Methods for treating hemophilia A

Abstract

The present disclosure provides a method of treating hemophilia A in a human subject in need thereof, the method comprising administering to the subject at a dosing interval (i) a factor VIII (FVIII) protein and (ii) a chimeric polypeptide comprising a von Willebrand factor (VWF) fragment comprising the D' domain of VWF and the D3 domain of VWF. For example, the present disclosure provides a method of treating hemophilia A in a human subject in need thereof, the method comprising administering to the subject at a dosing interval (i) a factor VIII (FVIII) polypeptide and (ii) a chimeric polypeptide comprising a von Willebrand factor (VWF) fragment comprising the D' domain of VWF and the D3 domain of VWF ... VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, the method comprising administering to the subject at a dosing interval (i) a factor VIII (FVIII) polypeptide and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, the method comprising administering to the subject at a dosing interval (i) a factor VIII (FVIII) polypeptide and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, the method comprising administering to the subject at a dosing interval (ii) a chimeric polypeptide comprising a von Willebrand factor (VWF) fragment comprising the D' domain of VWF and the D3 domain of VWF, the method comprising administering to the subject at a dosing interval (ii) a chimeric polypeptide comprising a von Willebrand factor (VWF) fragment comprising the D

Description

[Technical Field]

[0001] Reference to priority application This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 673,670, filed May 18, 2018, U.S. Provisional Patent Application No. 62 / 712,880, filed July 31, 2018, U.S. Provisional Patent Application No. 62 / 773,785, filed November 30, 2018, and U.S. Provisional Patent Application No. 62 / 801,576, filed February 5, 2019, each of which is incorporated by reference herein in its entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the Sequence Listing, submitted electronically in an ASCII text file (Name: SA9-461PC_SeqListing.txt; Size: 922 KB; Created: May 14, 2019), are incorporated herein by reference in their entirety. [Background technology]

[0003] Hemophilia A is a bleeding disorder caused by a defect in the gene encoding clotting factor VIII (FVIII) and affects 1 to 2 in every 10,000 male births. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients with hemophilia A can be treated with infusions of purified or recombinantly produced FVIII. Many commercially available FVIII products are known to have a half-life of approximately 8 to 12 hours, requiring frequent intravenous administration to patients. See Weiner MA and Cairo, MS, Pediatric Hematology Secrets; Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). Additionally, numerous approaches have been attempted to extend the half-life of FVIII. For example, approaches under development to extend the half-life of coagulation factors include PEGylation, glycoPEGylation, and conjugation with albumin. See Dumont et al., Blood. 119(13): 3024-3030 (2012). Consistent results have been demonstrated in humans; for example, rFVIIIFc was reported to improve half-life by approximately 1.7-fold compared to ADVATE® in patients with hemophilia A. See Powell et al., Blood. 119(13): 3031-3037 (2012). Thus, an increase in half-life, despite a modest improvement, indicates the presence of other half-life-limiting factors. See Liu, T. et al., 2007 ISTH meeting, abstract #PM-035; Henrik, A. et al., 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011 ISTH meeting abstract #P-WE-131.

[0004] The currently recommended standard of care involves regular administration of FVIII (routine prophylaxis) to minimize the number of bleeding episodes. Routine prophylaxis is associated with improvements in long-term outcomes but is a demanding regimen limited by the need for frequent intravenous (IV) administration. See Manco-Johnson et al., N Engl J Med. 357(6):535-44 (2007). Extended-half-life FVIII products reduce the frequency of prophylactic FVIII administration; however, all have comparable circulating half-lives, consistent with the upper limit in the half-life of rFVIII variants, which interact with von Willebrand factor (VWF) and are due to the half-life of endogenous VWF. See, e.g., Pipe et al., Blood. 128(16):2007-16 (2016). Prophylactic dosing for these FVIII products is every 3 to 5 days. Next-generation extended half-life FVIII products that prevent and control bleeding episodes for extended periods, resulting in less frequent dosing, could potentially address the challenge of adherence to demanding prophylaxis regimens, which in turn could improve the quality of life for people with hemophilia. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005) [Non-patent document 2] Weiner MA and Cairo, MS, Pediatric Hematology Secrets [Non-patent document 3] Lee, MT, 12. Disorders of Coagulation, Elsevier Health Sciences, 2001 Summary of the Invention [Means for solving the problem]

[0006] Certain embodiments of the present disclosure relate to a method of treating hemophilia A in a human subject in need thereof, comprising administering to the subject, at a dosing interval, multiple doses of a chimeric polypeptide comprising (i) a factor VIII (FVIII) protein and (ii) a von Willebrand factor (VWF) fragment comprising the D' domain of VWF and the D3 domain of VWF, wherein at least one of the multiple doses is from about 15 IU / kg to about 100 IU / kg, and the dosing interval is at least about every 7 days.

[0007] In some embodiments, the multiple doses include at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, at least 10 doses, at least 11 doses, at least 12 doses, at least 13 doses, at least 14 doses, at least 15 doses, at least 16 doses, at least 17 doses, at least 18 doses, at least 19 doses, at least 20 doses, or more doses.

[0008] In some embodiments, treating hemophilia A comprises controlling or reducing the incidence or frequency of bleeding episodes in a human subject in need thereof, hi some embodiments, treating hemophilia A comprises preventing or treating bleeding episodes in a human subject in need thereof.

[0009] In some embodiments, at least one of the multiple doses comprises about 20 IU / kg to about 95 IU / kg, about 20 IU / kg to about 90 IU / kg, about 20 IU / kg to about 85 IU / kg, about 20 IU / kg to about 80 IU / kg, about 20 IU / kg to about 75 IU / kg, about 20 IU / kg to about 70 IU / kg, about 20 IU / kg to about 65 IU / kg, about 20 IU / kg to about 60 IU / kg, about 20 IU / kg to about 55 IU / kg, about 20 IU / kg to about 50 IU / kg, about 20 IU / kg to about 45 IU / kg, about 20 IU / kg to about 40 IU / kg, about 20 IU / kg to about 35 IU / kg, about 20 IU / kg to about 30 IU / kg, or about 20 IU / kg to about 25 IU / kg. In some embodiments, at least one of the multiple doses comprises about 20 IU / kg to about 100 IU / kg, about 25 IU / kg to about 100 IU / kg, about 30 IU / kg to about 100 IU / kg, about 35 IU / kg to about 100 IU / kg, about 40 IU / kg to about 100 IU / kg, about 45 IU / kg to about 100 IU / kg, about 50 IU / kg to about 100 IU / kg, The dose is about 55 IU / kg to about 100 IU / kg, about 60 IU / kg to about 100 IU / kg, about 65 IU / kg to about 100 IU / kg, about 70 IU / kg to about 100 IU / kg, about 75 IU / kg to about 100 IU / kg, about 80 IU / kg to about 100 IU / kg, about 85 IU / kg to about 100 IU / kg, or about 90 IU / kg to about 100 IU / kg.

[0010] In some embodiments, at least one of the multiple doses is about 20 IU / kg to about 80 IU / kg, about 25 IU / kg to about 75 IU / kg, about 30 IU / kg to about 70 IU / kg, about 35 IU / kg to about 65 IU / kg, about 40 IU / kg to about 60 IU / kg, or about 45 IU / kg to about 55 IU / kg. In some embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg.

[0011] In some embodiments, at least one of the multiple doses is about 20 IU / kg, about 25 IU / kg, about 30 IU / kg, about 35 IU / kg, about 40 IU / kg, about 45 IU / kg, about 50 IU / kg, about 55 IU / kg, about 60 IU / kg, about 65 IU / kg, about 70 IU / kg, about 75 IU / kg, about 80 IU / kg, about 85 IU / kg, about 90 IU / kg, about 95 IU / kg, or about 100 IU / kg. In some embodiments, at least one of the multiple doses is about 25 IU / kg. In some embodiments, at least one of the multiple doses is about 50 IU / kg. In some embodiments, at least one of the multiple doses is about 65 IU / kg. In some embodiments, at least one of the multiple doses is about 80 IU / kg.

[0012] In some embodiments, the dosing interval is at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, or at least about 31 days.

[0013] In some embodiments, the dosing frequency is at least once per week, at least once per 2 weeks, at least once per 3 weeks, or at least once per 4 weeks. In some embodiments, the dosing interval is at least once per week. In some embodiments, the dosing interval is at least once per 2 weeks.

[0014] In some embodiments, the chimeric polypeptide is administered for prophylactic treatment.

[0015] In some embodiments, the FVIII protein is associated with the VWF fragment by a covalent bond, hi some embodiments, the covalent bond is a peptide bond or a disulfide bond.

[0016] In some embodiments, the FVIII protein comprises a FVIII polypeptide and a first half-life extension. In some embodiments, the first half-life extension is fused to the C-terminus or N-terminus of the FVIII polypeptide. In some embodiments, the first half-life extension is inserted within the FVIII polypeptide. In some embodiments, the first half-life extension is inserted within the B domain of the FVIII polypeptide. In some embodiments, the first half-life extension is inserted within the FVIII polypeptide immediately downstream of the amino acid corresponding to amino acid residue 745 of SEQ ID NO: 65. In some embodiments, the first half-life extension is fused to the FVIII polypeptide by a linker.

[0017] In some embodiments, the VWF fragment comprises a second half-life extending moiety. In some embodiments, the second half-life extending moiety is fused to the C-terminus or N-terminus of the VWF fragment. In some embodiments, the second half-life extending moiety is inserted within the VWF fragment. In some embodiments, the second half-life extending moiety is fused to the C-terminus of the VWF fragment. In some embodiments, the second half-life extending moiety is fused to the VWF fragment by a linker.

[0018] In some embodiments, the first half-life extending moiety, the second half-life extending moiety, or both, are selected from the group consisting of albumin, an immunoglobulin Fc region, an XTEN sequence, the C-terminal peptide of the beta subunit of human chorionic gonadotropin (CTP), a PAS sequence, a HAP sequence, transferrin, an albumin binding moiety, or any fragment, derivative, variant, and any combination thereof.

[0019] In some embodiments, the first half-life extending moiety comprises a first XTEN.

[0020] In some embodiments, the first XTEN is inserted within the FVIII polypeptide immediately downstream of the amino acid corresponding to amino acid residue 745 of SEQ ID NO:65.

[0021] In some embodiments, the second half-life extending moiety comprises a second XTEN. In some embodiments, the second XTEN is fused to the C-terminus of the VWF fragment.

[0022] In some embodiments, the FVIII protein comprises a first immunoglobulin (Ig) constant region or portion thereof. In some embodiments, the first Ig constant region or portion thereof is fused to the C-terminus or N-terminus of the FVIII polypeptide. In some embodiments, the first Ig constant region or portion thereof is inserted within the FVIII polypeptide. In some embodiments, the first Ig constant region or portion thereof is fused to the C-terminus of the FVIII polypeptide. In some embodiments, the first Ig constant region or portion thereof is fused to the FVIII polypeptide via a linker. In some embodiments, the first Ig constant region or portion thereof comprises a first Fc domain or portion thereof.

[0023] In some embodiments, the VWF fragment comprises a second Ig constant region or portion thereof. In some embodiments, the second Ig constant region or portion thereof is fused to the C-terminus or N-terminus of the VWF fragment. In some embodiments, the second Ig constant region or portion thereof is inserted within the VWF fragment. In some embodiments, the second Ig constant region or portion thereof is fused to the C-terminus of the VWF fragment. In some embodiments, the second Ig constant region or portion thereof is fused to the VWF fragment via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the second Ig constant region or portion thereof comprises a second Fc domain or portion thereof.

[0024] In some embodiments, the FVIII protein and the VWF fragment are associated with each other by a covalent bond between the first Fc domain and the second Fc domain, hi some embodiments, the FVIII protein and the VWF fragment are further associated with each other by a non-covalent interaction between the FVIII protein and the VWF fragment.

[0025] In one aspect, a method of treating hemophilia A in a human subject comprises administering, at a dosing interval, multiple doses of a chimeric polypeptide to a subject in need thereof, wherein the chimeric polypeptide comprises: (i) a first FVIII polypeptide fragment comprising the amino acid sequence of SEQ ID NO:215, a first XTEN sequence comprising the amino acid sequence of SEQ ID NO:8 (AE288), a second FVIII polypeptide fragment comprising the amino acid sequence of SEQ ID NO:216, and a first Fc region comprising the amino acid sequence of SEQ ID NO:217. and (ii) a VWF protein comprising a D' domain of VWF comprising the amino acid sequence of SEQ ID NO: 210, a D3 domain of VWF comprising the amino acid sequence of SEQ ID NO: 214, a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58 (AE144_5A), an a2 linker comprising the amino acid sequence of SEQ ID NO: 88, and a second Fc region comprising the amino acid sequence of SEQ ID NO: 217, wherein the first Fc region is covalently linked to the second Fc region by a disulfide bond.

[0026] In some embodiments, the chimeric polypeptide comprises a FVIII protein comprising a FVIII polypeptide, a first XTEN sequence, a first Fc region, and a VWF protein comprising a VWF D' domain, a VWF D3 domain, a second XTEN sequence, a FVIII a2 linker, and a second Fc region, wherein the FVIII polypeptide comprises the amino acid sequence of SEQ ID NO:215, the first XTEN sequence comprises the amino acid sequence of AE288 (SEQ ID NO:8) and is fused to the C-terminus of SEQ ID NO:215, the FVIII polypeptide further comprises the amino acid sequence of SEQ ID NO:216, and the first Fc region comprises the amino acid sequence of SEQ ID NO:217. the D' domain of VWF comprises the amino acid sequence of SEQ ID NO: 210, the D3 domain of VWF comprises the amino acid sequence of SEQ ID NO: 214, a second XTEN sequence comprises the amino acid sequence of AE144_5A (SEQ ID NO: 58) and is fused to the C-terminus of the D3 domain of VWF, an a2 linker comprises the amino acid sequence of SEQ ID NO: 88 and is fused to the C-terminus of the second XTEN sequence, a second Fc region comprises the amino acid sequence of SEQ ID NO: 217 and is fused to the C-terminus of the a2 linker, and the first Fc region is covalently linked to the second Fc region by a disulfide bond.

[0027] In some embodiments, the chimeric polypeptide comprises a FVIII protein comprising a FVIII signal peptide comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the chimeric polypeptide comprises a VWF protein comprising a VWF signal peptide comprising the amino acid sequence of SEQ ID NO: 208. In some embodiments, the chimeric polypeptide comprises a VWF protein comprising the D1D2 domains of VWF comprising the amino acid sequence of SEQ ID NO: 209.

[0028] In some embodiments, the chimeric polypeptide comprises a FVIII protein comprising an amino acid sequence at least about 80%, 90%, 95% or 100% identical to SEQ ID NO:201, SEQ ID NO:203 or SEQ ID NO:207, and a VWF protein comprising an amino acid sequence at least about 80%, 90%, 95% or 100% identical to SEQ ID NO:202 or SEQ ID NO:205.

[0029] In one embodiment, the chimeric polypeptide comprises a FVIII protein comprising the amino acid sequence of SEQ ID NO: 203 and a VWF protein comprising the amino acid sequence of SEQ ID NO: 205. In another embodiment, the chimeric polypeptide comprises a FVIII protein comprising the amino acid sequence of SEQ ID NO: 201 and a VWF protein comprising the amino acid sequence of SEQ ID NO: 202. In another embodiment, the chimeric polypeptide comprises a FVIII protein comprising the amino acid sequence of SEQ ID NO: 207 and a VWF protein comprising the amino acid sequence of SEQ ID NO: 202.

[0030] In some embodiments, the chimeric polypeptide is selected from the group consisting of FVIII-161 (SEQ ID NO: 69), FVIII-169 (SEQ ID NO: 70), FVIII-170 (SEQ ID NO: 71), FVIII-173 (SEQ ID NO: 72); FVIII-195 (SEQ ID NO: 73); FVIII-196 (SEQ ID NO: 74), FVIII199 (SEQ ID NO: 75), FVIII-201 (SEQ ID NO: 76); FVIII-203 (SEQ ID NO: 77), FVIII-204 (SEQ ID NO: 78), FVIII-205 (SEQ ID NO: 79), FVIII-266 (SEQ ID NO: 80), FVIII-267 (SEQ ID NO: 81), FVIII-268 (SEQ ID NO: 82), FVIII-269 (SEQ ID NO: 83), FVIII-270 (SEQ ID NO: 84), FVIII-271 (SEQ ID NO: 85), FVIII-272 (SEQ ID NO: 86), FVIII-273 (SEQ ID NO: 87), FVIII-274 (SEQ ID NO: 88), FVIII-275 (SEQ ID NO: 89), FVIII-276 (SEQ ID NO: 90), FVIII-277 (SEQ ID NO: 91), FVIII-278 (SEQ ID NO: 92), FVIII-279 (SEQ ID NO: 93), FVIII-280 (SEQ ID NO: 94), FVIII-281 (SEQ ID NO: 95), FVIII-282 (SEQ ID NO: 96), FVIII-283 (SEQ ID NO: 97), FVIII- FVIII proteins comprising an amino acid sequence at least about 80%, 90%, 95% or 100% identical to a sequence selected from VWF031 (SEQ ID NO: 86), VWF034 (SEQ ID NO: 87), VWF059 (SEQ ID NO: 197), VWF059A (SEQ ID NO: 202) or VWF036.

[0031] In some embodiments, the chimeric polypeptide is administered by a route selected from the group consisting of intravenous injection, intravenous infusion, subcutaneous administration, intramuscular administration, oral administration, intranasal administration, and pulmonary administration.

[0032] In some embodiments, the chimeric polypeptide after administration provides a plasma activity level of FVIII of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%. In some embodiments, the plasma activity level of FVIII is at least about 3%. In some embodiments, the plasma activity level of FVIII is at least about 5%.

[0033] In some embodiments, the chimeric polypeptide after administration provides a plasma activity level of FVIII of at least about 1 IU / dL, at least about 2 IU / dL, at least about 3 IU / dL, at least about 4 IU / dL, at least about 5 IU / dL, at least about 6 IU / dL, at least about 7 IU / dL, at least about 8 IU / dL, at least about 9 IU / dL, or at least about 10 IU / dL. In some embodiments, the plasma activity level of FVIII is at least about 3 IU / dL. In some embodiments, the plasma activity level of FVIII is at least about 5 IU / dL.

[0034] In some embodiments, the plasma activity level of FVIII is at least about 10 IU / dL at least about 5 days after administration of the chimeric polypeptide. In some embodiments, the plasma activity level of FVIII is at least about 5 IU / dL at least about 7 days after administration of the chimeric polypeptide. In some embodiments, the plasma activity level of FVIII is at least about 3 IU / dL at least about 8 days after administration of the chimeric polypeptide. In some embodiments, the plasma activity level of FVIII is at least about 1 IU / dL at least about 10 days after administration of the chimeric polypeptide.

[0035] In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg, and the interval between doses is at least about 7 days. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg, and the interval between doses is at least about 5 days. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg, and the interval between doses is at least about 5 days.

[0036] In some embodiments, at least one of the multiple doses is about 50 IU / kg. In some embodiments, the multiple doses are about 50 IU / kg and the dosing interval is about 5 days. In some embodiments, the multiple doses are about 50 IU / kg and the dosing interval is about 7 days. In some embodiments, the multiple doses are about 50 IU / kg and the dosing interval is about 14 days.

[0037] In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg, and the interval between doses is at least about 7 days. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg, and the interval between doses is at least about 7 days. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg, and the interval between doses is at least about 7 days.

[0038] In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg, and the interval between doses is at least about 10 days. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg, and the interval between doses is at least about 10 days. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg, and the interval between doses is at least about 10 days.

[0039] In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg and the interval between doses is at least about 14 days. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg and the interval between doses is at least about 14 days. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg and the interval between doses is at least about 14 days.

[0040] In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg and the dosing interval is at least about one week. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg and the dosing interval is at least about one week. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg and the dosing interval is at least about one week.

[0041] In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 80 IU / kg and the dosing interval is at least about two weeks. In some embodiments, at least one of the multiple doses is about 50 IU / kg to about 65 IU / kg and the dosing interval is at least about two weeks. In some embodiments, at least one of the multiple doses is about 65 IU / kg to about 80 IU / kg and the dosing interval is at least about two weeks.

[0042] In some embodiments, the dosing interval is at least about 5 days. In some embodiments, the dosing interval is at least about 7 days. In some embodiments, the dosing interval is about 5 to about 14 days. In some embodiments, the dosing interval is about 7 to about 14 days. In some embodiments, the dosing interval is at least about 10 days. In some embodiments, the dosing interval is about 10 to about 21 days. In some embodiments, the dosing interval is about 14 to about 21 days. In some embodiments, the dosing interval is about 14 days.

[0043] In some embodiments, the human subject is a female.In some embodiments, the human subject is a child.In some embodiments, the human subject is about 12 years old or younger, about 11 years old or younger, about 10 years old or younger, about 9 years old or younger, about 8 years old or younger, about 7 years old or younger, about 6 years old or younger, about 5 years old or younger, about 4 years old or younger, about 3 years old or younger, about 2 years old or younger, or younger than about 1 year old.

[0044] In some embodiments, the administration induces immune tolerance to FVIII in the human subject. In some embodiments, the administration reduces an inhibitory immune response to FVIII in the human subject. In some embodiments, the inhibitory immune response to FVIII comprises high titers of anti-FVIII antibodies in the human subject.

[0045] In some embodiments, administration of the chimeric polypeptide does not induce FVIII inhibitors after about 7 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 20 days, about 24 days, about 25 days, about 28 days, about 30 days, or about 35 days of administration. In some embodiments, administration of the chimeric polypeptide does not induce FVIII inhibitors after about 28 days of administration. [Brief explanation of the drawings]

[0046] [Figure 1]Figure 1 is a schematic diagram of rFVIIIFc-VWF-XTEN. FVIII: Factor VIII; VWF: von Willebrand factor; A1, A2, A3, C1, C2: domains of FVIII; D'D3: domains of VWF; Fc: Fc region of immunoglobulin constant region.

[0047] [Figure 2A] 2A and 2B show the protocol for testing the safety and efficacy of rFVIIIFc-VWF-XTEN in human patients in a low-dose cohort administered 25 IU / kg of rFVIIIFc-VWF-XTEN (FIG. 2A) and a high-dose cohort administered 65 IU / kg of rFVIIIFc-VWF-XTEN (FIG. 2B). [Figure 2B] 2A and 2B show the protocol for testing the safety and efficacy of rFVIIIFc-VWF-XTEN in human patients in a low-dose cohort administered 25 IU / kg of rFVIIIFc-VWF-XTEN (FIG. 2A) and a high-dose cohort administered 65 IU / kg of rFVIIIFc-VWF-XTEN (FIG. 2B).

[0048] [Figure 3A] 3A-3B are graphical representations of baseline-corrected FVIII activity levels based on activated partial thromboplastin time (aPTT) testing in human subjects with severe hemophilia A who received 25 IU / kg rFVIII followed by a washout period followed by 25 IU / kg rFVIIIFc-VWF-XTEN (FIG. 3A; low-dose cohort) or 65 IU / kg rFVIII followed by a washout period followed by 65 IU / kg rFVIIIFc-VWF-XTEN (FIG. 3B; high-dose cohort). Horizontal dashed lines indicate 3%, 5%, 10%, and 20% FVIII activity. [Figure 3B]3A-3B are graphical representations of baseline-corrected FVIII activity levels based on activated partial thromboplastin time (aPTT) testing in human subjects with severe hemophilia A who received 25 IU / kg rFVIII followed by a washout period followed by 25 IU / kg rFVIIIFc-VWF-XTEN (FIG. 3A; low-dose cohort) or 65 IU / kg rFVIII followed by a washout period followed by 65 IU / kg rFVIIIFc-VWF-XTEN (FIG. 3B; high-dose cohort). Horizontal dashed lines indicate 3%, 5%, 10%, and 20% FVIII activity.

[0049] [Figure 4] Figure 4 is a schematic diagram of the clinical trial design to evaluate the safety and tolerability of rFVIIIFc-VWF-XTEN at 50 IU / kg or 65 IU / kg doses administered weekly for a total of four doses in previously treated adult male patients (PTPs) aged 18 to 65 years (inclusive) with severe hemophilia A. EOS = end of study; ET = early termination; PK = pharmacokinetics. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present disclosure relates to a method of treating a bleeding disease or condition, e.g., hemophilia A, in a human subject in need thereof, comprising administering to the subject, at a dosing interval, multiple doses of (i) a factor VIII (FVIII) polypeptide and (ii) a chimeric polypeptide comprising a von Willebrand factor (VWF) fragment comprising the D' domain of VWF and the D3 domain of VWF. In some embodiments, at least one of the multiple doses is about 15 IU / kg to about 100 IU / kg. In some embodiments, the dosing interval is at least about every 5 days. In some embodiments, the dosing interval is at least about every 7 days.

[0051] I. Definition It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0052] Furthermore, "and / or," as used herein, should be interpreted as a specific disclosure of each of the two specified features or components, either with or without the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0053] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure relates.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0055] Units, prefixes, and symbols are shown in their International System of Units (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0056] The term "about" is used herein to mean approximately, roughly, roughly, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify a numerical value above and below the stated value by, for example, a variance above or below 10 percent (high or low).

[0057] The term "polynucleotide" or "nucleotide" is intended to encompass single and multiple nucleic acids and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, polynucleotides contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide, we mean a nucleic acid molecule, DNA, or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for purposes of the present disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid can include regulatory elements such as a promoter, an enhancer, a ribosomal binding site, or a transcription termination signal.

[0058] Certain proteins secreted by mammalian cells are associated with secretory signal peptides that are cleaved from the mature protein once transport of the growing protein chain across the rough endoplasmic reticulum has begun. Those skilled in the art will appreciate that signal peptides are generally fused to the N-terminus of a polypeptide and are cleaved from the complete or "full-length" polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, or a functional derivative of its sequence, that retains the ability to direct secretion of the polypeptide, is operably linked thereto. Alternatively, a heterologous mammalian signal peptide, such as the signal peptide of human tissue plasminogen activator (TPA) or mouse β-glucuronidase, or a functional derivative thereof, can be used.

[0059] As used herein, the term "polypeptide" is intended to encompass both "polypeptides" and "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from natural biological sources or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can arise in any manner, including by chemical synthesis.

[0060] An "isolated" polypeptide, or a fragment, variant, or derivative thereof, refers to a polypeptide that is not in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can be simply removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of this disclosure, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0061] The present disclosure also includes polypeptide fragments or variants, and any combination thereof. The term "fragment" or "variant," when referring to a polypeptide binding domain or binding molecule of the present disclosure, includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity for an FcRn-binding domain or Fc variant, clotting activity for an FVIII variant, or FVIII binding activity for a VWF fragment). Polypeptide fragments include proteolytic fragments and deletion fragments, as well as specific antibody fragments discussed elsewhere herein, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of the polypeptide binding domain or binding molecule of the present disclosure include the fragments described above, and also include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0062] As used herein, the term "VWF protein" or "VWF proteins" refers to any VWF fragment that interacts with FVIII and retains at least one or more properties normally provided to FVIII by full-length VWF, such as preventing premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that could result in premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but cannot bind VWF-bound FVIII, and / or stabilizing the interaction of the heavy and light chains of FVIII. A VWF fragment referred to herein is a VWF polypeptide that is less than a full-length VWF protein, wherein the VWF fragment retains the ability to interact with and / or bind to FVIII.

[0063] " Conservative amino acid substitution " refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. The family of amino acid residues with similar side chains has been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Therefore, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, this substitution is considered conservative. In other embodiments, strings of amino acids can be conservatively replaced with structurally similar strings that differ in the order and / or composition of side chain family members.

[0064] As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. As discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set, of course, so that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and allow gaps in homology of up to 5% of the total number of amino acids in the reference sequence.

[0065] As used herein, a "corresponding amino acid" or "equivalent amino acid" in a VWF or FVIII protein sequence is identified by alignment to maximize identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify the equivalent amino acid in the second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0066] As used herein, the term "insertion site" refers to a position in a FVIII polypeptide, or a fragment, variant, or derivative thereof, that is immediately downstream of a position at which a half-life extending moiety or heterologous moiety can be inserted. An "insertion site" is specified as a number, which is the number of amino acids in mature native FVIII (SEQ ID NO: 65) to which the insertion site corresponds that are immediately C-terminal to the position of insertion. For example, the phrase "a3 contains an XTEN at an insertion site corresponding to amino acid 1656 of SEQ ID NO: 65" indicates that the heterologous moiety is located between the two amino acids corresponding to amino acids 1656 and 1657 of SEQ ID NO: 65.

[0067] As used herein, the phrase "immediately downstream of an amino acid" refers to a position immediately adjacent to the terminal carboxyl group of an amino acid. For example, an insertion site immediately downstream of amino acid 745 corresponding to the mature wild-type FVIII protein means that the insertion site is between amino acids 745 and 746 corresponding to the mature wild-type FVIII protein. Similarly, the phrase "immediately upstream of an amino acid" refers to a position immediately adjacent to the terminal amine group of an amino acid.

[0068] As used herein, the phrase "between two amino acids at the insertion site" refers to the location where the XTEN or any other polypeptide is inserted between two adjacent amino acids. Thus, the phrases "inserted immediately downstream of an amino acid" and "inserted between two amino acids at the insertion site" are used interchangeably with "inserted at the insertion site."

[0069] As used herein, the terms "inserted," "is inserted," "inserted into," or grammatically related terms refer to the position of the XTEN in the chimeric polypeptide relative to the analogous position in native mature human FVIII. As used herein, the term refers to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII and does not indicate, imply, or infer any method or process by which the chimeric polypeptide is made. For example, with reference to the chimeric polypeptides provided herein, the phrase "XTEN is inserted immediately downstream of residue 745 of the FVIII polypeptide" means that the chimeric polypeptide comprises an XTEN immediately downstream of the amino acid corresponding to amino acid 745 in native mature human FVIII (e.g., bounded by amino acids corresponding to amino acids 745 and 746 of native mature human FVIII).

[0070] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked. Amino acid sequences normally present in different proteins may be combined in a fusion polypeptide, or amino acid sequences normally present in the same protein may be placed in a new arrangement in a fusion polypeptide, for example, in the fusion of a Factor VIII domain with an Ig Fc domain of the present disclosure. Fusion proteins are created, for example, by chemical synthesis or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric polypeptide can further comprise a second amino acid sequence linked to the first amino acid sequence by a covalent, non-peptide, or non-covalent bond.

[0071] As used herein, the term "linked" refers to a first amino acid sequence or nucleotide sequence being covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid sequence or nucleotide sequence can be directly joined or juxtaposed to the second amino acid sequence or nucleotide sequence, or an intervening sequence can covalently join the first sequence to the second sequence. The term "linked" not only refers to the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also includes the insertion of any two amino acids into the second amino acid sequence (or first amino acid sequence, respectively) across the first amino acid sequence (or second amino acid sequence). In some embodiments, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (for a polypeptide chain), a nucleotide or nucleotide chain (for a nucleotide chain), or any chemical moiety (for both polypeptide chains and polynucleotide chains). The term "coupled" may also be indicated by a hyphen (-).

[0072] As used herein, the term "associated with" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In some embodiments, the term "associated with" refers to a covalent bond, a non-peptide bond, or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In other embodiments, it refers to a covalent bond excluding a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a thiol group on a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 using the Kabat numbering system (positions 226 and 229 in the EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, agnostic bonds, flexural bonds, dipolar bonds, pi backbonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation / pi bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilicity, intercalation, stacking, entropic forces, or chemical polarity.

[0073] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. Certain enzymatic cleavage sites include intracellular processing sites. In some embodiments, a polypeptide has an enzymatic cleavage site that is cleaved by an enzyme activated during the coagulation cascade, such that cleavage of such a site occurs at the site of clot formation. Exemplary such sites include those recognized by thrombin, factor XIa, or factor Xa. Exemplary cleavage sites for FXIa include, for example, TQSFNDFTR (SEQ ID NO: 1) and SVSQTSKLTR (SEQ ID NO: 3). Exemplary cleavage sites for thrombin include, for example, DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG (SEQ ID NO: 6), ALRPR (SEQ ID NO: 7), ISDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 106), DKNTGDYYEDSYEDISAYLLSKNNAIEPRSFS (SEQ ID NO: 88), and IEPRSFS (SEQ ID NO: 194). Other enzyme cleavage sites are known in the art and are described elsewhere herein.

[0074] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide that is a target for an enzyme that functions after translation of the polypeptide. In some embodiments, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides before secretion of the protein from the cell. Examples of such processing sites include those targeted by the PACE / furin (PACE is an acronym for paired basic amino acid cleaving enzyme) family of endopeptidases. These enzymes are localized to the Golgi membrane and cleave proteins at the carboxy-terminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein, the "furin" family of enzymes includes, for example, PCSK1 (also known as PC1 / PC3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also known as PC7 / LPC, PC8, or SPC7). Other processing sites are known in the art.

[0075] It will be understood that in constructs which include more than one processing or cleavage site, such sites may be the same or different.

[0076] As used herein, a "processable linker" refers to a linker that includes at least one intracellular processing site, as described elsewhere herein.

[0077] As used herein, the term "half-life" refers to the biological half-life of a particular polypeptide in vivo. Half-life can be expressed as the time required for half of an administered dose to disappear from the circulation and / or other tissues in an animal. When constructing a clearance curve for a given polypeptide as a function of time, the curve is usually biphasic, with a rapid α-phase and a longer β-phase. The α-phase typically represents the equilibrium of the administered Fc polypeptide between the intravascular and extravascular spaces and is determined, in part, by the size of the polypeptide. The β-phase typically represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric polypeptides comprising FVIII are monophasic, thus having no alpha phase but only a single beta phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of a polypeptide in the β-phase. The typical beta-phase half-life of a human antibody in humans is 21 days. In certain embodiments, the half-life is expressed as the half-life of the terminal phase.

[0078] As used herein, hemostatic disorder refers to a genetically inherited or acquired condition characterized by a tendency to bleed, either spontaneously or as a result of trauma, due to impaired fibrin clot formation or an inability to form fibrin clots. Examples of such disorders include hemophilia. The three major forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency, or "Christmas disease"), and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities in fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, and Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb, the receptor for VWF, can be defective, resulting in a lack of primary clot formation (primary hemostasis) and an increased tendency to bleed, and Glanzmann and Naegeli thrombasthenia (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of clotting factors by the liver, which can increase the risk of bleeding.

[0079] As used herein, "administer" or "administering" refers to delivering a composition, e.g., a chimeric polypeptide, described herein to a subject. The composition, e.g., a chimeric polypeptide, can be administered to a subject using methods known in the art. In particular, the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, for example, via oral, sublingual, buccal, nasal, rectal, vaginal, or pulmonary routes. chimeric polypeptide. In some embodiments, administration is self-administration. In some embodiments, a parent administers the chimeric polypeptide to their child. In some embodiments, the chimeric polypeptide is administered to the subject by a medical professional, such as a doctor, medic, or nurse.

[0080] As used herein, the term "dose" refers to a single administration of a composition to a subject. A single dose can be administered all at once, for example, as a bolus, or over a period of time, for example, via intravenous infusion. The term "multiple doses" refers to two or more doses, for example, two or more administrations.

[0081] When referring to co-administration of two or more compositions, a dose of Composition A can be administered simultaneously with a dose of Composition B. Alternatively, a dose of Composition A can be administered before or after a dose of Composition B. In some embodiments, Composition A and Composition B are combined in a single formulation.

[0082] As used herein, the term "interval" or "dosing interval" refers to the length of time that elapses between a first dose of Composition A and a subsequent dose of the same composition administered to a subject. The dosing interval can refer to the time that elapses between the first dose and the second dose, or the dosing interval can refer to the length of time that elapses between multiple doses.

[0083] The term "dosing frequency" used herein refers to the number of doses administered per specific dosing interval.For example, dosing frequency can be described as once a week, once every two weeks, etc.Therefore, a 7-day dosing interval can also be described as once every 7 days, once every week, or once a week.

[0084] As used herein, the term "prophylactic treatment" refers to the administration of a therapy for the treatment of hemophilia, and such treatment is intended to prevent or reduce the severity of one or more symptoms of hemophilia, such as bleeding episodes, e.g., one or more spontaneous bleeding episodes, and / or joint damage. See Jimenez-Yuste et al., Blood Transfus. 12(3):314-19 (2014). To prevent or reduce the severity of such symptoms, e.g., bleeding episodes and the progression of joint disease, patients with hemophilia A may receive regular infusions of clotting factors as part of a prophylactic treatment regimen. The basis for such prophylactic treatment is the observation that hemophilia patients with 1% or higher levels of clotting factors, e.g., FVIII, rarely experience spontaneous bleeding episodes and have fewer hemophilia-related comorbidities compared to patients with severe hemophilia. See, e.g., Coppola A. et al, Semin. Thromb. Hemost. 38(1): 79-94 (2012). Medical professionals treating these hemophilia patients speculated that maintaining factor levels at approximately 1% through regular infusions could potentially reduce the risk of hemophilia symptoms, including bleeding episodes and joint damage. See ibid. Subsequent studies confirmed these benefits in pediatric hemophilia patients receiving prophylactic treatment with clotting factors, making prophylactic treatment a goal for people with severe hemophilia. See ibid.

[0085] "Prophylactic" treatment can also refer to the preemptive administration of a composition, e.g., a chimeric polypeptide, described herein to a subject to control, manage, prevent, or reduce the occurrence or severity of one or more symptoms of hemophilia A, e.g., bleeding episodes. Prophylactic treatment with a clotting factor, e.g., FVIII, is the standard of care for subjects with severe hemophilia A. See, e.g., Oldenburg, Blood 125:2038-44 (2015). In some embodiments, prophylactic treatment refers to administering a composition disclosed herein to a subject in need thereof to reduce the occurrence of one or more symptoms of hemophilia A. Prophylactic treatment can include administration of multiple doses. Multiple doses used in prophylactic treatment are typically administered at specific dosing intervals. In certain embodiments, the annualized bleeding rate can be reduced to less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1.

[0086] The terms "on-demand treatment" or "episodic treatment" refer to the administration of a chimeric molecule "as needed" in response to a symptom of hemophilia A, e.g., a bleeding episode, or before an activity that may cause bleeding. In one aspect, on-demand treatment can be given to a subject when bleeding begins, e.g., after an injury, or when bleeding is expected, e.g., before surgery. In another aspect, on-demand treatment can be given before an activity that increases the risk of bleeding, e.g., contact sports. In some embodiments, on-demand treatment is given as a single dose. In other embodiments, on-demand treatment is given as a first dose, followed by one or more additional doses. When the chimeric polypeptide is administered on-demand, the one or more additional doses can be administered at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, or at least about 120 hours after the first dose. It should be noted, however, that the dosage intervals associated with on-demand treatment are not the same as those used for prophylactic treatment.

[0087] In some embodiments, the subject requiring a general hemostatic agent is undergoing or about to undergo surgery. The chimeric polypeptide of the present disclosure can be administered before or after surgery. The chimeric polypeptide of the present disclosure can also be administered during or after surgery to control acute bleeding episodes. When the chimeric polypeptide is administered before surgery, administration can be at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, or at least about 72 hours before surgery. When the chimeric polypeptide is administered after surgery, administration can be at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, or at least about 72 hours after surgery. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedure, or stem cell transplantation.

[0088] As used herein, the term "acute bleeding" refers to a bleeding episode regardless of the underlying cause. For example, the subject may have trauma, uremia, inherited bleeding disorder (e.g., factor VII deficiency), platelet disorder, or tolerance due to the development of antibodies against coagulation factors.

[0089] "Treat", "treatment", "treating", as used herein, refers to, for example, reducing the severity of a disease or condition; reducing the duration of a disease course; ameliorating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition; or preventing one or more symptoms associated with a disease or condition. In some embodiments, the term "treating" or "treatment" refers to maintaining a FVIII trough level of at least about 1 IU / dL, 2 IU / dL, 3 IU / dL, 4 IU / dL, 5 IU / dL, 6 IU / dL, 7 IU / dL, 8 IU / dL, 9 IU / dL, 10 IU / dL, 11 IU / dL, 12 IU / dL, 13 IU / dL, 14 IU / dL, 15 IU / dL, 16 IU / dL, 17 IU / dL, 18 IU / dL, 19 IU / dL, or 20 IU / dL in a subject by administering a chimeric polypeptide or VWF fragment of the present disclosure. As used herein, a "trough level" in a hemophilia patient is a measurement of the lowest concentration reached by factor therapy, e.g., FVIII therapy, before the next dose is administered. In other embodiments, treating or treating means maintaining a trough level of FVIII of at least about 1 IU / dL between dosing intervals. In other embodiments, treating or treating means maintaining a trough level of FVIII of at least about 3 IU / dL between dosing intervals. In other embodiments, treating or treating means maintaining a trough level of FVIII of at least about 5 IU / dL between dosing intervals. In other embodiments, treating or treatment means maintaining a trough level of FVIII between about 1 and about 20 IU / dL, between about 2 and about 20 IU / dL, between about 3 and about 20 IU / dL, between about 4 and about 20 IU / dL, between about 5 and about 20 IU / dL, between about 6 and about 20 IU / dL, between about 7 and about 20 IU / dL, between about 8 and about 20 IU / dL, between about 9 and about 20 IU / dL, or between about 10 and about 20 IU / dL between dosing intervals.

[0090] "Treatment" or "treating" of a disease or condition can also include maintaining FVIII activity in a subject at a level equivalent to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophilic subject during a dosing interval. In other embodiments, treating or treating refers to maintaining an FVIII activity level of at least about 1% during a dosing interval. In other embodiments, treating or treating refers to maintaining an FVIII activity level of at least about 2% during a dosing interval. In other embodiments, treating or treating refers to maintaining an FVIII activity level of at least about 3% during a dosing interval. In other embodiments, treating or treating refers to maintaining an FVIII activity level of at least about 4% during a dosing interval. In another embodiment, treating or treating means maintaining an FVIII activity level of at least about 5% between dosing intervals. In another embodiment, treating or treating means maintaining an FVIII activity level of at least about 6% between dosing intervals. In another embodiment, treating or treating means maintaining an FVIII activity level of at least about 7% between dosing intervals. In another embodiment, treating or treating means maintaining an FVIII activity level of at least about 8% between dosing intervals. In another embodiment, treating or treating means maintaining an FVIII activity level of at least about 9% between dosing intervals. In another embodiment, treating or treating means maintaining an FVIII activity level of at least about 10% between dosing intervals. The minimum trough level required for treatment can be measured by one or more known methods (e.g., the aPTT assay or chromogenic assay described herein) and can be adjusted (increased or decreased) for each individual.

[0091] II. Methods of the Disclosure Certain aspects of the present disclosure relate to methods of treating hemophilia A in a subject in need thereof, comprising administering to the subject, at a dosage interval, a chimeric polypeptide comprising a FVIII protein and a VWF fragment. In some embodiments, the method comprises administering to the subject, at a dosage interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, e.g., rFVIIIFc-VWF-XTEN. In other aspects, the present disclosure relates to methods of treating a bleeding disease or condition, e.g., hemophilia A, in a subject in need thereof, comprising administering to the subject, at a dosage interval, multiple doses of a FVIII protein and multiple doses of a VWF fragment.

[0092] In some embodiments, the multiple doses include at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, at least 10 doses, at least 11 doses, at least 12 doses, at least 13 doses, at least 14 doses, at least 15 doses, at least 16 doses, at least 17 doses, at least 18 doses, at least 19 doses, at least 20 doses, or more. In some embodiments, the multiple doses are administered for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 10 years, at least about 15 years, at least about 20 years, or at least about 25 years.

[0093] In certain embodiments, the methods of the disclosure relate to treating hemophilia A. In some embodiments, treating hemophilia A comprises preventing a bleeding episode in a human subject in need thereof. In some embodiments, treating hemophilia A comprises treating a bleeding episode in a human subject in need thereof. In some embodiments, treating hemophilia A comprises controlling the incidence or frequency of bleeding episodes in a human subject in need thereof. In some embodiments, treating hemophilia A comprises reducing the incidence or frequency of bleeding episodes in a human subject in need thereof.

[0094] A. Dose In some embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered as a single dose or as multiple doses. In some embodiments, the amount of each of the multiple doses is the same. In other embodiments, one or more of the multiple doses is different from one or more of the other multiple doses. In some embodiments, at least one of the multiple doses of the chimeric polypeptide is about 5 IU / kg to about 200 IU / kg, or about 10 IU / kg to about 150 IU / kg. In certain embodiments, at least one of the multiple doses is about 15 IU / kg to about 100 IU / kg. In some embodiments, at least one of the multiple doses comprises about 20 IU / kg to about 95 IU / kg, about 20 IU / kg to about 90 IU / kg, about 20 IU / kg to about 85 IU / kg, about 20 IU / kg to about 80 IU / kg, about 20 IU / kg to about 75 IU / kg, about 20 IU / kg to about 70 IU / kg, about 20 IU / kg to about 65 IU / kg, about 20 IU / kg to about 60 IU / kg, about 20 IU / kg to about 55 IU / kg, about 20 IU / kg to about 50 IU / kg, about 20 IU / kg to about 45 IU / kg, about 20 IU / kg to about 40 IU / kg, about 20 IU / kg to about 35 IU / kg, about 20 IU / kg to about 30 IU / kg, or about 20 IU / kg to about 25 IU / kg.

[0095] In some embodiments, at least one of the multiple doses comprises about 20 IU / kg to about 100 IU / kg, about 25 IU / kg to about 100 IU / kg, about 30 IU / kg to about 100 IU / kg, about 35 IU / kg to about 100 IU / kg, about 40 IU / kg to about 100 IU / kg, about 45 IU / kg to about 100 IU / kg, about 50 IU / kg to about 100 IU / kg, about 55 IU / kg to about 100 IU / kg, about 60 IU / kg to about 100 IU / kg, about 65 IU / kg to about 100 IU / kg, about 70 IU / kg to about 100 IU / kg, about 75 IU / kg to about 100 IU / kg, about 80 IU / kg to about 100 IU / kg, about 85 IU / kg to about 100 IU / kg, or about 90 IU / kg to about 100 IU / kg. In some embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg.

[0096] In some embodiments, at least one of the multiple doses of the chimeric polypeptide is about 50 IU / kg to about 150 IU / kg, about 50 IU / kg to about 140 IU / kg, about 50 IU / kg to about 130 IU / kg, about 50 IU / kg to about 120 IU / kg, about 50 IU / kg to about 110 IU / kg, or about 50 IU / kg to about 100 IU / kg. In some embodiments, at least one of the multiple doses of the chimeric polypeptide is about 50 IU / kg to about 95 IU / kg, about 50 IU / kg to about 90 IU / kg, about 50 IU / kg to about 85 IU / kg, about 50 IU / kg to about 80 IU / kg, about 50 IU / kg to about 75 IU / kg, about 50 IU / kg to about 70 IU / kg, about 50 IU / kg to about 65 IU / kg, about 50 IU / kg to about 60 IU / kg, or about 50 IU / kg to about 55 IU / kg. In certain embodiments, at least one of the multiple doses of the chimeric polypeptide is about 50 IU / kg to about 80 IU / kg. In certain embodiments, at least one of the multiple doses of the chimeric polypeptide is about 50 IU / kg to about 65 IU / kg.

[0097] In some embodiments, at least one of the multiple doses of the chimeric polypeptide is about 60 IU / kg to about 150 IU / kg, about 60 IU / kg to about 140 IU / kg, about 60 IU / kg to about 130 IU / kg, about 60 IU / kg to about 120 IU / kg, about 60 IU / kg to about 110 IU / kg, or about 60 IU / kg to about 100 IU / kg. In some embodiments, at least one of the multiple doses of the chimeric polypeptide is about 60 IU / kg to about 95 IU / kg, about 60 IU / kg to about 90 IU / kg, about 60 IU / kg to about 85 IU / kg, about 60 IU / kg to about 80 IU / kg, about 60 IU / kg to about 75 IU / kg, about 60 IU / kg to about 70 IU / kg, or about 60 IU / kg to about 65 IU / kg.

[0098] In some embodiments, at least one of the multiple doses of the chimeric polypeptide is about 65 IU / kg to about 150 IU / kg, about 65 IU / kg to about 140 IU / kg, about 65 IU / kg to about 130 IU / kg, about 65 IU / kg to about 120 IU / kg, about 65 IU / kg to about 110 IU / kg, or about 65 IU / kg to about 100 IU / kg. In some embodiments, at least one of the multiple doses of the chimeric polypeptide is about 65 IU / kg to about 95 IU / kg, about 65 IU / kg to about 90 IU / kg, about 65 IU / kg to about 85 IU / kg, about 65 IU / kg to about 80 IU / kg, about 65 IU / kg to about 75 IU / kg, or about 65 IU / kg to about 70 IU / kg. In certain embodiments, at least one of the multiple doses of the chimeric polypeptide is about 65 IU / kg to about 80 IU / kg.

[0099] In some embodiments, at least one of the multiple doses is about 5 IU / kg, about 10 IU / kg, about 15 IU / kg, about 20 IU / kg, about 25 IU / kg, about 30 IU / kg, about 35 IU / kg, about 40 IU / kg, about 45 IU / kg, about 50 IU / kg, about 55 IU / kg, about 60 IU / kg, about 65 IU / kg, about 70 IU / kg, about 75 IU / kg, about 80 IU / kg, about 85 IU / kg, about 90 IU / kg, about 95 IU / kg, about 100 IU / kg, about 125 IU / kg, about 150 IU / kg, about 175 IU / kg, or about 200 IU / kg. In certain embodiments, at least one of the multiple doses is about 25 IU / kg. In certain embodiments, at least one of the multiple doses is about 30 IU / kg. In certain embodiments, at least one of the multiple doses is about 35 IU / kg. In certain embodiments, at least one of the multiple doses is about 40 IU / kg. In certain embodiments, at least one of the multiple doses is about 45 IU / kg. In certain embodiments, at least one of the multiple doses is about 50 IU / kg. In certain embodiments, at least one of the multiple doses is about 55 IU / kg. In certain embodiments, at least one of the multiple doses is about 60 IU / kg. In certain embodiments, at least one of the multiple doses is about 65 IU / kg. In certain embodiments, at least one of the multiple doses is about 70 IU / kg. In certain embodiments, at least one of the multiple doses is about 75 IU / kg. In certain embodiments, at least one of the multiple doses is about 80 IU / kg. In certain embodiments, at least one of the multiple doses is about 85 IU / kg. In certain embodiments, at least one of the multiple doses is about 90 IU / kg. In certain embodiments, at least one of the multiple doses is about 95 IU / kg. In certain embodiments, at least one of the multiple doses is about 100 IU / kg.

[0100] In some embodiments, each dose of the multiple doses is about 25 IU / kg. In some embodiments, each dose of the multiple doses is about 30 IU / kg. In some embodiments, each dose of the multiple doses is about 35 IU / kg. In some embodiments, each dose of the multiple doses is about 40 IU / kg. In some embodiments, each dose of the multiple doses is about 45 IU / kg. In some embodiments, each dose of the multiple doses is about 50 IU / kg. In some embodiments, each dose of the multiple doses is about 55 IU / kg. In some embodiments, each dose of the multiple doses is about 60 IU / kg. In some embodiments, each dose of the multiple doses is about 65 IU / kg. In some embodiments, each dose of the multiple doses is about 70 IU / kg. In some embodiments, each dose of the multiple doses is about 75 IU / kg. In some embodiments, each dose of the multiple doses is about 80 IU / kg. In some embodiments, each dose of the multiple doses is about 85 IU / kg. In some embodiments, each dose of the multiple doses is about 90 IU / kg. In some embodiments, each dose of the multiple doses is about 95 IU / kg. In some embodiments, each dose of the multiple doses is about 100 IU / kg.

[0101] In some embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered prophylactically. When administered prophylactically, at least one of the multiple doses can be about 15 IU / kg to about 100 IU / kg. In certain embodiments, at least one of the multiple doses administered prophylactically is about 25 IU / kg, about 30 IU / kg, about 35 IU / kg, about 40 IU / kg, about 45 IU / kg, about 50 IU / kg, about 55 IU / kg, about 60 IU / kg, about 65 IU / kg, about 70 IU / kg, about 75 IU / kg, about 80 IU / kg, about 85 IU / kg, about 90 IU / kg, about 95 IU / kg, or about 100 IU / kg. In some embodiments, at least one of the multiple doses administered prophylactically is about 25 IU / kg. In other embodiments, at least one of the multiple doses administered prophylactically is about 50 IU / kg. In other embodiments, at least one of the multiple doses administered prophylactically is about 65 IU / kg. In other embodiments, at least one of the multiple doses administered prophylactically is about 80 IU / kg.

[0102] In some embodiments, the chimeric polypeptide, for example, rFVIIIFc-VWF-XTEN, is administered on demand. When administered on demand, the chimeric polypeptide can be administered as a single dose or multiple doses. In some embodiments, the chimeric polypeptide is administered in one or multiple doses of about 15 IU / kg to about 100 IU / kg. In certain embodiments, the chimeric polypeptide is administered on demand in one or multiple doses of about 25 IU / kg, about 30 IU / kg, about 35 IU / kg, about 40 IU / kg, about 45 IU / kg, about 50 IU / kg, about 55 IU / kg, about 60 IU / kg, about 65 IU / kg, about 70 IU / kg, about 75 IU / kg, about 80 IU / kg, about 85 IU / kg, about 90 IU / kg, about 95 IU / kg, or about 100 IU / kg. In some embodiments, the chimeric polypeptide is administered on demand as one or more doses of about 25 IU / kg. In other embodiments, the chimeric polypeptide is administered on demand as one or more doses of about 50 IU / kg. In other embodiments, the chimeric polypeptide is administered on demand as one or more doses of about 65 IU / kg. In other embodiments, the chimeric polypeptide is administered on demand as one or more doses of about 80 IU / kg.

[0103] In some embodiments, administration of the chimeric polypeptide according to the present methods does not induce FVIII inhibitors after about 7 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 20 days, about 24 days, about 25 days, about 28 days, about 30 days, or about 35 days of administration. In some embodiments, administration of the chimeric polypeptide does not induce FVIII inhibitors after about 28 days of administration.

[0104] B. Dosage interval In certain embodiments, particularly for prophylactic treatment, the chimeric polypeptide, for example, rFVIIIFc-VWF-XTEN, is administered as multiple doses with a dosing interval. In some embodiments, the dosing interval is at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, or at least about 31 days.

[0105] In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 5 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 6 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 7 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 8 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 9 days. In certain embodiments, the dosing interval is at least about 10 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 11 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 12 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 13 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 14 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 21 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 27 days. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 30 days.

[0106] In some embodiments, the dosing frequency, e.g., for prophylactic treatment of hemophilia A, is at least twice per week, at least once per week, at least once per two weeks, at least once per three weeks, at least once per four weeks, at least once per five weeks, or at least once per six weeks. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least once per week. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least two weeks. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least three weeks. In certain embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least four weeks.

[0107] In some embodiments, the dosing frequency, e.g., for prophylactic treatment of hemophilia A, is about twice per three months, about once per month, about twice per month, about three times per month, about four times per month, about five times per month, about six times per month, about seven times per month, or about eight times per month. In certain embodiments, the dosing frequency is about once per month. In certain embodiments, the dosing frequency is about twice per month. In certain embodiments, the dosing frequency is about three times per month. In certain embodiments, the dosing frequency is about four times per month. In certain embodiments, the dosing frequency is about five times per month. In certain embodiments, the dosing frequency is about six times per month.

[0108] In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 3-5 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 4-6 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 4-7 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 4-8 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 4-9 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 4-10 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 5-7 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 5-8 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 5-9 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 5-10 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 6-8 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 6-9 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 6-10 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 7-9 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 7-10 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 7-11 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 7-12 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 7-13 days.In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 7-14 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 8-14 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 9-14 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 10-14 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 11-14 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 10-21 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 12-14 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 12-15 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 13-15 days. In some embodiments, the dosing interval, e.g., for prophylactic treatment of hemophilia A, is at least about 14-21 days.

[0109] In some embodiments, at least one of the multiple doses is administered according to the doses and dosing frequencies set forth in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0110] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 6-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 6-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 6-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 7-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 7-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 7-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 7-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 7-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 9-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 10-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 11-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 12-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 12-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg to about 65 IU / kg with an interval of at least about 14-21 days between doses.

[0111] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 25 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with a dosing interval of at least about 6-11 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 6-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 6-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 7-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 7-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 7-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 7-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 7-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval of at least about 9-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval between doses of at least about 10-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval between doses of at least about 10-21 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval between doses of at least about 11-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval between doses of at least about 12-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval between doses of at least about 12-15 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval between doses of at least about 13-15 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 25 IU / kg with an interval between doses of at least about 14-21 days.

[0112] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 50 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with a dosing interval of at least about 6-11 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 6-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 6-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 7-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 7-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 7-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 7-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 7-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 9-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 10-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 11-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 12-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 12-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg with an interval between doses of at least about 14-21 days.

[0113] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 65 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with a dosing interval of at least about 6-11 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 6-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 6-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 7-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 7-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 7-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 7-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 7-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 9-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 10-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 11-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 12-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 12-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg with an interval between doses of at least about 14-21 days.

[0114] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 6-11 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 6-12 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 6-13 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 7-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 7-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 7-11 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 7-12 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 7-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 9-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 10-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 11-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 12-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 12-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 80 IU / kg with a dosing interval of at least about 14-21 days.

[0115] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 6-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 6-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 6-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 7-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 7-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 7-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 7-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 7-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 9-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 10-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 11-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 12-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 12-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 80 IU / kg with an interval of at least about 14-21 days between doses.

[0116] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 6-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 6-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 6-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 7-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 7-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 7-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 7-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 7-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 9-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 10-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 11-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 12-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 12-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 50 IU / kg to about 65 IU / kg with an interval of at least about 14-21 days between doses.

[0117] In some embodiments, such as for the prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 5-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-14 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-10 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-8 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 6-9 days. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 6-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 6-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 6-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 7-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 7-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 7-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 7-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 7-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 7-14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 9-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 10-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 11-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 12-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 12-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 14-21 days between doses.

[0118] In some embodiments, such as for the prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 2 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 2 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 2 to 8 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 2 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 2 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 2-12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 2-13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 4-9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 4-10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 4-11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 4 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 4 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 4 to 14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 5-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 5-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 6-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 7-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 8-14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 10-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 10-21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 13-15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with an interval of at least about 14-21 days between doses.

[0119] In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 5 days. In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 7 days. In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 6 days. In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 5 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 4 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 3 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 2 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 8 days. In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 9 days. In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 10 days.In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 11 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 12 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 13 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing interval of at least about 14 days. In some embodiments, such as for prophylactic treatment of hemophilia A in pediatric subjects, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg, with a dosing interval of at least about 21 days.

[0120] In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing frequency of at least about one week. In some embodiments, such as for prophylactic treatment of hemophilia A in a pediatric subject, at least one of the multiple doses is administered at a dose of about 65 IU / kg to about 80 IU / kg with a dosing frequency of at least two times per week.

[0121] In certain embodiments, such as for the prophylactic treatment of hemophilia A, the method comprises administering to the subject, at a dosing interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, e.g., rFVIIIFc-VWF-XTEN, wherein at least one of the multiple doses is about 15 IU / kg to about 100 IU / kg, and the dosing interval is at least about 5 days. In certain embodiments, such as for the prophylactic treatment of hemophilia A, the method comprises administering to the subject, at a dosing interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, wherein at least one of the multiple doses is about 15 IU / kg to about 100 IU / kg, and the dosing interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 8 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 9 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 10 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 11 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 12 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 13 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg and the interval between doses is at least about 14 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg and the interval between doses is at least about 15 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg and the interval between doses is at least about 16 days.In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 17 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 18 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 19 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 20 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg to about 65 IU / kg, and the interval between doses is at least about 21 days.

[0122] In certain embodiments, the method includes administering to a subject, at a dosage interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, e.g., rFVIIIFc-VWF-XTEN, wherein at least one of the multiple doses is about 25 IU / kg, and the dosage interval is at least about 5 days. In certain embodiments, the method includes administering to a subject, at a dosage interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, wherein at least one of the multiple doses is about 25 IU / kg, and the dosage interval is at least about 6 days. In some embodiments, the method is for the prophylactic treatment of hemophilia A. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosage interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the interval between doses is at least about 8 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the interval between doses is at least about 9 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the interval between doses is at least about 10 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the interval between doses is at least about 11 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the interval between doses is at least about 12 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the interval between doses is at least about 13 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the interval between doses is at least about 14 days. In certain embodiments, at least one of the multiple doses is from about 25 IU / kg and the interval between doses is at least about 15 days. In certain embodiments, at least one of the multiple doses is from about 25 IU / kg and the interval between doses is at least about 16 days. In certain embodiments, at least one of the multiple doses is from about 25 IU / kg and the interval between doses is at least about 17 days.In certain embodiments, at least one of the multiple doses is from about 25 IU / kg and the interval between doses is at least about 18 days. In certain embodiments, at least one of the multiple doses is from about 25 IU / kg and the interval between doses is at least about 19 days. In certain embodiments, at least one of the multiple doses is from about 25 IU / kg and the interval between doses is at least about 20 days. In certain embodiments, at least one of the multiple doses is from about 25 IU / kg and the interval between doses is at least about 21 days.

[0123] In certain embodiments, the method includes administering to a subject, at a dosage interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, e.g., rFVIIIFc-VWF-XTEN, wherein at least one of the multiple doses is about 50 IU / kg, and the dosage interval is at least about 5 days. In certain embodiments, the method includes administering to a subject, at a dosage interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, wherein at least one of the multiple doses is about 50 IU / kg, and the dosage interval is at least about 6 days. In some embodiments, the method is for the prophylactic treatment of hemophilia A. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the dosage interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the interval between doses is at least about 8 days. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the interval between doses is at least about 9 days. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the interval between doses is at least about 10 days. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the interval between doses is at least about 11 days. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the interval between doses is at least about 12 days. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the interval between doses is at least about 13 days. In certain embodiments, at least one of the multiple doses is about 50 IU / kg, and the interval between doses is at least about 14 days. In certain embodiments, at least one of the multiple doses is from about 50 IU / kg and the interval between doses is at least about 15 days. In certain embodiments, at least one of the multiple doses is from about 50 IU / kg and the interval between doses is at least about 16 days. In certain embodiments, at least one of the multiple doses is from about 50 IU / kg and the interval between doses is at least about 17 days.In certain embodiments, at least one of the multiple doses is from about 50 IU / kg and the interval between doses is at least about 18 days. In certain embodiments, at least one of the multiple doses is from about 50 IU / kg and the interval between doses is at least about 19 days. In certain embodiments, at least one of the multiple doses is from about 50 IU / kg and the interval between doses is at least about 20 days. In certain embodiments, at least one of the multiple doses is from about 50 IU / kg and the interval between doses is at least about 21 days.

[0124] In certain embodiments, the method includes administering to a subject multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, e.g., rFVIIIFc-VWF-XTEN, at a dosage interval, wherein at least one of the multiple doses is about 65 IU / kg, and the dosage interval is at least about 5 days. In certain embodiments, the method includes administering to a subject multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, at a dosage interval, wherein at least one of the multiple doses is about 65 IU / kg, and the dosage interval is at least about 6 days. In some embodiments, the method is for the prophylactic treatment of hemophilia A. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the dosage interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the interval between doses is at least about 8 days. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the interval between doses is at least about 9 days. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the interval between doses is at least about 10 days. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the interval between doses is at least about 11 days. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the interval between doses is at least about 12 days. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the interval between doses is at least about 13 days. In certain embodiments, at least one of the multiple doses is about 65 IU / kg, and the interval between doses is at least about 14 days. In certain embodiments, at least one of the multiple doses is from about 65 IU / kg and the interval between doses is at least about 15 days. In certain embodiments, at least one of the multiple doses is from about 65 IU / kg and the interval between doses is at least about 16 days. In certain embodiments, at least one of the multiple doses is from about 65 IU / kg and the interval between doses is at least about 17 days.In certain embodiments, at least one of the multiple doses is from about 65 IU / kg and the interval between doses is at least about 18 days. In certain embodiments, at least one of the multiple doses is from about 65 IU / kg and the interval between doses is at least about 19 days. In certain embodiments, at least one of the multiple doses is from about 65 IU / kg and the interval between doses is at least about 20 days. In certain embodiments, at least one of the multiple doses is from about 65 IU / kg and the interval between doses is at least about 21 days.

[0125] In certain embodiments, the method includes administering to a subject, at a dosage interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, e.g., rFVIIIFc-VWF-XTEN, wherein at least one of the multiple doses is about 80 IU / kg, and the dosage interval is at least about 5 days. In certain embodiments, the method includes administering to a subject, at a dosage interval, multiple doses of a chimeric polypeptide comprising (i) a FVIII protein and (ii) a VWF fragment comprising the D' domain of VWF and the D3 domain of VWF, wherein at least one of the multiple doses is about 80 IU / kg, and the dosage interval is at least about 6 days. In some embodiments, the method is for the prophylactic treatment of hemophilia A. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosage interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the interval between doses is at least about 8 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the interval between doses is at least about 9 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the interval between doses is at least about 10 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the interval between doses is at least about 11 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the interval between doses is at least about 12 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the interval between doses is at least about 13 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the interval between doses is at least about 14 days. In certain embodiments, at least one of the multiple doses is from about 80 IU / kg and the interval between doses is at least about 15 days. In certain embodiments, at least one of the multiple doses is from about 80 IU / kg and the interval between doses is at least about 16 days. In certain embodiments, at least one of the multiple doses is from about 80 IU / kg and the interval between doses is at least about 17 days.In certain embodiments, at least one of the multiple doses is from about 80 IU / kg and the interval between doses is at least about 18 days. In certain embodiments, at least one of the multiple doses is from about 80 IU / kg and the interval between doses is at least about 19 days. In certain embodiments, at least one of the multiple doses is from about 80 IU / kg and the interval between doses is at least about 20 days. In certain embodiments, at least one of the multiple doses is from about 80 IU / kg and the interval between doses is at least about 21 days.

[0126] In some embodiments, the chimeric polypeptide is administered for prophylactic treatment of hemophilia A. Prophylactic treatment of hemophilia A involves alleviating or reducing the severity of symptoms of hemophilia A on a continuous or near-continuous basis. In some embodiments, prophylactic treatment is administered before the onset of symptoms of hemophilia A, e.g., before a bleeding event. In other embodiments, prophylactic treatment is administered on a regular basis, e.g., at a dosing interval described herein, to prevent the onset of symptoms or reduce the severity of symptoms before the onset of symptoms. Any dosing interval disclosed herein can be used in prophylactic treatment of hemophilia A.

[0127] In other embodiments, the chimeric polypeptide is administered before an activity that may cause one or more symptoms of hemophilia A, e.g., as an on-demand treatment. For example, a chimeric polypeptide of the present disclosure can be administered to a subject with hemophilia A before the subject undergoes surgery or combat in an activity that would otherwise increase the risk of physical trauma and / or bleeding events. When administered on-demand, the chimeric polypeptide can be administered as a single dose or as multiple doses. In some embodiments, the on-demand treatment involves administering multiple doses of the chimeric polypeptide with a dosing interval of at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, or at least about 120 hours. In certain embodiments, the on-demand treatment involves administering at least two doses, at least three doses, at least four doses, or at least five doses of the chimeric polypeptide.

[0128] In some embodiments, the subject has been previously treated with one or more FVIII replacement therapies. In certain embodiments, the subject did not respond to previous FVIII replacement therapies. In certain embodiments, the FVIII replacement therapy is ELOCTATE® or ADVATE®. In some embodiments, the subject is an adult. In some embodiments, the subject is an adult male. In some embodiments, the subject is an adult female. In other embodiments, the subject is a child, e.g., about 12 years old or younger, about 11 years old or younger, about 10 years old or younger, about 9 years old or younger, about 8 years old or younger, about 7 years old or younger, about 6 years old or younger, about 5 years old or younger, about 4 years old or younger, about 3 years old or younger, about 2 years old or younger, or about 1 year old or younger. In some embodiments, the subject is a female. In some embodiments, the subject is a male. In some embodiments, the subject is a female about 12 years old or younger. In some embodiments, the subject is a female about 11 years of age or younger. In some embodiments, the subject is a female about 10 years of age or younger.

[0129] The chimeric polypeptides described herein can be administered by any means known in the art. In some embodiments, the chimeric polypeptides are administered by a route selected from the group consisting of intravenous injection, intravenous infusion, subcutaneous administration, intramuscular administration, oral administration, intranasal administration, and pulmonary administration. In some embodiments, the chimeric polypeptides are administered intravenously. In other embodiments, the chimeric polypeptides are administered subcutaneously.

[0130] In some embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, after administration results in a plasma FVIII activity level of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%. In certain embodiments, the plasma FVIII activity level is at least about 3%. In some embodiments, the plasma FVIII activity level is at least about 4%. In some embodiments, the plasma FVIII activity level is at least about 5%. In some embodiments, the plasma FVIII activity level is at least about 4%. In some embodiments, the plasma FVIII activity level is at least about 5%. In some embodiments, the plasma FVIII activity level is at least about 5.6%. In some embodiments, the plasma FVIII activity level is at least about 7%. In some embodiments, the plasma FVIII activity level is at least about 10%. In some embodiments, the plasma activity level of FVIII is at least about 12%. In some embodiments, the plasma activity level of FVIII is at least about 12.95%. As used herein, plasma activity level is expressed as a percentage (%). Alternatively, plasma activity can be expressed in IU / dL, where 1% is equal to 1 IU / dL.

[0131] In some embodiments, the plasma activity level of FVIII is at least about 10% at least about 5 days after administration of the chimeric polypeptide, for example, at a dose of 25 IU / kg. In some embodiments, the plasma activity level of FVIII is at least about 12% at least about 5 days after administration of the chimeric polypeptide, for example, at a dose of 25 IU / kg. In some embodiments, the plasma activity level of FVIII is at least about 12.95% at least about 5 days after administration of the chimeric polypeptide, for example, at a dose of 25 IU / kg. In some embodiments, the plasma activity level of FVIII is at least about 5% at least about 7 days after administration of the chimeric polypeptide, for example, at a dose of 25 IU / kg. In some embodiments, the plasma activity level of FVIII is at least about 5.6% at least about 7 days after administration of the chimeric polypeptide, for example, at a dose of 25 IU / kg. In some embodiments, the plasma activity level of FVIII is at least about 3% at least about 8 days after administration of the chimeric polypeptide, e.g., at a dose of 25 IU / kg. In some embodiments, the plasma activity level of FVIII is at least about 1% at least about 10 days after administration of the chimeric polypeptide, e.g., at a dose of 25 IU / kg.

[0132] The present disclosure also includes a method for treating hemophilia A, comprising administering to a subject a composition comprising (i) a FVIII protein and (ii) a VWF fragment. In some embodiments, the FVIII protein is present in a first formulation, and the VWF fragment is present in a second formulation. In other embodiments, the FVIII protein and the VWF fragment are present in the same formulation. In certain embodiments, the FVIII protein and the VWF fragment are separate components of a single formulation. In certain embodiments, a FVIII protein, e.g., single-chain FVIII, FVIII-Fc, pegylated FVIII, full-length mature FVIII, or B-domain deleted FVIII, is administered at a dose disclosed herein, e.g., 25 IU / kg, 30 IU / kg, 35 IU / kg, 40 IU / kg, 45 IU / kg, 50 IU / kg, 55 IU / kg, 60 IU / kg, or 65 IU / kg, for 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, or or at least 14 days between doses, and the VWF fragments are administered at a dose of at least about 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 100:1, 200:1, 300:1, 400:1, 500:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 10,000:1, 50,000:1, 100,000:1, or 500,000:1 of the VWF fragment to FVIII molecule administered. In other embodiments, the VWF fragment is administered to a subject simultaneously with, immediately after, or immediately before the FVIII protein. In certain embodiments, the VWF fragment is administered about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours before the FVIII protein. In other embodiments, the FVIII protein is administered about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours before the VWF fragment.

[0133] C. Induction of immune tolerance Certain embodiments of the present disclosure relate to a method of treating hemophilia A in a subject in need thereof, comprising administering to the subject a chimeric polypeptide comprising a FVIII protein and a VWF fragment at a dosage interval, wherein the chimeric polypeptide induces immune tolerance to FVIII in the subject. Some embodiments of the present disclosure relate to a method of treating hemophilia A in a subject in need thereof, comprising administering to the subject a chimeric polypeptide comprising a FVIII protein and a VWF fragment at a dosage interval, wherein the chimeric polypeptide reduces an inhibitory response to FVIII in the subject. Some embodiments of the present disclosure relate to a method of treating hemophilia A in a subject in need thereof, comprising administering to the subject a chimeric polypeptide comprising a FVIII protein and a VWF fragment at a dosage interval, wherein the chimeric polypeptide does not induce an immune response to the chimeric polypeptide after administration. Another aspect of the present disclosure relates to a method of inducing immune tolerance in a human with hemophilia, comprising: (1) administering to the human an effective amount of a chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN, wherein the effective amount of the chimeric polypeptide induces immune tolerance in the human. Various methods of inducing immune tolerance using chimeric polypeptides comprising FVIII and Fc are disclosed in WO 2018 / 102760 A1, which is incorporated herein by reference in its entirety.

[0134] In certain embodiments, the method further comprises (2) administering a tapering regimen of the composition or chimeric polypeptide to the human after induction of immune tolerance. In certain embodiments, induction of immune tolerance occurs when the titer of the inhibitory antibody in the human is less than about 0.6 BU. In certain embodiments, induction of immune tolerance occurs when the titer of the inhibitory antibody in the human is less than about 0.6 BU and there is a 60% recovery of the activity of the coagulation factor monitored in plasma. In some embodiments of the present disclosure, the method further comprises (3) administering a prophylactic dose of a coagulation factor, e.g., a chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN, to the human after the tapering regimen.

[0135] In certain aspects, the human has not been treated with a previous immune tolerance therapy against a clotting factor, e.g., FVIII. The chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, can be administered to the human any time it is determined that the human has developed an inhibitory immune response, e.g., after measuring the level of the inhibitory immune response in the human. In other embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, can be administered to a human who has not yet developed one or more inhibitory immune responses to prevent the development of an inhibitory immune response. In some embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered to a human who has a high likelihood of developing an inhibitory immune response (e.g., exhibits a family history, genetic predisposition, or biomarkers). In some embodiments, the method further includes measuring the level of an inhibitory immune response or the likelihood of developing an inhibitory immune response prior to administration.

[0136] In some embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered to a human less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 2 weeks, less than about 3 weeks, less than about 4 weeks, less than about 2 months, less than about 3 months, less than about 4 months, less than about 5 months, less than about 6 months, less than about 1 year, less than about 2 years, less than about 3 years, less than about 4 years, or less than about 5 years after it has been determined that the human has developed an inhibitory immune response or has the potential to develop an inhibitory immune response, e.g., after measuring the level of an inhibitory immune response in the human or the potential to develop an inhibitory immune response. In certain embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered to a human immediately after it has been determined that the human has developed an inhibitory immune response or that the human has the potential to develop an inhibitory immune response, e.g., after measuring the level of an inhibitory immune response or the potential to develop an inhibitory immune response in the human. In certain embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered to a human less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 30 minutes, less than about 45 minutes, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 18 hours, or less than about 24 hours after it has been determined that the human has developed an inhibitory immune response or that the human has the potential to develop an inhibitory immune response, e.g., after measuring the level of an inhibitory immune response in the human or the potential to develop an inhibitory immune response. In certain embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered to a human about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, or about 24 hours after it has been determined that the human has developed an inhibitory immune response or that the human has the potential to develop an inhibitory immune response, e.g., after measuring the level of an inhibitory immune response in the human or the potential to develop an inhibitory immune response.In certain embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered to a human less than about one day after it has been determined that the human has developed an inhibitory immune response or has the potential to develop an inhibitory immune response, e.g., after measuring the level of an inhibitory immune response or the potential to develop an inhibitory immune response in the human.

[0137] The induction of immune response can continue until the level of inhibitor falls below a certain level or until the inhibitor is no longer detectable.In certain embodiments, the induction period can last at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, at least about 30 weeks, at least about 32 weeks, at least about 34 weeks, at least about 36 weeks, at least about 38 weeks, at least about 40 weeks, at least about 42 weeks, at least about 44 weeks, at least about 46 weeks, at least about 48 weeks, at least about 50 weeks, at least about 52 weeks, at least about 54 weeks, at least about 56 weeks, at least about 58 weeks, at least about 60 weeks, at least about 62 weeks, at least about 64 weeks, at least about 66 weeks, at least about 68 weeks, or at least about 70 weeks.In certain embodiments, the induction period is less than 60 weeks.

[0138] The inhibitory immune response treated by the methods of the present invention can include any response in a human that negatively impacts one or more effects of a coagulation factor treatment. In some embodiments, the inhibitory immune response includes the production of an inhibitory antibody against the coagulation factor, e.g., an inhibitory anti-FVIII antibody. In certain embodiments, the methods of the present disclosure further include measuring the titer of one or more inhibitory antibodies in a human before (e.g., baseline) or after administration of an effective amount of a chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN or a polynucleotide encoding same. In some embodiments, the titer of the inhibitory antibody before administration (e.g., baseline) is at least about 0.6 Bethesda Units (BU). In certain embodiments, the titer of the inhibitory antibody before administration (e.g., at baseline) is at least about 1 BU, at least about 2 BU, at least about 3 BU, at least about 4 BU, at least about 5 BU, at least about 6 BU, at least about 7 BU, at least about 10 BU, at least about 20 BU, at least about 30 BU, at least about 40 BU, at least about 50 BU, at least about 100 BU, at least about 150 BU, or at least about 200 BU. In one specific embodiment, the titer of the inhibitory antibody before administration (e.g., at baseline) is at least about 5 BU.

[0139] In some embodiments, the methods of the present invention reduce the titer of an inhibitory antibody in a human subject relative to the titer of the inhibitory antibody before administration. In certain embodiments, the titer of the inhibitory antibody after administration is less than about 0.6 BU. In some embodiments, the titer of the inhibitory antibody after administration is less than about 0.5 BU, less than about 0.4 BU, less than about 0.3 BU, less than about 0.2 BU, or less than about 0.1 BU. In a specific embodiment, the titer of the inhibitory antibody after administration is 0 BU. In other embodiments, the titer of the inhibitory antibody after administration is less than 5 BU, less than 4 BU, less than 3 BU, less than 2 BU, less than 1 BU, less than 0.9 BU, less than 0.8 BU, less than 0.7 BU, or less than 0.6 BU.

[0140] In some embodiments, administration of a chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN, increases the differentiation of macrophages in humans toward an M2-like phenotype compared to untreated controls and humans treated with coagulation factors alone. In some embodiments, the M2-like phenotype comprises upregulation of the NRF2 pathway, the PPAR gamma pathway, or both the NRF2 pathway and the PPAR gamma pathway. In some embodiments, the M2-like phenotype comprises upregulation of CD206 (MRC1). In some embodiments, the M2-like phenotype comprises upregulation of ARG1. In some embodiments, the M2-like phenotype comprises upregulation of CD206 (MRC1) and ARG1.

[0141] In some embodiments, administration of a chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN, results in higher expression of one or more genes in a human relative to expression of the one or more genes in an untreated subject or a subject treated with a coagulation factor alone. In some embodiments, administration results in higher expression of one or more genes in a human relative to expression of the one or more genes in an untreated subject or a subject treated with a coagulation factor alone. In some embodiments, administration results in higher expression of Hmox1, PPAR gamma, LPL, EGR2, SLCO4A1, heme oxygenase 1 (HO-1), oxidative stress-induced growth inhibitor 1 (OSGIN1), superoxide dismutase 1 (SOD1), glutathione-disulfide reductase (GSR), glutamate-cysteine ​​ligase catalytic subunit (GCLC), glutamate-cysteine ​​ligase modifying subunit (GCLM), NAD(P)H quinone dehydrogenase (NDH) kinase (NKK), NK cell proliferation inhibitor (NKC ... In some embodiments, administration results in increased expression of one or more genes selected from the group consisting of NQO1, fatty acid binding protein 5 (FABP5), B7-H3 (CD276), SLAM family member 3 (SLAMF3; lymphocyte antigen 9; LY9), SLAM family member 7 (SLAMF7), mannose receptor type C-1 (MRC1), solute carrier family 12 member 4 (SLC12A), neuropilin 1 (NRP1), and any combination thereof. In some embodiments, administration results in increased expression of one or more genes in the NRF2 pathway. In certain embodiments, the one or more genes in the NRF2 pathway are selected from the group consisting of HO-1, OSGIN1, SOD1, GSR, GCLC, GCLM, NQO1, and any combination thereof. In some embodiments, administration results in increased expression of one or more genes in the PPAR gamma pathway. In some embodiments, the one or more genes in the PPAR gamma pathway are selected from the group consisting of PPAR gamma, LPL, FABP5, EGR2, and any combination thereof. In some embodiments, the administration results in higher expression of one or more genes selected from the group consisting of B7-H3 (CD276), SLAMF3, SLAMF7, MRC1, SLC12A, NRP1, and any combination thereof.In certain embodiments, administration results in higher expression of one or more genes relative to expression of the one or more genes in an untreated human or a human administered the coagulation factor alone, wherein expression is at least about 1.5-fold higher, at least about 2-fold higher, at least about 2.5-fold higher, at least about 3-fold higher, at least about 3.5-fold higher, at least about 4-fold higher, at least about 4.5-fold higher, or at least about 5-fold higher.

[0142] In some embodiments, differential expression of one or more genes is observed less than 6 hours after administration of a chimeric polypeptide described herein, such as rFVIIIFc-VWF-XTEN. In some embodiments, differential expression is observed less than 12 hours after administration. In some embodiments, differential expression is observed less than 18 hours after administration. In some embodiments, differential expression is observed less than 24 hours after administration.

[0143] In some embodiments, the inhibitory immune response comprises a cell-mediated immune response. In certain embodiments, the cell-mediated immune response comprises the release of cytokines. In some embodiments, the cytokine is any cytokine associated with an increased immune response. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL-6, IL-16, IL-12, IL-4, IL-17, tumor necrosis factor alpha (TNF-α), interferon alpha, interferon gamma, and any combination thereof. In one embodiment, the cell-mediated immune response comprises an increased serum level of IL-12. In another embodiment, the cell-mediated immune response comprises an increased serum level of IL-4. In another embodiment, the cell-mediated immune response comprises an increased serum level of IL-17. In another embodiment, the cell-mediated immune response comprises an increased serum level of TNF-α.

[0144] Mutations in various genes are associated with an increased risk of developing an inhibitory immune response. For example, the TNF-α-308G>A polymorphism in Hap2, which is associated with increased constitutive and inducible transcription levels of TNF, is associated with an increased risk of developing an inhibitory immune response. See Astermark et al., Blood 108: 3739-3745 (2006), the entire contents of which are incorporated herein by reference. Thus, in some embodiments, the human has a genetic polymorphism associated with increased TNF-α. In some embodiments, the polymorphism is the TNF-α-308G>A polymorphism. In some embodiments, the human has a polymorphism in the IL10 gene, e.g., a polymorphism associated with increased IL10 secretion. In some embodiments, FVIII-Fc is administered to a subject having allele 134 of the IL10G microsatellite in the promoter region of the IL10 gene. See Astermark et al. Hemostatis, Thrombosis, and Vascular Biology 108: 3739-3745 (2006), which is incorporated by reference in its entirety.

[0145] In some embodiments, the human has a genetic polymorphism associated with decreased expression of CTLA-4 (cytotoxic T lymphocyte antigen 4). In some embodiments, the human has a mutation in DR15 (HLA-DR15) or DQB0602 MHC (major histocompatibility complex) class II molecule. Other MHC class II molecules associated with the development of inhibitory immune responses in subjects with hemophilia are A3, B7, C7, DQA0102, C2, DQA0103, DQB0603, and DR13 (see Inhibitors in Patients with Hemophilia, EC Rodriguez-Merchan & CA Lee, Eds., Blackwell Science, Ltd,, 2002).

[0146] In some embodiments, the disclosed method reduces the level of one or more cytokines in a subject compared to the level of one or more cytokines in the subject after a previous treatment with a polypeptide consisting of a FVIII protein. In another embodiment, the disclosed method reduces the level of one or more cytokines in a subject compared to the level of one or more cytokines in the subject before administration. In other embodiments, the expression of one or more tolerogenic molecules is increased after administration of the disclosed method relative to the expression level of the one or more tolerogenic molecules before administration. In certain embodiments, the one or more tolerogenic molecules are selected from IL-10, TGF-β, IL-35, IDO-1, and any combination thereof.

[0147] In other embodiments, the immune response comprises a clinical symptom selected from the group consisting of an increased tendency to bleed, a high consumption of clotting factors, a lack of response to clotting factor therapy, a decreased effectiveness of clotting factor therapy, a shortened half-life of clotting factors, and any combination thereof. In certain embodiments, the immune response comprises a clinical symptom selected from the group consisting of an increased tendency to bleed, a high consumption of clotting factors, a lack of response to clotting factor therapy, a decreased effectiveness of clotting factor therapy, a decreased recovery of clotting factor activity monitored in plasma, a shortened half-life of clotting factors, and any combination thereof.

[0148] In certain embodiments, the human has previously been diagnosed as having an inhibitory immune response. Such a diagnosis can be made using any method known in the art. For example, if the human has one or more of the following: (a) a titer of inhibitory antibodies against the coagulation factor of 0.6 BU or more; (b) an increase in serum levels of one or more cytokines selected from the group consisting of IL-12, IL-4, IL-17, and TNF-α; (c) an increased tendency to bleed; (d) a high consumption of coagulation factors; (e) a lack of response to coagulation factor therapy; (f) a decrease in the effectiveness of coagulation factor therapy; (g) a shortened half-life of the coagulation factor, and any combination thereof, the human can be characterized as having an immune response to the coagulation factor, for example, FVIII. In a specific embodiment, if the human has a titer of inhibitory antibodies against the coagulation factor of 0.6 BU or more, the human is characterized as having an immune response to the coagulation factor.

[0149] In some embodiments, the human is administered at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 24 months, at least about 25 months, at least about 26 months, at least about 27 months, at least about 28 months, at least about 29 months, at least about 30 months, at least about 31 months, at least about 32 months, at least about 33 months, at least about 34 months, at least about 35 months, at least about 36 months, at least about 37 months, at least about 38 months, at least about 39 months, at least about 40 months, at least about 41 months, at least about 42 months, at least about 43 months, at least about 44 months, at least about 45 months, at least about 46 months, at least about 47 months, at least about 48 months, at least about 49 months, at least about 50 months, at least about 51 months, at least about 52 months, at least about 53 months, at least about 54 months, at least about 55 months, at least about 56 months, at least about 57 months, at least about 58 months, at least about 59 months, at least about 60 months, at least about 61 months, at least about 62 months, at least about 63 months, at least about 64 months, at least about 65 months, In one embodiment, the human was previously diagnosed with developing an inhibitory immune response against the clotting factor at least about 5 months, at least about 23 months, at least about 24 months, at least about 27 months, at least about 30 months, at least about 33 months, at least about 36 months, at least about 39 months, at least about 42 months, at least about 45 months, at least about 48 months, at least about 51 months, at least about 54 months, at least about 57 months, at least about 60 months, at least about 6 years, at least about 7 years, at least about 8 years, at least about 10 years, at least about 15 years, or at least about 20 years prior to administration. In one embodiment, the human was previously diagnosed with developing an inhibitory immune response against the clotting factor at least about 5 years prior to administration.

[0150] In some embodiments, the methods of the present disclosure provide improved time to tolerance compared to standard treatments for inducing immune tolerance. As used herein, the term "time to tolerance" refers to the length of time between the administration of the first dose of a composition or chimeric protein comprising a coagulation factor and an Fc region and the development of immune tolerance in a human. A reduction in the time to tolerance can have significant benefits for humans, including, but not limited to, reducing the overall economic burden required to achieve tolerance. In some embodiments, the time to tolerance is about 1 to about 24 weeks, about 1 to about 23 weeks, about 1 to about 22 weeks, about 1 to about 21 weeks, about 2 to about 20 weeks, about 2 to about 19 weeks, about 2 to about 18 weeks, about 2 to about 17 weeks, about 3 to about 16 weeks, about 3 to about 15 weeks, about 3 to about 14 weeks, about 3 to about 13 weeks, about 4 to about 12 weeks, about 4 to about 11 weeks, about 4 to about 10 weeks, about 4 to about 9 weeks, about 5 to about 8 weeks, about 5 to about 7 weeks, about 5 to about 6 weeks, about 1 to about 12 weeks, about 1 to about 11 weeks, about 1 to about 10 weeks, about 1 to about 9 weeks, about 1 to about 8 weeks, about 1 to about 7 weeks, about 1 to about 6 weeks, about 1 to about 5 weeks, or about 1 to about 4 weeks. In some embodiments, the time to tolerance is less than about 70 weeks, less than about 65 weeks, less than about 60 weeks, less than about 58 weeks, less than about 56 weeks, less than about 54 weeks, less than about 52 weeks, less than about 50 weeks, less than about 48 weeks, less than about 46 weeks, less than about 44 weeks, less than about 42 weeks, less than about 40 weeks, less than about 38 weeks, less than about 36 weeks, less than about 34 weeks, less than about 32 weeks, less than about 30 weeks, less than about 28 weeks, less than about 26 weeks, less than about 24 weeks The time to tolerance is less than about 23 weeks, less than about 22 weeks, less than about 21 weeks, less than about 20 weeks, less than about 19 weeks, less than about 18 weeks, less than about 17 weeks, less than about 16 weeks, less than about 15 weeks, less than about 14 weeks, less than about 13 weeks, less than about 12 weeks, less than about 11 weeks, less than about 10 weeks, less than about 9 weeks, less than about 8 weeks, less than about 7 weeks, less than about 6 weeks, less than about 5 weeks, less than about 4 weeks, less than about 3 weeks, less than about 2 weeks, or less than about 1 week. In certain embodiments, the time to tolerance is about 4 to about 12 weeks. In one embodiment, the time to tolerance is about 4 weeks. In another embodiment, the time to tolerance is about 12 weeks. In some embodiments, the time to tolerance is less than about 10 months. In some embodiments, the time to tolerance is less than about 9 months.In some embodiments, the time to tolerance is less than about 8 months. In some embodiments, the time to tolerance is less than about 7 months. In some embodiments, the time to tolerance is less than about 6 months. In some embodiments, the time to tolerance is less than about 5 months. In some embodiments, the time to tolerance is less than about 4 months. In some embodiments, the methods of the disclosure result in a shorter time to tolerance in humans after treatment with a composition or chimeric protein comprising a clotting factor and an Fc region compared to the time to tolerance after treatment with the clotting factor alone.

[0151] In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies to the clotting factor of less than about 0.6 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies to the clotting factor of less than about 0.5 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies to the clotting factor of less than about 0.4 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies to the clotting factor of less than about 0.3 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies to the clotting factor of less than about 0.2 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies to the clotting factor of less than about 0.1 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies to the clotting factor of 0.0 BU. In certain embodiments, the titer of inhibitory antibodies is observed in two consecutive measurements, for example, in two consecutive weeks within a four-week period.

[0152] In some embodiments, the development of immune tolerance is characterized by an incremental recovery of >66% (e.g., an incremental recovery of about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%). As used herein, "incremental recovery" refers to peak FVIII levels 15-30 minutes after infusion.

[0153] After the induction and tapering periods are complete, the subject can then be given prophylactic treatment with the chimeric protein. The prophylactic dosing regimen can be any of the dosing regimens disclosed herein.

[0154] In some embodiments, the human treated using the methods of the present disclosure is currently undergoing or has recently undergone immunostimulatory therapy. For example, inhibitors have also been reported in HCV-positive hemophilia A patients undergoing interferon treatment and in HIV-positive hemophilia A patients with antiretroviral therapy-associated immune reconstitution inflammatory disease. See Report of Expert Meeting on FVIII Products and Inhibitor Development, European Medicines Agency (February 28, 2006-March 2, 2006). Thus, in some embodiments, the human is currently undergoing interferon therapy. In some embodiments, the human is currently undergoing antiviral therapy. In some embodiments, the human is currently undergoing antiretroviral therapy and has immune reconstitution inflammatory disease.

[0155] In certain embodiments, the human has had fewer than 150 exposure days (ED) to a clotting factor, such as FVIII. In one embodiment, the human has had fewer than 50 ED. In another embodiment, the human has had fewer than 20 ED.

[0156] Some aspects of the present disclosure relate to methods for reducing the severity or occurrence of an allergic or anaphylactic reaction to a clotting factor in a subject in need thereof, comprising administering to the subject a composition or chimeric protein comprising a clotting factor and an Fc region. In some embodiments, administration of the composition or chimeric protein reduces the severity of the anaphylactoid reaction to the clotting factor. In some embodiments, administration of the composition or chimeric protein reduces the severity of the allergic reaction to the clotting factor.

[0157] III. Chimeric Polypeptides Chimeric polypeptides useful for the present disclosure include a FVIII protein comprising a FVIII polypeptide and a VWF fragment comprising the D' and D3 domains of VWF. The VWF fragment is used to prevent or block the FVIII protein from interacting with endogenous VWF, thereby preventing the FVIII protein from being removed through the VWF clearance pathway. In some embodiments, to prevent the FVIII protein from dissociating from the VWF fragment, the FVIII protein and the VWF fragment are directly or indirectly linked to each other by a stronger bond than the natural interaction between FVIII and VWF.

[0158] In certain embodiments, the FVIII protein is bound to the VWF fragment by a covalent bond. The covalent bond can be any covalent bond known in the art. In some embodiments, the covalent bond is a disulfide bond. In some embodiments, the covalent bond is a peptide bond. In other embodiments, the FVIII protein is modified to increase the strength of the interaction between the FVIII protein and the VWF fragment. In other embodiments, the VWF fragment is modified to increase the strength of the interaction between the FVIII protein and the VWF fragment. In yet other embodiments, both the FVIII protein and the VWF protein are modified to increase the strength of the interaction between the FVIII protein and the VWF fragment.

[0159] In certain embodiments, the FVIII protein comprising a FVIII polypeptide and the VWF fragment comprising the D' and D3 domains of VWF are directly linked by at least one covalent bond between at least one amino acid in the FVIII polypeptide sequence and at least one amino acid in the D' and D3 domains of VWF. In other embodiments, the FVIII protein and the VWF fragment are indirectly linked by at least one covalent bond between at least one amino acid in the FVIII polypeptide sequence and at least one amino acid in a heterologous sequence fused directly or indirectly to the D' and D3 domains of VWF. In other embodiments, the FVIII protein and the VWF fragment are indirectly linked by at least one covalent bond between at least one amino acid in the VWF fragment sequence and at least one amino acid in a heterologous sequence fused directly or indirectly to the FVIII polypeptide. In yet other embodiments, the FVIII protein and the VWF fragment are indirectly linked by at least one covalent bond between at least one amino acid in the heterologous sequence directly or indirectly fused to the D' and D3 domains of VWF and at least one amino acid in the heterologous sequence directly or indirectly fused to the FVIII polypeptide.

[0160] The FVIII proteins of the present disclosure can comprise a FVIII polypeptide and one or more heterologous moieties, e.g., half-life extending moieties, fused directly or indirectly to the FVIII polypeptide. The VWF fragments of the present disclosure can also comprise the D' and D3 domains of VWF and one or more heterologous moieties, e.g., half-life extending moieties, fused directly or indirectly to the D' and D3 domains of VWF. In some embodiments, FVIII proteins useful for the present disclosure consist essentially of or consist of FVIII polypeptides, and VWF fragments useful for the present disclosure comprise the D' and D3 domains of VWF and one or more heterologous moieties, e.g., half-life extending moieties, fused directly or indirectly to the D' and D3 domains of VWF. In some embodiments, the FVIII protein comprises a FVIII polypeptide and one or more heterologous moieties, e.g., half-life extending moieties, fused directly or indirectly to the FVIII polypeptide, and a VWF fragment useful for the present disclosure consists essentially of or consists of the D' and D3 domains of VWF. In some embodiments, the chimeric polypeptide comprises a FVIII protein consisting essentially of or consisting of a FVIII polypeptide and a VWF fragment consisting essentially of or consisting of the D' and D3 domains of VWF.

[0161] In some embodiments, the chimeric polypeptide or protein disclosed herein is a FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer. In one embodiment, the FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimeric chimeric polypeptide comprises (i) an FVIII protein comprising a FVIII polypeptide, an XTEN inserted within the B domain of the FVIII polypeptide, and a first Fc region, and (ii) a VWF protein comprising a VWF fragment, a second XTEN sequence, an a2 linker, and a second Fc region. A schematic diagram of an exemplary FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer, rFVIIIFc-VWF-XTEN, is depicted in Figure 1.

[0162] In a particular embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN. In another particular embodiment, the rFVIIIFc-VWF-XTEN comprises (i) a FVIII protein comprising the amino acid sequence of SEQ ID NO: 203, and (ii) a VWF protein comprising the amino acid sequence of SEQ ID NO: 205. In yet another particular embodiment, the rFVIIIFc-VWF-XTEN comprises (i) a FVIII protein and (ii) a VWF protein covalently linked via a disulfide bond.

[0163] III.A. Factor VIII Polypeptides As used herein, "factor VIII," abbreviated throughout this application as "FVIII," refers to a functional FVIII polypeptide in its normal role in blood clotting, unless otherwise specified. Thus, the term FVIII includes functional variant polypeptides. "FVIII protein" is used to refer to a FVIII polypeptide (or protein) alone, a FVIII polypeptide fused to an additional polypeptide, and a FVIII polypeptide linked to one or more additional polypeptides, so long as the FVIII protein exhibits a FVIII function / activity. The terms "FVIII polypeptide," "FVIII portion," and "FVIII" refer to the FVIII polypeptide sequence alone. Examples of FVIII functions / activities include, but are not limited to, the ability to activate clots, act as a cofactor for factor IX, or inhibit Ca. 2+and the ability to form a tenase complex with factor IX in the presence of phospholipids, and then convert factor X to the activated form Xa. The FVIII polypeptide can be a human, porcine, canine, rat, or murine FVIII polypeptide. In addition, comparisons between human and other species-derived FVIII have identified conserved residues that may be necessary for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632). Full-length polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants, and modified versions. Various FVIII amino acid and nucleotide sequences are disclosed, for example, in U.S. Patent Application Publication Nos. 2015 / 0158929A1, 2014 / 0308280A1, and 2014 / 0370035A1, and International Publication No. WO 2015 / 106052A1. FVIII polypeptides include, for example, full-length FVIII, full-length FVIII without an N-terminal Met, mature FVIII polypeptides (without a signal sequence), mature FVIII polypeptides with an additional N-terminal Met, and / or FVIII polypeptides with a full or partial deletion of the B domain. FVIII variants include B domain deletions, whether partial or full deletions.

[0164] The FVIII polypeptide of the chimeric polypeptide used herein has FVIII activity in plasma. FVIII activity can be measured by any method known in the art. Numerous tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assays, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often by the Clauss method), platelet counting, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether abnormalities are corrected when the patient's plasma is mixed with normal plasma), clotting factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), various platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

[0165] The aPTT test is a performance indicator that measures the effectiveness of both the "intrinsic" (also referred to as the contact activation pathway) and the common coagulation pathway. This test is commonly used to measure the clotting activity of commercially available recombinant clotting factors, such as FVIII. It is typically used in conjunction with prothrombin time (PT), which measures the extrinsic pathway (see, e.g., Kamal et al., Mayo Clin Proc., 82(7):864-873 (2007)). In one embodiment, aPTT is tested using an assay in which FVIII activity is measured using Dade Actin FSL activated PTT reagent (Siemens Healthcare Diagnostics) on a BCS XP analyzer (Siemens Healthcare Diagnostics). In certain embodiments, the chimeric polypeptide has a plasma FVIII activity of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% as measured by an aPTT test.

[0166] The aPTT assay may also be used to assess the efficacy of a chimeric polypeptide prior to administration to a patient or subject (Hubbard AR, et al. J Thromb Haemost 11: 988-9 (2013)). The aPTT assay may also be used in conjunction with any of the assays described herein, either prior to or after administration to a patient or subject.

[0167] ROTEM analysis provides information on the overall dynamics of hemostasis: clotting time, clot formation, clot stability, and lysis. Different parameters in thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or the many factors that affect their interactions. This assay can provide a complete picture of secondary hemostasis.

[0168] The mechanism of the chromogenic assay is based on the principle of the blood coagulation cascade, in which activated FVIII promotes the conversion of factor X to factor Xa in the presence of activated factor IX, phospholipids, and calcium ions. Factor Xa activity is assessed by hydrolysis of the factor Xa-specific p-nitroanilide (pNA) substrate. The initial rate of release of p-nitroaniline, measured at 405 nM, is directly proportional to the activity of factor Xa and, therefore, to the FVIII activity in the sample. In one embodiment, the chromogenic assay is the BIOPHEN FVIII:C assay (Hyphen Biomed, Neurville-sur-Oise, France).

[0169] Chromogenic assays are recommended by the FVIII and Factor IX Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH). Since 1994, chromogenic assays have also been the reference method of the European Pharmacopoeia for determining the potency of FVIII concentrations. Thus, in some embodiments, chimeric polypeptides comprising a FVIII polypeptide have FVIII activity comparable to that of chimeric polypeptides comprising a mature FVIII polypeptide or a BDD FVIII polypeptide (e.g., RECOMBINATE®, KOGENATE FS®, HELIXATE FS®, XYNTHA / REFACTO AB®, HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®, ALPHANATE®, KOATE-DVI®, AFSTYLA®, and HYATE:C®).

[0170] Chromogenic assays may also be used to assess the efficacy of chimeric polypeptides prior to administration to a patient or subject (Hubbard AR, et al. J Thromb Haemost 11: 988-9 (2013)). Chromogenic assays may also be used in conjunction with any of the assays described herein, either prior to or after administration to a patient or subject.

[0171] In other embodiments, chimeric polypeptides comprising a FVIII polypeptide of the present disclosure have a factor Xa generation rate comparable to that of chimeric polypeptides comprising a mature FVIII polypeptide or a BDD FVIII polypeptide (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0172] To activate factor X to factor Xa, activated factor IX (factor IXa) is required to activate factor X. 2+ In the presence of FVIII cofactors, FVIII hydrolyzes one arginine-isoleucine bond in factor X to form factor Xa. Therefore, the interaction of FVIII with factor IX is important in the coagulation pathway. In certain embodiments, a chimeric polypeptide comprising a FVIII polypeptide can interact with factor IXa at a rate comparable to that of a chimeric polypeptide comprising a mature FVIII polypeptide sequence or a BDD FVIII polypeptide (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0173] In addition, FVIII binds to von Willebrand factor but is inactive in circulation. When not bound to VWF, FVIII is rapidly degraded and released from VWF by the action of thrombin. In some embodiments, a chimeric polypeptide comprising a FVIII polypeptide binds to von Willebrand factor, e.g., a VWF fragment disclosed herein, at a level comparable to that of a chimeric polypeptide comprising a mature FVIII polypeptide sequence or a BDD FVIII polypeptide (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0174] FVIII can be inactivated by activated protein C in the presence of calcium and phospholipids. Activated protein C cleaves the FVIII heavy chain after arginine 336 in the A1 domain, destroying the interaction site for factor X substrates, and after arginine 562 in the A2 domain, enhancing dissociation of the A2 domain and similarly destroying the interaction site with factor IXa. This cleavage also bisectes the A2 domain (43 kDa), generating the A2-N (18 kDa) and A2-C (25 kDa) domains. Thus, activated protein C can catalyze multiple cleavage sites in the heavy chain. In some embodiments, chimeric polypeptides comprising FVIII polypeptides are inactivated by activated protein C at levels comparable to chimeric polypeptides comprising mature FVIII polypeptide sequences or BDD FVIII polypeptides (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0175] In other embodiments, the chimeric polypeptide comprising a FVIII polypeptide has in vivo FVIII activity comparable to that of a chimeric polypeptide comprising a mature FVIII polypeptide sequence or a BDD FVIII polypeptide (e.g., ADVATE®, REFACTO®, or ELOCTATE®). In certain embodiments, the chimeric polypeptide comprising a FVIII polypeptide is capable of protecting HemA mice in a HemA mouse tail vein transection model at a level comparable to that of a chimeric polypeptide comprising a mature FVIII polypeptide sequence or a BDD FVIII polypeptide (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0176] An example of a human FVIII sequence (full length) is shown below. [Table 2] [Table 3-1] [Table 3-2] [Table 3-3]

[0177] FVIII polypeptides include full-length FVIII, full-length FVIII without an N-terminal Met, mature FVIII (without a signal sequence), mature FVIII with an additional N-terminal Met, and / or FVIII with a full or partial deletion of the B domain. In certain embodiments, the FVIII variant comprises a B domain deletion, whether a partial or full deletion.

[0178] The sequence of the native mature human FVIII polypeptide is set forth as SEQ ID NO: 65. The native FVIII polypeptide has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2, where A1, A2, and A3 are structurally related "A domains," B is the "B domain," C1 and C2 are structurally related "C domains," and a1, a2, and a3 are acidic spacer regions. With reference to the primary amino acid sequence positions in SEQ ID NO: 65, the A1 domain of human FVIII extends from Ala1 to about Arg336, the a1 spacer region extends from about Met337 to about Val374, the A2 domain extends from about Ala375 to about Tyr719, the a2 spacer region extends from about Glu720 to about Arg740, the B domain extends from about Ser741 to about Arg1648, the a3 spacer region extends from about Glu1649 to about Arg1689, the A3 domain extends from about Ser1690 to about Leu2025, the C1 domain extends from about Gly2026 to about Asn2072, and the C2 domain extends from about Ser2073 to Tyr2332. Other than specific proteolytic cleavage sites, the designation of the locations of boundaries between domains and regions of FVIII may vary in different literature references. Accordingly, the boundaries set forth herein are designated as approximate by use of the term "about."

[0179] The human FVIII gene was isolated and expressed in mammalian cells (Toole, JJ, et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, WI, et al., Nature 312:330-337 (1984); Vehar, GA, et al., Nature 312:337-342 (1984); WO 87 / 04187; WO 88 / 08035; WO 88 / 03558; and U.S. Pat. No. 4,757,006). The FVIII amino acid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199. Additionally, partially or completely B-domain deleted FVIII is shown in U.S. Patent Nos. 4,994,371 and 4,868,112. In some embodiments, the B-domain of human FVIII is replaced with the B-domain of human factor V, as shown in U.S. Patent No. 5,004,803. The cDNA and amino acid sequences encoding human factor VIII are shown in SEQ ID NOs: 1 and 2, respectively, of U.S. Patent Application Publication No. 2005 / 0100990.

[0180] The porcine FVIII sequence was published in Toole, JJ, et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). Furthermore, the complete porcine cDNA sequence obtained by PCR amplification of the FVIII sequence from a porcine spleen cDNA library was reported in Healey, JF, et al., Blood 88:4209-4214 (1996). Hybrid human / porcine FVIIIs with substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Patent No. 5,364,771 by Lollar and Runge and in WO 93 / 20093. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII, as well as chimeric FVIIIs using porcine A1 and / or A2 domains instead of the corresponding human domains, were reported in WO 94 / 11503. US Patent No. 5,859,204, Lollar, JS also discloses a porcine cDNA and deduced amino acid sequence. US Patent No. 6,458,563 discloses a B-domain deleted porcine FVIII.

[0181] U.S. Patent No. 5,859,204 to Lollar, JS reports functional mutants of FVIII with reduced antigenicity and reduced immunoreactivity. U.S. Patent No. 6,376,463 to Lollar, JS also reports mutants of FVIII with reduced immunoreactivity. U.S. Patent Application Publication No. 2005 / 0100990 to Saenko et al. reports functional mutations in the A2 domain of FVIII.

[0182] In some embodiments, the FVIII polypeptide (or FVIII portion of the chimeric polypeptide) can be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 67, or amino acids 1 to 2332 of SEQ ID NO: 65 (without the signal sequence), or the FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 64 and amino acids 1 to 1438 of SEQ ID NO: 67, or amino acids 1 to 19 of SEQ ID NO: 64 and amino acids 1 to 2332 of SEQ ID NO: 65 (with the signal sequence), where FVIII has coagulation activity, e.g., activates factor IX as a cofactor and converts factor X to activated factor X. The FVIII polypeptide (or the FVIII portion of the chimeric polypeptide) can be identical to the FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 67, or amino acids 1 to 2332 (without the signal sequence) of SEQ ID NO: 65. The FVIII polypeptide can further include a signal sequence.

[0183] As used herein, the "B domain" of FVIII is identical to B domains known in the art, defined by internal amino acid sequence identity and sites of proteolytic cleavage, e.g., residues Ser741 to Arg1648 of full-length human FVIII. Other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1 to Arg372; A2, residues Ser373 to Arg740; A3, residues Ser1690 to Asn2019; C1, residues Lys2020 to Asn2172; and C2, residues Ser2173 to Tyr2332. The A3-C1-C2 sequence includes residues Ser1690 to Tyr2332. The remaining sequence, residues Glu1649 to Arg1689, is commonly referred to as the a3 acidic region. The locations of all domain boundaries, including the B domain, for porcine, mouse, and canine FVIII are also known in the art. In some embodiments, the B domain of FVIII is deleted ("B domain deleted factor VIII" or "BDD FVIII"). An example of BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the same sequence as the factor VIII portion of the sequence in Table 4 (BDD FVIII heavy chain is double underlined, the B domain is in italics, and the BDD FVIII light chain is in standard text). In some embodiments, the B domain FVIII has a deletion of all but five amino acids of the B domain, as shown in Table 5 (SEQ ID NO: 68) (B domain is in italics). [Table 4] [Table 5]

[0184] "B domain deleted FVIII" is disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, and 5,171,844. Nos. 5,112,950, 4,868,112, and 6,458,563, U.S. Patent Application Publication Nos. 2017 / 0073393A1 and 2012 / 308641A1, and WO 2011 / 041770A1, WO 2015 / 106052A1 (PCT / US2015 / 010738), and WO 2016 / 025764. In some embodiments, the B domain-deleted FVIII sequence used in the disclosed methods comprises any one of the deletions disclosed in U.S. Patent No. 6,316,226 (also U.S. Patent No. 6,346,513) at column 4, line 4 to column 5, line 28 and in Examples 1-5. In other embodiments, the B-domain deleted factor VIII is S743 / Q1638 B-domain deleted factor VIII (SQ BDD FVIII) (e.g., a factor VIII having a deletion of amino acids 744 to 1637, e.g., a factor VIII having amino acids 1 to 743 and amino acids 1638 to 2332 of mature FVIII). In some embodiments, the B-domain deleted FVIII used in the methods of the disclosure has a deletion as disclosed in column 2, lines 26 to 51 and Examples 5 to 8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620).In some embodiments, the B-domain deleted factor VIII is described in U.S. Pat. No. 5,972,885, column 1, line 25 to column 2, line 40; U.S. Pat. No. 6,048,720, column 6, lines 1-22 and Example 1; U.S. Pat. No. 5,543,502, column 2, lines 17-46; U.S. Pat. No. 5,171,844, column 4, line 22 to column 5, line 36; U.S. Pat. No. 5,112,950, column 2, lines 55-68. No. 4,868,112; columns 2, lines 2-19, line 21 and Table 2 of U.S. Pat. No. 4,868,112; columns 2, lines 1-3, line 19, column 3, lines 40-4, line 67, column 7, lines 43-8, line 26, and columns 11, lines 5-13, line 39 of U.S. Pat. No. 7,041,635; or column 4, lines 25-53 of U.S. Pat. No. 6,458,563. In some embodiments, the B domain-deleted FVIII polypeptide has most of the B domain deleted, as disclosed in WO 91 / 09122, but still contains the amino-terminal sequence of the B domain that is essential for in vivo proteolytic processing of the primary translation product into two polypeptide chains. In some embodiments, a B-domain deleted FVIII polypeptide is constructed by deleting amino acids 747-1638, i.e., virtually the complete deletion of the B domain. Hoeben RC, et al. J. Biol. Chem. 265 (13): 7318-7323 (1990). B-domain deleted factor VIII polypeptides may also contain deletions of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P, et al. Protein Eng. 2(4): 301-6 (1988).Additional B domain deletions that are part of the present disclosure include, for example, deletions of amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, 5939-5942), 797 to 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347), 741 to 1646 (Kaufman (PCT Application Publication No. WO 87 / 04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564), 741 to 1648 (Pasek (PCT Application Publication No. 88 / 00831)), or 816 to 1598 or 741 to 1648 (Lagner (Behring Inst. Mitt. (1988) No. 82:16-25, EP 295597). In certain embodiments, the B-domain deleted FVIII polypeptide comprises a deletion of amino acid residues 746 to 1648 of mature FVIII (corresponding to a deletion of amino acid residues 765 to 1665 of full-length FVIII). In other embodiments, the B-domain deleted FVIII polypeptide comprises a deletion of amino acid residues 745 to 1648 of mature FVIII (corresponding to a deletion of amino acid residues 764 to 1665 of full-length FVIII).

[0185] In other embodiments, BDD FVIII comprises a FVIII polypeptide containing a fragment of the B domain corresponding to the amino acid sequence of the full-length FVIII sequence, but retaining one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or, optionally, 943. Examples of B domain fragments include 226 or 163 amino acids of the B domain (i.e., the first 226 or 163 amino acids of the B domain are retained) as disclosed in Miao, HZ, et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, SW, et al., J. Thromb. Haemost. 9: 2235-2242 (2011). In yet other embodiments, the BDD FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve expression of the BDD FVIII polypeptide. See Miao, HZ, et al., Blood 103(a): 3412-3419 (2004). In yet other embodiments, the BDD FVIII comprises a FVIII polypeptide containing a portion of the B domain but not one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, SW, et al., J. Thromb. Haemost. 9: 2235-2242 (2011). In some embodiments, the BDD FVIII comprises a single-chain FVIII containing a deletion at amino acids 765-1652 corresponding to mature full-length FVIII (also known as rFVIII-SingleChain and AFSTYLA®). See U.S. Patent No. 7,041,635. Each of the above deletions can be made in any FVIII sequence.

[0186] In some embodiments, the FVIII polypeptide has a partial B domain. In some embodiments, the FVIII polypeptide having a partial B domain is FVIII198. FVIII198 is a partial B domain containing single-chain FVIIIFc molecule-226N6. The number 226 represents the N-terminal 226 amino acids of the B domain of FVIII, and N6 represents the six N-glycosylation sites in the B domain.

[0187] In certain embodiments, the FVIII polypeptide is selected from the FVIII polypeptides disclosed in WO 2017 / 117630A1, WO 2018 / 087271A1, US9,878,017B2, US8,575,104B2, US8,754,194B2, US7,939,632B2, US2018 / 0161402A1, US9,956,269B2, US9,107,902B2, US2017 / 209546A1.

[0188] In some embodiments, FVIII is cleaved immediately after the arginine at amino acid 1648 (in full-length factor VIII or SEQ ID NO: 65), the arginine at amino acid 754 (in S743 / Q1638 B-domain deleted factor VIII or SEQ ID NO: 67), or the corresponding arginine residue (in other variants), thereby resulting in a heavy chain and a light chain. In other embodiments, the FVIII polypeptide comprises a heavy chain and a light chain that are linked or associated by a metal ion-mediated non-covalent bond.

[0189] In other embodiments, the FVIII is an arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 65), an arginine at amino acid 754 (in S743 / Q1638 B-domain deleted FVIII or SEQ ID NO: 67), or an uncleaved single-chain FVIII immediately following the corresponding arginine residue (in other variants). The single-chain FVIII can contain one or more amino acid substitutions. In some embodiments, the amino acid substitutions are at residues corresponding to residues 1648, 1645, or both, of the full-length mature factor VIII polypeptide (SEQ ID NO: 65), or residues 754, 751, or both, of the SQ BDD factor VIII (SEQ ID NO: 67). The amino acid substitution can be any amino acid other than arginine, for example, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine.

[0190] FVIII can be further cleaved by thrombin and then activated as FVIIIa, which can serve as a cofactor for activated factor IX (FIXa). Activated FIX then forms an Xase complex with activated FVIII, converting factor X to activated factor X (FXa). For activation, FVIII is cleaved by thrombin after three arginine residues at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain deleted FVIII sequence), resulting in FVIIIa with a 50 kDa A1 chain, a 43 kDa A2 chain, and a 73 kDa A3-C1-C2 chain. In some embodiments, the FVIII polypeptide useful for the present disclosure is inactive FVIII. In other embodiments, the FVIII polypeptide is activated FVIII.

[0191] A protein having a FVIII polypeptide linked or associated with a VWF protein can comprise a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65 or 67, wherein the sequence has the clotting activity of FVIII, for example, activating factor IX as a cofactor and converting factor X to activated factor X (FXa).

[0192] As used herein, "hybrid" or "chimeric" polypeptides and proteins include a combination of a first polypeptide and a second polypeptide. In some embodiments, a hybrid or chimeric polypeptide includes a single polypeptide chain, e.g., a chimeric polypeptide including a FVIII polypeptide and XTEN. See, e.g., US2015 / 0158929A1, the entire contents of which are incorporated herein by reference. In some embodiments, a hybrid or chimeric polypeptide includes a first polypeptide chain, e.g., a VWF fragment fused to an XTEN sequence and a first Ig constant region or portion thereof, and a second polypeptide chain, e.g., a FVIII polypeptide fused to a second Ig constant region or portion thereof, thereby forming a heterodimer. See, e.g., US2015 / 0266943A1, US2016 / 0251408A1, and US2017 / 0073393A1, the entire contents of which are incorporated herein by reference. In some embodiments, the first and second polypeptides in the hybrid are associated with each other through protein-protein interactions, such as charge-charge or hydrophobic interactions. In other embodiments, the first polypeptide comprises a VWF protein-XTEN-Fc fusion protein and the second polypeptide comprises a FVIII-Fc fusion protein, making the hybrid a heterodimer, where the XTEN contains fewer than 288 amino acids. In other embodiments, the first polypeptide comprises a VWF protein-XTEN-Fc fusion protein and the second polypeptide comprises a FVIII(X)-Fc fusion protein, making the hybrid a heterodimer, where the XTEN contains fewer than 288 amino acids. The first and second polypeptides can be associated with each other through a covalent bond, e.g., a disulfide bond, between the first and second Fc regions. The first and second polypeptides can also be associated with each other through a bond between the VWF fragment and the FVIII polypeptide.

[0193] In certain embodiments, the chimeric polypeptides disclosed herein comprise a FVIII protein comprising a first FVIII polypeptide fragment, an XTEN sequence, a second FVIII polypeptide fragment, and an Fc region. In some embodiments, the FVIII protein comprises, in N- to C-terminal order, a first FVIII polypeptide fragment fused to an XTEN sequence, a second FVIII polypeptide fragment, and an Fc region. In particular embodiments, the FVIII protein comprises a first FVIII polypeptide fragment comprising the amino acid sequence of SEQ ID NO:215, an XTEN sequence comprising the amino acid sequence of SEQ ID NO:8, a second FVIII polypeptide comprising the amino acid sequence of SEQ ID NO:216, and / or a first Fc region comprising the amino acid sequence of SEQ ID NO:217.

[0194] In one particular embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN and comprises a FVIII protein comprising the amino acid sequence of SEQ ID NO: 201. In another particular embodiment, the rFVIIIFc-VWF-XTEN comprises the amino acid sequence of SEQ ID NO: 207.

[0195] In some embodiments, the FVIII protein further comprises a FVIII signal peptide sequence. In one particular embodiment, the FVIII protein comprises a FVIII signal peptide comprising the amino acid sequence of SEQ ID NO: 64, a first FVIII polypeptide fragment comprising the amino acid sequence of SEQ ID NO: 215, an XTEN sequence comprising the amino acid sequence of SEQ ID NO: 8, a second FVIII polypeptide comprising the amino acid sequence of SEQ ID NO: 216, and / or a first Fc region comprising the amino acid sequence of SEQ ID NO: 217.

[0196] In one particular embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN and comprises a FVIII protein comprising the amino acid sequence of SEQ ID NO: 203. In another particular embodiment, the rFVIIIFc-VWF-XTEN comprises a FVIII protein encoded by the nucleic acid sequence of SEQ ID NO: 204, or a fragment thereof. Additional exemplary polypeptide sequences for FVIII proteins of the chimeric polypeptides disclosed herein are set forth in Tables 18-19.

[0197] As discussed above and below, a large number of functional FVIII variants are known.In addition, hundreds of non-functional mutations in FVIII have been identified in hemophilia patients, and it has been determined that the effect of these mutations on FVIII function is more due to their location in the three-dimensional structure of FVIII than to the nature of the substitution (Cutler et al., Hum. Mutat. 19:274-8 (2002)), which is incorporated herein by reference in its entirety.In addition, comparison between human-derived and other species-derived FVIII has identified conserved residues that may be necessary for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US6,251,632), which is incorporated herein by reference in its entirety.

[0198] III.B. Von Willebrand Factor (VWF) Fragments VWF (also known as F8VWF) is a large multimeric glycoprotein present in blood plasma and constitutively produced in endothelium (in Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2813-amino acid protein. All monomers contain a number of specific domains with specific functions: the D' / D3 domain (which binds to factor VIII), the A1 domain (which binds to platelet GPIb receptors, heparin, and / or possibly collagen), the A3 domain (which binds to collagen), the C1 domain (which, upon activation, binds the RGD domain to platelet integrin αIIbβ3), and a "cysteine ​​knot" domain at the C-terminus of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ), and β-human chorionic gonadotropin (βHCG)).

[0199] The term "VWF fragment" as used herein includes, but is not limited to, functional VWF fragments comprising the D' and D3 domains that are capable of inhibiting the binding of endogenous VWF to FVIII. In some embodiments, the VWF fragment binds to the FVIII protein. In other embodiments, the VWF fragment blocks the VWF-binding site on the FVIII protein, thereby inhibiting the interaction of the FVIII protein with endogenous VWF. In other embodiments, the VWF fragment blocks the binding of FVIII to endogenous VWF, thereby preventing the clearance of the FVIII protein through the VWF clearance pathway. VWF fragments include derivatives, variants, mutants, or analogs that retain these activities of VWF.

[0200] The 2813 monomer amino acid sequence for human VWF is reported in Genbank under accession number NP000543.2. The nucleotide sequence encoding human VWF is reported in Genbank under accession number NM000552.3. The nucleotide sequence of human VWF is designated as SEQ ID NO: 20. SEQ ID NO: 21 is the amino acid sequence of full-length VWF. Each domain of VWF is listed in Table 6. [Table 6-1] [Table 6-2] [Table 6-3]

[0201] The VWF protein used herein may be a VWF fragment containing the D' and D3 domains of VWF, wherein the VWF fragment binds to factor VIII (FVIII) and inhibits the binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment containing the D' and D3 domains may further contain a VWF domain selected from the group consisting of the A1 domain, the A2 domain, the A3 domain, the D1 domain, the D2 domain, the D4 domain, the B1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain, the CK domain, one or more fragments thereof, and any combination thereof. In some embodiments, the VWF fragment comprises, consists essentially of, or consists of: (1) the D' and D3 domains of VWF, or a fragment thereof; (2) the D1, D', and D3 domains of VWF, or a fragment thereof; (3) the D2, D', and D3 domains of VWF, or a fragment thereof; (4) the D1, D2, D', and D3 domains of VWF, or a fragment thereof; or (5) the D1, D2, D', D3, and A1 domains of VWF, or a fragment thereof. The VWF fragments described herein do not contain a site that binds to a VWF clearance receptor. In other embodiments, the VWF fragments described herein are not amino acids 764-1274 of SEQ ID NO: 21. The VWF fragments of the present disclosure can include any other sequence linked or fused to the VWF fragment. For example, the VWF fragments described herein can further include a signal peptide.

[0202] In some embodiments, the VWF fragment comprising the D' and D3 domains binds to or associates with a FVIII protein, for example, US2015 / 0023959A1, US2015 / 0266943A1, US2016 / 0251408A1, US2017 / 0073393A1, US2018 / 185455A1, US2018 / 0051067A1, US2017 / 0152300A1, US9, See US 878,017B2, US 9,458,223B2, US 8,575,104B2, WO 2017 / 117630A1, US 2018 / 0161402A1, WO 2017 / 117631A1, WO 2018 / 087271A1, US 9,107,902B2, WO 2017 / 222337A1, WO 2015 / 185758A1. By binding to or associating with the FVIII protein, the VWF fragments of the present disclosure protect FVIII from protease cleavage and activation, stabilize the heavy and light chains of FVIII, and prevent clearance of FVIII by scavenger receptors. In other embodiments, the VWF fragment binds to or associates with the FVIII protein, blocking or preventing the binding of the FVIII protein to phospholipids and activated protein C. By preventing or inhibiting the binding of the FVIII protein to endogenous full-length VWF, the VWF fragment of the present disclosure reduces the clearance of FVIII by the VWF clearance receptor, thus extending the half-life of the chimeric polypeptide. Thus, the extension of the half-life of the chimeric polypeptide results from the binding or association of the FVIII protein with the VWF fragment lacking the binding site for the VWF clearance receptor and the shielding or protection of the FVIII protein by the VWF fragment from endogenous VWF containing the binding site for the VWF clearance receptor. The FVIII protein bound to or protected by the VWF fragment can also enable the recycling of the FVIII protein.By eliminating the binding site for the VWF clearance pathway receptor contained in the full-length VWF molecule, the FVIII / VWF heterodimer of the present disclosure is shielded from the VWF clearance pathway, further extending the half-life of FVIII.

[0203] In some embodiments, a VWF protein useful for the present disclosure comprises the D' domain and D3 domain of VWF, wherein the D' domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-866 of SEQ ID NO: 21, and the VWF protein prevents or inhibits endogenous VWF from binding to FVIII. In other embodiments, a VWF protein comprises the D' domain and D3 domain of VWF, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867-1240 of SEQ ID NO: 21, and the VWF protein prevents or inhibits endogenous VWF from binding to FVIII. In some embodiments, the VWF proteins described herein comprise, consist essentially of, or consist of the D' and D3 domains of VWF, which are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-1240 of SEQ ID NO: 21, and wherein the VWF protein prevents or inhibits binding of endogenous VWF to FVIII. In other embodiments, the VWF protein comprises, consists essentially of, or consists of D1, D2, D', and D3 domains at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23-1240 of SEQ ID NO: 21, wherein the VWF protein prevents or inhibits binding of endogenous VWF to FVIII. In yet other embodiments, the VWF protein further comprises a signal peptide operably linked thereto.

[0204] In some embodiments, a VWF protein useful for the present disclosure comprises (1) a D'D3 domain, a D1D'D3 domain, a D2D'D3 domain, or a D1D2D'D3 domain, and (2) up to about 10 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1250 of SEQ ID NO:21), up to about 15 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1255 of SEQ ID NO:21), up to about 20 ... The VWF protein may consist essentially of, or consist of, an additional VWF sequence of up to about 25 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1260 of SEQ ID NO:21), up to about 25 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1265 of SEQ ID NO:21), or up to about 30 amino acids (e.g., any sequence from amino acids 764-1240 of SEQ ID NO:21 to amino acids 764-1260 of SEQ ID NO:21). In some embodiments, the VWF protein comprising or consisting essentially of the D' and D3 domains is neither amino acids 764-1274 of SEQ ID NO:21 nor full-length mature VWF. In some embodiments, the D1D2 domains are expressed in trans with the D' and D3 domains. In some embodiments, the D1D2 domains are expressed in cis with the D' and D3 domains.

[0205] In other embodiments, the VWF protein comprising the D'D3 domain linked to the D1D2 domain further comprises an intracellular cleavage site, e.g., a PACE (furin) or PC5 cleavage site, which allows cleavage of the D1D2 domain from the D'D3 domain upon expression. Non-limiting examples of intracellular cleavage sites are disclosed elsewhere herein.

[0206] In yet another embodiment, the VWF protein comprises the D' domain and the D3 domain but does not comprise an amino acid sequence selected from the group consisting of: (1) amino acids 1241 to 2813 corresponding to SEQ ID NO: 21; (2) amino acids 1270 to 2813 corresponding to SEQ ID NO: 21; (3) amino acids 1271 to 2813 corresponding to SEQ ID NO: 21; (4) amino acids 1272 to 2813 corresponding to SEQ ID NO: 21; (5) amino acids 1273 to 2813 corresponding to SEQ ID NO: 21; (6) amino acids 1274 to 2813 corresponding to SEQ ID NO: 21, and any combination thereof.

[0207] In still other embodiments, a VWF protein of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence corresponding to the D' domain, the D3 domain, and the A1 domain, wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764-1479 of SEQ ID NO: 21, and the VWF protein prevents endogenous VWF from binding to FVIII. In certain embodiments, the VWF protein is not amino acids 764-1274 of SEQ ID NO: 21.

[0208] In some embodiments, the VWF protein of the present disclosure comprises a D' domain and a D3 domain, but is not limited to: (1) an A1 domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a B1 domain, (6) a B2 domain, (7) a B3 domain, (8) a C1 domain, (9) a C2 domain, (10) a CK domain, (11) a CK domain and a C2 domain, (12) a CK domain, a C2 domain, and a C1 domain, (13) a CK domain, a C2 domain, a C1 domain, and a B3 domain, (14) a CK domain, a C2 domain, a C1 domain, a B3 domain, and a B2 domain, (15) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, and a B1 domain, (16) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, and a B1 domain, (17) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, and a B1 domain, (18) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B1 domain, and a B1 domain, (19) a CK domain, a C2 domain, a C1 domain, a B1 domain, a B2 domain, and a B1 domain, (20) a CK domain, a C2 domain, a C1 domain, a B1 domain, a B2 domain, and a B1 domain, (21) a CK domain, a C2 domain, a C1 domain, a B1 domain, a B2 domain, and a B1 domain, (22) a CK domain, a C1 domain, a B1 domain, a B2 domain, and a B1 domain, (23) a CK domain, a C2 domain, a C1 domain, a B1 domain, a B2 domain, and a B1 domain, ( (6) does not contain at least one VWF domain selected from the group consisting of a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, and a D4 domain; (17) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, and an A3 domain; (18) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, and an A2 domain; (19) a CK domain, a C2 domain, a C1 domain, a B3 domain, a B2 domain, a B1 domain, a D4 domain, an A3 domain, an A2 domain, and an A1 domain; and (20) any combination thereof.

[0209] In yet other embodiments, the VWF protein comprises a D'D3 domain and one or more domains or modules. Examples of such domains or modules include, but are not limited to, the domains and modules disclosed in Zhour et al., Blood, published online April 6, 2012: DOI 10.1182 / blood-2012-01-405134, the entire contents of which are incorporated herein by reference. For example, the VWF protein may comprise a D'D3 domain and one or more domains or modules selected from the group consisting of an A1 domain, an A2 domain, an A3 domain, a D4N module, a VWD4 module, a C8-4 module, a TIL-4 module, a C1 module, a C2 module, a C3 module, a C4 module, a C5 module, a C5 module, a C6 module, and combinations thereof.

[0210] In yet another embodiment, the VWF protein is linked to a heterologous moiety, wherein the heterologous moiety is linked to the N-terminus or C-terminus of the VWF protein, or inserted immediately downstream of one or more amino acids in the VWF protein (e.g., one or more XTEN insertion sites). For example, the insertion site for the heterologous moiety in the VWF protein can be the D' domain, the D3 domain, or both. The heterologous moiety can be a half-life extender.

[0211] In certain embodiments, VWF proteins useful for the present disclosure form multimers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, or higher multimers. In other embodiments, the VWF protein is a monomer having only one VWF protein. In some embodiments, the VWF protein of the present disclosure can have one or more amino acid substitutions, deletions, additions, or modifications. In some embodiments, the VWF protein can include amino acid substitutions, deletions, additions, or modifications that prevent the VWF protein from forming disulfide bonds or from forming dimers or multimers. In other embodiments, the amino acid substitutions are within the D' domain and D3 domain. In certain embodiments, the VWF protein useful for the present disclosure contains at least one amino acid substitution at residue 1099, residue 1142, or both residues 1099 and 1142, corresponding to SEQ ID NO: 21. The at least one amino acid substitution can be any amino acid that does not naturally occur in wild-type VWF. For example, the amino acid substitution can be any amino acid other than cysteine, such as isoleucine, alanine, leucine, asparagine, lysine, aspartic acid, methionine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, or histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit the VWF protein from forming multimers.

[0212] In certain embodiments, the VWF protein useful herein may be further modified to improve its interaction with FVIII, for example, to improve binding affinity to FVIII. As a non-limiting example, the VWF protein contains a serine residue at the residue corresponding to amino acid 764 of SEQ ID NO: 21 and a lysine residue at the residue corresponding to amino acid 773 of SEQ ID NO: 21. Residues 764 and / or 773 may contribute to the binding affinity of the VWF protein to FVIII. In other embodiments, the VWF protein useful for the present disclosure may have other modifications, for example, the protein may be pegylated, glycosylated, hesylated, or polysialylated.

[0213] In certain embodiments, the chimeric polypeptides disclosed herein comprise a VWF protein comprising a VWF fragment, an XTEN sequence, an FVIII a2 linker, and an Fc region. In some embodiments, the VWF protein comprises a VWF fragment fused, in N- to C-terminal order, to an XTEN sequence, an a2 linker, and an Fc region. In certain embodiments, the VWF protein comprises a VWF D' domain comprising the amino acid sequence of SEQ ID NO: 210, a VWF D3 domain comprising the amino acid sequence of SEQ ID NO: 214, an XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58 (AE144_5A), an a2 linker comprising the amino acid sequence of SEQ ID NO: 88, and / or an Fc region comprising the amino acid sequence of SEQ ID NO: 217.

[0214] In one particular embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN and comprises a VWF protein comprising the amino acid sequence of SEQ ID NO:202.

[0215] In some embodiments, the VWF protein comprises a VWF fragment comprising the D1, D2, D', and / or D3 domains of VWF. In one embodiment, the VWF fragment comprises the D1D2 domains of VWF comprising the amino acid sequence of SEQ ID NO: 209. In some embodiments, the VWF protein further comprises a VWF signal peptide sequence. In one embodiment, the VWF signal peptide comprises the amino acid sequence of SEQ ID NO: 208. In a particular embodiment, the VWF protein comprises a VWF signal peptide comprising the amino acid sequence of SEQ ID NO: 208, a VWF D1D2 region comprising the amino acid sequence of SEQ ID NO: 209, a VWF D' domain comprising the amino acid sequence of SEQ ID NO: 210, a VWF D3 domain comprising the amino acid sequence of SEQ ID NO: 214, an XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58 (AE144_5A), an a2 linker comprising the amino acid sequence of SEQ ID NO: 88, and / or an Fc region comprising the amino acid sequence of SEQ ID NO: 217.

[0216] In one particular embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN and comprises a VWF protein comprising the amino acid sequence of SEQ ID NO: 205. In another particular embodiment, the rFVIIIFc-VWF-XTEN comprises a VWF protein encoded by the nucleic acid sequence of SEQ ID NO: 206, or a fragment thereof. Additional exemplary polypeptide sequences for the VWF protein of the chimeric polypeptides disclosed herein are set forth in Tables 18-19.

[0217] III.C. Half-life extending moieties In some embodiments, the chimeric polypeptide of the present disclosure comprises one or more half-life extenders. In some embodiments, the FVIII polypeptide of the chimeric polypeptide is fused or linked to one or more half-life extenders (i.e., FVIII proteins). In other embodiments, the chimeric polypeptide comprises at least two half-life extenders: a first half-life extender fused to the FVIII polypeptide and a second half-life extender fused to the D'D3 domain of VWF. In some embodiments, the first half-life extender is fused to the C-terminus or N-terminus of the FVIII polypeptide. In some embodiments, the first half-life extender is inserted within the FVIII polypeptide. In some embodiments, the first half-life extender is inserted within the B domain of the FVIII polypeptide. In some embodiments, the first half-life extender is inserted within the FVIII polypeptide immediately downstream of the amino acid corresponding to amino acid residue 745 of SEQ ID NO: 65. In some embodiments, the first heterologous moiety is fused to the FVIII polypeptide by a linker.

[0218] In certain embodiments, the D'D3 domain of VWF in the VWF fragment can be fused or linked to a second half-life extending moiety. In some embodiments, the second half-life extending moiety is fused to the C-terminus or N-terminus of the VWF fragment. In some embodiments, the second half-life extending moiety is inserted within the VWF fragment. In some embodiments, the second half-life extending moiety is fused to the C-terminus of the VWF fragment. In certain embodiments, the second half-life extending moiety is fused to the VWF fragment by a linker.

[0219] In other embodiments, the chimeric polypeptide comprises a FVIII polypeptide and a VWF fragment comprising the D' and D3 domains of VWF, wherein the VWF D' and D3 domains are fused to one or more half-life extending moieties, and wherein the VWF D' and D3 domains and the FVIII polypeptide are associated with an bond stronger than the naturally occurring bond between FVIII and VWF.

[0220] In some embodiments, the chimeric polypeptide comprises a FVIII polypeptide and a VWF fragment comprising the D' and D3 domains of VWF, wherein the VWF D' and D3 domains are fused to the FVIII polypeptide by a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the VWF D' and D3 domains are further linked to the FVIII polypeptide by at least one non-covalent bond. In certain embodiments, the VWF D' and D3 domains are further fused to an Fc. In some embodiments, the VWF D' and D3 domains are fused to an Fc by a linker. In some embodiments, the Fc is fused to a second Fc by an additional linker. In certain embodiments, the Fc and the second Fc are linked to each other by a covalent bond, for example, a disulfide bond. In certain embodiments, the chimeric polypeptide comprises a FVIII polypeptide and a VWF fragment comprising the D' domain and the D3 domain of VWF, as disclosed in International Application Publication No. WO2017 / 222337A1, the entire contents of which are incorporated herein by reference.

[0221] The first half-life extending moiety, the second half-life extending moiety, or both, can be selected from the group consisting of an FcRn binding partner, such as albumin or an immunoglobulin Fc region, an XTEN sequence, the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, a PAS sequence, an HAP sequence, transferrin, an albumin binding moiety, or any fragment, derivative, variant, and any combination thereof.

[0222] III.C.1. Ig constant region or portion thereof In some embodiments, the chimeric polypeptide of the present disclosure also comprises a first Ig constant region or portion thereof fused to the FVIII polypeptide, optionally via a linker. The first Ig constant region or portion thereof can be inserted into the FVIII polypeptide or fused to the C-terminus or N-terminus of the FVIII polypeptide. In some embodiments, the chimeric polypeptide further comprises a second Ig constant region or portion thereof fused to a VWF protein. The first Ig constant region or portion thereof can be inserted into the VWF fragment or fused to the C-terminus or N-terminus of the VWF fragment. In certain embodiments, the first Ig constant region is linked or attached to the second Ig constant region by a covalent bond, such as a disulfide bond.

[0223] The Ig constant region or portion thereof can be combined with additional heterologous moieties, such as an XTEN sequence and a VWF protein, to improve the pharmacokinetic or pharmacodynamic properties of the chimeric polypeptide. In certain embodiments, the Ig constant region or portion thereof extends the half-life of the molecule fused to the Ig constant region or portion thereof.

[0224] The Ig constant region is composed of domains designated as CH (constant heavy chain) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA, IgD, or IgE), the constant region may be composed of three or four CH domains. Some isotype (e.g., IgG) constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, NY, NY.

[0225] The Ig constant region or portion thereof for producing the chimeric polypeptides of the present disclosure can be obtained from several different sources. In some embodiments, the Ig constant region or portion thereof is derived from human Ig. However, it is understood that the Ig constant region or portion thereof can be derived from the Ig of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques). Furthermore, the Ig constant region or portion thereof can be derived from any Ig class, including IgM, IgG, IgD, IgA, and IgE, and any Ig isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human isotype IgG1 is used.

[0226] A variety of Ig constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domain sequences can be selected that have specific effector functions (or lack specific effector functions) or have specific modifications that reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences, or portions thereof) can be derived from these sequences using art-recognized techniques. The genetic material obtained using any of the aforementioned methods can then be altered or synthesized to obtain the polypeptides of the present disclosure. It is further recognized that the scope of the present disclosure encompasses alleles, variants, and mutations of constant region DNA sequences.

[0227] The sequence of an Ig constant region or a portion thereof can be cloned, for example, using the polymerase chain reaction and primers selected to amplify the domain of interest. To clone the sequence of an Ig constant region or a portion thereof from an antibody, mRNA can be isolated from hybridomas, spleen, or lymphocytes, reverse transcribed into DNA, and the antibody gene can be amplified by PCR. PCR amplification methods are described in detail in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188, as well as in, for example, "PCR Protocols: A Guide to Methods and Applications" (Innis et al., eds., San Diego, CA (1990); Ho et al., 1989, Gene 77:51; Horton et al., 1993, Methods Enzymol. 217:270). PCR can be initiated by consensus constant region primers or by more specific primers based on the published DNA and amino acid sequences of heavy and light chains. As discussed above, PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, libraries can be screened with consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250)). Cloning of antibody sequences is further described in Newman et al., US Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated herein by reference.

[0228] As used herein, an Ig constant region can include all domains and hinge regions or portions thereof. In some embodiments, an Ig constant region or portion thereof includes the CH2 domain, CH3 domain, and hinge region, i.e., the Fc region or FcRn binding partner.

[0229] As used herein, the term "Fc region" is defined as the portion of a polypeptide corresponding to the Fc region of a native Ig, i.e., formed by the dimeric association of the Fc domains of each of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, as used herein, the term "genetically fused Fc region," or "single-chain Fc region" (scFc region), refers to a synthetic dimeric Fc region composed of genetically linked Fc domains within a single polypeptide chain (i.e., encoded in a single contiguous gene sequence).

[0230] In some embodiments, "Fc region" refers to that portion of a single Ig heavy chain beginning at the hinge region just upstream of the papain cleavage site (i.e., residue 216 of IgG, assuming the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge, CH2, and CH3 domains.

[0231] The Fc region of an Ig constant region can include CH2, CH3, and CH4 domains, as well as a hinge region, depending on the Ig isotype. Chimeric polypeptides containing an Ig Fc region confer several desirable properties to the chimeric polypeptide, including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525), and binding to Fc receptors, such as neonatal Fc receptors (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO03 / 077834; US2003-0235536A1, which are incorporated by reference in their entireties).

[0232] The Ig constant region or its part can be an FcRn binding partner.FcRn is active in adult epithelial tissues and is expressed in the lumen of the intestine, the lung airways, the nasal surface, the vaginal surface, the colon and the rectal surface (U.S. Patent No. 6,485,726).The FcRn binding partner is the part of the Ig that binds to FcRn.Another example of an FcRn binding partner is albumin, which will be further described below.

[0233] FcRn receptors have been isolated from several mammalian species, including humans. The sequences of human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al., 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG at relatively low pH (but does not bind other Ig classes, such as IgA, IgM, IgD, and IgE), actively transports IgG across cells from the lumen to the serosal membrane, and then releases IgG at the relatively high pH found in interstitial fluid. It is expressed in adult epithelial tissues (U.S. Patent Nos. 6,485,726, 6,030,613, 6,086,875; WO03 / 077834; US2003-0235536A1), including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69), renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358), as well as nasal epithelium, vaginal surface, and bile duct surface.

[0234] FcRn binding partners useful in the present disclosure include molecules to which the FcRn receptor can specifically bind, including whole IgG, Fc fragments of IgG, and other fragments containing the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact region between Fc and FcRn is near the junction of the CH2 and CH3 domains. All Fc-FcRn contact is within a single Ig heavy chain. FcRn binding partners include whole IgG, Fc fragments of IgG, and other fragments of IgG containing the complete binding region of the FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 in the CH2 domain, and amino acid residues 385-387, 428, and 433-436 in the CH3 domain. All references to amino acid numbering of Igs or Ig fragments or regions are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, US Department of Public Health, Bethesda, MD.

[0235] Fc regions or FcRn binding partners bound to FcRn can effectively cross epithelial barriers via FcRn, thus providing a non-invasive means of systemically administering desired therapeutic molecules. Additionally, fusion proteins containing Fc regions or FcRn binding partners are endocytosed by cells expressing FcRn. However, these fusion proteins are recycled back into the circulation rather than targeted for degradation, thus increasing the in vivo half-life of these proteins. In certain embodiments, the portion of the Ig constant region is an Fc region or FcRn binding partner that associates with another Fc region or another FcRn binding partner, typically via disulfide bonds and other non-specific interactions, to form dimers and higher-order multimers.

[0236] Two FcRn receptors can bind to a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds to a single polypeptide of an Fc homodimer. In some embodiments, linking an FcRn binding partner, such as an Fc fragment of IgG, to a biologically active molecule provides a means for delivering the biologically active molecule orally, bucally, sublingually, rectally, vaginally, as an aerosol administered intranasally, via the pulmonary route, or via the ocular route. In other embodiments, the chimeric polypeptide can be administered invasively, for example, subcutaneously or intravenously.

[0237] An FcRn binding partner region is a molecule or portion thereof that can specifically bind to an FcRn receptor and then be actively transported by the FcRn receptor in the Fc region. Specific binding refers to the formation of a relatively stable complex between two molecules under physiological conditions. Specific binding is characterized by high affinity and low to moderate binding capacity, and is distinguished from nonspecific binding, which typically has low affinity and moderate to high binding capacity. Typically, binding occurs when the affinity constant KA is greater than or equal to 10. 6 M -1 Higher, or 10 8 M -1 A higher binding concentration is considered specific. If necessary, non-specific binding can be reduced by changing the binding conditions without substantially affecting specific binding. Those skilled in the art can optimize appropriate binding conditions, such as the concentration of the molecule, the ionic strength of the solution, temperature, the time allowed for binding, and the concentration of the blocking agent (e.g., serum albumin, milk casein), using routine techniques.

[0238] In certain embodiments, the chimeric polypeptides of the present disclosure comprise one or more truncated Fc regions that are still sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of the Fc region that binds to FcRn (i.e., the FcRn-binding portion) comprises approximately amino acids 282-438 (EU numbering) of IgG1, with primary contact sites at amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus, the Fc region of the present disclosure can comprise or consist of an FcRn-binding portion. The FcRn-binding portion can be derived from a heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In some embodiments, an FcRn-binding portion from an antibody of human isotype IgG1 is used. In other embodiments, an FcRn-binding portion from an antibody of human isotype IgG4 is used.

[0239] In other embodiments, an "Fc region" comprises the amino acid sequence of an Fc domain or an amino acid sequence derived from an Fc domain. In certain embodiments, an Fc region comprises at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain (approximately amino acids 216-230 of an antibody Fc region according to EU numbering), a CH2 domain (approximately amino acids 231-340 of an antibody Fc region according to EU numbering), a CH3 domain (approximately amino acids 341-438 of an antibody Fc region according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, an Fc region comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, the Fc region comprises, consists essentially of, or consists of a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), a hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), or a CH2 domain (or portion thereof) fused to both a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In still other embodiments, the Fc region lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). In certain embodiments, the Fc region comprises or consists of amino acids corresponding to EU numbers 221-447.

[0240] The Fc region, designated herein as F, F1, or F2, can be obtained from a number of different sources. In some embodiments, the Fc region of the polypeptide is derived from human Ig. However, it is understood that the Fc region can be derived from Ig of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, or guinea pigs) or non-human primates (e.g., chimpanzees, macaques). Furthermore, the Fc domain polypeptide or portion thereof can be derived from any Ig class, including IgM, IgG, IgD, IgA, and IgE, and any Ig isotype, including IgG1, IgG2, IgG3, and IgG4. In other embodiments, the human isotype IgG1 is used.

[0241] In certain embodiments, the Fc variant confers an alteration in at least one effector function conferred by an Fc region comprising said wild-type Fc domain (e.g., an improved or reduced ability of the Fc region to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or to induce antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine ​​residue.

[0242] The Fc regions of the disclosure can utilize art-recognized Fc variants known to confer altered (e.g., enhanced or diminished) effector function and / or FcR or FcRn binding. Specifically, binding molecules of the disclosure can utilize Fc variants described in, for example, International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, and WO03 / 074569, each of which is incorporated herein by reference. A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO 04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 08 5967A2; U.S. Patent Publication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; The polypeptides may include alterations (e.g., substitutions) at one or more of the amino acid positions disclosed in Nos. 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956; and 7,317,091.In some embodiments, a specific change (e.g., a specific substitution of one or more amino acids disclosed in the art) can be made at one or more of the amino acid positions of the present disclosure. In other embodiments, a different change (e.g., a different substitution of one or more amino acid positions disclosed in the art) can be made at one or more of the amino acid positions of the present disclosure.

[0243] The Fc region of IgG or FcRn binding partners can be modified according to well-recognized procedures, such as site-directed mutagenesis, to produce modified IgGs or Fc fragments or portions thereof that bind FcRn. Such modifications include modifications away from the FcRn contact site as well as modifications within the contact site that preserve or even enhance binding to FcRn. For example, human IgG1 The following single amino acid residues in Fc (Fcγ1): P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R291A, and R292A. 2A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N3 15A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K3 38A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D 376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D Substitutions such as 413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A can be made without significant loss of Fc binding affinity for FcRn, e.g., P238A represents the wild-type proline substituted with alanine at position 238. As an example, some embodiments incorporate the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine, other amino acids can be substituted for the wild-type amino acids at the above-specified positions.A single mutation can be introduced into the Fc, resulting in more than 100 Fc regions that differ from the native Fc. In addition, combinations of two, three, or more of these individual mutations can be introduced together to generate hundreds more Fc regions. Moreover, one of the Fc regions of the constructs of the present disclosure may be mutated and the other Fc region of the construct may not be mutated at all, or both may be mutated, but with different mutations.

[0244] Certain of the above mutations can confer new functionality to the Fc region or FcRn binding partner. For example, some embodiments incorporate N297A, eliminating a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby increasing the circulating half-life of the Fc region, and to prevent the Fc region from binding to Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, and Fc gamma RIIIA without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As a further example of the new functionality resulting from the above mutations, the affinity for FcRn can be increased in some cases beyond that of the wild type. This increased affinity may reflect an increased "on" rate, a decreased "off" rate, or both an increased "on" rate and a decreased "off" rate. Examples of mutations that may confer increased affinity for FcRn include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0245] Additionally, at least three human Fc gamma receptors appear to recognize binding sites on IgG within the lower hinge region, generally amino acids 234-237. Therefore, another example of novel functionality and potentially reduced immunogenicity could result from mutation of this region, for example, by replacing amino acids 233-236 of human IgG1 "ELLG" with the corresponding sequence from IgG2 "PVA" (with a single amino acid deletion). It has been shown that Fcγ RI, Fcγ RII, and Fcγ RIII, which mediate various effector functions, do not bind to IgG1 when such mutations are introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77, and Armour et al. 1999, Eur. J. Immunol. 29:2613.

[0246] In some embodiments, the Ig constant region or portion thereof, e.g., the Fc region, is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO: 89 or SEQ ID NO: 3 of U.S. Pat. No. 5,739,277), and optionally further comprising a sequence selected from HQSLGTQ (SEQ ID NO: 90), HQNLSDGK (SEQ ID NO: 91), HQNISDGK (SEQ ID NO: 92), or VISSHLGQ (SEQ ID NO: 93) (or SEQ ID NOs: 11, 1, 2, and 31, respectively, of U.S. Pat. No. 5,739,277).

[0247] In other embodiments, the immunoglobulin constant region or portion thereof includes an amino acid sequence in the hinge region or portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or portion thereof. The disulfide bond formed by the immunoglobulin constant region or portion thereof positions the first polypeptide comprising the FVIII polypeptide and the second polypeptide comprising the VWF fragment together, thereby preventing endogenous VWF from exchanging the VWF fragment and binding to the FVIII polypeptide. Thus, the disulfide bond between the first immunoglobulin constant region or portion thereof and the second immunoglobulin constant region or portion thereof prevents interaction between endogenous VWF and the FVIII polypeptide. This inhibition of the interaction between VWF and the FVIII polypeptide allows the half-life of the chimeric polypeptide to exceed the two-fold limit. The hinge region or portion thereof can be further linked to one or more domains of CH1, CH2, CH3, fragments thereof, and any combination thereof. In certain embodiments, the immunoglobulin constant region or portion thereof is a hinge region and CH2.

[0248] In certain embodiments, the Ig constant region or portion thereof is hemiglycosylated. For example, a chimeric polypeptide comprising two Fc regions or FcRn binding partners may contain a first glycosylated Fc region (e.g., a glycosylated CH2 region) or FcRn binding partner and a second non-glycosylated Fc region (e.g., a non-glycosylated CH2 region) or FcRn binding partner. In some embodiments, a linker can be interposed between the glycosylated and non-glycosylated Fc regions. In other embodiments, the Fc region or FcRn binding partner is fully glycosylated, i.e., the entire Fc region is glycosylated. In other embodiments, the Fc region can be aglycosylated, i.e., none of the Fc portions are glycosylated.

[0249] In certain embodiments, the chimeric polypeptides of the present disclosure comprise amino acid substitutions to an Ig constant region or portion thereof (e.g., Fc variants) that alter the antigen-independent effector function of the Ig constant region, particularly the circulating half-life of the protein.

[0250] Such proteins exhibit either increased or decreased binding to FcRn compared to proteins lacking these substitutions, and therefore have increased or decreased serum half-lives, respectively. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals in which a long half-life of the administered polypeptide is desirable, for example, for treating chronic diseases or disorders (see U.S. Patents 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful for administration to mammals in situations where a shortened circulation time may be advantageous, such as for in vivo diagnostic imaging, or in situations where the starting polypeptide has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta, and are therefore useful in treating diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity may be desirable include applications in which localization in the brain, kidney, and / or liver is desirable. In one exemplary embodiment, a chimeric polypeptide of the present disclosure exhibits reduced transport from the vasculature across the epithelium of renal glomeruli. In other embodiments, a chimeric polypeptide of the present disclosure exhibits reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In some embodiments, a protein with altered FcRn binding comprises at least one Fc region or FcRn binding partner (e.g., one or two Fc regions or FcRn binding partners) with one or more amino acid substitutions within the "FcRn-binding loop" of an Ig constant region. The FcRn-binding loop is composed of amino acid residues 280 to 299 (according to EU numbering) of a wild-type, full-length Fc region. In other embodiments, the Ig constant region or portion thereof in the chimeric polypeptide of the present disclosure with altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner with one or more amino acid substitutions within the 15 Å FcRn "contact region."As used herein, the term "contact region" of 15 Å FcRn includes residues at the following positions of the wild-type full-length Fc portion: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering). In other embodiments, an Ig constant region or portion thereof of the present disclosure with altered FcRn-binding affinity comprises at least one Fc region or FcRn-binding partner with one or more amino acid substitutions at amino acid positions corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions with altered FcRn-binding activity are disclosed in International PCT Publication No. WO 05 / 047327, which is incorporated herein by reference.

[0251] The Fc region or FcRn binding partner used in the present disclosure may also contain art-recognized amino acid substitutions that alter the glycosylation of the chimeric polypeptide. For example, the Fc region or FcRn binding partner of a chimeric polypeptide linked to the D'D3 domain of VWF or a FVIII polypeptide may contain an Fc region with mutations that lead to reduced glycosylation (e.g., N- or O-linked glycosylation) or may contain an altered glycoform of the wild-type Fc moiety (e.g., low-fucose or fucose-free glycans).

[0252] In some embodiments, the unprocessed chimeric polypeptides of the present disclosure may comprise a genetically fused Fc region (i.e., an scFc region) having two or more of its constituent Ig constant regions or portions thereof independently selected from the Ig constant regions or portions thereof described herein. In some embodiments, the Fc regions of the dimeric Fc region are the same. In other embodiments, at least two of the Fc regions are different. For example, the Fc regions or FcRn binding partners of the proteins of the present disclosure may comprise the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet other embodiments, the Fc regions or FcRn binding partners of the proteins of the present disclosure may differ at one or more amino acid positions in sequence. For example, at least two of the Fc regions or FcRn binding partners can differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions).

[0253] III.C.2. XTEN Sequences As used herein, "XTEN sequence" refers to an extended-length polypeptide having a non-naturally occurring, substantially non-repetitive sequence composed primarily of small, hydrophilic amino acids, which sequence has little or no secondary or tertiary structure under physiological conditions. As a partner in a chimeric polypeptide, XTEN can serve as a carrier and, when linked to a VWF protein or FVIII sequence of the present disclosure to create a chimeric polypeptide, impart certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties. Such desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics. As used herein, "XTEN" specifically excludes antibodies or antibody fragments, such as single-chain antibodies or Fc fragments of the light or heavy chains.

[0254] In some embodiments, shorter XTEN sequences provide improved half-life extension properties compared to longer XTEN sequences when the XTEN sequence is fused to a VWF protein and / or a second Ig constant region or portion thereof. Accordingly, in some embodiments, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or portion thereof contains less than, i.e., is shorter than, 288 amino acids in length. In some embodiments, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or portion thereof consists of an amino acid sequence having a length between 12 and 287 amino acids. In other embodiments, the XTEN sequence fused to a VWF protein and / or a second Ig constant region or portion thereof comprises at least about 36 amino acids, at least about 42 amino acids, at least about 72 amino acids, or at least about 144 amino acids, but less than 288 amino acids. In other embodiments, the XTEN sequence fused to the VWF protein and / or second Ig constant region or portion thereof is selected from AE36, AG36, AE42, AG42, AE72, AG72, AE144, or AG144. In some embodiments, the XTEN sequence fused to the VWF protein and / or second Ig constant region or portion thereof is an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:14, and the chimeric polypeptide exhibits improved half-life compared to a chimeric polypeptide without the XTEN sequence.

[0255] The chimeric polypeptides of the present disclosure can further comprise additional (second, third, or more) XTEN sequences. The additional XTEN sequences can be further fused to the FVIII polypeptide or the first Ig constant region or portion thereof. The additional XTEN sequences can be of any length. For example, the additional XTEN sequence fused to the FVIII polypeptide or the first Ig constant region or portion thereof is a peptide or polypeptide having more than about 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, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, the additional XTEN sequence is a peptide or polypeptide having more than about 20 amino acid residues to about 3000 amino acid residues, more than about 30 residues to about 2500 residues, more than about 40 residues to about 2000 residues, more than about 50 residues to about 1500 residues, more than about 60 residues to about 1000 residues, more than about 70 residues to about 900 residues, more than about 80 residues to about 800 residues, more than about 90 residues to about 700 residues, more than about 100 residues to about 600 residues, more than about 110 residues to about 500 residues, or more than about 120 residues to about 400 residues. In certain embodiments, the additional XTEN fused to the FVIII polypeptide comprises at least about 288 amino acids. In certain embodiments, the additional XTEN fused to the FVIII polypeptide comprises about 288 amino acids. In certain embodiments, the additional XTEN fused to the FVIII polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, 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% identical to the amino acid sequence of AE288 (SEQ ID NO: 8).

[0256] The XTEN sequence (i.e., the XTEN sequence fused to a VWF protein and / or a second Ig constant region or portion thereof, or the XTEN sequence fused to a FVIII polypeptide and / or a first Ig constant region or portion thereof, or inserted into one or more insertion sites within a FVIII polypeptide) can comprise one or more sequence motifs of 9 to 14 amino acid residues, or an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence motif, wherein the motif comprises, consists essentially of, or consists of four to six types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P). See US2010-0239554A1.

[0257] In some embodiments, the XTEN sequence comprises non-overlapping sequence motifs, whereby at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% of the sequence is composed of multiple units of non-overlapping sequence selected from a single motif family selected from Table 7, thereby resulting in a family sequence. As used herein, "family" means that the XTEN has motifs selected from only the single motif categories of Table 7; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in the XTEN that are not from the family motif are selected to achieve required properties, for example, to allow for the incorporation of a restriction site by the encoding nucleotides, the incorporation of a cleavage sequence, or to achieve better linkage to FVIII or VWF. In some embodiments of the XTEN family, the XTEN sequence comprises multiple units of non-overlapping sequence motifs from the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, and the resulting XTEN exhibits the ranges of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families in Table 7. These sequences can be selected to achieve desired physical / chemical characteristics, including properties such as net charge, hydrophilicity, lack of secondary structure, or lack of repetition conferred by the amino acid composition of the motif, as described in more detail below. In the embodiments described above in this paragraph, motifs to be incorporated into the XTEN can be selected and assembled using methods described herein to achieve XTENs of from about 36 to about 3,000 amino acid residues. [Table 7]

[0258] In some embodiments, the XTEN sequences used in this disclosure are AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396 , AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF5 76, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE 828, AG828, AD836, AE864, AF864, AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE104 4, AE1140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1 044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US2010-0239554A1.

[0259] In some embodiments, the XTEN sequence is selected from the group consisting of AE42 (SEQ ID NO: 9), AE72 (SEQ ID NO: 10), AE144_2A (SEQ ID NO: 55), AE144_3B (SEQ ID NO: 56), AE144_4A (SEQ ID NO: 57), AE144_5A (SEQ ID NO: 58), AE144_6B (SEQ ID NO: 59), AG144_A (SEQ ID NO: 60), AG144_B (SEQ ID NO: 61), AG144_C (SEQ ID NO: 62), AG144_F (SEQ ID NO: 63), AE864 (SEQ ID NO: 64), AG144_G (SEQ ID NO: 65), AG144_H (SEQ ID NO: 66), AG144_J (SEQ ID NO: 67), AG144_L (SEQ ID NO: 68), AG144_L (SEQ ID NO: 69), AG144_M (SEQ ID NO: 70), AG144_N (SEQ ID NO: 71), AG144_O (SEQ ID NO: 72), AG144_P (SEQ ID NO: 73), AG144_P (SEQ ID NO: 74), AG144_R (SEQ ID NO: 75), AG144_S (SEQ ID NO: 76), AG144_T (SEQ ID NO: 77), AG144_V (SEQ ID NO: 78), AG144_V (SEQ ID NO: 79), AG144_V (SEQ ID NO: 80), AG144_V (SEQ ID NO: 81), AG144_ No. 15), AE576 (SEQ ID NO: 16), AE288 (SEQ ID NO: 8), AE288_2 (SEQ ID NO: 54), AE144 (SEQ ID NO: 11), AG864 (SEQ ID NO: 17), AG576 (SEQ ID NO: 18), AG288 (SEQ ID NO: 19), AG144 (SEQ ID NO: 14), and any combination thereof. In other embodiments, the XTEN sequence is selected from the group consisting of AE42 (SEQ ID NO: 9), AE72 (SEQ ID NO: 10), AE144_2A (SEQ ID NO: 55), AE144_3B (SEQ ID NO: 56), AE144_4A (SEQ ID NO: 57), AE144_5A (SEQ ID NO: 58), AE144_6B (SEQ ID NO: 59), AG144_A (SEQ ID NO: 60), AG144_B (SEQ ID NO: 61), AG144_C (SEQ ID NO: 62), AG144_F (SEQ ID NO: 63), AE864 (SEQ ID NO: 15), AE576 (SEQ ID NO: 16), AE288 (SEQ ID NO: 8), AE288_2 (SEQ ID NO: 54), AE144 (SEQ ID NO: 11), AG864 (SEQ ID NO: 17), AG576 (SEQ ID NO: 18), AG288 (SEQ ID NO: 19), AG144 (SEQ ID NO: 14), and any combination thereof. In some embodiments, the XTEN sequence is AE 288. The amino acid sequences of certain XTEN sequences of the present disclosure are shown in Table 8. [Table 8-1] [Table 8-2] [Table 8-3] Table 8-4

[0260] In embodiments in which the XTEN component has less than 100% of its amino acids consisting of four, five, or six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), or less than 100% of the sequence consists of a sequence motif of Table 7 or an XTEN sequence of Table 8, other amino acid residues of the XTEN are selected from any of the other 14 naturally occurring L-amino acids, but preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) may be interspersed throughout the XTEN sequence, located within or between sequence motifs, or concentrated in one or more short stretches of the XTEN sequence, e.g., creating a linker between the XTEN and a FVIII or VWF component. In such cases where the XTEN component includes amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), it is preferred that less than about 2% of the amino acids or less than about 1% of the amino acids be hydrophobic residues, such that the resulting sequence generally lacks secondary structure, e.g., does not have more than 2% alpha helices or more than 2% beta sheets as determined by the methods disclosed herein. Hydrophobic residues that are less favorable for XTEN construction include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. In addition, XTEN sequences can be designed to contain less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or none of the following amino acids: cysteine ​​(to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine, and glutamine (to avoid deamidation).Thus, in some embodiments, XTEN components containing other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) have sequences with less than 5% of residues contributing to alpha helices and beta sheets as measured by the Chou-Fasman algorithm, and at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm.

[0261] In further embodiments, the XTEN sequences used in the present disclosure affect the physical or chemical properties, such as pharmacokinetics, of the chimeric polypeptides of the present disclosure. The XTEN sequences used in the present disclosure can exhibit one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius. In some embodiments, the XTEN sequences linked to the FVIII proteins of the present disclosure increase pharmacokinetic properties, such as a longer terminal half-life or an increased area under the curve (AUC), thereby causing the chimeric polypeptides described herein to persist in vivo for an increased period compared to wild-type FVIII. In further embodiments, the XTEN sequences used in the present disclosure increase pharmacokinetic properties, such as a longer terminal half-life or an increased area under the curve (AUC), thereby causing the FVIII proteins to persist in vivo for an increased period compared to wild-type FVIII.

[0262] A variety of methods and assays can be used to determine the physical / chemical properties of proteins containing XTEN sequences. Such methods include, but are not limited to, analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV / visible spectroscopy. Additional methods are disclosed in Amau et al., Prot Expr and Purif 48, 1-13 (2006).

[0263] Additional examples of XTEN sequences that can be used in accordance with the present disclosure are disclosed in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or International Patent Publication Nos. WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, WO2011028344A2, or WO20130122617A1.

[0264] III.C.2. Albumin Albumin or a portion thereof can be an FcRn binding partner. In certain embodiments, the chimeric polypeptide used in the methods of the present disclosure comprises at least one albumin polypeptide or a fragment, variant, or derivative thereof. Human serum albumin (HSA, or HA) is a 609 amino acid protein in its full-length form, which is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. As used herein, the term "albumin" includes full-length albumin, or a functional fragment, variant, derivative, or analog thereof. Examples of albumin or fragments or variants thereof are disclosed in U.S. Patent Publication Nos. 2008 / 0194481A1, 2008 / 0004206A1, 2008 / 0161243A1, 2008 / 0261877A1, or 2008 / 0153751A1, or PCT Application Publication Nos. 2008 / 033413A2, 2009 / 058322A1, or 2007 / 021494A2, which are incorporated by reference in their entireties.

[0265] Albumin-binding polypeptides (ABPs) can include, but are not limited to, bacterial albumin-binding domains, albumin-binding peptides, or albumin-binding antibody fragments that can bind to albumin.The domain 3 from streptococcal protein G disclosed in Kraulis et al., FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci. 11:206-213 (2002) is an example of a bacterial albumin-binding domain.An example of an albumin-binding peptide is disclosed in Dennis et al., J. Biol. Chem. 2002, 277: 35035-35043 (2002). Examples of albumin-binding antibody fragments are disclosed in Muller and Kontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Roovers et al., Cancer Immunol. Immunother. 56:303-317 (2007), and Holt et al., Prot. Eng. Design Sci., 21:283-288 (2008), the entire contents of which are incorporated herein by reference.

[0266] In certain embodiments, the chimeric polypeptides used in the methods of the present disclosure comprise at least one binding site for a non-polypeptide small molecule, variant, or derivative thereof, capable of binding to albumin. For example, the chimeric polypeptides may comprise one or more organic albumin-binding moieties. An example of such an albumin-binding moiety is 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate ("Albu" tag), as disclosed in Trussel et al., Bioconjugate Chem. 20:2286-2292 (2009).

[0267] In certain embodiments, the VWF fragment described herein is fused to albumin. In some embodiments, the VWF fragment-albumin construct forms a homodimer. In some embodiments, the D'D3 domain of VWF fused to albumin associates with a FVIII polypeptide. In certain embodiments, the D'D3 domain of VWF fused to albumin associates with a FVIII polypeptide through a stronger interaction than the natural non-covalent interaction between wild-type FVIII and VWF. In certain embodiments, the D'D3 domain of VWF and / or the FVIII polypeptide comprises one or more mutations that increase the affinity between the D'D3 domain of VWF and the FVIII polypeptide. In certain embodiments, the D'D3 domain of VWF and / or the FVIII polypeptide comprises one or more mutations that allow disulfide bond formation between the D'D3 domain of VWF and / or the FVIII polypeptide.

[0268] III.C.3.CTP In certain embodiments, the chimeric polypeptide used in the methods of the present disclosure comprises at least one C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, or a fragment, variant, or derivative thereof. CTP peptides are known to increase the half-life of the protein. See, for example, U.S. Patent No. 5,712,122, which is incorporated herein by reference in its entirety. Non-limiting exemplary CTP peptides are disclosed in U.S. Patent Application Publication No. US2009 / 0087411A1, which is incorporated herein by reference.

[0269] III.C.4.PAS In certain embodiments, the chimeric polypeptide used in the methods of the present disclosure comprises at least one PAS peptide, or a fragment, variant, or derivative thereof. As used herein, a PAS peptide or PAS sequence refers to an amino acid sequence containing primarily alanine and serine residues, or primarily alanine, serine, and proline residues, that forms a random coil conformation under physiological conditions. Thus, a PAS sequence is a building block, amino acid polymer, or sequence cassette that contains, consists essentially of, or consists of alanine, serine, and proline, and can be used as part of a heterologous moiety in a chimeric polypeptide. An amino acid polymer can also form a random coil conformation when residues other than alanine, serine, and proline are added as minor components in the PAS sequence. By "minor components" is meant that amino acids other than alanine, serine, and proline can be added to the PAS sequence to some extent, e.g., up to about 12%, i.e., about 12 out of 100 amino acids of the PAS sequence, up to about 10%, up to about 9%, up to about 8%, about 6%, about 5%, about 4%, about 3%, i.e., about 2%, or about 1% of the amino acids. Amino acids other than alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Under physiological conditions, the PAS peptide forms a random coil conformation, which can mediate increased in vivo and / or in vitro stability for the recombinant protein of the present disclosure and has procoagulant activity.

[0270] Non-limiting examples of PAS peptides are disclosed, for example, in U.S. Patent Publication No. 2010 / 0292130A1; PCT Application Publication No. WO2008 / 155134A1; and European Patent Publication No. EP2173890.

[0271] III.C.5.HAP In certain embodiments, the chimeric polypeptide used in the methods of the present disclosure comprises at least one homoamino acid polymer (HAP) peptide, or a fragment, variant, or derivative thereof. The HAP peptide may comprise a repeat sequence of glycine having a length of at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids. The HAP sequence can extend the half-life of the moiety fused or linked to the HAP sequence. Non-limiting examples of HAP sequences include, but are not limited to, (Gly)n, (Gly4Ser)n, or S(Gly4Ser)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In other embodiments, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. See, e.g., Schlapschy M et al., Protein Eng. Design Selection, 20: 273-284 (2007).

[0272] III.C.6. Transferrin In certain embodiments, the chimeric polypeptide used in the methods of the present disclosure comprises at least one transferrin peptide, or a fragment, variant, or derivative thereof. Any transferrin can be fused to the chimeric polypeptide used in the methods of the present disclosure. As an example, wild-type human Tf (Tf) is a 679-amino acid protein of approximately 75 kDa (without considering glycosylation), with two major domains, N (approximately 330 amino acids) and C (approximately 340 amino acids), which appears to have originated from gene duplication. See GenBank Accession Nos. NM001063, XM002793, M12530, XM039845, XM039847, and S95936 (www.ncbi.nlm.nih.gov), all of which are incorporated herein by reference in their entirety.

[0273] Transferrin transports iron through transferrin receptor (TfR)-mediated endocytosis. After iron is released into endosomal compartments and the Tf-TfR complex is recycled to the cell surface, Tf is released back into the extracellular space for the next cycle of ion transport. Tf possesses a long half-life of more than 14-17 days (Li et al., Trends Pharmacol. Sci. 23:206-209 (2002)). Transferrin fusion proteins have been tested for half-life extension, targeted delivery for cancer therapy, oral delivery, and sustained activation of proinsulin (Brandsma et al., Biotechnol. Adv., 29: 230-238 (2011); Bai et al., Proc. Natl. Acad. Sci. USA 102:7292-7296 (2005); Kim et al., J. Pharmacol. Exp. Ther., 334:682-692 (2010); Wang et al., J. Controlled Release 155:386-392 (2011)).

[0274] III.C.7.PEG In certain embodiments, a chimeric polypeptide used in the methods of the present disclosure comprises at least one binding site for a non-polypeptide heterologous moiety, or a fragment, variant, or derivative thereof. For example, a chimeric polypeptide used in the methods of the present disclosure may comprise one or more polyethylene glycol (PEG) moieties attached to one or more amino acid residues in a coagulation factor and / or Fc region.

[0275] PEGylation of a protein can refer to a conjugate formed between a protein and at least one polyethylene glycol (PEG) molecule. PEGs are commercially available in a wide variety of molecular weights and average molecular weight ranges. Typical examples of average molecular weight ranges for PEG include, but are not limited to, about 200, about 300, about 400, about 600, about 1000, about 1300-1600, about 1450, about 2000, about 3000, about 3000-3750, about 3350, about 3000-7000, about 3500-4500, about 5000-7000, about 7000-9000, about 8000, about 10000, about 8500-11500, about 16000-24000, about 35000, about 40000, about 60000, and about 80000 daltons. These average molecular weights are provided as examples only and are not meant to be limiting in any way.

[0276] The chimeric polypeptides used in the methods of the present disclosure can be PEGylated to contain mono- or poly(e.g., 2-4) PEG moieties. PEGylation can be carried out by any PEGylation reaction known in the art. Methods for preparing PEGylated protein products generally include (i) reacting a polypeptide with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions in which the peptide of the present disclosure is conjugated to one or more PEG groups; and (ii) obtaining the reaction product. Generally, optimal reaction conditions for the reaction are determined on a case-by-case basis based on known parameters and the desired results.

[0277] There are many PEG conjugation methods available to those skilled in the art, for example, Malik F et al., Exp. Hematol. 20:1028-35 (1992); Francis, Focus on Growth Factors 3(2):4-10 (1992); European Patent Publication Nos. EP0401384, EP0154316, and EP0401384; and International Patent Application Publication Nos. WO92 / 16221 and WO95 / 34326. As a non-limiting example, FVIII variants can contain cysteine ​​substitutions, and the cysteine ​​can be further conjugated to a PEG polymer. See Mei et al., Blood 116:270-279 (2010), and U.S. Patent No. 7,632,921, the entire contents of which are incorporated herein by reference.

[0278] In certain embodiments, the chimeric polypeptide comprises a single-chain FVIII polypeptide covalently linked to a VWF fragment comprising the D' and D3 domains of VWF, wherein the FVIII polypeptide comprises a deletion of all or part of the B domain, the FVIII polypeptide is PEGylated, and the VWF fragment is fused directly or indirectly to the N-terminus of the FVIII polypeptide (e.g., PEG-scFVIII-D'D3, "MG1121," or LAFATE). In other embodiments, the chimeric polypeptide comprises a single-chain FVIII polypeptide covalently linked to a VWF fragment comprising the D' and D3 domains of VWF, wherein the FVIII polypeptide comprises a deletion of all or part of the B domain, the VWF fragment is PEGylated, and the VWF fragment is fused directly or indirectly to the N-terminus of the FVIII polypeptide (e.g., scFVIII-D'D3-PEG). In other embodiments, the chimeric polypeptide comprises a single-chain FVIII polypeptide covalently linked to a VWF fragment comprising the D' and D3 domains of VWF, the FVIII polypeptide comprising a deletion of all or part of the B domain, the FVIII polypeptide and the VWF fragment are PEGylated, and the VWF fragment is fused directly or indirectly to the N-terminus of the FVIII polypeptide (e.g., scFVIII-D'D3-PEG).

[0279] III.C.8.HES In certain embodiments, the chimeric polypeptide used in the method of the present disclosure comprises at least one hydroxyethyl starch (HES) polymer. HES is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body. HES exhibits advantageous biological properties and is used as a blood replacement agent and in hemodilution therapy in clinics. See, for example, Sommermeyer et al., Krankenhauspharmazie 8:271-278 (1987); and Weidler et al., Arzneim.-Forschung / Drug Res. 41: 494-498 (1991).

[0280] HES is primarily characterized by its molecular weight distribution and degree of substitution. HES has an average molecular weight (weight average) of 1-300 kD, 2-200 kD, 3-100 kD, or 4-70 kD. Hydroxyethyl starch can further exhibit a molar substitution of 0.1-3, 0.1-2, 0.1-0.9, or 0.1-0.8, and a C2:C6 substitution ratio with respect to the hydroxyethyl group ranging from 2 to 20. An example of HES with an average molecular weight of approximately 130 kD is Fresenius' VOLUVEN®. VOLUVEN® is an artificial colloid used in replacement fluids for therapeutic applications, such as the treatment and prevention of hypovolemia. Numerous HES conjugation methods are available to those skilled in the art, including the same PEG conjugation methods described above.

[0281] III.C.9.PSA In certain embodiments, the chimeric polypeptide used in the methods of the present disclosure comprises at least one polysialic acid (PSA) polymer. PSA is a naturally occurring, unbranched polymer of sialic acid produced by certain bacterial strains and in certain mammalian cells. See, for example, Roth J. et al. (1993) in Polysialic Acid: From Microbes to Man, eds. Roth J., Rutishauser U., Troy FA (BirkhaeuserVerlag, Basel, Switzerland), pp. 335-348. PSA can be produced with various degrees of polymerization, from n=about 80 or more sialic acid residues to n=2, by limited acid hydrolysis, digestion with neuraminidase, or fractionation of the naturally occurring bacterial form of the polymer. There are several methods of PSA conjugation available to those skilled in the art, such as the same PEG conjugation method described above. In certain embodiments, activated PSA can also be conjugated to cysteine ​​amino acid residues within coagulation factors, such as FVIII, or within the Fc region. See, for example, U.S. Patent No. 5,846,951.

[0282] III.C.10. Clearance receptors In certain embodiments, when the coagulation factor of the chimeric polypeptide comprises a FVIII polypeptide and at least one fragment of an FVIII clearance receptor, or a FVIII-binding fragment, variant, or derivative thereof, the half-life of the chimeric polypeptide used in the method of the present disclosure can be extended. Insertion of a clearance receptor, such as a soluble form of the low-density lipoprotein-related protein receptor LRP1 or a fragment thereof, can block the binding of FVIII to the clearance receptor, thereby extending its half-life, for example, in vivo half-life. LRP1 is a 600-kDa integral membrane protein involved in the receptor-mediated clearance of various proteins, including FVIII. See, for example, Lenting et al., Haemophilia 16:6-16 (2010). Other suitable FVIII clearance receptors are, for example, LDLR (low-density lipoprotein receptor), VLDLR (very-low-density lipoprotein receptor), and megalin (LRP-2), or fragments thereof. See, e.g., Bovenschen et al., Blood 106:906-912 (2005); Bovenschen, Blood 116:5439-5440 (2010); Martinelli et al., Blood 116:5688-5697 (2010).

[0283] III.D. Insertion Site In some embodiments, the insertion site in the FVIII polypeptide is located in one or more domains of the FVIII polypeptide, such as the N-terminus, the A1 domain, the A2 domain, the A3 domain, the B domain, the C1 domain, the C2 domain, the C-terminus, or a combination of two or more thereof, or between two domains of the FVIII polypeptide, such as the A1 domain and the a1 acidic region, and the a1 acidic region and the A2 domain, the A2 domain and the a2 acidic region, the a2 acidic region and the B domain, the B domain and the A3 domain, and the A3 domain and the C1 domain, the C1 domain and the C2 domain, or any combination thereof. For example, insertion sites into which an XTEN sequence can be inserted are selected from the group consisting of the N-terminus and the A1 domain, the N-terminus and the A2 domain, the N-terminus and the A3 domain, the N-terminus and the B domain, the N-terminus and the C1 domain, the N-terminus and the C2 domain, the N-terminus and the C-terminus, the A1 and the A2 domains, the A1 and the A3 domains, the A1 and the B domains, the A1 and the C1 domains, the A1 and the C2 domains, the A1 domain and the C-terminus, the A2 and the A3 domains, the A2 and the B domains, the A2 and the C1 domains, the A2 and the C2 domains, the A2 domain and the C-terminus, the A3 and the B domains, the A3 and the C1 domains, the A3 and the C2 domains, the A3 domain and the C-terminus, the B and the C1 domains, the B and the C2 domains, the B domain and the C-terminus, the C1 and the C2 domains, the C1 and the C-terminus, the C2 domain and the C-terminus, and combinations of two or more thereof.

[0284] FVIII polypeptides in which an XTEN sequence has been inserted immediately downstream of, or linked to the C-terminus or N-terminus of, one or more amino acids in the FVIII polypeptide (e.g., one or more XTEN insertion sites) retain FVIII activity after linkage to or insertion with the XTEN sequence. The XTEN sequence can be inserted once, or more than once, two, three, four, five, or six times in the FVIII polypeptide such that the insertion does not affect FVIII activity (i.e., such that the FVIII protein still retains its clotting properties).

[0285] FVIII polypeptides useful in the present disclosure can be linked to one or more XTEN polypeptides at the N-terminus or C-terminus of the FVIII polypeptide by an optional linker, or can be inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII polypeptide by one or more optional linkers.

[0286] In still other embodiments, one or more XTENs are inserted into the B domain of FVIII. In one example, XTEN is inserted between amino acids 740 and 1640 corresponding to SEQ ID NO: 65, with the FVIII sequence between amino acids 740 and 1640 optionally being absent. In another example, XTEN is inserted between amino acids 741 and 1690 corresponding to SEQ ID NO: 65, with the FVIII sequence between amino acids 740 and 1690 optionally being absent. In another example, XTEN is inserted between amino acids 741 and 1648 corresponding to SEQ ID NO: 65, with the FVIII sequence between amino acids 741 and 1648 optionally being absent. In yet another example, XTEN is inserted between amino acids 743 and 1638 corresponding to SEQ ID NO: 65, with the FVIII sequence between amino acids 743 and 1638 optionally being absent. In yet another example, XTEN is inserted between amino acids 745 and 1656 corresponding to SEQ ID NO: 65, with the FVIII sequence between amino acids 745 and 1656 optionally being absent. In some examples, XTEN is inserted between amino acids 745 and 1657 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 745 and 1657 is optionally absent. In certain examples, XTEN is inserted between amino acids 745 and 1667 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 745 and 1667 is optionally absent. In yet other examples, XTEN is inserted between amino acids 745 and 1686 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 745 and 1686 is optionally absent. In some other examples, XTEN is inserted between amino acids 747 and 1642 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 747 and 1642 is optionally absent. In yet other examples, XTEN is inserted between amino acids 751 and 1667 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 751 and 1667 is optionally absent. In some embodiments, the XTEN sequence is inserted between amino acids 745 and 746 of the mature FVIII polypeptide, or at the corresponding insertion site of the B-domain deleted FVIII polypeptide.In certain embodiments, an XTEN sequence comprising an amino acid sequence having at least about 70%, at least about 75%, 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 of AE288 (SEQ ID NO: 8) is inserted between amino acids 745 and 746 of mature FVIII, wherein the mature FVIII has a deletion of amino acid residues 746-1648. In certain embodiments, the XTEN is inserted immediately downstream of amino acid 745 of a B-domain deleted FVIII (SEQ ID NO: 68) of Table 5.

[0287] III.E. Linker In certain embodiments, the chimeric polypeptide of the present disclosure further comprises one or more linkers in the FVIII protein and / or VWF fragment. One type of linker is a cleavable linker that can be cleaved by various proteases when administered to a subject in vivo, for example, at the site of a blood clot. In some embodiments, the cleavable linker allows for cleavage of a moiety, such as a VWF protein, from the XTEN sequence, thus from the chimeric polypeptide at the site of the blood clot cascade, thereby allowing activated FVIII (FVIIIa) to retain its FVIIIa activity. Another type of linker is a processable linker that contains an intracellular cleavage site and can therefore be cleaved by intracellular processing enzymes in host cells, thereby allowing for convenient expression of the polypeptide and formation of the chimeric polypeptide.

[0288] One or more linkers can be present between any two proteins in the chimeric polypeptide. In some embodiments, the chimeric polypeptide comprises (i) a FVIII polypeptide and (ii) a first polypeptide comprising a first Ig constant region or a portion thereof, and (iii) a VWF protein, (iv) a linker (e.g., a cleavable linker), (v) an XTEN sequence, and (vi) a second polypeptide comprising a second Ig constant region or a portion thereof. In other embodiments, the chimeric polypeptide comprises (i) a FVIII polypeptide and (ii) a first polypeptide comprising a first Ig constant region or a portion thereof, and (iii) a VWF protein, (iv) an XTEN sequence, (v) a linker (e.g., a cleavable linker), and (vi) a second Ig constant region or a portion thereof. In other embodiments, the chimeric polypeptide comprises (i) a FVIII polypeptide, and (ii) a first polypeptide comprising a first Ig constant region or portion thereof, and (iii) a VWF protein, (iv) a first linker (e.g., a cleavable linker), (v) an XTEN sequence, (vi) a second linker (e.g., a cleavable linker), and (vii) a second Ig constant region or portion thereof. In some embodiments, the first polypeptide comprises a linker, e.g., a cleavable linker, between the FVIII polypeptide and the first Ig constant region.

[0289] In certain embodiments, the chimeric polypeptide comprises a single chain comprising (i) a FVIII polypeptide, (ii) a first Ig constant region or portion thereof, (iii) a linker (e.g., a processable linker), (iv) a VWF protein, (v) an XTEN sequence, and (vi) a second Ig constant region or portion thereof. In other embodiments, the chimeric polypeptide comprises a single chain comprising (i) a FVIII polypeptide, (ii) a first Ig constant region or portion thereof, (iii) a first linker (e.g., a processable linker), (iv) a VWF protein, (v) a second linker (e.g., a cleavable linker), (vi) an XTEN sequence, and (vii) a second Ig constant region or portion thereof. The processable linker can be processed after the chimeric polypeptide is expressed in a host cell; the chimeric polypeptide thus produced in the host cell can have a final form comprising two or three polypeptide chains.

[0290] The linker useful in the present disclosure can comprise any organic molecule. Examples of linkers that can be used in the chimeric polypeptide of the present disclosure are disclosed in, for example, US2011 / 0183907A1, US2016 / 0229903A1, and US2016 / 0251408A1, each of which is incorporated herein by reference in its entirety. In some embodiments, the linker comprises a polymer, such as polyethylene glycol (PEG) or hydroxyethyl starch (HES). In other embodiments, the linker comprises an amino acid sequence. The linker may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1 to 5 amino acids, 1 to 10 amino acids, 1 to 20 amino acids, 10 to 50 amino acids, 50 to 100 amino acids, 100 to 200 amino acids, 200 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 600 amino acids, 600 to 700 amino acids, 700 to 800 amino acids, 800 to 900 amino acids, or 900 to 1000 amino acids. In some embodiments, the linker comprises an XTEN sequence. Additional examples of XTENs that can be used in accordance with the present disclosure are disclosed in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or International Patent Publication Nos. WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, or WO2011028344A2. In other embodiments, the linker is a PAS sequence.

[0291] In certain embodiments, the linker is a polymer, such as polyethylene glycol (PEG) or hydroxyethyl starch (HES). In other embodiments, the linker is an amino acid sequence. The linker may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker may contain 1 to 5 amino acids, 1 to 10 amino acids, 1 to 20 amino acids, 10 to 50 amino acids, 50 to 100 amino acids, 100 to 200 amino acids, 200 to 300 amino acids, 300 to 400 amino acids, 400 to 500 amino acids, 500 to 600 amino acids, 600 to 700 amino acids, 700 to 800 amino acids, 800 to 900 amino acids, or 900 to 1000 amino acids.

[0292] Examples of linkers are well known in the art. In certain embodiments, the linker has the sequence G n The linker comprises the sequence (GA) n The linker may comprise the sequence (GGS) n In other embodiments, the linker may comprise (GGGS) n (SEQ ID NO: 101). In yet another embodiment, the linker comprises the sequence (GGS) n (GGGGS) n(SEQ ID NO: 95). In these examples, n can be an integer between 1 and 100. In other examples, n can be an integer between 1 and 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 96), GGSGGSGGSGGSGGG (SEQ ID NO: 97), GGSGGSGGGGSGGGGS (SEQ ID NO: 98), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 99), or GGGSGGGGSGGGGGS (SEQ ID NO: 100). The linker does not eliminate or impair VWF protein activity or Factor VIII clotting activity. Optionally, the linker enhances the VWF protein activity or the coagulation activity of the Factor VIII protein, for example, by further reducing the effect of steric hindrance and by making the VWF protein or Factor VIII moiety more accessible to its target binding site.

[0293] In certain embodiments, linkers useful for chimeric polypeptides are 15-25 amino acids in length. In other embodiments, linkers useful for chimeric polypeptides are 15-20 amino acids in length. In some embodiments, linkers useful for chimeric polypeptides are 10-25 amino acids in length. In other embodiments, linkers for chimeric polypeptides are 15 amino acids in length. In still other embodiments, linkers for chimeric polypeptides are (GGGGS) n (SEQ ID NO: 94), in which G represents glycine, S represents serine, and n is an integer of 1 to 20.

[0294] III.F. Cutting site Cleavable linkers can incorporate a moiety that can be cleaved chemically (e.g., hydrolysis of an ester bond), enzymatically (i.e., by incorporating a protease cleavage sequence), or photolytically (e.g., a chromophore such as 3-amino-3-(2-nitrophenyl)propionic acid (ANP)) to release one molecule from another. Examples of suitable cleavable linkers for the chimeric polypeptides of the present disclosure can be found, for example, in US2016 / 0229903A1 and US2016 / 0251408A1, each of which is incorporated by reference in its entirety.

[0295] In certain embodiments, the cleavable linker comprises one or more cleavage sites at the N-terminus, C-terminus, or both. In other embodiments, the cleavable linker consists essentially of or consists of one or more cleavable sites. In other embodiments, the cleavable linker comprises a heterologous amino acid linker sequence or polymer described herein and one or more cleavable sites.

[0296] In certain embodiments, the cleavable linker comprises one or more cleavage sites (i.e., intracellular processing sites) that can be cleaved in a host cell. Non-limiting examples of c...

Claims

1. 1. A composition for treating hemophilia A in a human subject, comprising a chimeric polypeptide: the chimeric polypeptide comprises (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:207, and (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO:202, wherein the first polypeptide is linked to the second polypeptide by a disulfide bond; multiple doses of the chimeric polypeptide are administered intravenously to the subject with an interval between doses of at least about 7 days; The composition, wherein each of said multiple doses is from about 50 IU / kg to about 65 IU / kg.

2. The composition of claim 1 , wherein the FVIII polypeptide is linked to the VWF polypeptide by two disulfide bonds.

3. 3. The composition of claim 1 or 2, wherein each of the multiple doses is 50 IU / kg and the dosing interval is at least 7 days.

4. The composition of any one of claims 1 to 3, wherein at least one of the multiple doses is 50 IU / kg.

5. The composition of any one of claims 1 to 3, wherein at least one of the multiple doses is 65 IU / kg.

6. The composition according to any one of claims 1 to 5, which is for preventive treatment.

7. The composition of any one of claims 1 to 6, wherein the subject has severe hemophilia A.

8. The composition according to any one of claims 1 to 7, characterized in that the multiple doses are administered for at least 6 months.

9. The composition according to any one of claims 1 to 8, wherein said multiple doses are administered over a period of at least 12 months.

10. 10. The composition of any one of claims 1-9, wherein the subject has previously received treatment for hemophilia A with any recombinant FVIII, plasma-derived FVIII, or cryoprecipitate for at least 150 exposure days (ED).

11. The subject: (a) currently receiving a prophylactic treatment regimen with a marketed FVIII product and has had at least four bleeding episodes in the 12 months prior to administration of the composition; or (b) currently receiving an on-demand treatment regimen with a marketed FVIII product and have had at least 12 bleeding episodes in the 12 months prior to administration of the composition; The composition according to any one of claims 1 to 10,

12. The composition of any one of claims 1 to 11, wherein the subject has a platelet count of at least 100,000 cells / μL.

13. The composition of any one of claims 1 to 12, wherein the subject does not have other clotting disorders in addition to hemophilia A.

14. The composition of any one of claims 1 to 13, wherein the subject does not have a history of developing an inhibitor to a FVIII product.

15. 1. A composition for treating hemophilia A in a subject in need thereof, comprising a chimeric polypeptide, the chimeric polypeptide comprising: (i) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 207; and (ii) a second polypeptide comprising the D' domain of VWF and the D3 domain of VWF, wherein the VWF comprises the amino acid sequence set forth in SEQ ID NO: 202; wherein the chimeric polypeptide is administered intravenously as a dose of 50 IU / kg once weekly for at least six months.

16. 16. The composition of claim 15, wherein the subject has severe hemophilia A.

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