. In some aspects, a disorder of platelet function is an acquired disorder of platelet function due to liver disease. In some aspects, a disorder of platelet function is an acquired disorder of platelet function due to uremia. In some aspects, a disorder of platelet function is an acquired disorder of platelet function due to a myeloproliferative disorder (e.g., essential thrombocythemia). In some aspects, a disorder of platelet function is idiopathic, but evident in one or more assays used to assess platelet function. In some aspects, a blood disorder characterized by excessive bleeding is a connective tissue disorder. In some aspects, a connective tissue disorder is HHT. In some aspects, the HHT is type 1 HHT. In some aspects, the HHT is type 2 HHT. In some aspects, the HHT is HHT with juvenile polyposis coli.

[0272] The antibody or the pharmaceutical composition disclosed herein are, in some aspects, administered to the subject in need thereof in an amount and for a time sufficient to treat one or more symptoms selected from the group consisting of epistaxis, cutaneous bleeding, bleeding from minor wounds, oral-cavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, and central nervous system (CNS) bleeding.

[0273] Disclosed herein, in certain aspects, are methods of accumulating VWF protein in blood plasma of a subject in need thereof, comprising administering to the subject a composition comprising an antibody or pharmaceutical composition disclosed herein. In some aspects, the accumulation of the VWF protein results in a blood plasma level of the VWF protein that is at least 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater as compared to a blood plasma level of the VWF protein in the absence of the antibody. In some aspects, the accumulation of the VWF protein results in a blood plasma level of the VWF protein that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%,800%, 850%, 900%, 950%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, 5,000%, 5,500%, 6,000%, 6,500%, 7,000%, 7,500%, 8,000%, 8,500%, 9,000%, 9,500%, 10,000%, or greater as compared to a blood plasma level of the VWF protein in the absence of the antibody. In some aspects, the degree of accumulation of a VWF protein depends on the effective amount of an antibody or antigen-binding fragment thereof administered to a subject, as described herein.

[0274] Disclosed herein, in certain aspects, are methods of accumulating Factor VIII protein in blood plasma of a subject in need thereof, comprising administering to the subject a composition comprising an antibody or pharmaceutical composition disclosed herein. In some aspects, the accumulation of the Factor VIII protein results in a blood plasma level of the Factor VIII protein that is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold greater as compared to a blood plasma level of the Factor VIII protein in the absence of the antibody. In some aspects, the accumulation of the Factor VIII protein results in a blood plasma level of the Factor VIII protein that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, 5,000%, 5,500%, 6,000%, 6,500%, 7,000%, 7,500%, 8,000%, 8,500%, 9,000%, 9,500%, 10,000%, or greater as compared to the blood plasma level of a VWF protein in the absence of the antibody. In some aspects, the degree of accumulation of a FVIII protein depends on the effective amount of an antibody or antigen-binding fragment thereof administered to a subject, as described herein.

[0275] Also disclosed herein, in certain aspects, are methods of increasing blood-plasma half-life of a VWF protein in a subject in need thereof, comprising administering to the subject a composition comprising an antibody or pharmaceutical composition disclosed herein. In some aspects, the antibody increases the blood plasma half-life of the VWF protein by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to the blood plasma halflife of the VWF protein in the absence of the antibody. In some aspects, the antibody increases the blood plasma half-life of the VWF protein by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1,000%, 1,500%, 2,000%, 2,500%, 3,000%, 3,500%, 4,000%, 4,500%, 5,000%, 5,500%, 6,000%, 6,500%, 7,000%, 7,500%, 8,000%,8,500%, 9,000%, 9,500%, or 10,000% as compared to the blood plasma half-life of the VWF protein in the absence of the antibody.

[0276] In some aspects, the antibody does not bind dog, mouse, or rat VWF CK domains. In some aspects, the antibody increases VWF antigen by about 1.5 fold or more. In some aspects, the antibody increases VWF antigen by about 2 fold or more within the first week post-initial treatment.

[0277] In some aspects, the antibody does not elicit a cytokine release.

[0278] In some aspects, the antibody does not prevent or inhibit the binding of VWF to GPlba. In some aspects, the antibody results in greater binding of VWF to GPlba than a comparator or an anti-VWF-Al domain antibody. In some aspects, the antibody does not prevent or inhibit the binding of VWF to collagen. In some aspects, the antibody results in greater binding of VWF to collagen than a comparator or an anti-VWF-A3 domain antibody.

[0279] In some aspects, the antibody does change the VWF multimer distribution (e.g. low, medium and / or high molecular weight VWF fraction) in subject plasma post administration.

[0280] In some aspects, the antibody binds FcRn, e.g., with a binding affinity similar to an IgG4 control antibody.

[0281] Any of the methods disclosed herein, in some instances, further include administering to the subject an additional therapeutic modality. Non-limiting examples of additional therapeutic modalities include therapeutic agents other than the antibody of the disclosure (e.g., compounds, peptides, nucleic acids, gene therapy, cell therapy etc.). In some aspects, other antibodies, small molecule therapeutics, and / or other agents are combined in separate compositions for simultaneous or sequential administration. In one aspect, simultaneous administration comprises one or more compositions that are administered at the same time, or within 30 minutes of each other. In some aspects, administration occurs at the same or different sites.Route of Administration

[0282] Antibodies or antigen-binding fragments thereof are formulated for any suitable route of administration to a subject including, but not limited to injection (e.g., intravenous injection), in some aspects. Injection includes, e.g., subcutaneous, peritoneal, intravenous injection, intramuscular injection, or spinal injection into the cerebrospinal fluid (CSF). In some aspects, the antibodies of the disclosure are formulated for subcutaneous administration. In some aspects, the antibodies of the disclosure are formulated for peritoneal administration. In some aspects, the antibodies of the disclosure are formulated for intravenous administration (e.g., intravenousinjection or infusion). In some aspects, the antibodies of the disclosure are formulated for intramuscular administration. In some aspects, the antibodies of the disclosure are formulated for spinal injection into the cerebrospinal fluid (CSF) administration. In some aspects, administration is in one, two, three, four, five, six, seven, or more injection sites. In some aspects, administration is in one injection site. In some aspects, administration is in two injection sites. In some aspects, administration is in three injection sites. In some aspects, administration is in four injection sites. In some aspects, administration is in five injection sites. In some aspects, administration is in six injection sites.

[0283] For in vivo applications, contacting occurs, e.g., via administration of a composition (e.g., a composition described herein) to a subject by any suitable means. An antibody described herein, in some aspects, is administered, e.g., either systemically or locally, including via parenteral, subcutaneous, intraperitoneal, intracerebrospinal, intrapulmonary, and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral routes include, e.g., intravenous, intraarterial, intraperitoneal, epidural, intramuscular, and intrathecal administration. Such administration, in some aspects, is as a bolus, continuous infusion, or pulse infusion. In some aspects, compositions are administered by injection depending in part on whether the administration is brief or chronic. Other modes of administration methods are contemplated, including topical, particularly transdermal, transmucosal, rectal, oral, or local administration e.g., through a catheter placed close to the desired site.Dosing Regimens

[0284] The antibodies described herein are particularly attractive medicaments for the prophylactic, subcutaneous treatment of subjects that have a bleeding disorder, such as VWD. The antibodies described herein may be administered as a single dose administration. The antibodies described herein may be administered at a regular interval. For example, the antibodies described herein may be administered about once weekly (“Q1W”), about once every two weeks (“Q2W”), about once every three weeks (“Q3W”), about once every four weeks (“Q4W”), about once a month, or about once every two months.

[0285] In some aspects of the disclosure, the antibody is administered at a flat dose from about 10 mg to about 300 mg. In some aspects of the disclosure, the antibody is administered at a flat dose from about 20 mg to about 300 mg. In some aspects of the disclosure, the antibody is administered at a flat dose of about 10 mg. In some aspects of the disclosure, the antibody is administered at a flat dose of about 300 mg. In some aspects of the disclosure, the antibody isadministered at a flat dose of about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 mg, about 215 mg, about 220 mg, about 225 mg, about 230 mg, about 235 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 315 mg, or about 320 mg. In some aspects of the disclosure, the antibody is administered at a flat dose of from about 15 mg to about 25 mg, about 20 mg to about 40 mg, about 40 mg to about 60 mg, about 60 mg to about 80 mg, about 80 mg to about 100 mg, about 100 mg to about 120 mg, about 120 mg to about 140 mg, about 140 mg to about 160 mg, about 160 mg to about 180 mg, about 180 mg to about 200 mg, about 200 mg to about 220 mg, about 220 mg to about 240 mg, about 240 mg to about 260 mg, about 260 mg to about 280 mg, about 280 mg to about 300 mg, or about 300 mg to about 320 mg. In some aspects of the disclosure, the antibody is administered at a flat dose of from about 20 mg to about 70 mg, about 70 mg to about 120 mg, about 120 mg to about 170 mg, about 170 mg to about 220 mg, about 220 mg to about 270 mg, or about 270 mg to about 320 mg.

[0286] In some aspects the antibody is administered at a flat dose of 20 mg. In some aspects, the antibody is administered at a flat dose of 20 mg once weekly ("Q1W"). In some aspects, the antibody is administered at a flat dose of 20 mg about once every two weeks ("Q2W"). In some aspects, the antibody is administered at a flat dose of 20 mg about once every three weeks ("Q3W"). In some aspects, the antibody is administered at a flat dose of 20 mg about once every four weeks ("Q4W"). In some aspects, the antibody is administered at a flat dose of 20 mg about once a month. In some aspects, the antibody is administered at a flat dose of 20 mg about once every two months.

[0287] In some aspects, the antibody is administered at a flat dose of 50 mg. In some aspects, the antibody is administered at a flat dose of 50 mg once weekly ("Q1W"). In some aspects, the antibody is administered at a flat dose of 50 mg about once every two weeks ("Q2W"). In some aspects, the antibody is administered at a flat dose of 50 mg about once every three weeks ("Q3W"). In some aspects, the antibody is administered at a flat dose of 50 mg about once every four weeks ("Q4W"). In some aspects, the antibody is administered at a flat dose of 50 mg aboutonce a month. In some aspects, the antibody is administered at a flat dose of 50 mg about once every two months.

[0288] In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 20 mg. In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 20 mg once weekly ("Q1W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 20 mg about once every two weeks ("Q2W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 20 mg about once every three weeks ("Q3W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 20 mg about once every four weeks ("Q4W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 20 mg about once a month. In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 20 mg about once every two months.

[0289] In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 50 mg. In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 50 mg once weekly ("Q1W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 50 mgabout once every two weeks ("Q2W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 50 mg about once every three weeks ("Q3W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 50 mg about once every four weeks ("Q4W"). In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 50 mg about once a month. In some aspects, an antibody comprising a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10 is administered at a flat dose of 50 mg about once every two months.

[0290] In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 20 mg. In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 20 mg once weekly ("Q1W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 20 mg about once every two weeks ("Q2W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 20 mg about once every three weeks ("Q3W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of20 mg about once every four weeks ("Q4W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 20 mg about once a month. In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 20 mg about once every two months.

[0291] In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 50 mg. In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 50 mg once weekly ("Q1W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 50 mg about once every two weeks ("Q2W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 50 mg about once every three weeks ("Q3W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 50 mg about once every four weeks ("Q4W"). In some aspects, an antibody comprising a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 50 mg about once a month. In some aspects, an antibody comprising a first heavy chain (HC)comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14 is administered at a flat dose of 50 mg about once every two months.

[0292] In some aspects of the disclosure, the antibody is administered at a weight-based dose. In some aspects of the disclosure, the antibody is administered at a dose from about 0.1 mg / kg to about 6.5 mg / kg. In some aspects of the disclosure, the antibody is administered at a dose of about 0.1 mg / kg. In some aspects of the disclosure, the antibody is administered at a dose of about 0.5 mg / kg. In some aspects of the disclosure, the antibody is administered at a dose of about 6 mg / kg. In some aspects of the disclosure, the antibody is administered at a dose from about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3 mg / kg, about 3.1 mg / kg, about 3.2 mg / kg, about 3.3 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, about 4.5 mg / kg, about 4.6 mg / kg, about 4.7 mg / kg, about 4.8 mg / kg, about 4.9 mg / kg, about 5 mg / kg, about 5.1 mg / kg, about 5.2 mg / kg, about 5.3 mg / kg, about 5.4 mg / kg, about 5.5 mg / kg, about 5.6 mg / kg, about 5.7 mg / kg, about 5.8 mg / kg, about 5.9 mg / kg, about 6 mg / kg, about 6.1 mg / kg, about 6.2 mg / kg, about 6.3 mg / kg, about 6.4 mg / kg, about 6.5 mg / kg. In some aspects of the disclosure, the antibody is administered at a dose from about 0.1 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 1.5 mg / kg, about 1.5 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 2.5 mg / kg, about 2.5 mg / kg to about 3.0 mg / kg, about 3.0 mg / kg to about 3.5 mg / kg, about 3.5 mg / kg to about 4.0 mg / kg, about 4.0 mg / kg to about 4.5 mg / kg, about 4.5 mg / kg to about 5.0 mg / kg, about 5.0 mg / kg to about 5.5 mg / kg, about 5.5 mg / kg to about 6.0 mg / kg, or about 6.0 mg / kg to about 6.5 mg / kg. In some aspects of the disclosure, the antibody is administered at a dose from about 0.1 mg / kg to about 1.0 mg / kg, about 1.0 mg / kg to about 2.0 mg / kg, about 2.0 mg / kg to about 3.0 mg / kg, about 3.0 mg / kg to about 4.0 mg / kg, about 4.0 mg / kg to about 5.0 mg / kg, about 5.0 mg / kg to about 6.0 mg / kg, or about 6.0 mg / kg to about 6.5 mg / kg.Combination Therapy

[0293] In some aspects of the disclosure, the antibodies and compositions described herein are administered in combination with one or more (e.g., 1, 2, 3, 4, 5 or more) additional therapeutic agents or modalities for treatment of a disease or disorder described herein (e.g., a blood disorder characterized by excessive bleeding), in some aspects.

[0294] In some aspects, the one or more additional therapeutic agents is desmopressin (DDAVP), tranexamic acid (TXA), thalidomide, tamoxifen, ocreotide, a concentrate of plasma-derived VWF protein, a concentrate of VWF protein in complex with FVIII (VWF :F VIII; e.g., Humate P and Wilate), or a concentrate of recombinant VWF protein (e.g., Vonicog alfa), or a concentrate of FVIII protein (e.g., Alphanate and Koate-HP). In cases where the blood disorder is heavy menstrual bleeding, the one or more additional therapeutic agents is a hormonal treatment, such as, e.g., an oral contraceptive pill (e.g., an estrogen-containing contraceptive) or an intrauterine device, in some aspects. In some aspects, the one or more additional therapeutic modalities is gene therapy. In some aspects, the gene therapy is FVIII gene therapy. For example, an antibody or composition of the disclosure may be administered in combination with a FVIII gene therapy to extend the half-life of FVIII and / or promote accumulation of FVIII in blood plasma of a subject. In some aspects, an antibody or composition of the disclosure is administered to a patient for whom FVIII gene therapy alone is insufficient to attain therapeutically effective levels of FVIII in blood plasma. In some aspects, an antibody or composition of the disclosure achieves therapeutically effective levels of FVIII in blood plasma.

[0295] In some aspects, the one or more additional therapeutic modalities include blood transfusion, platelet transfusion, surgery, or cauterization. In some aspects, the one or more additional therapeutic modalities include blood transfusion. In some aspects, the one or more additional therapeutic modalities include platelet transfusion. In some aspects, the one or more additional therapeutic modalities include surgery. In some aspects, the one or more additional therapeutic modalities include cauterization.

[0296] In some aspects, a composition (an antibody or an antigen-binding fragment described herein) is administered alone or in combination with a second composition either simultaneously or sequentially dependent upon the condition to be treated.

[0297] Disclosed herein, in certain aspects, are methods for the treatment of blood disorders in a patient in need thereof, comprising administering to the patient (a) a first pharmaceutical composition comprising a monospecific antibody targeted against von Willebrand factor (VWF),wherein binding of the antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium. In some aspects, the mature VWF is a recombinant human von Willebrand Factor (rhVWF). In some aspects, the mature VWF is plasma-derived human von Willebrand Factor (pdVWF). In some aspects, the agent that induces the release of VWF from the vascular endothelium is DDAVP.

[0298] In some aspects, the monospecific antibody targeted against von Willebrand factor (VWF) and the mature VWF protein or functional fragment thereof are administered simultaneously or sequentially. In some aspects, the monospecific antibody targeted against von Willebrand factor (VWF) is administered prior to the mature VWF protein or functional fragment thereof. In some aspects, the monospecific antibody targeted against von Willebrand factor (VWF) is administered after the mature VWF protein or functional fragment thereof.

[0299] Blood disorders characterized by excessive bleeding arise, in part, due to dysregulation of hemostasis, which can occur when proteins involved in the clotting cascades are disrupted. For example, von Willebrand disease (VWD) is an inherited bleeding disorder that results from deficiency in VWF. Such deficiency can arise from low levels of VWF (i.e., quantitative effect) or from presence of dysfunctional VWF (i.e., qualitative effect). It is one of the most common inherited blood clotting disorders in humans. Hereditary forms of VWD include type 1, type 2, type 3, and platelet-type VWD (PT-VWD). In addition to inherited pathology, VWD exists in acquired forms. Generally, VWD is characterized by tendency to bleed, easy bruising, nosebleeds, bleeding gums, heavy menstrual bleeding, blood loss during childbirth, and bleeding following surgical intervention. Recently, another diagnostic category of “low VWF” has emerged, corresponding to individuals with VWF levels that are below normal but insufficient to meet diagnostic criteria for VWD. VWF deficiency in VWD presents primarily in organs with small vessels, including skin, gastrointestinal tract, uterus, and colon. Low VWF is often defined as 30-50% VWF activity level, though definitions can vary from country to country.

[0300] As mentioned above, hereditary VWD occurs in multiple forms, including type 1, type 2, type 3, and platelet-type. Type 1 VWD is the predominant form (40-80% of all cases). While precise definitions may vary from country to country, Type 1 VWD is often defined as < 30% VWF or < 50% VWF as compared to a healthy subject. Type 1 VWD patients can be asymptomatic and lead a normal life without complications. Therefore, although VWD occurs inabout 1% of the population, clinically significant cases are observed in about 1 / 10,000 cases. Type 2 VWD corresponds to 15-50% of all cases and generally presents as a bleeding tendency that varies between patients. Type 2 VWD can be further classified into subtypes based on the presence and behavior of VWF multimers, including Types 2A, 2B, 2M, and 2N. Type 2A is characterized by a defect in the ability to form large VWF multimers. Type 2M VWD results from reduced binding between VWF and the GPlb platelet receptor or collagen, resulting in reduced platelet adhesion and aggregation. Type 2N VWD corresponds to a deficiency in binding between VWF and FVIII, resulting in reduced blood plasma levels of F VIII similar to those seen in hemophilia A. Type 3 VWD is characterized by complete or almost complete absence of VWF in plasma, resulting in the most severe form of VWD. Type 3 VWD is also associated with very low blood plasma levels of FVIII. Platelet-type VWD presents with qualitatively and quantitatively normal VWF protein, but features a defect in the platelet GPlb receptor that results in increased affinity to VWF. This increased affinity to VWF produces abnormally large platelet aggregates that are cleared from circulation along with VWF multimers. The pathogenesis of acquired VWD is variable. It can be associated with the presence of autoantibodies to VWF, resulting in rapid clearance of VWF-autoantibody complexes from the circulation, or interference with its function. Acquired VWD can also occur with adsorption of VWF by tumor cells and non-immunologic mechanisms of destruction. Current treatments for VWD include use of desmopressin, tranexamic acid (TXA), and concentrates of plasma-derived VWF and / or FVIII or recombinant VWF. However, these therapies suffer from several shortcomings, including but not limited to short half-lives which pose challenges to use in bleeding prophylaxis, gastrointestinal side effects, limited efficacy in major bleeds, high cost (e.g., VWF / FVIII supplementation), inconvenient intravenous administration (e.g., VWF and FVIII supplementation), and / or risk of blood-transmitted diseases. Therefore, there is an unmet need for the treatment of VWF deficiency that cannot be remedied by currently available therapies.

[0301] Hemophilia A is another disease characterized by disrupted hemostasis resulting from a genetic deficiency in functional FVIII. Such a deficiency can arise from low levels of normal FVIII protein and / or the presence of a dysfunctional variant of FVIII. Hemophilia A presents with internal or external bleeding episodes, with bleeding severity corresponding to the degree of FVIII deficiency. Patients with hemophilia A have an elevated risk for excessive bleeding from common injuries and often exhibit spontaneous bleeding without cause. Sites of bleeding may include skin, joints, muscles, digestive tract, and brain. Various mutations in FVIII can result in a positive diagnosis, with the severity of disease resulting from differences in amount of functionalFVIII protein. Patients with less than 1% of normal levels of active FVIII exhibit severe hemophilia, those with 1-5% exhibit moderate hemophilia, and those with 5-40% have mild hemophilia.

[0302] Hey des syndrome is a syndrome featuring abnormal hemostasis from acquired VWD and gastrointestinal bleeding from angiodysplasia, in the presence of aortic stenosis. Patients with aortic valve stenosis have a narrowed valve, resulting in increased sheer stress on blood passing through it. The increase in shear stress causes platelet and VWF activation, further resulting in the activation of ADAMTS13 and degradation of VWF. Accordingly, the reduced levels of VWF in the blood increase the propensity to bleed. Surgical correction of aortic stenosis results in resolution of the acquired VWD in most cases, but not all patients are suitable for surgery.

[0303] Abnormal uterine bleeding (AUB) is a disorder featuring vaginal bleeding from the uterus that is frequent, long-lasting, heavier than normal, and / or irregular, which may lead to anemia and reduced quality of life. AUB affects as many as 25% of women and may be associated with structural and non-structural causes. Examples of structural causes for AUB include leiomyoma (fibroids), adenomyosis (endometrial invasion of myometrium), polyps, and cancer. Non-structural causes for AUB may be medical (e.g., hypothyroidism) or may result from a disorder of hemostasis (accounting for about 10% of all AUB cases). Due to its heterogenous etiology, treatment of AUB is complicated, but generally involves use of hormonal therapies, non-steroidal anti-inflammatory drugs (NSAIDS), and TXA. A recent UK survey of AUB patients (Cox et al., BMC Health Serv Res, 13 :491 (2013)) has reported a low rate of satisfaction with treatment outcomes after one year, highlighting an unmet need for improved therapies to treat AUB.

[0304] Alpha-2-antiplasmin (A2AP / SERPINF2) deficiency is yet another blood disorder characterized by excessive bleeding. A2AP is a serine protease inhibitor that functions in inactivating plasmin and thereby inhibits fibrinolysis. A2AP deficiency is an especially rare disorder with a prevalence of less than 1 in 1,000,000 that occurs in a homozygous or heterozygous form. In the homozygous form, A2AP deficiency presents with severe bleeding characterized by spontaneous hemorrhage and hemarthrosis. The heterozygous form has variable presentation that can be associated with bleeding post-surgery / injury, and spontaneous musculoskeletal bleeds. No effective, long-lasting prophylactic treatments exist for the treatment of A2AP deficiency.

[0305] Disrupted hemostasis can also result from disorders of platelet function. As discussed above, platelet activation, adhesion, and aggregation are critical steps in clot formation andstabilization. Abnormal levels and / or functioning of platelets can result in excessive bleeding (e.g., epistaxis, nosebleeds, gum bleeding, menorrhagia, and prolonged bleeding after cuts or surgery). Platelet disorders can be heritable or acquired. Examples of congenital disorders of platelet function include but are not limited to Glanzmann’s thrombasthenia, Bernard-Soulier syndrome, Chromosome 22qll.2 deletion syndromes, platelet-type VWD, gray platelet syndrome, May -Hegglin anomaly, Epstein syndrome, Fechter syndrome, Sebastian syndrome, Wiskott-Aldrich syndrome, Chediak-Higashi syndrome, Hermansky-Pudlak syndrome, and storage pool / secretion disorder. Non-limiting examples of acquired disorders of platelet function include use of certain medications (e.g., aspirin, dipyridamole, clopidogrel, and ibuprofen), liver disease, uremia, and myeloproliferative disorders (e.g., essential thrombocythemia).

[0306] Abnormal bleeding can also be hallmarks of certain heritable connective tissue disorders, resulting from abnormal vasculature with increased vulnerability to injury and abnormal interactions with platelets and coagulation factors. Examples of such disorders include hereditary hemorrhagic telangiectasia (HHT) / Osler-Weber-Rendu (OWR) disease, which is characterized by formation of anteriovenous malformations (AVM) in internal organs, and telangiectasia on mucocutaneous surfaces including skin, gastrointestinal mucosa, and upper respiratory tract. HHT features an autosomal dominant pattern of inheritance and has a prevalence of 1 / 5000. The majority of HHT patients (80%) have an identifiable causative mutation in ENG, ACVRL1, or MADH4 genes, all of which are involved in TGF-p / BMP signaling pathway required for smooth muscle differentiation and vascular modeling. Mutations in each gene are distinctly associated with particular clinical subtypes of HHT. Specifically, HHT Type 1, accounting for about 60% of all HHT cases, is associated with mutations in ENG (chromosome 9q34.11) and presents with AVM in lungs and brain. HHT Type 2, accounting for about 38% of all HHT cases, is associated with mutations in ACVRL1 (chromosome 12ql3.13) and presents with AVM in liver, lungs, and spine. The third subtype, HHT with juvenile polyposis coli, is associated with mutations in MADH4 (chromosome 18q21.2), presents with AVM and gastrointestinal polyps, and accounts for about 2% of all HHT cases. Among HHT patients, clinical presentation is highly variable and is associated with an increased risk for cerebral abscess, migraine, ischemic / embolic stroke, heart failure, colon cancer, and bleeding complications such as, e.g., epistaxis, gastrointestinal bleeding, hemorrhagic stroke secondary to CNS malformation, pulmonary hemorrhage, increased risk of mortality in pregnancy associated with pulmonary hemorrhage and CNS bleeds, and iron deficiency. Available treatment options for HHT include invasive techniques such as surgery cryotherapy, and ablation, treatment withTXA, hormonal therapy (e.g., estrogen, tamoxifen, or danazol), or an anti-angiogenic therapy (e.g., thalidomide and bevacizumab).

[0307] In some aspects, a blood disorder characterized by excessive bleeding is selected from a von Willebrand disease (VWD), hemophilia A, disorders of platelet function, and connective tissue disorders. In some aspects, a blood disorder characterized by excessive bleeding is VWD. In some aspects, the VWD is congenital VWD (cVWD). In some aspects, the VWD is acquired VWD (a VWD). In some aspects, the aVWD includes Heydes syndrome. In some aspects, the VWD is type 1 VWD. In some aspects, the VWD is type 2 A VWD. In some aspects, the VWD is type 2M VWD. In some aspects, the VWD is type 2N VWD. In some aspects, the VWD is type 3 VWD. In some aspects, a blood disorder characterized by excessive bleeding is hemophilia A. In some aspects, a blood disorder characterized by excessive bleeding is a disorder of platelet function. In some aspects, a blood disorder characterized by excessive bleeding is abnormal uterine bleeding (AUB). In some aspects, a blood disorder characterized by excessive bleeding is alpha-2-antiplasmin (A2AP) deficiency. In some aspects, a disorder of platelet function is Glanzmann’s thrombasthenia. In some aspects, a disorder of platelet function is Bernard-Soulier syndrome. In some aspects, a disorder of platelet function is a chromosome 22ql 1.2 deletion syndrome. In some aspects, a disorder of platelet function is platelet type VWD. In some aspects, a disorder of platelet function is gray platelet syndrome. In some aspects, a disorder of platelet function is May-Hegglin anomaly. In some aspects, a disorder of platelet function is Epstein syndrome. In some aspects, a disorder of platelet function is Fechter syndrome. In some aspects, a disorder of platelet function is Sebastian syndrome. In some aspects, a disorder of platelet function is Wiskott-Aldrich syndrome. In some aspects, a disorder of platelet function is Chediak-Higashi syndrome. In some aspects, a disorder of platelet function is Hermansky-Pudlak syndrome. In some aspects, a disorder of platelet function is a storage pool / secretion disorder. In some aspects, a disorder of platelet function is an acquired disorder of platelet function resulting from use of certain medications (e.g., aspirin, dipyridamole, clopidogrel, and ibuprofen. In some aspects, a disorder of platelet function is an acquired disorder of platelet function due to liver disease. In some aspects, a disorder of platelet function is an acquired disorder of platelet function due to uremia. In some aspects, a disorder of platelet function is an acquired disorder of platelet function due to a myeloproliferative disorder (e.g., essential thrombocythemia). In some aspects, a disorder of platelet function is idiopathic, but evident in one or more assays used to assess platelet function. In some aspects, a blood disorder characterized by excessive bleeding is a connective tissue disorder. In some aspects, a connective tissue disorder is HHT. In someaspects, the HHT is type 1 HHT. In some aspects, the HHT is type 2 HHT. In some aspects, the HHT is HHT with juvenile polyposis coli.

[0308] In some aspects, the blood disorder is an episode of unwanted or excessive bleeding. In some aspects, the unwanted or excessive bleeding is minor bleeding. In some aspects, the minor bleeding is readily managed epistaxis, oral bleeding, or menorrhagia. In some aspects, the unwanted or excessive bleeding is major bleeding. In some aspects, the major bleeding is severe or refractory epistaxis, menorrhagia, GI bleeding, CNS trauma, hemarthrosis, or traumatic hemorrhage. In some aspects, the unwanted or excessive bleeding is associated with von Willebrand disease (VWD), or hemophilia A.Bleeding Events

[0309] In some aspects, the method comprises prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF. In some aspects, the monospecific antibody targeted against VWF is administered regularly to the patient to reduce the frequency of a bleeding event, inhibit the occurrence of a bleeding event, or reduce the severity of a bleeding event. In some aspects, the monospecific antibody targeted against VWF is administered to the patient on any suitable schedule. For example, the antibody may be administered weekly, every other week, or once a month.Minor Bleeding Events - VWF on demand

[0310] In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising a mature VWF protein or functional fragment thereof. Where the bleeding event is a minor bleeding event, in some aspects, mature VWF protein is administered to the patient at an initial dose of 30 to 50 lU / kg of patient body weight. In some aspects, a subsequent dose of mature VWF protein is administered at a dose of 30 to 50 lU / kg of patient body weight every 8, 12 or 24 hours as clinically required.Major Bleeding Events - VWF on demand

[0311] In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising a mature VWF protein or functional fragment thereof. Where the bleeding event is a major bleeding event, in some aspects,mature VWF protein or functional fragment thereof is administered to the patient at an initial dose of 35 to 80 lU / kg of patient body weight. In some aspects, a subsequent dose of mature VWF protein or functional fragment thereof is administered at a dose of 35 to 80 lU / kg of patient body weight every 8, 12 or 24 hours for approximately 2 to 4 days as clinically required.Bleeding Event - DDAVP on demand

[0312] In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising a therapeutic agent that induces the release of VWF from the vascular endothelium, e.g. DDAVP. In some aspects, an initial dose of DDAVP is administered by IV infusion over 15-30 min at a dose of 0.3 micrograms / kg of patient body weight (to a maximum dosage of 20 micrograms) diluted in 50 mL in 0.9% saline. In some aspects, an initial dose of DDAVP is administered by inhalation at a dose of 150 micrograms in one nostril. In some aspects, an initial dose of DDAVP is administered by inhalation at a dose of 150 micrograms in each nostril. In some aspects, a subsequent dose of DDAVP is administered after 8 hours to 12 hours and once daily thereafter, based upon clinical condition and von Willebrand factor and factor VIII levels.VWF Levels

[0313] In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium when the blood plasma level of VWF of the patient is indicative of an increased risk for unwanted or excessive bleeding event.

[0314] von Willebrand Factor (VWF) levels in patients are determined by measuring the patient’s plasma VWF antigen (VWF:Ag) level or VWF activity level. Exemplary VWF activity assays include the GPlbR and GPlbM assays, the VWF:CB assay, and the VWF:RCo assay. Generally, a plasma VWF activity of less than 50 lU / dL is indicative of a bleeding disorder or an increased risk for unwanted bleeding events. While definitions may vary from country to country, generally patients with plasma VWF levels of between 30-50 lU / dL may not be associated with VWF mutations and can be considered as representing “low” VWF as a risk factor for bleeding, von Willebrand Factor levels will vary between bleeding disorders and evenwithin bleeding disorders, with the common theme being that the lower the VWF levels, the greater the risk for bleeding. Exemplary diseases that can be identified by plasma VWF levels include Type 1 VWD, which generally has a plasma VWF level of below 30 lU / dL, for example 15 lU / dL (i.e., partial quantitative deficiency of VWF). Plasma VWF level of less than 2 lU / dL (i.e., virtually complete deficiency of VWF) are generally classified as Type 3 VWD.VWF Levels - VWF on Demand

[0315] In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium when the blood plasma level of VWF of the patient is below 50 lU / dL. In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium when the blood plasma level of VWF of the patient is below 30 lU / dL. In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium when the blood plasma level of VWF of the patient is below 20 lU / dL. In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium when the blood plasma level of VWF of the patient is below 10 lU / dL.

[0316] In some aspects, mature VWF protein is administered to the patient at an initial dose of 20 to 60 lU / kg of patient body weight. In some aspects, a subsequent dose of mature VWF protein is administered at a dose of 20 to 60 lU / kg of patient body weight every 8, 12 or 24 hours as clinically required.VWF Levels - DDAPV on Demand

[0317] In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising a therapeutic agent that induces the release of VWF from the vascular endothelium, e.g. DDAVP, when the blood plasma level of VWF of the patient is below 50 lU / dL. In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising a therapeutic agent that induces the release of VWF from the vascular endothelium, e.g. DDAVP, when the blood plasma level of VWF of the patient is below 30 HJ / dL. In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising a therapeutic agent that induces the release of VWF from the vascular endothelium, e.g. DDAVP, when the blood plasma level of VWF of the patient is below 20 lU / dL. In some aspects, the method comprises (a) prophylactically administering to the patient the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising a therapeutic agent that induces the release of VWF from the vascular endothelium, e.g. DDAVP, when the blood plasma level of VWF of the patient is below 10 lU / dL.

[0318] In some aspects, an initial dose of DDAVP is administered by IV infusion over 15-30 min at a dose of 0.3 micrograms / kg of patient body weight (to a maximum dosage of 20 micrograms) diluted in 50 mL in 0.9% saline. In some aspects, an initial dose of DDAVP is administered by inhalation at a dose of 150 micrograms in one nostril. In some aspects, an initial dise of DDAVP is administered by inhalation at a dose of 150 micrograms in each nostril. In some aspects, a subsequent dose of DDAVP is administered after 8 hours to 12 hours and once daily thereafter, based upon clinical condition and von Willebrand factor and factor VIII levels.Prophylaxis with 2nd Composition

[0319] In some aspects, the method comprises prophylactically administering to the patient the second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium. In some aspects, the second pharmaceutical composition is administered regularlyto the patient to reduce the frequency of a bleeding event, inhibit the occurrence of a bleeding event, or reduce the severity of a bleeding event. In some aspects, the second pharmaceutical composition is administered to the patient on any suitable schedule.

[0320] In some aspects, mature VWF protein is administered twice a week, weekly, every other week, or once a month. In some aspects, the method comprises prophylactically administering mature VWF protein at a dose of between 20-60 lU / kg of patient body weight. In some aspects, mature VWF protein is administered 2 to 3 times per week. In some aspects, mature VWF protein is administered twice per week. In some aspects, mature VWF protein is administered every 2 to 3 days. In individuals diagnosed with type 3 VWD, in some aspects the method comprises prophylactically administering the second pharmaceutical composition comprising the mature VWF protein at a dose of between 40-60 lU / kg of patient body weight of twice per week.

[0321] In some aspects, DDAVP is administered by IV infusion over 15-30 min at a dose of 0.3 micrograms / kg of patient body weight (to a maximum dosage of 20 micrograms) diluted in 50 mL in 0.9% saline. In some aspects, DDAVP is administered by inhalation at a dose of 150 micrograms in one nostril. In some aspects, DDAVP is administered by inhalation at a dose of 150 micrograms in each nostril. Dosages of DDAVP are based on clinical condition and von Willebrand factor and factor VIII levels.Prophylaxis with both Compositions

[0322] In some aspects, the method comprises prophylactically administering to the patient (a) the first pharmaceutical composition comprising the monospecific antibody targeted against VWF and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium.Von Willebrand Factor

[0323] Disclosed herein, in certain aspects, are methods for the treatment of blood disorders in a patient in need thereof, comprising administering to the patient (a) a first pharmaceutical composition comprising a monospecific antibody targeted against von Willebrand factor (VWF), wherein binding of the antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or functional fragment thereof or (ii) a therapeutic agent that induces the release of VWFfrom the vascular endothelium. In some aspects, the von Willebrand factor is a recombinant human von Willebrand Factor (rhVWF). In some aspects, the von Willebrand factor is plasma-derived VWF (pdVWF). In some aspects, the agent that induces the release of VWF from the vascular endothelium is desmopressin (DDAVP).

[0324] VWF is synthesized by endothelial cells and megakaryocytes as pre-pro-VWF with a 22- amino acid (aa) signal peptide, 741 aa propeptide, and 2050 aa mature VWF protein (SEQ ID NO: 1).MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYL LAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLY LETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEG TLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARC HPLVDPEPFVALCEKTLCECAGGLECACP ALLEY ARTCAQEGMVLYGWTDHSACSPVC PAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSG KRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLA RDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQL PLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGN QGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLT SPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPG RCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKA QCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSZ SCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALER CPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPG ECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKD ETHFEWESGRYIILLLGKALSWWDRHLSISWLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQ VEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLV DPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKWTWRTATLCPQSCEERNLREN GYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVC EVAGRRFASGKKVTLNPSDPEHCQICHCDWNLTCEACQEPGGLWPPTDAPVSPTTLYVEDI SEPPLHDFYCSRLLDL VFLLDGSSRLSEAEFEVLKAFWDMMERLRJSQKWVRVA WEYHDGS HAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRITLLEMASQEPQ RMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVS YLCDLAPEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRS KEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQWPIGVGPNANVQELERIG WPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFD EMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNWPEKAHLLSLVDVMQREGGPSQIGD ALGFAVRYLTSEMHGARPGASKAWILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQL RILAGPAGDSNWKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQ CHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVT FDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEV TVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGL CGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLP LFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSL VYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQF LEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCD PVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCD EYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTC TDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEWTGSPRGDSQSSW KSVGSQWASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCE RMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGE CCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHK CLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSID INDVQDQCSCCSPTRTEPMQVALHCTNGSWYHEVLNAMECKCSPRKCSK(SEQ ID NO: 1; UniProtNo. P04275-1; signal peptide: amino acids 1-22; propeptide: amino acids 23-763; mature VWF amino acids 764-2813).

[0325] The mature VWF polypeptide is a large (2,050 amino acid residues) blood glycoprotein that functions in hemostasis and platelet adhesion. VWF is generally localized to the blood plasma, vascular endothelium, megakaryocytes, and subendothelial connective tissue, and exists in multimers of various sizes ranging from about 500 to 20,000 kDa. VWF multimer size typically scales with its role in hemostasis, i.e., larger VWF multimers are more effective at promoting clotting. Different domains of VWF are involved in different functions. VWF contains the following domains, from N-terminus to C-terminus: D1-D2-D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK. The D1-D2 domains, corresponding to the VWF propeptide, are proteolytically cleaved during processing of VWF into mature VWF. The D’-D3 domain mediates multimerization of VWF dimers and also binds to Factor VIII. The Al domain binds to platelet GPlb receptor, heparin, and collagen. The A2 domain is cleaved by ADAMTS13,resulting in generation of smaller VWF multimers. The A3 domain binds to collagen. The C4 domain binds to platelet to GPIIa / IIIb. The CK domain is important for tail-to-tail homodimerization of VWF monomers. Binding of VWF to other proteins, such as, e.g., FVIII, results in the stabilization of the binding partner and increases its blood plasma half-life. For example, in the absence of VWF, FVIII has a half-life of about 1-2 hours, but extends to 8-12 hours when bound by VWF. In addition to acting as a carrier of FVIII, VWF promotes clotting by facilitating platelet adhesion, activation, and aggregation. Deficiency or dysfunction of VWF is associated with a tendency to bleed and occurs in a number of blood disorders characterized by excessive bleeding, including, e.g., VWD and Hey des syndrome.

[0326] In some aspects, the mature von Willebrand Factor (VWF) has an amino acid sequence according to the amino acid sequence of SEQ ID NO: 16.SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRC VALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFD GLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDG EVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGN FDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMV DSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGK VVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCH AHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLT CEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFE VLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGS QVASTSEVLKYTLFQIFSKIDRPEASRITLLLMASQEPQRMSRNFVRYVQGLKKKKVIVIP VGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPDMA QVTVGPGLLGVSTLGPKRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQ DSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFL VSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILI QDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSF AKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDA LGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYD AAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGD VWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCV CTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVK HSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQ PILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLC RTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKV MLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKA PTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTC ACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCG CTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSC RSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQWASPENPCLINECVRV KEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERMEACMLNGTVIGPGKTV MIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLR GGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIP GTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQC SCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK(SEQ ID NO: 16).

[0327] In some aspects, the mature VWF protein has a polypeptide sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 16. In some aspects, the mature VWF protein has polypeptide sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16. In some aspects, the mature VWF protein has a polypeptide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In some aspects, the mature VWF protein has a polypeptide sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0328] In some aspects, the mature VWF is recombinant human VWF (rhVWF). In some aspects, the rhVWF has a polypeptide sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Chinese hamster ovary (CHO) cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Chinese hamster ovary cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Chinese hamster ovary cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology inthe Chinese hamster ovary cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Chinese hamster ovary cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Chinese hamster ovary cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence 100% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Chinese hamster ovary cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF shows differences from plasma-derived VWF in glycosylation and sialylation. In some aspects, ABO(H) blood group antigens are not expressed on the rhVWF product, and sialylation levels are increased as compared to plasma-derived VWF.

[0329] In some aspects, the mature VWF is recombinant human VFW (rhVWF). In some aspects, the rhVWF has a polypeptide sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Human embryonic kidney (HEK) cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Human embryonic kidney (HEK) cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Human embryonic kidney (HEK) cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Human embryonic kidney (HEK) cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Human embryonic kidney (HEK) cell line without the addition of exogenous human or animal-derived VWF. In some aspects, the rhVWF has a polypeptide sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Human embryonic kidney (HEK) cell line without the addition of exogenous human or animal-derived VWF. In someaspects, the rhVWF has a polypeptide sequence 100% identical to the amino acid sequence of SEQ ID NO: 16 and is manufactured by recombinant DNA technology in the Human embryonic kidney (HEK) cell line without the addition of exogenous human or animal-derived VWF.

[0330] In some aspects, the mature VWF is plasma-derived human von Willebrand Factor (pdVWF). In some aspects, the mature VWF is allogeneic pdVWF. In some aspects, the pdVWF is derived from pooled human plasma. In some aspects, the second pharmaceutical composition comprises pdVWF and Factor VIII. In some aspects, the second pharmaceutical composition comprises pdVWF does not comprise Factor VIII.

[0331] In some aspects, the blood disorder is an episode of unwanted or excessive bleeding. In some aspects, the unwanted or excessive bleeding is minor bleeding. In some aspects, the minor bleeding is readily managed epistaxis, oral bleeding, or menorrhagia. In some aspects, the unwanted or excessive bleeding is major bleeding. In some aspects, the major bleeding is severe or refractory epistaxis, menorrhagia, GI bleeding, CNS trauma, hemarthrosis, or traumatic hemorrhage. In some aspects, the unwanted or excessive bleeding is associated with von Willebrand disease (VWD), or hemophilia A.Desmopressin

[0332] Desmopressin (DDAVP; l-deamino-8-D-arginine vasopressin) is a synthetic peptide that induces release of endogenous VWF from Weibel-Palade bodies of the endothelial cells into the circulation. DDAVP is a first-line treatment for Type 1 VWD (though not for severe forms). Under current protocols, DDAVP is administered by IV infusion 15-30 min at a dose of 0.3 micrograms / kg of patient body weight (to a maximum dosage of 20 micrograms) diluted in 50 mL in 0.9% saline. If used prior to a procedure expected to induce a bleeding event, DDAVP is administer 30 min prior to the procedure. DDAVP may also be administered by inhalation. For a patient weighing less than 50 kg, the recommended dosage is 150 micrograms in one nostril. For a patient weighing more than 50 kg, the recommended dosage is 150 micrograms in each nostril. If used to reduce spontaneous or traumatic bleeding, doses may be repeated after 8 hours to 12 hours and once daily thereafter, if needed, based upon clinical condition and von Willebrand factor and factor VIII levels.EXAMPLES

[0333] The following examples are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their invention.Example 1: Effect of AB001 on the VWF:FVIII interaction

[0334] VWF is a carrier of coagulation Factor VIII (FVIII) in circulation and is critical for maintaining its half-life and concentration in plasma. To investigate the effect of antibody binding on the VWF FVIII interaction, the capacity of VWF to bind FVIII in the presence of monovalent (one-arm) anti -VWF CK domain antibody AB001 was determined.

[0335] Briefly, the ASSERACHROM® VWF:FVIIIB assay (Diagnostica Stago) was performed according to the manufacturer’s instructions:1) Immuno-capture of VWF from citrated normal human pooled plasma onto immobilized anti-VWF antibody (VWF capture was performed at a 15-fold dilution of plasma);2) Binding of recombinant human FVIII in presence of 0-600 nM AB001 or64 pg / ml polyclonal rabbit anti-human VWF antibody; and3) Quantification of bound FVIII using horseradish peroxidase (HRP)-conjugated anti-FVIII antibody.

[0336] Exemplary results are shown in FIG. 1. In presence of AB001, binding of FVIII to plasma VWF remained unaffected. In contrast, the polyclonal antibody against VWF prevented FVIII: VWF complex formation.Example 2: Effect of AB001 on the cleavage of VWF by ADAMTS13

[0337] The plasma protease ADAM Metallopeptidase with Thrombospondin Type 1 Motif 13 (ADAMTS13) is responsible for processing of VWF into shorter multimers. The cleavage site for ADAMTS13 within the VWF A2 domain is cryptic under static conditions and is exposed when VWF is subjected to shear stress. Under static conditions, shear stress is mimicked by ristocetin, which in turn triggers a reduction in VWF multimer size upon addition to human plasma.

[0338] The effect of monovalent (one-arm) anti-VWF CK domain antibody AB001 on the proteolytic processing of VWF by ADAMTS13 was evaluated in human plasma using ristocetin. Briefly, citrated plasma (3.2% v / v) from three healthy human donors was diluted 5-fold in buffer (10 mM HEPES, 6.5 mM BaCh, pH 7.4 with or without 1.5 mg / ml ristocetin) and then incubated with AB001 (0 and 400 nM) or AB001 non-binding isotype control (400 nM) at 37°C for 0 and 2 h. After incubation, reactions were stopped with 10 mM EDTA and the multimer pattern was determined using gel electrophoresis separation followed by immunodetection of VWF and quantification by densitometry. In the resulting intensity profile, the fastest migrating peaks 1-3 and 4-8 are defined as low molecular-weight and medium molecular-weight VWF, respectively, while the remaining slowest migrating peaks are defined as high molecular- weight VWF. The intensity of the peaks directly correlated with the multimer concentration. The fractional content (%) of low, medium, and high molecular-weight multimers was calculated, and exemplary results are shown in FIG.2. In the presence of AB001, processing of VWF is retained, indicating that AB001 does not interfere with ADAMTS13.Example 3: Binding of AB001 to human plasma-derived VWF

[0339] Binding of monovalent (one-arm) anti-VWF CK domain antibody AB001 to human plasma-derived VWF was measured using SPR analysis at pH 7.4 and 37°C. Briefly, anti-VWF-A3 domain antibody, recognizing the collagen-binding site in the A3 domain of human VWF, was immobilized on a CM4 sensor chip using standard amine coupling chemistry. Human plasma-derived VWF was then captured by exposure to 50 nM VWF in running buffer (10 mM HEPES, 150 mM NaCl, 0.05 % v / v polysorbate 20, pH 7.4 supplemented with 1 mg / ml BSA) for 60 s at a flow rate of 10 pl / min. The kinetics of AB001 binding were measured at 37°C, with concentrations from 0 to 4 nM in running buffer and a flow rate of 30 pl / min. The association and dissociation phases lasted 400 and 1800 s, respectively. Sensorgrams were analyzed according to a Langmuir 1 : 1 binding model. The equilibrium binding constant (Kd) was calculated from the estimated on (KOn) and off (KOff) rate constants and determined to be 0.5 nM.Example 4: Effect of AB001 on the VWF:platelet gplb interaction

[0340] Glycoprotein lb (gplb) is a key receptor for VWF on the platelet. The interaction with gplb is dependent upon the exposure of a binding site in the Al domain of VWF. Under physiological conditions, the gplb binding site becomes accessible when VWF is subjected to shear stress at sites of vascular injury. Under static conditions, such as those prevailing in vitro,the effect of shear stress can be recapitulated by addition of the peptide antibiotic ristocetin, which enhances the binding of VWF to gplb receptors on platelets.

[0341] To evaluate the effect of monovalent (one-arm) anti-VWF CK domain antibodies on the ability of VWF to engage the gplb receptor on platelets, plasma from three healthy human donors (stabilized in 3.2% sodium citrate) was incubated with 0 nM (“untreated”), 300 nM, or 600 nM of antibody for 10-30 mins followed by quantitative determination of the ristocetin cofactor activity of VWF using the STA-VWF:RCo assay. This assay is based on the change in turbidity of a platelet suspension, as measured by photometry. A suspension of reconstituted lyophilized platelets is added to the test plasma followed by the addition of ristocetin to induce VWF- and gplb-dependent platelet aggregation. Aggregation induces a decrease in turbidity and, consequently, a decrease in absorbance, which can be measured photometrically. The decrease in absorbance is a function of the VWF activity in the plasma sample.

[0342] For each combination of donor plasma, antibody, and antibody concentration, the ristocetin cofactor activity of VWF was measured and normalized to that of a donor-matched control plasma without antibody.

[0343] VWF:RCo assay results for AB001 are shown in FIG. 3A. AB001 did not affect the gplba-dependent RCo activity of VWF. In contrast, the activity of VWF was inhibited in presence of antibodies blocking the gplba binding site in the VWF Al domain or the VWF binding site in gplba.

[0344] Additionally, the effect of monovalent (one-arm) anti-VWF CK domain antibody AB001 on the binding of human plasma-derived VWF to the recombinant extracellular domain of gplba was evaluated using an ELISA.

[0345] The ELISA was prepared by coating of a 96-well plate with 2 pg / ml mouse anti-StrepTag antibody in coating buffer (TBS containing 5 mM CaCh) overnight at 5°C. All subsequent steps were performed at room temperature with washing of the plate in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 5 mM CaCh between each step. The plate was blocked by incubation with blocking buffer (TBS containing 5 mM CaCh and 1% [w / v] BSA) for 1 h, followed by capture of the gplba ectodomain (100 nM) for 2 h. Titration series of 0-80 nM purified plasma-derived VWF were prepared with 1 mg / ml ristocetin, AB001 (0-400 nM) or a control antibody (AB001 non-binding isotype control, anti-gplba antibody, or anti-VWF-Al domain antibody) (400 nM) in dilution buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCh, 1% [w / v] BSA, pH 7.3) and incubated for 30 min before transfer to the ELISA plate and a further 30-min incubation. Each well was washed and subsequently exposed to 400 nM AB001 in dilutionbuffer for 30 min before detection of bound VWF with 1.3 mg / ml HRP-conjugated polyclonal rabbit anti-VWF antibody diluted 1:3,000 in blocking buffer. After 5 min incubation, chromogenic substrate was added and, following color development for 4 min, the reaction was quenched and absorbance measured at 450 nm.

[0346] Exemplary results are shown in FIG. 3B. Similar to the VWF:RCo assay, binding of VWF to gplba was not affected by AB001 but prevented by antibodies blocking the VWF:gplba interaction.Example 5: Effect of anti-VWF CK domain antibodies on the VWF:collagen III interaction

[0347] The effect of monovalent (one-arm) anti-VWF CK domain antibody AB001 on the binding of VWF to collagen III was evaluated using an ELISA with collagen III coated to the plate surface and incubation with a fixed concentration of plasma-derived VWF (2 nM) preincubated with 0 or 300 nM monovalent antibody. VWF binding was detected using a HRP-conjugated anti-VWF antibody. By relating the readout in the presence of monovalent antibody with that in the absence, the effect of monovalent antibody on collagen binding was determined.

[0348] Human placenta collagen III was dissolved in 0.05 M acetic acid to a concentration of 1 mg / mL followed by extensive dialysis against TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5). The ELISA assay was prepared by coating a 96-well plate with 100 pL of 50 pg / mL collagen III in coating buffer (TBS containing 5 mM CaCh) overnight at 5°C. Following a single wash of the plate with 200 pL of wash buffer (TBS containing 0.05% (v / v) Tween-20 and 5 mM CaCh), the plate was blocked by incubation with 200 pL blocking buffer (TBS containing 5 mM CaCh and 1% (w / w) BSA) for 1 hour at room temperature. After wash with 200 pL wash buffer, the plate was incubated with 100 pL of plasma-derived VWF (2 nM) pre-incubated for 30-min with 0, 200, or 400 nM monovalent antibody. Incubation lasted 60 min and was followed by consecutive 5-min incubations with 100 pL of monoclonal rabbit anti-VWF antibody (1 mg / mL diluted 1:1000) and 100 pL of HRP-conjugated goat anti-rabbit IgG antibody (1 mg / mL diluted 1:2000). Before and after addition of a primary and secondary antibody, the plate was washed 5 times with 200 pL wash buffer. A chromogenic horseradish peroxidase (HRP) substrate (100 pL) was then added and, following color development for 8 min, ELISA stop solution (100 pL) was added to quench the reaction. Absorbance was measured at 450 nm with subtraction of the absorbance at 620 nm.

[0349] ELISA assay results for AB001 are shown in FIG. 4A. In the presence of AB001, VWF retained near-normal binding to collagen.

[0350] Additionally, the effect of AB001 on the collagen-binding activity of human plasma-derived VWF was quantified using the Zymutest™ VWF:CBA ELISA assay (Hypen Biomed) performed at room temperature according to manufacturer’s instructions with modifications. Normal human plasma diluted 50-fold in sample diluent was supplemented with AB001 (0-600 nM) or a control antibody (AB001 non-binding isotype control or anti-VWF-A3 domain antibody) (400 nM) and incubated for 30 min. Samples were then transferred to the ELISA plate coated with fibrillar collagen (equine type I and III) and incubated another 2 h. Before detection of bound VWF with HRP-conjugated polyclonal anti -VWF antibody, the plate was washed and exposed to 600 nM AB001 for 30 min. Exemplary results are shown in FIG. 4B.Supplementation of citrated healthy human plasma with AB001 did not affect VWF binding, whereas an antibody blocking the VWF A3 domain prevented VWF:collagen complex formation.Example 6: Fc gamma and FcRn receptor binding of anti- VWF CK antibody

[0351] Binding of monovalent (one-armed) antibody AB001 to a panel of human Fc gamma receptors (FcyRs) and the neonatal fragment crystallizable receptor (FcRn) was assessed by SPR (Biacore), employing single-cycle analysis for Fc gamma receptors and steady-state affinity analysis for FcRn. Binding to FcRn supports the molecular mechanism of A06 P1 C12 to accumulate VWF via endosomal recycling.

[0352] For FcyRs, AB001 showed either no binding (FcyRs: I, IIIA176F, IIA167R, and IIIB) or binding that was too weak (FcyRs: IIIA176V, IIA167H, and IIB) to determine a Kd (data not shown). For FcRn, AB001 binding was similar to IgG4 control antibodies and is pH-dependent (data not shown). The results support that AB001 engages the FcRn pathway to protect VWF from degradation.Example 7: Cytokine release induced by anti- VWF CK antibody

[0353] The potential for monovalent (one-armed) antibody AB001 to induce release of cytokines IL-2, IL-6, IL- 10, IFN-y, and TNF-a was investigated in a Blood Outgrowth Endothelial Cells (BOEC) liquid co-culture cytokine release assay with whole blood overlaying the endothelial cells. The target of AB001, VWF, is present in this assay, as VWF is expressed in endothelial cells. AB001 concentrations tested in the assay were 2, 20, and 100 pg / mL [20, 199, and 994 nM], Whole blood samples from 10 healthy human donors were analyzed after incubation with AB001 for 24 hours.

[0354] AB001 did not elicit cytokine release in human whole blood in the BOEC co-culture cytokine release assay at any of the concentrations tested (data not shown).Example 8: Dose selection in humansExposure-based considerations

[0355] The starting dose of AB001 for administering in humans was selected as 20 mg, and a maximum expected clinical dose of 300 mg was selected in a predictive single dose study in humans. In FIG. 5A and FIG. 5B, predicted PK profiles areas shown for the two doses for participants with varying baseline residual VWF:Ag levels.VWF antigen and activity-based considerations

[0356] In FIGS. 6A and 6B, the absolute peak VWF:Ag is illustrated, as predicted by both ratio and allometric scaling for participants with a range of residual VWF levels and receiving doses of AB001 of 20 mg and 300 mg, respectively.Example 9: Multiple-Dose Administration

[0357] A multiple-dose administration study is conducted in humans. The AB001 doses selected are projected based on the PK / PD data and the analysis and modeling of data collected in the single ascending dose part of the clinical study.Example 10: Extension of the half-life of exogenous VWF in the presence of AB001

[0358] The effect of AB001 on the pharmacokinetics of recombinant human VWF (rhVWF) was assessed in cynomolgus monkeys.

[0359] Two groups (1 female and 1 male / group) of cynomolgus monkeys were dosed as described in Table 4. In Group 1, animals (710M, 730F) received 400 lU / kg rhVWF (3.17 ml / kg) intravenously (IV) at day -7. After washout of rhVWF, 30 mg / kg AB001 (0.193 ml / kg) was administered subcutaneously (SC) at day 1, followed by vehicle (3.17 ml / kg; 20 mM Histidine / Histidine-HCl pH 5.90) at day 6. In Group 2, animals (714M, 73 IF) received vehicle (3.17 ml / kg) at day -7. This was followed by SC administration of 30 mg / kg AB001 (0.193 ml / kg) at day 1, and IV administration of 400 lU / kg rhVWF (3.17 ml / kg) at day 6. The dose of AB001 was selected to ensure that enough free AB001 would be present in circulation to bind the additional VWF present in circulation after administration of rhVWF.

[0360] For both groups, plasma sampling was performed at the following timepoints (Table 3):Table 3

[0361] Blood was collected into polypropylene test tubes with 3.2% sodium citrate as anticoagulant. Platelet poor plasma was prepared by two rounds of centrifugation (2840 g for 10 min) followed by aliquotation into fresh polypropylene tubes and immediate freezing on dry ice. Samples were stored at -80°C until measurement.

[0362] An assay based on the change in turbidity of a microparticle suspension, as measured by photometry, was used to quantify VWF antigen (VWF:Ag) in plasma samples from cynomolgus monkeys. A suspension of latex microparticles, coated with antibody specific for VWF, is mixed with the test plasma to be quantified. An antigen-antibody reaction takes place, leading to an agglutination of the latex microparticles which induces an increase in turbidity of the reaction medium. This increase in turbidity is reflected by an increase in absorbance, the latter being measured photometrically. The increase in absorbance is a function of the VWF:Ag level present in the test sample. The measurement of VWF:Ag is presented in FIGs. 8A and 8B.

[0363] Quantification of human IgG4 anti-VWF antibody in plasma samples was done by immunoaffinity capture followed by tryptic digest and quantification using LC-MS / MS. Using biotinylated goat-anti-human IgG antibody coupled to streptavidin-coated high-capacity magnetic beads, human anti-VWF antibody was selectively captured from cynomolgus monkey plasma samples and combined with an antibody standard. Following rounds of washing of the beads to remove non-specific plasma constituents, bound human antibody was subjected to tryptic digest and then quantified by LC-MS / MS. For the quantification, the isotope labelled tryptic peptide VVSVLTVLHQDWLNGK was used. This peptide is derived from the antibody heavy chain and is conserved across IgGl-4.Table 4

[0364] As shown in FIG. 7, the PK profile of AB001 was comparable between animals (102M, 710M, 714M, 722F, 730F, and 73 IF) and groups and present in excess throughout the study.

[0365] In Group 1, IV administration of rhVWF at day -7 resulted in a transient increase in plasma levels of VWF with an estimated elimination half-life of 4.1 to 4.3 hours. At day 1, after complete washout of rhVWF, administration of AB001 resulted in a 2 to 3-fold accumulation of endogenous VWF, which was sustained until study end at day 16 (FIG. 8A). In Group 2, rhVWF was administered five days after AB001 at which time accumulation of endogenous VWF had plateaued (FIG. 8B). As seen for Group 1, this led to an instantaneous increase in plasma levels of VWF. However, in the presence of AB001, the elimination of rhVWF was significantly prolonged, with an estimated half-life of 10.3 to 10.7 hours, i.e., approximately 2.5-times longer than the half-life of free rhVWF (FIG. 9). Additionally, after washout of rhVWF after about 48 hours, VWF levels returned to a level 2.1 to 2.4-fold above the pre-dose level consistent with the maintained accumulation of endogenous VWF in the presence of AB001 (FIGs. 8B and 9).Example 11: Evaluation of pharmacokinetics and pharmacodynamics (PK / PD) and safety of AB001 following a single flat dose

[0366] AB001 is a monospecific antibody designed to bind and accumulate endogenous circulating Von Willebrand Factor (VWF) and increase FVIII levels. A Phase 1 / 2 study of AB001 was conducted in two cohorts (Al and A2).

[0367] Key inclusion criteria in cohorts Al and A2 included individuals diagnosed with Type 1 Von Willebrand Disease (VWD), exhibiting VWF activity <40% at baseline, FVIII activity <70% at baseline, both males and females, and age group between 18 and 65 years old. Cohort Al (n=3) received a flat dose at 20 mg of AB001 through subcutaneous injection. Cohort A2 (n=3) received a flat dose at 50 mg of AB001 through subcutaneous injection. This adaptive protocol design enables future modifications to dose and dose regimen and duration of follow upto match the PD response. Detailed baseline and demographics of cohorts Al and A2 are shown in Table 5.Table 5: Baseline and Demographics of Cohorts Al and A2

[0368] Initial safety data demonstrated that no safety concerns were identified with AB001. In cohort Al (20mg of AB001), no treatment-emergent adverse events (TEAEs) were reported. In cohort A2 (50mg of AB001), Two Grade 1 TEAEs was observed, both assessed as unrelated. No thrombotic events were observed. No thrombocytopenia or changes in D-dimer levels was observed. No injection site or hypersensitivity reactions was observed. Anti-drug antibody (ADA) testing were found negative at all time-points. No changes in inflammatory markers (C3a, C5a, and cytokines) were observed.

[0369] Pharmacokinetics of ^kBOOl are shown in FIGI* 10* A dose— dependent increase in (Dmax was observed. Tmax was observed earlier in cohort A2 (50mg) than in cohort Al (20mg). Cohort A2 (50mg) was found to maintain higher plasma levels through to Day 15 of the study period, suggesting extended duration potential with increased doses of AB001.

[0370] Pharmacodynamics of AB001 are shown in FIGs. 11A-11C. Dose dependent increase and prolonged duration of accumulation of VWF antigen (FIG. 11 A), VWF activity (FIG. 11B), and FVIII activity (FIG. 11C) were observed. In the cohort A2 (50mg), >1.5-fold accumulation is observed for all three PD parameters, maintained for at least 8-10 days.

[0371] Improved Thrombin Generation (TG) and activated partial thromboplastin time (APTT) was observed with AB001 in cohort Al (FIGs. 12A-12C). Methods performed in FIGs.12A-12C include Calibrated Automated Thrombogram, 0.5 pM tissue factor, platelet-poor plasma. Exploratory endpoint in Phase 1 was observed (n=2 evaluable; one participant excluded due to baseline interference). Peak TG corrected to levels equivalent to normal plasma (with approximately 55-65% FVIII) (FIG. 12A). The thrombin generation assay measures overall potential of a plasma sample to produce thrombin after coagulation is activated. The FVIII was based on control experiments where control plasma was spiked with increasing FVIII concentrations to establish Peak Thrombin Generation and endogenous thrombin potential.

[0372] The coagulation effect of AB001 was measurable using standard APTT laboratory tests (FIG. 12B). The Activated Partial Thromboplastin Time (aPTT) test is generally used to assess the blood's ability to clot. It evaluates the intrinsic pathway of coagulation and detecting deficiencies or inhibitors of clotting factors, including Factor VIII, Factor IX, and Factor XI.

[0373] Finally, the VWF multimer distribution remained stable (FIG. 12C) in subjects administered AB001. FIG. 12C depicts the proteolytic processing of VWF multimers in human plasma. The distribution of VWF into low, medium, and high molecular weight fractions (LMW, IMW, and HMW) is shown as percent of total VWF for each timepoint (at pre-dose, day 8, and day 29). In the presence of AB001, processing of VWF is retained, indicating that AB001 does not interfere with the proteolytic processing of VWF.

[0374] In summary, a dose dependent increase in PK and PD parameters were observed with AB001. With a dose of 50 mg of AB001 (cohort A2), >1.5-fold elevation VWF and FVIII activity were achieved and this was maintained for at least 8-10 days. No treatment-emergent adverse events were recorded. Safety and PK / PD data supported continued dose escalation to explore increased accumulation and duration of PD response with higher doses of AB001. These disclosures supported development of AB001 as a prophylactic therapy for Von Willebrand Disease (VWD).OTHER EMBODIMENTS

[0375] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific aspects, it should be understood that the disclosure as claimed should not be unduly limited to such specific aspects. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. Certain aspects are in the claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a bleeding disorder in a subject in need thereof, comprising administering to the subject a flat dose of a pharmaceutical composition comprising an antibody or antigen binding site thereof that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region (“anti-VWF antibody), wherein the flat dose is about 10 mg to about 300 mg of the anti-VWF antibody.

2. The method of claim 1, wherein the flat dose is about 20 mg to 100 mg of the anti-VWF antibody.

3. The method of claim 1, wherein the flat dose is about 20 mg of the anti-VWF antibody.

4. The method of claim 1, wherein the flat dose is about 30 mg of the anti-VWF antibody.

5. The method of claim 1, wherein the flat dose is about 50 mg of the anti-VWF antibody.

6. The method of claim 1, wherein the flat dose is about 100 mg of the anti-VWF antibody.

7. The method of claim 1, wherein the flat dose is about 150 mg of the anti-VWf antibody.

8. A method of treating a bleeding disorder in a subject in need thereof, comprising administering to the subject a weight-based dose of a pharmaceutical composition comprising an antibody or antigen binding portion thereof that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region (“anti- VWF antibody), wherein the weight-based dose is about 0.1 mg / kg to about 6 mg / kg of the anti-VWF antibody.

9. The method of any one of claims 1 to 8, wherein the bleeding disorder comprises von Willebrand disease (VWD), Heydes syndrome, hemophilia A, disorders of platelet function, connective tissue disorders, or any combination thereof.

10. The method of claim 9, wherein the bleeding disorder comprises Von Willebrand disease type 1, type 2A, type 2B, type 2M, type 2N, or type 3.

11. The method of claim 9, wherein the bleeding disorder comprises congenital Von Willebrand disease (cVWD) or acquired Von Willebrand disease (aVWD).

12. The method of any one of claims 1 to 11, wherein the bleeding disorder in the subject comprises symptoms of epistaxis, cutaneous bleeding, bleeding from minor wounds, oralcavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, central nervous system (CNS) bleeding, or any combination thereof.

13. The method of any one of claims 1 to 12, wherein the pharmaceutical composition is administered subcutaneously.

14. The method of any one of claims 1 to 13, wherein the pharmaceutical composition is administered repeatedly at a regular interval.

15. The method of claim 14, wherein the regular interval is about once weekly.

16. The method of claim 14, wherein the regular interval is about once every two weeks.

17. The method of claim 14, wherein the regular interval is about once every three weeks.

18. The method of claim 14, wherein the regular interval is about once every four weeks.

19. The method of claim 14, wherein the regular interval is about once a month.

20. The method of claim 14, wherein the regular interval is about once every two months.

21. A method of treating one or more symptoms of Von Willebrand disease (VWD) in a subject in need thereof, comprising administering to the subject a dose of a pharmaceutical composition comprising an antibody or an antigen-binding site that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region, wherein the one more symptoms comprise epistaxis, cutaneous bleeding, bleeding from minor wounds, oral-cavity bleeding, angiodysplasia, gastrointestinal bleeding, bleeding from tooth extraction, postoperative bleeding, heavy menstrual bleeding, obstetric hemorrhage, hematuria, muscle hematoma, joint bleeding, visceral bleeding, central nervous system (CNS) bleeding, or any combination thereof.

22. A method of accumulating a von Willebrand Factor (VWF) protein in blood plasma, accumulating a FVIII protein in blood plasma, and / or increasing blood plasma half-life of a VWF protein in a subject in need thereof, comprising administering to the subject a dose of a pharmaceutical composition comprising an antibody or an antigen-binding site thereof that specifically binds to an epitope of a von Willebrand Factor (VWF) protein positioned at a cysteine knot (CK) domain and a fragment crystallizable (Fc) region.

23. The method of any one of claims 1 to 22, wherein the anti-VWF antibody comprises a variable heavy chain (VH) region having an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 7 and a variable light chain (VL) region having an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 10.

24. The method of any one of claims 1 to 23, wherein the anti-VWF antibody comprises a VH comprising a complementarity determining region (CDR) Hl having the amino acid sequence set forth in SEQ ID NO: 2, a CDR H2 having the amino acid sequence set forth in SEQ ID NO: 3, and a CDR H3 having the amino acid sequence set forth in SEQ ID NO: 8; and a VL comprising a CDR LI having the amino acid sequence set forth in SEQID NO: 11, a CDR L2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR L3 having the amino acid sequence set forth in SEQ ID NO: 9.

25. The method of any one of claims 1 to 24, wherein the anti-VWF antibody comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10.

26. The method of any one of claims 1 to 25, wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13, a second HC comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4, and a light chain (LC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 14.

27. The method of any one of claims 1 to 26, wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14.

28. The method of claim 27, wherein the second HC does not comprise a variable heavy chain (VH) region.

29. The method of claim 28, wherein the second HC chain does not comprise a VH region and also does not comprise a CHI domain.

30. The method of claim 29, wherein the second HC consists of the amino acid sequence set forth in SEQ ID NO: 4.

31. The method of any one of claims 1 to 30, further comprising administering to the subject a second pharmaceutical composition comprising a mature VWF protein or a functional fragment thereof or a therapeutic agent that induces the release of VWF from the vascular endothelium.

32. A method of treating a bleeding disorder in a subject in need thereof, the method comprising administering to the patient (a) a first pharmaceutical composition comprising an anti-VWF antibody targeted against von Willebrand factor (VWF), wherein binding of the anti-VWF antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or a functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium.

33. A method of treating an episode of unwanted or excessive bleeding in a subject in need thereof, the method comprising administering to the subject (a) a first pharmaceutical composition comprising an anti-VWF antibody targeted against von Willebrand factor (VWF), wherein binding of the anti-VWF antibody to a VWF protein results in extension of the half-life of the VWF protein, and (b) a second pharmaceutical composition comprising (i) a mature VWF protein or a functional fragment thereof or (ii) a therapeutic agent that induces the release of VWF from the vascular endothelium.

34. The method of any one of claims 31 to 33, wherein the agent that induces the release of VWF from the vascular endothelium is desmopressin (DDAVP).

35. The method of any one of claims 31 to 33, wherein the anti-VWF antibody is administered prior to administration of the second pharmaceutical composition.

36. The method of any one of claims 31 to 33, wherein the anti-VWF antibody is administered after administration of the second pharmaceutical composition.

37. The method of any one of claims 31 to 36 , comprising prophylactically administering to the patient the anti-VWF antibody.

38. The method of any one of claims 31 to 37, comprising (a) prophylactically administering to the patient the anti-VWF antibody and (b) in response to a minor bleeding event, a major bleeding event, or the blood plasma level of VWF in the subject dropping below 100 lU / dL, administering to the subject the second pharmaceutical composition.

39. The method of any one of claims 31 to 38, wherein the second composition further comprises FVIII.

40. The method of any one of claims 31, 32, and 34 to 39, wherein the bleeding disorder comprises von Willebrand disease (VWD), Heydes syndrome, hemophilia A, disorders of platelet function, connective tissue disorders, or any combination thereof.

41. The method of any one of claims 33 to 40, wherein the unwanted or excessive bleeding episode is associated with von Willebrand disease (VWD), Heydes syndrome, hemophilia A, disorders of platelet function, connective tissue disorders, or any combination thereof.

42. The method of any one of claims 31 to 41, wherein the mature VWF protein comprises a polypeptide comprising (i) an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, recombinant human VWF (rhVWF), or plasma- derived VWF (pdVWF) or (ii) a functional fragment thereof.

43. The method of any one of claims 31 to 42, wherein the anti-VWF antibody comprises a variable heavy chain (VH) region comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 7 and a variable light chain (VL) region comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 10.

44. The method of any one of claims 31 to 43, wherein the anti-VWF antibody comprises a VH comprising a complementarity determining region (CDR) Hl having the amino acid sequence set forth in SEQ ID NO: 2, a CDR H2 having the amino acid sequence set forth in SEQ ID NO: 3, and a CDR H3 having the amino acid sequence set forth in SEQ ID NO: 8; and a VL comprising a CDR LI having the amino acid sequence set forth in SEQ ID NO: 11, a CDR L2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR L3 having the amino acid sequence set forth in SEQ ID NO: 9.

45. The method of any one of claims 31 to 44, wherein the anti-VWF antibody comprises a variable heavy chain (VH) region comprising the amino acid sequence of SEQ ID NO: 7 and a variable light chain (VL) region comprising of the amino acid sequence of SEQ ID NO: 10.

46. The method of any one of claims 31 to 45, wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13, a second HC comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4, and a light chain (LC) comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 14.

47. The method of any one of claims 31 to 46, wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 14.

48. The method of claim 47, wherein the second HC does not comprise a variable heavy chain (VH) region.

49. The method of claim 48, wherein the second HC chain does not comprise a VH region and also does not comprise a CHI domain.

50. The method of claim 49, wherein the second HC consists of the amino acid sequence set forth in SEQ ID NO: 4.

51. A method of treating a bleeding disorder in a subject in need thereof comprising subcutaneously administering to the subject a flat dose of 20 mg of an anti-VWF antibody,wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising a VH- CHl-hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising a hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising a VL-CL polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14,wherein, the CHI domain of the first HC is operably linked to the CL region of the LC via a disulfide bridge, andwherein the hinge region of the first HC and the hinge region of the second HC are operably linked to one another by a disulfide bridge.

52. A method of treating a bleeding disorder in a subject in need thereof comprising subcutaneously administering to the subject a flat dose of 50 mg of an anti-VWF antibody,wherein the anti-VWF antibody comprises a first heavy chain (HC) comprising a VH- CHl-hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13, a second HC comprising a hinge-CH2-CH3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain (LC) comprising a VL-CL polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14,wherein, the CHI domain of the first HC is operably linked to the CL region of the LC via a disulfide bridge, andwherein the hinge region of the first HC and the hinge region of the second HC are operably linked to one another by a disulfide bridge.

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