Treatment methods for hemophilia A

Abstract

To provide a method for treating hemophilia A in human subjects who require it. [Solution] The present disclosure provides a method for treating hemophilia A in a human subject in need, comprising administering to the subject at a certain interval between doses a chimeric polypeptide comprising (i) factor VIII (FVIII) protein and (ii) a VWF fragment comprising the D' domain of von Willebrand factor (VWF) and the D3 domain of VWF.

Description

[Technical Field]

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

[0002] References to electronically submitted sequence listings The contents of the sequence listing submitted electronically in an ASCII text file (name: SA9-461PC_SeqListing.txt; size: 922 kilobytes; creation date: May 14, 2019) are incorporated herein by reference in their entirety. [Background technology]

[0003] Hemophilia A is a bleeding disorder caused by a deficiency in the gene encoding coagulation factor VIII (FVIII), and it affects 1 to 2 out of every 10,000 male births. et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients with hemophilia A may be treated with infusions of purified or recombinantly produced FVIII. Many commercially available FVIII products are known to have a half-life of approximately 8–12 hours, requiring frequent intravenous administration to the patient. 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). In addition, numerous approaches have been attempted to extend the half-life of FVIII. For example, approaches in the development of extending 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 has been reported to improve the half-life by approximately 1.7 times compared to ADVATE® in patients with hemophilia A. See Powell et al., Blood. 119(13): 3031-3037 (2012). Therefore, the increase in half-life, despite the minor 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 treatment includes routine administration of FVIII (routine prophylaxis) to minimize the number of bleeding episodes. Routine prophylaxis is associated with improved 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). FVIII products with extended half-lives reduce the frequency of prophylactic FVIII administration; however, all interact with von Willebrand factor (VWF), and this is due to the half-life of endogenous VWF. These variants have an equivalent circulating half-life, consistent with the upper limit of the half-life of the rFVIII variant. See, for example, Pipe et al., Blood. 128(16):2007-16 (2016). Prophylactic dosing of these FVIII products is every 3-5 days. The next-generation, extended-half-life FVIII product, which allows for less frequent administration and long-term prevention and control of bleeding episodes, potentially addresses the challenges of adhering to demanding prophylactic regimens, which in turn could improve the quality of life for hemophilia patients. [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 [Overview of the project] [Means for solving the problem]

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

[0007] In some embodiments, multiple doses include at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least ten doses, at least eleven doses, at least twelve doses, at least thirteen doses, at least fourteen doses, at least fifteen doses, at least sixteen doses, at least seventeen doses, at least eighteen doses, at least nineteen doses, at least twenty doses, or more doses.

[0008] In some embodiments, treatment for hemophilia A includes controlling or reducing the incidence or frequency of bleeding episodes in human subjects requiring treatment. In some embodiments, treatment for hemophilia A includes preventing or treating bleeding episodes in human subjects requiring treatment.

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

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

[0011] In some embodiments, at least one of the plurality of dosages 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 plurality of dosages is about 25 IU / kg. In some embodiments, at least one of the plurality of dosages is about 50 IU / kg. In some embodiments, at least one of the plurality of dosages is about 65 IU / kg. In some embodiments, at least one of the plurality of dosages 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 every two weeks, at least once every three weeks, or at least once every four weeks. In some embodiments, the dosing interval is at least once per week. In some embodiments, the dosing interval is at least once every two weeks.

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

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

[0016] In some embodiments, the FVIII protein comprises an FVIII polypeptide and a first half-life extension portion. In some embodiments, the first half-life extension portion is fused to the C-terminus or N-terminus of the FVIII polypeptide. In some embodiments, the first half-life extension portion is inserted into the FVIII polypeptide. In some embodiments, the first half-life extension portion is inserted into the B domain of the FVIII polypeptide. In some embodiments, the first half-life extension portion is inserted into 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 portion is fused to the FVIII polypeptide by a linker.

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

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

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

[0020] In some embodiments, the first XTEN is inserted into 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 extension portion includes 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 includes a first immunoglobulin (Ig) constant region or a portion thereof. In some embodiments, the first Ig constant region or a portion thereof is fused to the C-terminus or N-terminus of the FVIII polypeptide. In some embodiments, the first Ig constant region or a portion thereof is inserted into the FVIII polypeptide. In some embodiments, the first Ig constant region or a portion thereof is fused to the C-terminus of the FVIII polypeptide. In some embodiments, the first Ig constant region or a portion thereof is fused to the FVIII polypeptide by a linker. In some embodiments, the first Ig constant region or a portion thereof includes a first Fc domain or a portion thereof.

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

[0024] In some embodiments, the FVIII protein and the VWF fragment are linked to each other by a covalent bond between the first and second Fc domains. In some embodiments, the FVIII protein and the VWF fragment are further linked to each other by non-covalent interactions between the FVIII protein and the VWF fragment.

[0025] In one embodiment, a method for treating hemophilia A in a human subject comprises administering multiple doses of a chimeric polypeptide to the subject in need at a certain interval, wherein the chimeric polypeptide comprises (i) an FVIII protein comprising a first FVIII polypeptide fragment containing the amino acid sequence of SEQ ID NO: 215, a first XTEN sequence containing the amino acid sequence of SEQ ID NO: 8 (AE288), a second FVIII polypeptide fragment containing the amino acid sequence of SEQ ID NO: 216, and a first Fc region containing the amino acid sequence of SEQ ID NO: 217, and (ii) the D' domain of VWF containing the amino acid sequence of SEQ ID NO: 210, and the amino acid sequence of SEQ ID NO: 214. A method is disclosed herein comprising a VWF protein comprising a D3 domain of VWF containing an acid sequence, a second XTEN sequence containing the amino acid sequence of SEQ ID NO: 58 (AE144_5A), an a2 linker containing the amino acid sequence of SEQ ID NO: 88, and a second Fc region containing 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 an FVIII protein comprising an FVIII polypeptide, a first XTEN sequence, and a first Fc region, and a VWF protein comprising the D' domain of VWF, the D3 domain of VWF, a second XTEN sequence, the α2 linker of FVIII, 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, and the FVIII polypeptide further comprises the amino acid sequence of SEQ ID NO: 216, and the first Fc region is fused to the amino acid sequence of SEQ ID NO: 217 The first Fc region contains an amino acid sequence and is fused to the C-terminus of SEQ ID NO 216. The D' domain of VWF contains the amino acid sequence of SEQ ID NO 210, the D3 domain of VWF contains the amino acid sequence of SEQ ID NO 214, the second XTEN sequence contains the amino acid sequence of AE144_5A (SEQ ID NO 58) and is fused to the C-terminus of the D3 domain of VWF, the a2 linker contains the amino acid sequence of SEQ ID NO 88 and is fused to the C-terminus of the second XTEN sequence, the second Fc region contains 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 an FVIII protein containing an FVIII signal peptide having the amino acid sequence of SEQ ID NO: 64. In some embodiments, the chimeric polypeptide comprises a VWF protein containing a VWF signal peptide having the amino acid sequence of SEQ ID NO: 208. In some embodiments, the chimeric polypeptide comprises a VWF protein containing the D1D2 domain of VWF having the amino acid sequence of SEQ ID NO: 209.

[0028] In some embodiments, the chimeric polypeptide includes an FVIII protein having 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 having 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 an FVIII protein containing the amino acid sequence of SEQ ID NO: 203 and a VWF protein containing the amino acid sequence of SEQ ID NO: 205. In another embodiment, the chimeric polypeptide comprises an FVIII protein containing the amino acid sequence of SEQ ID NO: 201 and a VWF protein containing the amino acid sequence of SEQ ID NO: 202. In yet another embodiment, the chimeric polypeptide comprises an FVIII protein containing the amino acid sequence of SEQ ID NO: 207 and a VWF protein containing the amino acid sequence of SEQ ID NO: 202.

[0030] In some embodiments, the chimeric polypeptide is 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), FVIII-199 (SEQ ID NO: 75), FVIII-201 (SEQ ID NO: 76); FVIII-203 (SEQ ID NO: 77), FVIII-204 (SEQ ID NO: 78) , a sequence selected from FVIII-205 (sequence number 79), FVIII-266 (sequence number 80), FVIII-267 (sequence number 81), FVIII-268 (sequence number 82), FVIII-269 (sequence number 83), FVIII-271 (sequence number 84), FVIII-272 (sequence number 85), FVIII-312 (sequence number 173), or FVIII-312A (sequence number 203) and at least approximately 80%, 90%, 95%, or 1 The product includes an FVIII protein containing 00% identical amino acid sequences, and a VWF protein containing at least approximately 80%, 90%, 95%, or 100% identical amino acid sequences to sequences 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, nasal administration, and pulmonary administration.

[0032] In some embodiments, the administered chimeric polypeptide results in plasma activity levels 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 administered chimeric polypeptide results in plasma activity levels 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 approximately 50 IU / kg to approximately 80 IU / kg. In some embodiments, at least one of the multiple doses is approximately 50 IU / kg to approximately 65 IU / kg. In some embodiments, at least one of the multiple doses is approximately 65 IU / kg to approximately 80 IU / kg. In some embodiments, at least one of the multiple doses is approximately 50 IU / kg to approximately 80 IU / kg, and the dosing interval is at least approximately 7 days. In some embodiments, at least one of the multiple doses is approximately 50 IU / kg to approximately 65 IU / kg, and the dosing interval is at least approximately 5 days. In some embodiments, at least one of the multiple doses is approximately 65 IU / kg to approximately 80 IU / kg, and the dosing interval is at least approximately 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 approximately 50 IU / kg to approximately 80 IU / kg. The dose is 65 IU / kg. In some embodiments, at least one of the multiple doses is approximately 65 IU / kg to approximately 80 IU / kg. In some embodiments, at least one of the multiple doses is approximately 50 IU / kg to approximately 80 IU / kg, and the dosing interval is at least approximately 7 days. In some embodiments, at least one of the multiple doses is approximately 50 IU / kg to approximately 65 IU / kg, and the dosing interval is at least approximately 7 days. In some embodiments, at least one of the multiple doses is approximately 65 IU / kg to approximately 80 IU / kg, and the dosing interval is at least approximately 7 days.

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

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

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

[0041] In some embodiments, at least one of the multiple doses is approximately 50 IU / kg to approximately 80 IU / kg, and the dosing interval is at least approximately 2 weeks. In some embodiments, at least one of the multiple doses is approximately 50 IU / kg to approximately 65 IU / kg, and the dosing interval is at least approximately 2 weeks. In some embodiments, at least one of the multiple doses is approximately 65 IU / kg to approximately 80 IU / kg, and the dosing interval is at least approximately 2 weeks.

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

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

[0044] In some embodiments, the administration induces immune tolerance to FVIII in human subjects. In some embodiments, the administration reduces the inhibitory immune response to FVIII in human subjects. In some embodiments, the inhibitory immune response to FVIII includes high-titer anti-FVIII antibodies in human subjects.

[0045] In some embodiments, the administration of the chimeric polypeptide lasts for approximately 7 days, 10 days, and 11 days. In between, FVIII inhibitors are not induced after administration for approximately 12, 13, 14, 15, 20, 24, 25, 28, 30, or 35 days. In some embodiments, administration of the chimeric polypeptide does not induce FVIII inhibitors after approximately 28 days of administration. [Brief explanation of the drawing]

[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: Domain of VWF; Fc: Fc region of the constant immunoglobulin region.

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

[0048] [Figure 3A] Figures 3A and 3B are graphical representations of baseline-adjusted FVIII activity levels based on activated partial thromboplastin time (aPTT) tests in human subjects with severe hemophilia A. These regimens were either administered 25 IU / kg of rFVIII followed by a drug-free period and then 25 IU / kg of rFVIIIFc-VWF-XTEN (Figure 3A; low-dose cohort), or 65 IU / kg of rFVIII followed by a drug-free period and then 65 IU / kg of rFVIIIFc-VWF-XTEN (Figure 3B; high-dose cohort). Horizontal dashed lines represent FVIII activity at 3%, 5%, 10%, and 20%. [Figure 3B]Figures 3A and 3B are graphical representations of baseline-adjusted FVIII activity levels based on activated partial thromboplastin time (aPTT) tests in human subjects with severe hemophilia A. These regimens were either administered 25 IU / kg of rFVIII followed by a drug-free period and then 25 IU / kg of rFVIIIFc-VWF-XTEN (Figure 3A; low-dose cohort), or 65 IU / kg of rFVIII followed by a drug-free period and then 65 IU / kg of rFVIIIFc-VWF-XTEN (Figure 3B; high-dose cohort). Horizontal dashed lines represent FVIII activity at 3%, 5%, 10%, and 20%.

[0049] [Figure 4] Figure 4 is a schematic diagram of the design of a clinical trial evaluating the safety and tolerability of rFVIIIFc-VWF-XTEN administered at a dose of 50 IU / kg or 65 IU / kg once weekly for a total of four doses in adult male patients (PTP) aged 18 to 65 years (inclusive) with a history of treatment for severe hemophilia A. EOS = End of Study; ET = Early Termination; PK = Pharmacokinetics. [Modes for carrying out the invention]

[0050] This disclosure relates to a method for treating a hemorrhagic disorder or condition, such as hemophilia A, in a human subject in need thereof, comprising administering to the subject multiple doses of (i) factor VIII (FVIII) polypeptide and (ii) a chimeric polypeptide comprising a VWF fragment including the D' domain of von Willebrand factor (VWF) and the D3 domain of VWF, at a certain dosing interval. 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 every 5 days. In some embodiments, the dosing interval is at least every 7 days.

[0051] I. Definition It should be noted that the terms “a” or “an” entity refer to one or more such entities; for example, “a nucleotide sequence” is understood to represent one or more nucleotide sequences. Thus, the terms “a” (or “an”), “one or more” and “at least one” may be used interchangeably herein.

[0052] Furthermore, when used herein, “and / or” should be interpreted as a specific disclosure of each of two identified features or components, either together with or without the other. Thus, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B and / or C,” the term “and / or” is intended to include 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] Wherever an aspect is described herein using the word “including,” it will be understood that other similar aspects described in terms of “consisting of” and / or “essentially consisting of” are also provided.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to whom this disclosure relates. For example, see 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 a general dictionary of many of the terms used in this disclosure to those skilled in the art.

[0055] Units, prefixes, and symbols are shown in the form permitted by their International System of Units (SI). Numerical ranges include the number defining the range. Unless otherwise indicated, amino acid sequences are described from left to right in the direction of amino to carboxyl. The headings provided herein are not limitations on the various aspects of this disclosure. Thus, the terms defined immediately thereunder are more completely defined by referring to this specification in their entirety.

[0056] The term “approximately” is used herein to mean roughly, about, approximately, or in that range. When the term “approximately” is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the numerical value being expressed. Generally, the term “approximately” can modify the numerical value above and below the value being referred to, for example, by a 10 percent upper or lower (higher or lower) variance.

[0057] The terms “polynucleotide” or “nucleotide” are intended to encompass single nucleic acids and multiple nucleic acids and refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, polynucleotides include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term "acid" refers to any one or more nucleic acid segments, e.g., fragments of DNA or RNA, present in a polynucleotide. With respect to "isolated" nucleic acids or polynucleotides, a nucleic acid molecule, DNA, or RNA that has been removed from its native environment is intended. For example, a recombinant polynucleotide encoding a factor VIII polypeptide contained in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells, or recombinant polynucleotides purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of this disclosure. Isolated polynucleotides or nucleic acids according to this disclosure further include synthetically produced such molecules. In addition, polynucleotides or nucleic acids may include regulatory elements such as promoters, enhancers, ribosome-binding sites, or transcription termination signals.

[0058] Certain proteins secreted by mammalian cells bind to secretory signal peptides, which are cleaved from the mature protein once transport across the rough endoplasmic reticulum of the growing protein chain begins. Those skilled in the art know that the signal peptide is generally fused to the N-terminus of the polypeptide and cleaved from the complete or "full-length" polypeptide to produce 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 polypeptide secretion is operably bound to it. Alternatively, signal peptides from other mammalian species, such as human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptides, or functional derivatives thereof, can be used.

[0059] As used herein, the term “polypeptide” is intended to encompass one and more “polypeptides” and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or more of two or more amino acids and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, “proteins,” “amino acid chains,” or any other terms used to refer to chains or more of two or more amino acids are within the scope of 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 amino acids that do not exist in nature. Polypeptides may originate from natural biological sources or may be produced by recombinant techniques but are not necessarily translated from a given nucleic acid sequence. They may be produced by any method, including by chemical synthesis.

[0060] An “isolated” polypeptide, or its fragments, variants, or derivatives, refers to a polypeptide that does not exist in its natural environment. No specific level of purification is required. For example, an isolated polypeptide can be readily extracted from its native or natural environment. Recombinantly generated polypeptides and proteins expressed in host cells are considered to be isolated for the purposes of this disclosure, like native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any preferred technique.

[0061] This disclosure also includes polypeptide fragments or variants, and any combination thereof. The terms “fragment” or “variant” refer to polypeptide binding domains in this disclosure. Where referring to a binding molecule, it includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., FcRn binding affinity to an Fc variant or FcRn binding domain, coagulation activity for an FVIII variant, or FVIII binding activity to a VWF fragment). Polypeptide fragments include proteolytic and deletion fragments, in addition to specific antibody fragments discussed elsewhere in this specification, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of polypeptide binding domains or binding molecules in this disclosure include the fragments described above, and also include polypeptides having altered amino acid sequences resulting from amino acid substitutions, deletions, or insertions. Variants may or may not be naturally occurring. Variants that are not naturally occurring can be generated using mutagenic techniques known in the art. Variant polypeptides may include conserved or non-conserved amino acid substitutions, deletions, or additions.

[0062] As used herein, the terms “VWF protein” or “multiple VWF proteins” mean any VWF fragment that interacts with FVIII and retains at least one of the 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 may result in premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but not to VWF-bound FVIII, and / or stabilizing the heavy- and light-chain interactions of FVIII. VWF fragments as referred herein are VWF polypeptides that are less than full-length VWF proteins, where the VWF fragment retains the ability to interact with and / or bind to FVIII.

[0063] A "conservative amino acid substitution" is the replacement of an amino acid residue with an amino acid residue having a similar side chain. In this technology, families of amino acid residues having similar side chains are defined, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar 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, when 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, the amino acid string can be conservatively replaced with a structurally similar string that differs in the order and / or composition of the side-chain family members.

[0064] As is known in the art, “sequence identity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of the second polypeptide. In the context of this specification, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide is not limited to the BESTFIT program (Wisconsin Sequence Analysis Package, version 8 for Unix®, Genetics Computer Group, University Research Park, 575 Science). This can be determined using methods and computer programs / software known in the art, such as those by 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 the reference sequence in this disclosure, the parameters are, of course, set so that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and a homology gap of up to 5% of the total number of amino acids in the reference sequence is permitted.

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

[0066] As used herein, the term “insertion site” refers to a location in an FVIII polypeptide, or its fragment, variant, or derivative, immediately downstream of a site where a half-life extension or heterologous moiety can be inserted. “Insertion site” is identified as a number, which is the number of amino acids in the corresponding mature native FVIII (SEQ ID NO: 65) that the insertion site is immediately C-terminal to the insertion site. For example, the phrase “a3 contains XTEN at the 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 the position immediately adjacent to the terminal carboxyl group of an amino acid. For example, the 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 the 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 position where XTEN or any other polypeptide is inserted between two adjacent amino acids. Therefore, the phrases "inserted immediately downstream of an amino acid" and "inserted between two amino acids at the insertion site" are used synonymously 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 XTEN in the chimeric polypeptide relative to a similar position in native mature human FVIII. As used herein, these terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII, and do not indicate, imply, or infer any method or process by which the chimeric polypeptide is made. For example, referring to the chimeric polypeptide provided herein, the phrase “XTEN is inserted immediately downstream of residue 745 of the FVIII polypeptide” means that the chimeric polypeptide contains XTEN immediately downstream of the amino acid corresponding to amino acid 745 in native mature human FVIII (e.g., the amino acids corresponding to amino acids 745 and 746 of native mature human FVIII are bounded).

[0070] A "fusion" or "chimeric" protein contains a first amino acid sequence linked to a second amino acid sequence that is not naturally linked. Amino acid sequences that are normally present in another protein can be combined in a fusion polypeptide or are normally present in the same protein. The amino acid sequence may be placed in a new configuration in the fusion polypeptide, for example, in a fusion with the Ig Fc domain of the factor VIII domain of the present disclosure. The fusion protein is created, for example, by chemical synthesis or by creating and translating polynucleotides in which the peptide region encodes in a desired relationship. The chimeric polypeptide may further include a second amino acid sequence linked to the first amino acid sequence by covalent, non-peptide, or non-covalent bonds.

[0071] As used herein, the term “linked” refers to the covalent or noncovalent conjugation of a first amino acid sequence or nucleotide sequence to a second amino acid sequence or nucleotide sequence. The first amino acid sequence or nucleotide sequence may be directly conjugated or juxtaposed with the second amino acid sequence or nucleotide sequence, or an intervening sequence may covalently conjugate the first sequence to the second sequence. The term “linked” includes not only the fusion of the first amino acid sequence to the second amino acid sequence at the C-terminus or N-terminus, but also the insertion of the entire first amino acid sequence (or second amino acid sequence) into any two amino acids in the second amino acid sequence (or each of the first amino acid sequences). In some embodiments, the first amino acid sequence may be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence may be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker may be a peptide or polypeptide (for polypeptide chains), or a nucleotide or nucleotide chain (for nucleotide chains), or any chemical moiety (for both polypeptide and polynucleotide chains). The term "concatenated" is also indicated by a hyphen (-).

[0072] As used herein, the term “bonded to” 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 “bonded to” means a covalent, non-peptide, or non-covalent bond. This bond can be indicated by a colon, i.e., (:). In other embodiments, this means a covalent bond other than a peptide bond. For example, the amino acid cysteine ​​contains a thiol group on a second cysteine ​​residue that can form a disulfide bond or bridge with the thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 using the Kabat numbering system (positions 226 or 229 in the EU numbering system). Examples of covalent bonds, though not limited to them, include peptide bonds, metallic bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidyl bonds, agnostic bonds, bent bonds, bipolar bonds, Pi backbonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugations, hyperconjugations, aromaticity, haptonumber, or antibonding. Non-exclusive examples of non-covalent bonds include ionic bonds (e.g., cation / pi bonds, or salt bonds), metallic 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 terms “cleavage site” or “enzyme cleavage site” refer to a site recognized by an enzyme. Certain enzyme cleavage sites include intracellular processing sites. In some embodiments, polypeptides have enzyme cleavage sites that are cleaved by enzymes activated during the coagulation cascade, such that the cleavage of such sites occurs at the site of blood clot formation. Exemplary such sites include, for example, thrombin, factor XIa, or Examples of enzymes recognized by factor Xa include TQSFNDFTR (SEQ ID NO: 1) and SVSQTSKLTR (SEQ ID NO: 3). Examples of thrombin cleavage sites include 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 in this specification.

[0074] As used herein, the terms “processing site” or “intracellular processing site” refer to the type of enzymatic cleavage site in a polypeptide that is targeted by an enzyme that functions after the polypeptide’s translation. In some embodiments, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides before the protein is secreted from the cell. Examples of such processing sites include those targeted by the PACE / furin family of endopeptidases (PACE is an acronym for paired basic amino acid cleavage enzymes). These enzymes are localized to the Golgi membrane and cleave proteins at the carboxyl terminus 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] In a structure that includes two or more processing or cutting sites, it is understood that such sites may be identical or different.

[0076] As used herein, “processable linker” refers to a linker that includes at least one intracellular processing site, which is described somewhere in this specification.

[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 the amount administered to a subject to be eliminated from the circulation and / or other tissues in the animal. When constructing a clearance curve for a given polypeptide as a function of time, the curve is typically biphasic, having a rapid α-phase and a longer β-phase. The α-phase typically represents the equilibrium of the administered Fc polypeptide between intravascular and extravascular spaces, and is partly determined 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 containing FVIII are monophasic and therefore have only a single beta-phase, but no α-phase. Thus, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase. The typical beta-phase half-life of a human antibody in humans is 21 days. In certain embodiments, half-life is expressed as the half-life of the final phase.

[0078] When used herein, hemostatic disorder means a genetically inherited or acquired condition characterized by a tendency to bleed, either spontaneously or as a result of trauma, due to impaired ability to form fibrin clots or inability to form fibrin clots. An example of such disorder is hemophilia. The three main forms are hemophilia A ( These include 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 disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiencies or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X or XIII, and Bernard-Soulier syndrome, which is a deficiency or absence of GPIb. GPIb, the receptor for VWF, can be deficient, leading to a lack of primary clot formation (primary hemostasis) and increased bleeding tendency, and Glanzmann and Naegeli thrombasthenia (Glanzmann thrombasthenia). In hepatic failure (acute and chronic types), there is insufficient production of coagulation factors by the liver, which can increase the risk of bleeding.

[0079] As used herein, “administer” or “give administration” refers to delivering the compositions described herein, for example, chimeric polypeptides, to a subject. The compositions, for example, chimeric polypeptides, may be administered to a subject using methods known in the art. In particular, the compositions may be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, for example, via oral, sublingual, buccal, nasal, rectal, vaginal, or pulmonary routes. In some embodiments, administration is self-administered. In some embodiments, a parent administers the chimeric polypeptide to a child. In some embodiments, the chimeric polypeptide is administered to a subject by a healthcare professional, such as a physician, medic, or nurse.

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

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

[0082] As used herein, the terms “interval” or “medication interval” refer to the length of time elapsed between a first dose of composition A administered to a subject and a subsequent dose of the same composition. A medication interval may refer to the time elapsed between a first dose and a second dose, or a medication interval may refer to the length of time elapsed between multiple doses.

[0083] As used herein, the term “medication frequency” refers to the number of doses administered per specific medication interval. For example, medication frequency can be described as once a week, once every two weeks, etc. Thus, a 7-day medication 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 therapies for the treatment of hemophilia, such treatments intended to prevent or reduce the severity of one or more symptoms of hemophilia, e.g., bleeding episodes, e.g., one or more spontaneous bleeding episodes, and / or joint injuries. See Jimenez-Yuste et al., Blood Transfus. 12(3):314-19 (2014). To prevent or reduce the progression of such symptoms, e.g., bleeding episodes and joint diseases, patients with hemophilia A may receive regular infusions of clotting factors as part of a prophylactic treatment regimen. The basis of such prophylactic treatment is having a clotting factor at a level of 1% or higher, e.g., FVIII Observations include the rare occurrence of spontaneous bleeding episodes in hemophilia patients and fewer hemophilia-related comorbidities compared to patients with severe hemophilia. See, for example, Coppola A. et al, Semin. Thromb. Hemost. 38(1): 79-94 (2012). Healthcare professionals treating these hemophilia patients have estimated that maintaining factor levels at approximately 1% through regular infusions may potentially reduce the risk of hemophilia symptoms, including bleeding episodes and joint injuries. See ibid. Subsequent studies have confirmed these benefits in pediatric hemophilia patients receiving prophylactic treatment with coagulation factors, making prophylactic treatment a target for individuals with severe hemophilia. See ibid.

[0085] "Prophylactic" treatment can also refer to a preemptive administration to a subject of a composition described herein, e.g., a chimeric polypeptide, 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 coagulation factor, e.g., FVIII, is standard treatment for subjects with severe hemophilia A. See, for example, Oldenburg, Blood 125:2038-44 (2015). In some embodiments, prophylactic treatment refers to administering a composition disclosed herein to a subject in need to reduce the occurrence of one or more symptoms of hemophilia A. Prophylactic treatment may include multiple dose administrations. 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 could cause bleeding. In one embodiment, on-demand treatment may be given to a subject when bleeding begins, e.g., after an injury, or when bleeding is expected, e.g., before surgery. In another embodiment, on-demand treatment may 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, one or more additional doses may 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. However, it should be noted that the medication intervals associated with on-demand treatments are not the same as those used for prophylactic treatments.

[0087] In some embodiments, subjects requiring a general hemostatic agent are undergoing or about to undergo surgery. The chimeric polypeptides of this disclosure may be administered before or after surgery. The chimeric polypeptides of this disclosure may also be administered during or after surgery to control acute bleeding episodes. When the chimeric polypeptide is administered before surgery, the administration may 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, the administration may 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. Surgery may be limited to While not typically performed, examples include liver transplantation, hepatectomy, dental procedures, or stem cell transplantation.

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

[0089] As used herein, “to treat,” “to treat,” and “to treat” refer, for example, to reduce the severity of a disease or condition; to reduce the duration of the disease course; to improve one or more symptoms associated with a disease or condition; to provide a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition; or to prevent one or more symptoms associated with a disease or condition. In some embodiments, the terms “treatment” or “treatment” mean maintaining in a subject a trough level of FVIII 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 by administering the chimeric polypeptide or VWF fragment of this disclosure. As used herein, “trough level” in a hemophilia patient is the lowest concentration measured by factor therapy, e.g., FVIII therapy, before the next dose is administered. In other embodiments, treatment means maintaining a trough level of FVIII of at least about 1 IU / dL during the dosing interval. In other embodiments, treatment means maintaining a trough level of FVIII of at least about 3 IU / dL during the dosing interval. In other embodiments, treatment means maintaining a trough level of FVIII of at least about 5 IU / dL during the dosing interval. In other embodiments, treatment, or treatment, means maintaining a trough level of FVIII between approximately 1 to approximately 20 IU / dL, between approximately 2 to approximately 20 IU / dL, between approximately 3 to approximately 20 IU / dL, between approximately 4 to approximately 20 IU / dL, between approximately 5 to approximately 20 IU / dL, between approximately 6 to approximately 20 IU / dL, between approximately 7 to approximately 20 IU / dL, between approximately 8 to approximately 20 IU / dL, between approximately 9 to approximately 20 IU / dL, or between approximately 10 to approximately 20 IU / dL during the interval between doses.

[0090] "Treatment" or "treatment" of a disease or condition may also include maintaining FVIII activity in the subject at a level comparable 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 FVIII activity in a non-hemophilic subject during the dosing interval. In other embodiments, treating or treatment means maintaining an FVIII activity level of at least about 1% during the dosing interval. In other embodiments, treating or treatment means maintaining an FVIII activity level of at least about 2% during the dosing interval. In other embodiments, treating or treatment means maintaining an FVIII activity level of at least about 3% during the dosing interval. In other embodiments, treating or treatment means maintaining an FVIII activity level of at least about 4% during the dosing interval. In other embodiments, treatment means maintaining an activity level of at least about 5% of FVIII during the dosing interval. In other embodiments, treatment means maintaining an activity level of at least about 6% of FVIII during the dosing interval. In other embodiments, treatment means maintaining an activity level of at least about 7% of FVIII during the dosing interval. In other embodiments, treatment means maintaining an activity level of at least about 8% of FVIII during the dosing interval. In other embodiments, treatment means maintaining an activity level of at least about 9% of FVIII during the dosing interval. In other embodiments, treatment means maintaining an activity level of at least about 10% of FVIII during the dosing interval. The minimum trough level required for treatment can be measured by one or more known methods (e.g., the aPTT assay or colorimetric assay described herein) and can be adjusted (increased or decreased) for each individual.

[0091] II. Method of Disclosure Certain embodiments of the present disclosure relate to a method for treating hemophilia A in a subject requiring such treatment, comprising administering a chimeric polypeptide comprising FVIII protein and VWF fragments to the subject at certain dosing intervals. In some embodiments, the method comprises administering to the subject multiple doses of a chimeric polypeptide comprising (i) FVIII protein and (ii) VWF fragments comprising the D' domain of VWF and the D3 domain of VWF, e.g., rFVIIIFc-VWF-XTEN, at certain dosing intervals. In other embodiments, the present disclosure relates to a method for treating a bleeding disorder or condition, e.g., hemophilia A, in a subject requiring such treatment, comprising administering to the subject multiple doses of FVIII protein and multiple doses of VWF fragments at certain dosing intervals.

[0092] In some embodiments, the multiple doses include at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least ten doses, at least eleven doses, at least twelve doses, at least thirteen doses, at least fourteen doses, at least fifteen doses, at least sixteen doses, at least seventeen doses, at least eighteen doses, at least twenty doses, or more doses. In some embodiments, the multiple doses are administered for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about twelve months, at least about eighteen months, at least about two years, at least about three years, at least about four years, at least about five years, at least about ten years, at least about fifteen years, at least about twenty years, or at least about twenty-five years.

[0093] In certain embodiments, the methods of the present disclosure relate to treating hemophilia A. In some embodiments, treatment of hemophilia A includes preventing bleeding episodes in human subjects who require it. In some embodiments, treatment of hemophilia A includes treating bleeding episodes in human subjects who require it. In some embodiments, treatment of hemophilia A includes controlling the incidence or frequency of bleeding episodes in human subjects who require it. In some embodiments, treatment of hemophilia A includes reducing the incidence or frequency of bleeding episodes in human subjects who require it.

[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 differs 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 a particular embodiment, 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 is approximately 20 IU / kg to approximately 95 IU / kg, approximately 20 IU / kg to approximately 90 IU / kg, approximately 20 IU / kg to approximately 85 IU / kg, approximately 20 IU / kg to approximately 80 IU / kg, approximately 20 IU / kg to approximately 75 IU / kg, approximately 20 IU / kg to approximately 70 IU / kg, and approximately 20 IU The possible IU / kg levels are approximately 65 IU / kg, 60 IU / kg, 55 IU / kg, 50 IU / kg, 45 IU / kg, 40 IU / kg, 35 IU / kg, 30 IU / kg, or 25 IU / kg.

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

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

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

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

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

[0101] In some embodiments, a chimeric polypeptide, such as rFVIIIFc-VWF-XTEN, is administered prophylactically. When administered prophylactically, at least one of the multiple doses may range from 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 is approximately 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 as multiple doses. In some embodiments, the chimeric polypeptide is administered as one or more doses ranging from about 15 IU / kg to about 100 IU / kg. In certain embodiments, the chimeric polypeptide is administered on demand as one or more doses ranging from 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 approximately 25 IU / kg. In other embodiments, the chimeric polypeptide is administered on demand as one or more doses of approximately 50 IU / kg. In other embodiments, the chimeric polypeptide is administered on demand as one or more doses of approximately 65 IU / kg. In other embodiments, the chimeric polypeptide is administered on demand as one or more doses of approximately 80 IU / kg.

[0103] In some embodiments, administration of the chimeric polypeptide by this method does not induce FVIII inhibitors after approximately 7, 10, 11, 12, 13, 14, 15, 20, 24, 25, 28, 30, or 35 days of administration. In some embodiments, administration of the chimeric polypeptide does not induce FVIII inhibitors after approximately 28 days of administration.

[0104] B. Medication interval In certain embodiments, particularly for prophylactic measures, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered as multiple doses at intervals between doses. In some embodiments, the intervals between doses are 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 interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 5 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 6 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 7 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 8 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 9 days. In certain embodiments, the interval between doses is at least about 10 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 11 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 12 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 13 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 14 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about It is 21 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 27 days. In certain embodiments, the interval between doses, for example, the interval between doses for prophylactic treatment of hemophilia A, is at least about 30 days.

[0106] In some embodiments, the frequency of medication, for example, the frequency of medication for prophylactic treatment of hemophilia A, is at least twice every week, at least once every week, at least once every two weeks, at least once every three weeks, at least once every four weeks, at least once every five weeks, or at least once every six weeks. In certain embodiments, the interval between medications, for example, the interval between medications for prophylactic treatment of hemophilia A, is at least once every week. In certain embodiments, the interval between medications, for example, the interval between medications for prophylactic treatment of hemophilia A, is at least two weeks. In certain embodiments, the interval between medications, for example, the interval between medications for prophylactic treatment of hemophilia A, is at least three weeks. In certain embodiments, the interval between medications, for example, the interval between medications for prophylactic treatment of hemophilia A, is at least four weeks.

[0107] In some embodiments, the frequency of medication, for example, for prophylactic treatment of hemophilia A, is approximately twice every three months, once every month, twice every month, three times every month, four times every month, five times every month, six times every month, seven times every month, or eight times every month. In a particular embodiment, the frequency of medication is approximately once every month. In a particular embodiment, the frequency of medication is approximately twice every month. In a particular embodiment, the frequency of medication is approximately three times every month. In a particular embodiment, the frequency of medication is approximately four times every month. In a particular embodiment, the frequency of medication is approximately five times every month. In a particular embodiment, the frequency of medication is approximately six times every month.

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

[0109] In some embodiments, at least one of the multiple doses is administered according to the doses and dosing frequencies listed 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 prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 5 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 8 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 8 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 9 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with an interval of at least approximately 10 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 6. The drug is administered at a dose of 5 IU / kg with at least an interval of approximately 10 to 21 days. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with at least an interval of approximately 11 to 14 days. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with at least an interval of approximately 12 to 14 days. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with at least an interval of approximately 12 to 15 days. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg with at least an interval of approximately 13 to 15 days. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 25 IU / kg to approximately 65 IU / kg, with an interval of at least approximately 14 to 21 days between doses.

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

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

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

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

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

[0116] In some embodiments, such as for prophylactic treatment of hemophilia A, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 5 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 8 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 6 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 7 to 14 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 8 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 9 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 10 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 10 to 21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 11 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 12 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 12 to 15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 13 to 15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 50 IU / kg to approximately 65 IU / kg with an interval of at least approximately 14 to 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 approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 5 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 6 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 6 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 6 It is administered at intervals of up to 8 days. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with at least 6 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with at least 6 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with at least 6 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with at least 6 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with at least 7 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 7 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 7 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 7 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 7 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 7 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 8 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 9 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 10 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 10 to 21 days between doses.In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 11 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 12 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 12 to 15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 13 to 15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 14 to 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 approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 2 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 2 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 2 to 8 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 2 to 9 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 2 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 2 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 2 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 4 to 9 days between doses. In some embodiments, At least one dose is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg, with an interval of at least 4 to 10 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg, with an interval of at least 4 to 11 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg, with an interval of at least 4 to 12 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg, with an interval of at least 4 to 13 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg, with an interval of at least 4 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 5 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 5 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 6 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 7 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 8 to 14 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 10 to 15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 10 to 21 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 13 to 15 days between doses. In some embodiments, at least one of the multiple doses is administered at a dose of approximately 65 IU / kg to approximately 80 IU / kg with an interval of at least approximately 14 to 21 days between doses.

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

[0120] 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 approximately 65 IU / kg to approximately 80 IU / kg with at least one week between doses. 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 approximately 65 IU / kg to approximately 80 IU / kg with at least two doses approximately one week apart.

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

[0122] In certain embodiments, the method involves administering multiple doses of a chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, comprising (i) FVIII protein and (ii) a VWF fragment containing the D' domain and D3 domain of VWF, to a subject at certain dosing intervals, where at least one of the multiple doses is about 25 IU / kg and the dosing interval is at least about 5 days. In certain embodiments, the method involves administering multiple doses of a chimeric polypeptide, comprising (i) FVIII protein and (ii) a VWF fragment containing the D' domain and D3 domain of VWF, to a subject at certain dosing intervals, where at least one of the multiple doses is about 25 IU / kg and the dosing 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 dosing interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosing interval is at least about 8 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosing interval is at least about 9 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosing interval is at least about 10 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosing interval is at least about 11 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosing interval is at least about 12 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosing interval is at least about 13 days. In certain embodiments, at least one of the multiple doses is about 25 IU / kg, and the dosing interval is at least about 14 days. In certain embodiments, at least one of the multiple doses is approximately 25 IU / kg, and the dosing interval is at least approximately 15 days. In certain embodiments, at least one of the multiple doses is approximately 25 IU / kg, and the dosing interval is at least approximately 16 days. In certain embodiments, at least one of the multiple doses is approximately 25 IU / kg, and the dosing interval is at least approximately 17 days.In certain embodiments, at least one of the multiple doses is approximately 25 IU / kg, and the dosing interval is at least approximately 18 days. In certain embodiments, at least one of the multiple doses is approximately 25 IU / kg, and the dosing interval is at least approximately 19 days. In certain embodiments, at least one of the multiple doses is approximately 25 IU / kg, and the dosing interval is at least approximately 20 days. In certain embodiments, at least one of the multiple doses is approximately 25 IU / kg, and the dosing interval is at least approximately 21 days.

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

[0124] In certain embodiments, the method involves administering multiple doses of a chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, comprising (i) FVIII protein and (ii) a VWF fragment containing the D' domain and D3 domain of VWF, to a subject at certain dosing intervals, where at least one of the multiple doses is about 65 IU / kg and the dosing interval is at least about 5 days. In certain embodiments, the method involves administering multiple doses of a chimeric polypeptide, comprising (i) FVIII protein and (ii) a VWF fragment containing the D' domain and D3 domain of VWF, to a subject at certain dosing intervals, where at least one of the multiple doses is about 65 IU / kg and the dosing 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 dosing interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 8 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 9 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 10 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 11 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 12 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 13 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 14 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 15 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 16 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 17 days.In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 18 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 19 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 20 days. In certain embodiments, at least one of the multiple doses is approximately 65 IU / kg, and the dosing interval is at least approximately 21 days.

[0125] In certain embodiments, this method involves (i) the FVIII protein, and (ii) ) The method comprises administering multiple doses of a chimeric polypeptide comprising a VWF fragment containing the D' domain of VWF and the D3 domain of VWF, for example, rFVIIIFc-VWF-XTEN, to a subject at a certain dosing interval, where at least one of the multiple doses is about 80 IU / kg and the dosing interval is at least about 5 days. In certain embodiments, the method comprises administering multiple doses of a chimeric polypeptide comprising (i) FVIII protein and (ii) a VWF fragment containing the D' domain of VWF and the D3 domain of VWF, to a subject at a certain dosing interval, where at least one of the multiple doses is about 80 IU / kg and the dosing 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 dosing interval is at least about 7 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosing interval is at least about 8 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosing interval is at least about 9 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosing interval is at least about 10 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosing interval is at least about 11 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosing interval is at least about 12 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosing interval is at least about 13 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg, and the dosing interval is at least about 14 days. In certain embodiments, at least one of the multiple doses is approximately 80 IU / kg, and the dosing interval is at least approximately 15 days. In certain embodiments, at least one of the multiple doses is approximately 80 IU / kg, and the dosing interval is at least approximately 16 days. In certain embodiments, at least one of the multiple doses is approximately 80 IU / kg, and the dosing interval is at least approximately 17 days.In certain embodiments, at least one of the multiple doses is about 80 IU / kg and the dosing interval is at least about 18 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg and the dosing interval is at least about 19 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg and the dosing interval is at least about 20 days. In certain embodiments, at least one of the multiple doses is about 80 IU / kg and the dosing interval 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 includes alleviating or reducing the severity of symptoms of hemophilia A on a continuous or substantially continuous basis. In some embodiments, the prophylactic treatment is administered before the onset of symptoms of hemophilia A, for example, before a bleeding event. In other embodiments, the prophylactic treatment is administered on a regular basis, for example, at the dosing intervals disclosed herein, before the onset of symptoms, to prevent the onset of symptoms, or to reduce the severity of symptoms. Any dosing interval disclosed herein may be used in prophylactic treatment of hemophilia A.

[0127] In other embodiments, the chimeric polypeptide is administered, for example, as an on-demand treatment, before an activity that could cause one or more symptoms of hemophilia A. For example, the chimeric polypeptide of this disclosure may be administered to a subject with hemophilia A before the subject undergoes surgery or combat in an activity that otherwise increases the risk of physical trauma and / or bleeding events. When administered on demand, the chimeric polypeptide may 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 at dosing intervals 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 at least two doses, at least three doses, at least four doses, or administering at least five doses of 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 the previous FVIII replacement therapy. 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, for example, a child about 12 years or younger, about 11 years or younger, about 10 years or younger, about 9 years or younger, about 8 years or younger, about 7 years or younger, about 6 years or younger, about 5 years or younger, about 4 years or younger, about 3 years or younger, about 2 years or younger, or about 1 year or younger). In some embodiments, the subject is female. In some embodiments, the subject is male. In some embodiments, the subject is a female about 12 years old or younger. In some embodiments, the subjects are females about 11 years of age or younger. In some embodiments, the subjects are females about 10 years of age or younger.

[0129] The chimeric polypeptides described herein may be administered by any means known in the art. 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, nasal administration, and pulmonary administration. In some embodiments, the chimeric polypeptide is administered intravenously. In other embodiments, the chimeric polypeptide is administered subcutaneously.

[0130] In some embodiments, the administered chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, results in plasma activity levels 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 certain 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 4%. In some embodiments, the plasma activity level of FVIII is at least about 5%. In some embodiments, the plasma activity level of FVIII is at least about 4%. In some embodiments, the plasma activity level of FVIII is at least about 5%. In some embodiments, the plasma activity level of FVIII is at least about 5.6%. In some embodiments, the plasma activity level of FVIII is at least about 7%. In some embodiments, the plasma activity level of FVIII 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, the plasma activity level is expressed as a percentage (%). Alternatively, plasma activity can be expressed in units of 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 at a dose of, for example, 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 at a dose of, for example, 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 at a dose of, for example, 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 at a dose of, for example, 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 at a dose of, for example, 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 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 at a dose of 25 IU / kg.

[0132] This disclosure also includes methods for treating hemophilia A, comprising administering a composition comprising (i) FVIII protein and (ii) VWF fragment to a subject. 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, FVIII protein, e.g., single-stranded FVIII, FVIII-Fc, pegylated FVIII, full-length mature FVIII, or FVIII with a deleted B-domain, is administered at doses 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, and Alternatively, the medication may be administered at intervals of at least 14 days, with the VWF fragment being administered in a ratio of at least approximately 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 the FVIII molecule. In other embodiments, the VWF fragment is administered to the subject simultaneously with the FVIII protein, immediately afterward, or immediately beforeward. In certain embodiments, the VWF fragment is administered approximately 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, or 6 hours before the FVIII protein. In other embodiments, the FVIII protein is administered approximately 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, or 6 hours before the VWF fragment.

[0133] C. Induction of immune tolerance Certain aspects of this disclosure relate to a method for treating hemophilia A in a subject requiring such treatment, comprising administering a chimeric polypeptide comprising FVIII protein and VWF fragments to the subject at a certain dosing interval, wherein the chimeric polypeptide induces immune tolerance to FVIII in the subject. Parts of this disclosure relate to a method for treating hemophilia A in a subject requiring such treatment, comprising administering a chimeric polypeptide comprising FVIII protein and VWF fragments to the subject at a certain dosing interval, wherein the chimeric polypeptide reduces the inhibitory response to FVIII in the subject. Parts of this disclosure relate to a method for treating hemophilia A in a subject requiring such treatment, comprising administering a chimeric polypeptide comprising FVIII protein and VWF fragments to the subject at a certain dosing interval, wherein the chimeric polypeptide does not induce an immune response to the chimeric polypeptide after administration. Another aspect of this disclosure relates to a method for inducing immune tolerance in a person with hemophilia, comprising (1) administering to the person an effective amount of the chimeric polypeptide described herein, for example, rFVIIIFc-VWF-XTEN, wherein the effective amount of the chimeric polypeptide induces immune tolerance in the person. Various methods for inducing immune tolerance using a chimeric polypeptide comprising FVIII and Fc are disclosed in International Publication No. 2018 / 102760A1, 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 a 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 being 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 a human after the tapering regimen.

[0135] In certain embodiments, a person has not been treated with prior immunotolerance therapy for a coagulation factor, e.g., FVIII. The chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, can be administered to a person at any time it is determined that the person has developed an inhibitory immune response, e.g., after measuring the level of inhibitory immune response in the person. In other embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, can be administered to a person who has not yet developed an immune response to one or more inhibitors in order to prevent the development of an inhibitory immune response. In some embodiments, the chimeric polypeptide, e.g., rFVIIIFc-VWF-XTEN, is administered to a person who has a high probability of developing an inhibitory immune response (e.g., exhibiting a family history, genetic predisposition, or biomarker). In some embodiments, the method further includes measuring the level of inhibitory immune response or the probability of developing an inhibitory immune response prior to administration.

[0136] In some embodiments, a chimeric polypeptide, for example, rFVIIIFc-VWF-XTEN, is administered to a human after, for example, measuring the level of inhibitory immune response or the likelihood of developing an inhibitory immune response in a human, and after it is determined that the human has developed an inhibitory immune response or is likely to develop an inhibitory immune response, within approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years. In certain embodiments, a chimeric polypeptide, such as rFVIIIFc-VWF-XTEN, is administered to a human, for example, immediately after it is determined that the human has developed an inhibitory immune response or is likely to develop one, following a measurement of the level of inhibitory immune response or the likelihood of developing an inhibitory immune response in the human. In certain embodiments, a chimeric polypeptide, for example, rFVIIIFc-VWF-XTEN, is administered to a human after, for example, measuring the level of inhibitory immune response or the likelihood of developing an inhibitory immune response in a human, and after it is determined that the human has developed an inhibitory immune response or is likely to develop an inhibitory immune response, in less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 20 minutes, less than 30 minutes, less than 45 minutes, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, less than 5 hours, less than 6 hours, less than 7 hours, less than 8 hours, less than 9 hours, less than 10 hours, less than 11 hours, less than 12 hours, less than 18 hours, or less than 24 hours. In certain embodiments, a chimeric polypeptide, for example, rFVIIIFc-VWF-XTEN, is administered to a human at intervals of approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, or 24 hours, after measuring the level of inhibitory immune response or the likelihood of developing an inhibitory immune response in a human, and after determining that the human has developed an inhibitory immune response or is likely to develop one.In certain embodiments, a chimeric polypeptide, for example, rFVIIIFc-VWF-XTEN, is administered to a human less than one day after it is determined that the human has developed an inhibitory immune response or is likely to develop one, after measuring the level of inhibitory immune response or the likelihood of developing an inhibitory immune response in the human. It can be done.

[0137] The induction of an immune response can continue until the level of the inhibitor falls below a certain level or until the inhibitor becomes undetectable. In certain embodiments, the induction period may continue for 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 method of the present invention may include any response in humans that negatively affects one or more effects of treatment of coagulation factors. In some embodiments, the inhibitory immune response includes the production of inhibitory antibodies against coagulation factors, e.g., inhibitory anti-FVIII antibodies. In certain embodiments, the method of the present disclosure further includes measuring the titer of one or more inhibitory antibodies in humans before (e.g., at baseline) or after administration of an effective amount of the chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN or the polynucleotide encoding it. In some embodiments, the titer of the inhibitory antibody before administration (e.g., at baseline) is at least about 0.6 bethesda units (BU). In a particular embodiment, the titer of the inhibitory antibody before administration (e.g., 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 a particular embodiment, the titer of the inhibitory antibody before administration (e.g., baseline) is at least about 5 BU.

[0139] In some embodiments, the method of the present invention reduces the titer of the 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 particular 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 the chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN, increases the differentiation of macrophages in humans to an M2-like phenotype compared to macrophage differentiation in untreated controls and in humans treated with coagulation factors alone. In some embodiments, the M2-like phenotype includes upregulation of the NRF2 pathway, the PPAR-gamma pathway, or both the NRF2 and PPAR-gamma pathways. In some embodiments, the M2-like phenotype includes upregulation of CD206(MRC1). In some embodiments, the M2-like phenotype includes upregulation of ARG1. In some embodiments, the M2-like phenotype includes upregulation of both CD206(MRC1) and ARG1.

[0141] In some embodiments, administration of the chimeric polypeptide described herein, e.g., rFVIIIFc-VWF-XTEN, affects the expression of one or more genes in untreated subjects or subjects treated with coagulation factors alone, compared to the expression of one or more genes in humans. 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 modified subunit (GCLM), and NAD(P)H quinone dehydrogenase. In some embodiments, administration results in higher expression of one or more genes selected from the group consisting of Nase 1 (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 C-1 type (MRC1), solute transporter family 12 member 4 (SLC12A), neuropilin 1 (NRP1), and any combination thereof. In some embodiments, administration results in higher expression of one or more genes in the NRF2 pathway. In certain embodiments, 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 higher expression of one or more genes in the PPAR gamma pathway. In some embodiments, one or more genes of the PPAR-gamma pathway are selected from the group consisting of PPAR-gamma, LPL, FABP5, EGR2, and any combination thereof. In some embodiments, 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 compared to the expression of one or more genes in untreated humans or humans administered with a coagulation factor alone, with expression being at least about 1.5 times higher, at least about 2 times higher, at least about 2.5 times higher, at least about 3 times higher, at least about 3.5 times higher, at least about 4 times higher, at least about 4.5 times higher, or at least about 5 times higher.

[0142] In some embodiments, differential expression of one or more genes is observed less than 6 hours after administration of the chimeric polypeptide described herein, e.g., 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 includes a cell-mediated immune response. In certain embodiments, the cell-mediated immune response includes the release of cytokines. In some embodiments, the cytokines are any cytokines associated with an increase in the immune response. In some embodiments, the cytokines are selected from the group consisting of IL-1, IL-6, IL-16, IL-12, IL-4, IL-17, tumor necrosis factor α (TNF-α), interferon α, interferon γ, and any combination thereof. In one embodiment, the cell-mediated immune response includes an increase in serum levels of IL-12. In another embodiment, the cell-mediated immune response includes an increase in serum levels of IL-4. In yet another embodiment, the cell-mediated immune response includes an increase in serum levels of IL-17. In yet another embodiment, the cell-mediated immune response includes an increase in serum levels of TNF-α.

[0144] Mutations in various genes are associated with an increased risk of developing inhibitory immune responses. For example, the TNF-α-308G>A polymorphism in Hap2, associated with increased constitutive and inductive transcription levels of TNF, is associated with an increased risk of developing inhibitory immune responses. See Astermark et al., Blood 108: 3739-3745 (2006), which is incorporated herein by reference in its entirety. For this reason, in some embodiments, humans have a genetic polymorphism associated with increased TNF-α. In some embodiments, the polymorphism is the TNF-α-308G>A polymorphism. In some embodiments, humans It has polymorphisms in the IL10 gene, such as polymorphisms associated with increased IL10 secretion. In some embodiments, FVIII-Fc is administered to subjects 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 herein by reference in its entirety.

[0145] In some embodiments, humans have a genetic polymorphism associated with reduced expression of CTLA-4 (cytotoxic T lymphocyte antigen 4). In some embodiments, humans have mutations in the 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 method of the Disclosure reduces the level of one or more cytokines in a subject compared to the level of one or more cytokines in the subject after prior treatment with a polypeptide comprising FVIII protein. In other embodiments, the method of the Disclosure 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 immunotolerogenic molecules increases after administration of the method of the Disclosure compared to the expression level of one or more immunotolerogenic molecules before administration. In certain embodiments, one or more immunotolerogenic molecules are selected from IL-10, TGF-β, IL-35, IDO-1, and any combination thereof.

[0147] In other embodiments, the immune response includes clinical symptoms selected from the group consisting of increased bleeding tendency, high consumption of coagulation factors, lack of response to coagulation factor therapy, decreased effectiveness of coagulation factor therapy, shortening of the half-life of coagulation factors, and any combination thereof. In certain embodiments, the immune response includes clinical symptoms selected from the group consisting of increased bleeding tendency, high consumption of coagulation factors, lack of response to coagulation factor therapy, decreased effectiveness of coagulation factor therapy, decreased recovery of the activity of coagulation factors monitored in plasma, shortening of the half-life of coagulation factors, and any combination thereof.

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

[0149] In some embodiments, humans take at least approximately one month, at least approximately two months, at least approximately three months, at least approximately four months, at least approximately five months, at least approximately six months, at least approximately seven months, at least approximately eight months, at least approximately nine months, and at least Both individuals were previously diagnosed with an inhibitory immune response to coagulation factors approximately 10 months ago, at least 11 months ago, at least 12 months ago, at least 13 months ago, at least 14 months ago, at least 15 months ago, at least 16 months ago, at least 17 months ago, at least 18 months ago, at least 19 months ago, at least 20 months ago, at least 21 months ago, at least 22 months ago, at least 23 months ago, at least 24 months ago, at least 27 months ago, at least 30 months ago, at least 33 months ago, at least 36 months ago, at least 39 months ago, at least 42 months ago, at least 45 months ago, at least 48 years ago, at least 51 months ago, at least 54 months ago, at least 57 months ago, at least 60 months ago, at least 6 years ago, at least 7 years ago, at least 8 years ago, at least 10 years ago, at least 15 years ago, or at least 20 years ago. In one embodiment, the human was previously diagnosed with an inhibitory immune response to coagulation factors at least about five years prior to administration.

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

[0151] In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against coagulation factors of less than approximately 0.6 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against coagulation factors of less than approximately 0.5 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against coagulation factors of less than approximately 0.4 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against coagulation factors of less than approximately 0.3 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against coagulation factors of less than approximately 0.2 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against coagulation factors of less than approximately 0.1 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against coagulation factors of 0.0 BU. In certain embodiments, the titer of the inhibitory immune antibody is observed in two consecutive measurements, for example, in two consecutive weeks within a period of four weeks.

[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%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%). As used herein, “incremental recovery” refers to the peak FVIII level 15–30 minutes after infusion.

[0153] Following the completion of the induction and tapering periods, subjects may subsequently receive prophylactic treatment with the chimeric protein. The prophylactic drug regimen may be any of the drug regimens disclosed herein.

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

[0155] In one particular embodiment, the human had less than 150 exposure days (ED) to a coagulation factor, e.g., FVIII. In another embodiment, the human had less than 50 ED. In yet another embodiment, the human had less than 20 ED.

[0156] Some aspects of this disclosure relate to a method for reducing the severity or occurrence of allergic or anaphylactic reactions to coagulation factors in subjects requiring such reduction, the method comprising administering a composition or chimeric protein comprising a coagulation factor and an Fc region to the subject. In some embodiments, the administration of the composition or chimeric protein reduces the severity of anaphylactic-like reactions to coagulation factors. In some embodiments, the administration of the composition or chimeric protein reduces the severity of allergic reactions to coagulation factors.

[0157] III. Chimera polypeptide A chimeric polypeptide useful for this disclosure comprises an FVIII protein containing the FVIII polypeptide, and a VWF fragment containing 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 allowing the FVIII protein to bypass VWF clearance. To prevent removal through the pathway, in some embodiments, the FVIII protein and the VWF fragment are linked to each other directly or indirectly by a stronger bond than the innate interaction between FVIII and VWF, in order to prevent the dissociation of the FVIII protein from the VWF fragment.

[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 another embodiment, 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, an FVIII protein containing the FVIII polypeptide, and a VWF fragment containing 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 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 that is directly or indirectly fused with the D' and D3 domains of VWF. In other embodiments, the FVIII protein and 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 that is directly or indirectly fused with the FVIII polypeptide. In yet another embodiment, the FVIII protein and the VWF fragment are indirectly linked by at least one covalent bond between at least one amino acid in a heterologous sequence directly or indirectly fused with the D' domain and D3 domain of the VWF, and at least one amino acid in a heterologous sequence directly or indirectly fused with the FVIII polypeptide.

[0160] The FVIII protein of this disclosure may include an FVIII polypeptide and one or more heterologous moieties, such as half-life extension moieties, directly or indirectly fused with the FVIII polypeptide. The VWF fragment of this disclosure may also include the D' and D3 domains of the VWF, and one or more heterologous moieties, such as half-life extension moieties, directly or indirectly fused with the D' and D3 domains of the VWF. In some embodiments, the FVIII protein useful for this disclosure is essentially or consists of an FVIII polypeptide, and the VWF fragment useful for this disclosure includes the D' and D3 domains of the VWF, and one or more heterologous moieties, such as half-life extension moieties, directly or indirectly fused with the D' and D3 domains of the VWF. In some embodiments, the FVIII protein comprises an FVIII polypeptide and one or more heterologous moieties, such as half-life extension moieties, directly or indirectly fused with the FVIII polypeptide, and the VWF fragment useful for this disclosure consists essentially of the D' and D3 domains of the VWF or comprises the D' and D3 domains of the VWF. In some embodiments, the chimeric polypeptide comprises an FVIII protein consisting essentially of or comprising the FVIII polypeptide, and a VWF fragment consisting essentially of or comprising the D' and D3 domains of the VWF.

[0161] In some embodiments, the chimeric polypeptide or protein disclosed herein is FV This is a III-XTEN-Fc / D'D3-XTEN-Fc heterodimer. In one embodiment, the FVIII-XTEN-Fc / D'D3-XTEN-Fc heterodimer chimeric polypeptide comprises (i) an FVIII protein containing an FVIII polypeptide, XTEN inserted into the B domain of the FVIII polypeptide, and a first Fc region, and (ii) a VWF protein containing 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 shown in Figure 1.

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

[0163] III.A. Factor VIII Polypeptide As used herein, “Factor VIII,” abbreviated throughout this application as “FVIII,” means the functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. For this purpose, the term FVIII includes functional variant polypeptides. “FVIII protein” is used to refer to the FVIII polypeptide (or protein) alone, the FVIII polypeptide fused with an additional polypeptide, and the FVIII polypeptide bound to one or more additional polypeptides, insofar as the FVIII protein exhibits the function / activity of FVIII. The terms “FVIII polypeptide,” “FVIII portion,” and “FVIII” refer to the FVIII polypeptide sequence alone. Examples of FVIII function / activity, but not limited to, include the ability to activate coagulation, the ability to function as a cofactor for factor IX, or Ca 2+This includes the ability to form a tenase complex with factor IX in the presence of phospholipids, and subsequently convert factor X to its active form, Xa. FVIII polypeptides can be human, porcine, canine, rat, or mouse. 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); US6,251,632). Full-length polypeptide and polynucleotide sequences are known as well as many functional fragments, variants, and modified versions. Sequences of various FVIII amino acids and nucleotides are disclosed, for example, in U.S. Patent Application Publication 2015 / 0158929A1, U.S. Patent Application Publication 2014 / 0308280A1, and U.S. Patent Application Publication 2014 / 0370035A1, and International Publication 2015 / 106052A1. Examples of FVIII polypeptides include full-length FVIII, full-length FVIII without a Met at the N-terminus, mature FVIII polypeptide (without a signal sequence), mature FVIII polypeptide with an additional Met at the N-terminus, and / or FVIII polypeptides with a complete or partial deletion of the B-domain. FVIII variants include B-domain deletions, whether partial or complete.

[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 assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often by the Krauss method), platelet counting method, platelet function test (often by PFA-100), TCT, bleeding time, mixed test (when the patient's plasma is normal) (whether the abnormality is corrected when mixed with plasma), coagulation factor assays, antiphospholipid antibodies, D-dimers, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), diluted 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 called the contact activation pathway) and common coagulation pathways. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors, such as FVIII. It is typically used in conjunction with prothrombin time (PT), which measures the exogenous pathway (see, e.g., Kamal et al., Mayo Clin Proc., 82(7):864-873 (2007)). In one embodiment, the aPTT is tested using an assay in which FVIII activity is measured using Dade®Actin®FSL-activated PTT reagent (Siemens Health Care Diagnostics) in a BCS®XP analyzer (Siemens Healthcare Diagnostics). To a certain extent, the chimeric polypeptide has 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 the aPTT test.

[0166] The aPTT assay may also be used to evaluate the potency 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 further be used in conjunction with any of the assays described herein, either before or after administration to a patient or subject.

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

[0168] The mechanism of the colorimetric 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. The activity of factor Xa is assessed by the hydrolysis of a factor Xa-specific p-nitroanilide (pNA) substrate. The initial rate of p-nitroaniline release, 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 colorimetric assay is the BIOPHEN FVIII:C assay (Hyphen Biomed, Neurville sur Oise, France).

[0169] The chromogenic assay is recommended by the FVIII and Factor IX Subcommittee of the Scientific Standardization Committee (SSC) of the International Society on Thrombosis and Hemostasis (ISTH). Since 1994, the chromogenic assay has also been the European Pharmacopoeia reference method for determining the potency of FVIII concentrations. For this reason, in some embodiments, a chimeric polypeptide containing an FVIII polypeptide is used instead of a mature FVIII polypeptide or a BDD FVIII polypeptide (e.g., REC). It possesses FVIII activity comparable to chimeric polypeptides containing OMBINATE(registered trademark), KOGENATE FS(registered trademark), HELIXATE FS(registered trademark), XYNTHA / REFACTO AB(registered trademark), HEMOFIL-M(registered trademark), MONARC-M(registered trademark), MONOCLATE-P(registered trademark), HUMATE-P(registered trademark), ALPHANATE(registered trademark), KOATE-DVI(registered trademark), AFSTYLA(registered trademark), and HYATE:C(registered trademark).

[0170] Chromogenic assays may also be used to evaluate the potency of chimeric polypeptides prior to administration to patients or subjects (Hubbard AR, et al. J Thromb Haemost 11: 988-9 (2013)). Chromogenic assays may further be used in conjunction with any of the assays described herein, either before or after administration to patients or subjects.

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

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

[0173] In addition, FVIII binds to von Willebrand factor but is inactive during circulation. If FVIII is not bound to VWF, it is rapidly degraded and released from VWF by the action of thrombin. In some embodiments, chimeric polypeptides containing the FVIII polypeptide bind to von Willebrand factor, e.g., VWF fragments disclosed herein, at levels comparable to chimeric polypeptides or BDD FVIII polypeptides containing a mature FVIII polypeptide sequence (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 behind arginine 336 in the A1 domain, disrupting the interaction site with the factor X substrate, and cleaves behind arginine 562 in the A2 domain, enhancing the dissociation of the A2 domain and similarly disrupting the interaction site with factor IXa. This cleavage also bifurcates the A2 domain (43 kDa), producing 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 containing the FVIII polypeptide are inactivated by activated protein C to a level comparable to chimeric polypeptides or BDD FVIII polypeptides containing a mature FVIII polypeptide sequence (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0175] In other embodiments, the chimeric polypeptide containing the FVIII polypeptide is a chimeric polypeptide containing a mature FVIII polypeptide sequence or a BDD FVIII polypeptide (e.g., ADVATE®, REFACTO®, or ELOCT). It has in vivo FVIII activity comparable to that of ATE®. In certain embodiments, chimeric polypeptides containing FVIII polypeptides have the ability to protect HemA mice in a HemA mouse tail vein transection model at a level comparable to chimeric polypeptides or BDD FVIII polypeptides containing mature FVIII polypeptide sequences (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] Examples of FVIII polypeptides include full-length FVIII, full-length FVIII without N-terminus Met, mature FVIII (without signal sequence), mature FVIII with additional N-terminus Met, and / or FVIII with total or partial deletion of the B domain. In certain embodiments, the FVIII variant includes a B domain deletion, whether partial or total.

[0178] The sequence of the native mature human FVIII polypeptide is shown as Sequence ID No. 65. The native FVIII polypeptide has the following formula: A1-a1-A2-a2-B-a3-A3-C1-C2 (wherein A1, A2 and A3 are structurally related "A domains", B is a "B domain", C1 and C2 are structurally related "C domains", and a1, a2 and a3 are acidic spacer regions). Referring to the primary amino acid sequence positions in Sequence ID No. 65, the A1 domain of human FVIII extends from Ala1 to approximately Arg336, the a1 spacer region extends from approximately Met337 to approximately Val374, the A2 domain extends from approximately Ala375 to approximately Tyr719, the a2 spacer region extends from approximately Glu720 to approximately Arg740, the B domain extends from approximately Ser741 to approximately Arg1648, the a3 spacer region extends from approximately Glu1649 to approximately Arg1689, the A3 domain extends from approximately Ser1690 to approximately Leu2025, the C1 domain extends from approximately Gly2026 to approximately Asn2072, and the C2 domain extends from approximately Ser2073 to Tyr2332. Aside from specific proteolytic cleavage sites, the designation of the boundaries between domains and regions of FVIII may differ in different literature references. Therefore, the boundaries described herein are approximate, indicated by the use of the term "approximately."

[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); International Publication No. 87 / 04187; International Publication No. 88 / 08035; International Publication No. 88 / 03558; and U.S. Patent No. 4,757,006). The FVIII amino acid sequence was determined from cDNA as shown in U.S. Patent No. 4,965,199. In addition, FVIII with partial or complete deletion of the B domain is shown in U.S. Patents 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 5,004,803. The cDNA and amino acid sequences encoding human factor VIII are shown in Sequence IDs 1 and 2 of U.S. Patent Application Publication 2005 / 0100990, respectively.

[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 from 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 FVIII with substitutions of all domains, all subunits and specific amino acid sequences was disclosed by Lollar and Runge in U.S. Patent No. 5,364,771 and WO93 / 20093. More recently, the nucleotides and corresponding amino acid sequences of the A1 and A2 domains of porcine FVIII, as well as chimeric FVIII using the porcine A1 and / or A2 domains instead of the corresponding human domain, were reported in WO94 / 11503. U.S. Patent No. 5,859,204, by Lollar, JS, also discloses porcine cDNA, and its amino acid sequence has been estimated. U.S. Patent No. 6,458,563 discloses B-domain deletion porcine FVIII.

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

[0182] In some embodiments, the FVIII polypeptide (or the FVIII portion of a chimeric polypeptide) may 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 1-1438 of SEQ ID NO: 67, or amino acids 1-2332 (without a signal sequence) of SEQ ID NO: 65, or amino acids 1-19 of SEQ ID NO: 64 and amino acids 1-1438 of SEQ ID NO: 67, or amino acids 1-19 of SEQ ID NO: 64 and amino acids 1-2332 (with a signal sequence) of SEQ ID NO: 65, where FVIII has coagulation activity and, for example, activates factor IX as a cofactor to convert factor X into activated factor X. The FVIII polypeptide (or the FVIII portion of a chimeric polypeptide) may be identical to the FVIII amino acid sequence of amino acids 1-1438 of SEQ ID NO: 67, or amino acids 1-2332 (without a signal sequence) of SEQ ID NO: 65. The FVIII polypeptide may further contain a signal sequence.

[0183] The “B domain” of FVIII, as used herein, is the same as the B domain known in the art, defined by the identity of its internal amino acid sequence and the site of proteolytic cleavage, for example, residues Ser741–Arg1648 in full-length human FVIII. Other human FVIII domains are defined by the following amino acid residues: A1, residues Ala1–Arg372; A2, residues Ser373–Arg740; A3, residues Ser1690–Asn2019; C1, residues Lys2020–Asn2172; C2, residues Ser2173–Tyr2332. The A3-C1-C2 sequence includes residues Ser1690–Tyr2332. The remaining sequence, residues Glu1649–Arg1689, is commonly referred to as the a3 acidic region. The locations of the boundaries of all domains, including the B domain, for pig, 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 portion of factor VIII in Table 4 (the BDD FVIII heavy chain is double-underlined, the B domain is italicized, and the BDD FVIII light chain is in standard letters). In some embodiments, the B-domain FVIII has a deletion of the entire B domain except for 5 amino acids, as shown in Table 5 (SEQ ID NO: 68) (the B domain is italicized). [Table 4] [Table 5]

[0184] "B-domain deletion FVIII" refers to U.S. patents 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. The B-domain deletion FVIII sequence may have a total or partial deletion as disclosed in U.S. Patent No. 5,112,950, U.S. Patent No. 4,868,112, and U.S. Patent No. 6,458,563, U.S. Patent Publication No. 2017 / 0073393A1 and U.S. Patent Publication No. 2012 / 308641A1, and International Publication No. 2011 / 041770A1, International Publication No. 2015 / 106052A1 (PCT / US2015 / 010738) and International Publication No. 2016 / 025764. In some embodiments, the B-domain deletion FVIII sequence used in the method of the present disclosure includes any one of the deletions disclosed in U.S. Patent No. 6,316,226 (also U.S. 6,346,513), column 4, line 4 to column 5, line 28 and Examples 1 to 5. In other embodiments, the B-domain deletion factor VIII is S743 / Q1638 B-domain deletion factor VIII (SQ BDD FVIII) (e.g., factor VIII having a deletion between amino acids 744 and 1637, e.g., factor VIII having amino acids 1 to 743 and amino acids 1638 to 2332 of mature FVIII). In some embodiments, the B-domain deletion FVIII used in the methods of this disclosure has the deletion disclosed in column 2, lines 26 to 51 and Examples 5 to 8 of U.S. Patent No. 5,789,203 (also U.S. 6,060,447, U.S. 5,595,886, and U.S. 6,228,620).In some embodiments, B-domain deletion factor VIII is located in the first column, lines 25-40 of U.S. Patent No. 5,972,885; the sixth column, lines 1-22 and Example 1 of U.S. Patent No. 6,048,720; the second column, lines 17-46 of U.S. Patent No. 5,543,502; the fourth column, lines 22-5 and line 36 of U.S. Patent No. 5,171,844; and the second column, lines 55-68 of U.S. Patent No. 5,112,950. See the eye, Figure 2 and Example 1; columns 2, lines 2-19, line 21 and Table 2 of U.S. Patent 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, 5-13, line 39 of U.S. Patent No. 7,041,635; or have the deletion described in column 4, lines 25-53 of U.S. Patent No. 6,458,563. In some embodiments, the B-domain deletion FVIII polypeptide has a deletion of most of the B-domain, as disclosed in WO91 / 09122, but still contains the amino-terminal sequence of the B-domain which is essential for the in vivo proteolytic processing of the primary translation product into two polypeptide chains. In some embodiments, the B-domain deletion FVIII polypeptide is constructed by the deletion of amino acids 747–1638, i.e., a virtually complete deletion of the B-domain. Hoeben RC, et al. J. Biol. Chem. 265 (13): 7318-7323 (1990). The B-domain deletion factor VIII polypeptide may also contain deletions of amino acids 771–1666 or 868–1562 of FVIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988). Additional B-domain deletions that are part of this disclosure are, for example, amino acids 982 to 1562 or 760 to 1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, This includes deletions of 5939-5942), 797-1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347), 741-1646 (Kaufman (PCT application publication WO87 / 04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564), 741-1648 (Pasek (PCT application publication 88 / 00831)), or 816-1598 or 741-1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP295597)). In certain embodiments, the B-domain deletion FVIII polypeptide contains deletions of amino acid residues 746-1648 of mature FVIII (corresponding to deletions of 765-1665 in full-length FVIII). In other embodiments, the B-domain deletion FVIII polypeptide contains deletions of amino acid residues 745-1648 of mature FVIII (corresponding to deletions of 764-1665 in full-length FVIII).

[0185] In other embodiments, BDD FVIII comprises an FVIII polypeptide containing a fragment of the B domain that holds one or more N-linked glycosylation sites corresponding to the amino acid sequence of the full-length FVIII sequence, for example, residues 757, 784, 828, 900, 963, or optionally 943. Examples of B domain fragments are given in Miao, HZ, et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6: In yet another embodiment, BDD FVIII comprises 226 or 163 amino acids of the B domain, as disclosed in 1352-1359 (2008) and Pipe, SW, et al., J. Thromb. Haemost. 9: 2235-2242 (2011) (i.e., the first 226 or 163 amino acids of the B domain are retained). In yet another embodiment, BDD FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve the expression of the BDD FVIII polypeptide. See Miao, HZ, et al., Blood 103(a): 3412-3419 (2004). In yet another embodiment, BDD FVIII comprises an FVIII polypeptide that contains a portion of the B domain but does not contain one or more furin cleavage sites (e.g., Arg1313 and Arg1648). Pipe, SW, See et al., J. Thromb. Haemost. 9: 2235-2242 (2011). In some embodiments, BDD FVIII includes single-chain FVIII containing deletions 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 aforementioned deletions can be made in any FVIII sequence.

[0186] In some embodiments, FVIII 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 a single-chain FVIIIFc molecule-226N6. The number 226 represents the 226th amino acid at the N-terminus 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 International Publication No. 2017 / 117630A1, International Publication No. 2018 / 087271A1, US9,878,017B2, US8,575,104B2, US8,754,194B2, US7,939,632B2, US2018 / 0161402A1, US9,956,269B2, US9,107,902B2, and US2017 / 209546A1.

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

[0189] In other embodiments, FVIII is arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 65), arginine at amino acid 754 (in S743 / Q1638 B-domain deletion FVIII or SEQ ID NO: 67), or an uncleaved single-stranded FVIII immediately following the corresponding arginine residue (in other variants). The single-stranded FVIII may 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 may be any amino acid other than arginine, such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolicin, or taurine.

[0190] FVIII can be further cleaved by thrombin and subsequently activated as FVIIIa, which can act as a cofactor for activated factor IX (FIXa). Activated FIXa then forms a Xase complex with activated FVIII, converting factor X to activated factor X (FXa). For activation, FVIII is cleaved by thrombin behind three arginine residues at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain deletion FVIII sequence). The cleavage yields FVIIIa having 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 this disclosure is inactive FVIII. In other embodiments, the FVIII polypeptide is activated FVIII.

[0191] Proteins having an FVIII polypeptide linked to or associated with a VWF protein may contain a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 65 or 67, where the sequence has the coagulation activity of FVIII, for example, activating factor IX as a cofactor to convert factor X to activated factor X (FXa).

[0192] When used herein, "hybrid" or "chimeric" polypeptides and proteins include a combination of a first polypeptide and a second polypeptide. In some embodiments, the hybrid or chimeric polypeptide includes a chimeric polypeptide comprising a single polypeptide chain, for example, FVIII polypeptide and XTEN. See US2015 / 0158929A1, which is incorporated herein by reference in its entirety. In some embodiments, the hybrid or chimeric polypeptide comprises a combination of a first polypeptide chain, e.g., a VWF fragment fused with an XTEN sequence and a first Ig constant region or portion thereof, and a second polypeptide chain, e.g., an FVIII polypeptide fused with a second Ig constant region or portion thereof, thereby forming a heterodimer. See, for example, US2015 / 0266943A1, US2016 / 0251408A1, and US2017 / 0073393A1, which are incorporated herein by reference in their entirety. In some embodiments, the first and second polypeptides in the hybrid are linked to each other via 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 an FVIII-Fc fusion protein to form a heterodimer hybrid, where XTEN contains fewer than 288 amino acids. The first polypeptide and the second polypeptide can be linked together through a covalent bond between the first Fc region and the second Fc region, for example, a disulfide bond. The first polypeptide and the second polypeptide can further be linked together through a bond between the VWF fragment and the FVIII polypeptide.

[0193] In certain embodiments, the chimeric polypeptide disclosed herein comprises an 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 a first FVIII polypeptide fragment fused with the XTEN sequence, then with the second FVIII polypeptide fragment, and finally with the Fc region, in the order of N-terminus to C-terminus. In detailed 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 detailed embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN, which comprises an FVIII protein containing the amino acid sequence of SEQ ID NO: 201. In another detailed embodiment, rFVIIIFc-VWF-XTEN contains the amino acid sequence of SEQ ID NO: 207.

[0195] In some embodiments, the FVIII protein further comprises an FVIII signal peptide sequence. In one detailed embodiment, the FVIII protein comprises an 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 detailed embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN and comprises an FVIII protein having the amino acid sequence of SEQ ID NO: 203. In another detailed embodiment, rFVIIIFc-VWF-XTEN comprises an FVIII protein encoded by the nucleic acid sequence or fragment thereof of SEQ ID NO: 204. Additional exemplary polypeptide sequences relating to the FVIII protein of the chimeric polypeptides disclosed herein are shown in Tables 18-19.

[0197] As discussed above and below, a great many 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 attributable to their location within the three-dimensional structure of FVIII than to the nature of the substitutions (Cutler et al., Hum. Mutat. 19:274-8 (2002)), which is incorporated herein by reference in whole. 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); US6,251,632), which is also incorporated herein by reference in whole.

[0198] III.B. Von Willebrand Factor (VWF) Fragments VWF (also known as F8VWF) is a large, multimeric glycoprotein present in blood plasma, constitutively generated in the endothelium (in Viber-Parade bodies), megakaryocytes (in platelet α-granules), and subendothelial connective tissue. The basic VWF monomer is a 2813-amino acid protein. All monomers contain numerous specific domains with particular 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 (when activated, the RGD domain binds to platelet integrin αIIbβ3), and the "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] As used herein, the term “VWF fragment” includes, but is not limited to, functional VWF fragments containing D' and D3 domains 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 between the FVIII protein and 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. Examples of VWF fragments include derivatives, variants, mutants, or analogs that retain these activities of VWF.

[0200] The 2813 monomeric amino acid sequence for human VWF is reported under Genbank accession number NP000543.2. The nucleotide sequence encoding human VWF is reported under Genbank 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. The respective domains of VWF are 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, where 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 include a VWF domain selected from the group consisting of the A1 domain, A2 domain, A3 domain, D1 domain, D2 domain, D4 domain, B1 domain, B2 domain, B3 domain, C1 domain, C2 domain, CK domain, one or more fragments thereof, and any combination thereof. In some embodiments, the VWF fragment may be (1) the D' and D3 domains of VWF, or fragments thereof, or (2) the D1 domain, D' domain, and D3 domain of VWF (3) the D2 domain, D' domain and D3 domain of VWF, or fragments thereof, (4) the D1 domain, D2 domain, D' domain and D3 domain of VWF, or fragments thereof, or (5) the D1 domain, D2 domain, D' domain, D3 domain and A1 domain of VWF, or fragments thereof, comprising, essentially consisting of, or consisting of. The VWF fragments described herein do not contain a site that binds to the 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 this disclosure may include any other sequences linked to or fused to the VWF fragment. For example, the VWF fragments described herein may further include a signal peptide.

[0202] In some embodiments, VWF fragments containing the D' domain and the D3 domain bind to or conjugate with the FVIII protein. For example, each of these is incorporated herein by reference in its entirety in US2015 / 0023959A1, US2015 / 0266943A1, US2016 / 0251408A1, US2017 / 0073393A1, US2018 / 185455A1, US2018 / 0051067A1, US2017 / 0152300A1, US9, See 878,017B2, US9,458,223B2, US8,575,104B2, WO2017 / 117630A1, US2018 / 0161402A1, WO2017 / 117631A1, WO2018 / 087271A1, US9,107,902B2, WO2017 / 222337A1, and WO2015 / 185758A1. By binding to or associating with the FVIII protein, the VWF fragments of this 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 conjugates with the FVIII protein, blocking or preventing the FVIII protein from binding to phospholipids and activated protein C. By preventing or inhibiting the binding of the FVIII protein to endogenous full-length VWF, the VWF fragments of this disclosure reduce the clearance of FVIII by the VWF clearance receptor, thus extending the half-life of the chimeric polypeptide. Therefore, the extension of the half-life of the chimeric polypeptide is due to the binding or conjugation with the VWF fragment, which lacks the VWF clearance receptor binding site to the FVIII protein, and the shielding or protection of the FVIII protein by the VWF fragment from endogenous VWF containing the VWF clearance receptor binding site. The FVIII protein bound to or protected by the VWF fragment may also allow for the reuse of the FVIII protein.By removing the binding site of the VWF clearance pathway receptor contained in the full-length VWF molecule, the FVIII / VWF heterodimer of this disclosure is shielded from the VWF clearance pathway and further extends the half-life of FVIII.

[0203] In some embodiments, the VWF protein useful for this 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 the binding of endogenous VWF to FVIII. In other embodiments, the 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 the binding of endogenous VWF to FVIII. In some embodiments, the VWF protein described herein comprises, essentially consists of, or consists of the D' and D3 domains of VWF, which are amino acids 764-1240 of SEQ ID NO: 21 and at least 60%, 70%, 80%, 85%, 90%, 95%, and 96%. In other embodiments, the VWF protein is 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23-1240 of SEQ ID NO: 21, or is essentially composed of or composed of D1, D2, D', and D3 domains, which are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these, where the VWF protein prevents or inhibits the binding of endogenous VWF to FVIII. In yet another embodiment, the VWF protein further comprises signal peptides operably linked thereto.

[0204] In some embodiments, VWF proteins useful for this disclosure include (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 to 764-1250 of SEQ ID NO: 21), up to about 15 amino acids (e.g., any sequence from amino acids 764-1240 to 764-1255 of SEQ ID NO: 21), and about 20 amino acids. The VWF protein consists essentially of, or comprises, additional VWF sequences up to acid (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 approximately 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 approximately 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 containing, or essentially consisting 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 domain is expressed in trans with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in cis with the D'D3 domain.

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

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

[0207] In further embodiments, the VWF protein of this disclosure comprises, essentially consists of, or consists of amino acid sequences corresponding to the D' domain, D3 domain, and A1 domain, wherein the amino acid sequences are 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 the binding of endogenous VWF 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 this disclosure includes a D' domain and a D3 domain, but also includes (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, and (8) a C1 domain. Domains, (9) C2 domains, (10) CK domains, (11) CK domains and C2 domains, (12) CK domains, C2 domains and C1 domains, (13) CK domains, C2 domains, C1 domains and B3 domains, (14) CK domains, C2 domains, C1 domains, B3 domains and B2 domains, (15) CK domains, C2 domains, C1 domains, B3 domains, B2 domains and B1 domains, (16) CK domains, C2 domains, C1 domains, B3 domains, B2 domains, B1 domains and D4 domains, (17) C (18) Not including K domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, and A3 domain, (19) Not including CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, A3 domain, and A2 domain, and (20) Not including at least one VWF domain selected from the group consisting of CK domain, C2 domain, C1 domain, B3 domain, B2 domain, B1 domain, D4 domain, A3 domain, A2 domain, and A1 domain, and any combination thereof.

[0209] In further 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, those disclosed in Zhour et al., Blood published online April 6, 2012: DOI 10.1182 / blood-2012-01-405134, which are incorporated herein by reference in their entirety. For example, the VWF protein may include a D'D3 domain and one or more domains or modules selected from the group consisting of A1, A2, A3, D4N module, VWD4 module, C8-4 module, TIL-4 module, C1 module, C2 module, C3 module, C4 module, C5 module, C6 module, and combinations thereof.

[0210] In yet another embodiment, the VWF protein is ligated to a heterologous moiety, which is either ligated 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 may be a D' domain, a D3 domain, or both. The heterologous moiety may be a half-life extender.

[0211] In certain embodiments, the VWF protein useful for this disclosure forms a multimer, e.g., a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or a higher-order multimer. In other embodiments, the VWF protein is a monomer having only one VWF protein. In some embodiments, the VWF protein of this disclosure may have one or more amino acid substitutions, deletions, additions, or modifications. In some embodiments, the VWF protein may include amino acid substitutions, deletions, additions, or modifications such that the VWF protein cannot form disulfide bonds or dimers or multimers. In other embodiments, the amino acid substitution is within the D' domain and D3 domain. In certain embodiments, the VWF protein useful for this 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 may be any amino acid not naturally present in wild-type VWF. For example, the amino acid substitution could 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 formation of polymers in the VWF protein.

[0212] In certain embodiments, the VWF protein useful herein may be further modified to improve its interaction with FVIII, for example, to improve its binding affinity to FVIII. As a non-limiting example, the VWF protein may contain 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 this disclosure may have other modifications, for example, the protein may be pegylated, glycosylated, hesylated or polysialated.

[0213] In certain embodiments, the chimeric polypeptide disclosed herein comprises a VWF protein comprising a VWF fragment, an XTEN sequence, an a2 linker of FVIII, and an Fc region. In some embodiments, the VWF protein comprises a VWF fragment fused with the XTEN sequence, the a2 linker, and the Fc region, in the order of N-terminus to C-terminus. In certain embodiments, the VWF protein comprises 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, 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 detailed embodiment, the chimeric polypeptide is rFVIIIFc-VWF-XTEN and comprises a VWF protein containing the amino acid sequence of SEQ ID NO: 202.

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

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

[0217] III.C. Half-life extension portion In some embodiments, the chimeric polypeptide of the Disclosure comprises one or more half-life extension moieties. In some embodiments, the FVIII polypeptide of the chimeric polypeptide is fused to or bound to one or more half-life extension moieties (i.e., FVIII proteins). In other embodiments, the chimeric polypeptide comprises at least two half-life extension moieties, i.e., a first half-life extension moiety fused to the FVIII polypeptide, and a second half-life extension moiety fused to the D'D3 domain of the VWF. In some embodiments, the first half-life extension moiety is fused to the C-terminus or N-terminus of the FVIII polypeptide. In some embodiments, the first half-life extension moiety is fused within the FVIII polypeptide. It is inserted. In some embodiments, the first half-life extension portion is inserted into the B domain of the FVIII polypeptide. In some embodiments, the first half-life extension portion is inserted into 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 portion 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 to or linked to a second half-life extension portion. In some embodiments, the second half-life extension portion is fused to the C-terminus or N-terminus of the VWF fragment. In some embodiments, the second half-life extension portion is inserted into the VWF fragment. In some embodiments, the second half-life extension portion is fused to the C-terminus of the VWF fragment. In certain embodiments, the second half-life extension portion is fused to the VWF fragment by a linker.

[0219] In other embodiments, the chimeric polypeptide comprises an FVIII polypeptide and a VWF fragment containing the D' and D3 domains of VWF, wherein the D'D3 domain of VWF is fused to one or more half-life extension portions, and the D'D3 domain of VWF and the FVIII polypeptide are linked by a stronger bond than the naturally occurring bond between FVIII and VWF.

[0220] In some embodiments, the chimeric polypeptide comprises an FVIII polypeptide and a VWF fragment containing the D' and D3 domains of VWF, where the D' and D3 domains of VWF are fused to the FVIII polypeptide by a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the D' and D3 domains of VWF are further linked to the FVIII polypeptide by at least one non-covalent bond. In certain embodiments, the D' and D3 domains of VWF are further fused to Fc. In some embodiments, the D' and D3 domains of VWF are fused to Fc by a clinker. In some embodiments, Fc is fused to a second Fc by an additional linker. In certain embodiments, Fc and the second Fc are covalently linked to each other, for example, by a disulfide bond. In certain embodiments, the chimeric polypeptide comprises an FVIII polypeptide, as well as a VWF fragment containing the D' and D3 domains of the VWF, as disclosed in International Patent Publication WO2017 / 222337A1, which is incorporated herein by reference in whole.

[0221] The first half-life extension portion, the second half-life extension portion, or both can be selected from the group consisting of FcRn binding partners, such as albumin or immunoglobulin Fc regions, XTEN sequences, C-terminal peptides (CTPs) of the β-subunit of human chorionic gonadotropin, PAS sequences, HAP sequences, transferrin, albumin-binding portions, or any fragments, derivatives, variants thereof, or any combination thereof.

[0222] III.C.1. Ig steady-state region or part thereof In some embodiments, the chimeric polypeptide of the present disclosure also includes a first Ig constant region or portion thereof fused to the FVIII polypeptide by a linker as needed. 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 includes a second Ig constant region or portion thereof fused to the 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 a portion thereof can be combined with additional heterologous regions, such as XTEN sequences and VWF proteins, to improve the pharmacokinetic or pharmacodynamic properties of the chimeric polypeptide. In certain embodiments, the Ig constant region or a portion thereof extends the half-life of the molecule fused to the Ig constant region or a portion thereof.

[0224] The Ig constant region consists 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 consist of three or four CH domains. The constant region of some isotypes (e.g., IgG) also contains a hinge region. Janeway See et al. 2001, Immunobiology, Garland Publishing, NY, NY.

[0225] The Ig constant region or portion thereof for producing the chimeric polypeptides of this disclosure can be obtained from a number of 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 may be derived from 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 may 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, human isotype IgG1 is used.

[0226] A diverse array 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 with specific effector functions (or lack thereof) or specific modifications that reduce immunogenicity can be selected. Many antibody and antibody-coding gene sequences have been published, and suitable Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences, or parts thereof) can be derived from these sequences using techniques recognized in the art. The genetic material obtained using one of the aforementioned methods can then be modified or synthesized to obtain polypeptides of the present disclosure. The scope of the present disclosure is further recognized to encompass alleles, variants, and mutations of constant region DNA sequences.

[0227] The sequence of the Ig constant region or a portion thereof can be cloned, for example, using polymerase chain reaction and primers selected to amplify the domain of interest. To clone the sequence of the Ig constant region or a portion thereof from an antibody, mRNA can be isolated from hybridomas, spleens, 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. Patents 4,683,195; 4,683,202; 4,800,159; 4,965,188, and, for example, “PCR Protocols: A Guide to Methods and Applications” Innis et al. eds., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270). PCR can be initiated with consensus constant-region primers or with more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the antibody light and heavy chains. In this case, the library can be isolated with consensus primers or larger homologous probes, such as mouse constant-region probes. It can be cleaned. Numerous primer sets suitable for the amplification of 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.) (al. 1994. J. Immunol. Methods 173:33); Antibody leader sequence (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250)). Cloning of the antibody sequence is further described in Newman et al., U.S. Patent No. 5,658,570, filed on 25 January 1995, which is incorporated herein by reference.

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

[0229] As used herein, the term “Fc region” is defined as the polypeptide portion corresponding to the Fc region of native Ig, which is formed by the dimeric association of the respective Fc domains of its two heavy chains. Native Fc regions form homodimers with other Fc regions. In contrast, as used herein, the term “gene fusion Fc region” or “single-stranded Fc region” (scFc region) refers to a synthetic dimeric Fc region consisting of genetically linked Fc domains within a single polypeptide chain (i.e., encoded in a single, continuous gene sequence).

[0230] In some embodiments, the “Fc region” refers to a portion of a single Ig heavy chain that begins in the hinge region immediately upstream of the papain cleavage site (i.e., IgG residue 216, with the first residue of the heavy chain constant region being 114) and terminates at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge domain, the CH2 domain, and the CH3 domain.

[0231] The Fc region of the Ig constant region can include the CH2, CH3, and CH4 domains, as well as the hinge region, depending on the Ig isotype. Chimeric polypeptides containing the Fc region of Ig confer several desirable properties on 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 the neonatal Fc receptor (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO03 / 077834; US2003-0235536A1, which are hereby incorporated by reference in their entirety).

[0232] The Ig constant region or a portion thereof can be an FcRn binding partner. FcRn is active in adult epithelial tissues and is expressed in the intestinal lumen, pulmonary airways, nasal surface, vaginal surface, colonic and rectal surfaces (U.S. Patent No. 6,485,726). The FcRn binding partner is the portion of Ig that binds to FcRn. Another example of an FcRn binding partner is albumin, which is 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 to IgG at relatively low pH (but not to other Ig classes, such as IgA, IgM, IgD, and IgE), actively transports IgG across cells from the lumen to the serosal direction, and then releases IgG at the relatively high pH found in the interstitial fluid. It is secreted. This is expressed in adult epithelial tissues (U.S. Patent Nos. 6,485,726, 6,030,613, 6,086,875; WO03 / 077834; US2003-0235536A1) including the 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 the entire IgG, the Fc fragment 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 regions between Fc and FcRn are near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are within a single Ig heavy chain. FcRn-binding partners include the entire IgG, the Fc fragment of IgG, and other fragments of IgG containing the complete binding region of FcRn. The major contact sites include amino acid residues 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. Whenever reference is made to the amino acid numbering of an Ig or Ig fragment or region, it is based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

[0235] An Fc region or FcRn-binding partner bound to FcRn can effectively cross the epithelial barrier mediated by FcRn, thus providing a non-invasive means of systemically delivering a desired therapeutic molecule. In addition, fusion proteins containing an Fc region or FcRn-binding partner undergo endocytosis by FcRn-expressing cells. However, these fusion proteins are not targeted for degradation but are recycled again during circulation, thus increasing the in vivo half-life of these proteins. In certain embodiments, a portion of the Ig constant region is an Fc region or FcRn-binding partner that typically associates with another Fc region or another FcRn-binding partner via disulfide bonds and other nonspecific 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 bioactive molecule provides a means of delivering the bioactive molecule as an aerosol administered orally, intraoral, sublingually, rectally, vaginally, or intranasally, or via the pulmonary or ocular pathway. In other embodiments, the chimeric polypeptide can be administered invasively, for example, subcutaneously or intravenously.

[0237] The FcRn binding partner region is a molecule or portion of the Fc region that can be specifically bound to an FcRn receptor and subsequently actively transported by the FcRn receptor. 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 usually distinguished from nonspecific binding, which has low affinity and moderate to high binding capacity. Typically, binding occurs when the affinity constant KA is 10⁻¹⁰. 6 M -1 higher, or 10 8 M -1It is considered specific at higher temperatures. If necessary, nonspecific bonding can be reduced without substantially affecting specific bonding by changing the bonding conditions. Those skilled in the art can determine appropriate bonding conditions, such as molecular concentration, ionic strength of the solution, and temperature. The degree of binding, the time required for binding, and the concentration of blocking agents (e.g., serum albumin, casein lactate) can be optimized using conventional techniques.

[0238] In certain embodiments, the chimeric polypeptide of the Disclosure comprises one or more cleaved 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 282–438 amino acids according to the EU numbering of IgG1, and the primary contact sites are 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 Disclosure may or may consist of an FcRn binding portion. The FcRn binding portion may originate from the 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, the FcRn binding moiety from an antibody of human isotype IgG4 is used.

[0239] In other embodiments, the “Fc region” includes the amino acid sequence of the Fc domain or an amino acid sequence derived from the Fc domain. In certain embodiments, the Fc region includes at least one of the following: hinge domains (e.g., upper, middle, and / or lower hinge regions) (approximately amino acids 216–230 of the antibody Fc region according to EU numbering), CH2 domain (approximately amino acids 231–340 of the antibody Fc region according to EU numbering), CH3 domain (approximately amino acids 341–438 of the antibody Fc region according to EU numbering), CH4 domain, or variants, parts, or fragments thereof. In other embodiments, the Fc region includes the complete Fc domain (i.e., the hinge domain, CH2 domain, and CH3 domain). In some embodiments, the Fc region includes, is essentially, 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 yet other embodiments, the Fc region lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). In certain embodiments, the Fc region includes or consists of amino acids corresponding to EU numbers 221-447.

[0240] The Fc region, as indicated 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 may 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 polypeptide or a portion of the Fc domain may 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, human isotype IgG1 is used.

[0241] In certain embodiments, the Fc variant provides an alteration of at least one effector function conferred by the Fc region containing the wild-type Fc domain (e.g., the 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 an improvement or reduction in the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or complement-dependent cell-mediated cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine ​​residue.

[0242] The Fc regions of this disclosure may use Fc variants known in the art to impart effector functions and / or alterations (e.g., enhancement or reduction) of FcR or FcRn binding. Specifically, the binding molecules of this disclosure may be, for example, those incorporated herein by reference, from the 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, WO03 / 074569 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 Publications US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or U.S. Patent No. 5,648,260; No. 5,739,277; No. 5,834,250; This may include changes (e.g., substitutions) at one or more amino acid positions disclosed in Patent 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, specific changes (e.g., specific substitutions of one or more amino acids disclosed in the art) can be produced at one or more of the amino acid positions disclosed herein. In other embodiments, different changes (e.g., different substitutions of one or more amino acid positions disclosed in the art) can be produced at one or more of the amino acid positions disclosed herein.

[0243] The Fc region or FcRn binding partner of IgG can be modified using well-recognized procedures such as site-directed mutagenesis to produce modified IgG or its Fc fragment or portion to which FcRn binds. Such modifications include modifications away from the FcRn contact site, as well as modifications within the contact site that preserve or further enhance binding to FcRn. For example, the following single amino acid residues in human IgG1 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, T289 A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309 A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333 A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K36 0A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N39 0A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A can be replaced without significantly losing the binding affinity of Fc to FcRn. For example, P238A represents wild-type proline substituted with alanine at position 238. As an example, some embodiments incorporate the N297A mutation to remove a highly conserved N-glycosylation site. In addition to alanine, other amino acids can be used in place of the wild-type amino acid at the positions specified above. Introducing a single mutation into Fc can produce more than 100 Fc regions different from the native Fc. In addition, introducing two, three, or more combinations of these individual mutations together can produce hundreds or even more Fc regions. Furthermore, one of the Fc regions of a construct of the present disclosure may be mutated while the other Fc regions of the construct are not mutated at all, or both may be mutated, but by different mutations.

[0244] Certain mutations described above can confer novel functionality to the Fc region or FcRn binding partners. For example, some embodiments incorporate N297A, removing a highly conserved N-glycosylation site. The effects of this mutation include reducing immunogenicity, thereby increasing the circulating half-life of the Fc region, and preventing 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 novel functionality resulting from the above mutations, the affinity for FcRn can be increased beyond the wild-type affinity in some cases. This increase in affinity may reflect an increase in the "on" rate, a decrease in the "off" rate, or both. Examples of mutations thought to 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] In addition, at least three human Fc gamma receptors appear to recognize binding sites on IgG in the lower hinge region, generally within amino acids 234–237. Therefore, another example of novel functionality and potential immunogenicity reduction can arise from mutations in this region (with a one-amino acid deletion), for example, by replacing amino acids 233–236 of human IgG1 “ELLG” with the corresponding sequence from IgG2 “PVA”. When such mutations are introduced, it has been shown that Fcγ RI, Fcγ RII, and Fcγ RIII, which mediate various effector functions, do not bind to IgG1. 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 a portion thereof, for example, the Fc region, is a polypeptide comprising the sequence PKNSSMISNTP (SEQ ID NO: 89 or SEQ ID NO: 3 of U.S. Patent No. 5,739,277) and optionally further comprising sequences 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. Patent No. 5,739,277).

[0247] In other embodiments, the immunoglobulin constant region or portion thereof includes a hinge region or portion thereof that contains an amino acid sequence that forms one or more disulfide bonds with another immunoglobulin constant region or portion thereof. The disulfide bonds formed by the immunoglobulin constant region or portion thereof allow the first polypeptide comprising the FVIII polypeptide and the second polypeptide comprising the VWF fragment to interact with endogenous VWF without exchanging the VWF fragment. The hinge region or its portion is positioned so as not to bind to the FVIII polypeptide. Thus, the disulfide bond between the first immunoglobulin constant region or its portion and the second immunoglobulin constant region or its portion prevents interaction between the endogenous VWF and the FVIII polypeptide. This inhibition of interaction between VWF and the FVIII polypeptide allows the half-life of the chimeric polypeptide to exceed the 2x limit. The hinge region or its portion can be further linked to CH1, CH2, CH3, one or more domains of its fragment, and any combination thereof. In certain embodiments, the immunoglobulin constant region or its portion is the hinge region and CH2.

[0248] In certain embodiments, the Ig constant region or a 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 may be interposed between the glycosylated and non-glycosylated Fc regions. In other embodiments, the Fc region or FcRn binding partner is completely glycosylated, i.e., the entire Fc region is glycosylated. In other embodiments, the Fc region may not be glycosylated, i.e., none of the Fc portions are glycosylated.

[0249] In certain embodiments, the chimeric polypeptides of the present disclosure include amino acid substitutions to the Ig constant region or a portion thereof (e.g., an Fc variant) that alter the antigen-independent effector function of the Ig constant region, particularly altering the half-life during the circulation 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 where a longer 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 where a shorter circulation time may be advantageous, for example, for in vivo diagnostic imaging, or in situations where the starting polypeptide has toxic side effects if it remains in circulation for a long period of time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta, and for this reason are useful in treating diseases or disorders in pregnant women. In addition, other applications where reduced FcRn binding affinity may be desirable include applications where localization in the brain, kidney, and / or liver is desirable. In one exemplary embodiment, the chimeric polypeptide of this disclosure exhibits reduced transport from the vascular system across the renal glomerular epithelium. In other embodiments, the chimeric polypeptide of this disclosure exhibits reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular lumen. In some embodiments, proteins having altered FcRn binding include at least one Fc region or FcRn binding partner (e.g., one or two Fc regions or FcRn binding partners) having one or more amino acid substitutions within an "FcRn binding loop" of the Ig constant region. The FcRn binding loop consists of amino acid residues 280-299 (according to EU numbering) of the wild-type, full-length Fc region. In other embodiments, the Ig constant region or portion of the chimeric polypeptide of the disclosure having altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions within a 15 Å FcRn "contact region".As used herein, the term "contact region" of 15ÅFcRn refers to the following positions in the Fc portion of the full length of the wild type: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 3. The residues include 69, 372-389, 391, 393, 408, 424, 425-440 (EU numbering). In other embodiments, the Ig constant region or portion of the present disclosure having altered FcRn binding affinity includes at least one Fc region or FcRn binding partner having 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 that alter FcRn binding activity are disclosed in International PCT Publication WO05 / 047327, which is incorporated herein by reference.

[0251] The Fc region or FcRn binding partner used in this disclosure may also include amino acid substitutions recognized in the art 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 FVIII polypeptide may include an Fc region having a mutation that leads to a reduction in glycosylation (e.g., N or O linked glycosylation) or may include a modified glycoform of the wild-type Fc moiety (e.g., a low-fucose or fucose-free glycan).

[0252] In some embodiments, the unprocessed chimeric polypeptide of the present disclosure may comprise a genetically fused Fc region (i.e., 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 contain 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 still 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 the sequence. For example, at least two of the Fc regions or FcRn binding partners may 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 Sequence As used herein, "XTEN sequence" refers to an extended-length polypeptide having a sequence that is composed primarily of small hydrophilic amino acids and that, under physiological conditions, has a low degree of secondary or tertiary structure or no secondary or tertiary structure, and has a substantially non-repetitive sequence that does not occur in nature. As a partner for a chimeric polypeptide, XTEN can serve as a carrier and impart certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties when linked to the VWF protein or FVIII sequence of the present disclosure to create a chimeric polypeptide. 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 the VWF protein and / or the second Ig constant region or a portion thereof. Therefore, in some embodiments, the XTEN sequence fused to the VWF protein and / or the second Ig constant region or a portion thereof contains less than 288 amino acids in length, i.e., shorter than 288 amino acids. In one embodiment, the XTEN sequence fused to the VWF protein and / or the second Ig constant region or a portion thereof consists of an amino acid sequence having a length between 12 and 287 amino acids. In another embodiment, the XTEN sequence fused to the VWF protein and / or the second Ig constant region or a portion thereof contains 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 yet another embodiment, the XTEN sequence fused to the VWF protein and / or the second Ig constant region or a 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 a 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 an improved half-life compared to a chimeric polypeptide without the XTEN sequence.

[0255] The chimeric polypeptides of this disclosure may further comprise additional (second, third, or more) XTEN sequences. These additional XTEN sequences may be further fused to or to the FVIII polypeptide or the first Ig constant region. The additional XTEN sequences may be of any length. For example, an additional XTEN sequence fused to or to the FVIII polypeptide or the first Ig constant region is a peptide or polypeptide having more than approximately 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 up to about 3000 amino acid residues, more than about 30 residues up to about 2500 residues, more than about 40 residues up to about 2000 residues, more than about 50 residues up to about 1500 residues, more than about 60 residues up to about 1000 residues, more than about 70 residues up to about 900 residues, more than about 80 residues up to about 800 residues, more than about 90 residues up to about 700 residues, more than about 100 residues up to about 600 residues, more than about 110 residues up to about 500 residues, or more than about 120 residues up to about 400 residues. In certain embodiments, the additional XTEN fused to the FVIII polypeptide contains at least about 288 amino acids. In certain embodiments, the additional XTEN fused to the FVIII polypeptide contains about 288 amino acids. In certain embodiments, the additional XTEN fused to the FVIII polypeptide includes 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] An XTEN sequence (i.e., an XTEN sequence fused to the VWF protein and / or the second Ig constant region or a portion thereof, or an XTEN sequence fused to or inserted into one or more insertion sites within the FVIII polypeptide and / or the first Ig constant region or a portion thereof) may contain one or more sequence motifs of 9 to 14 amino acid residues, or amino acid sequences that are at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence motif, the motifs containing, essentially consisting of, or comprising 4 to 6 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 at least about 80% of the sequence, or at least Approximately 85%, or at least approximately 90%, or at least approximately 91%, or at least approximately 92%, or at least approximately 93%, or at least approximately 94%, or at least approximately 95%, or at least approximately 96%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99%, or at least approximately 100%, consists of multiple units of non-overlapping sequences selected from single-motif families selected from Table 7, thereby resulting in a family sequence. As used herein, “family” means that XTEN has motifs selected only from the single-motif categories of Table 7; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and any other amino acids in XTEN that are not from the family motif are selected to achieve the desired properties, such as enabling the incorporation of a restriction site by the encoding nucleotide, enabling the incorporation of a cleavage sequence, or achieving 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 range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more 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 repeatability given by the amino acid composition of the motif, as described in more detail below. In the embodiments described above in this paragraph, the motifs to be incorporated into the XTEN can be selected and assembled using the methods described herein to obtain an XTEN of about 36 to about 3000 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, and 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 The sequence is at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of 044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US2010-0239554A1.

[0259] In some embodiments, the XTEN sequence is AE42 (sequence number 9), AE72 (sequence number 10), AE144_2A (sequence number 55), AE144_3B (sequence number 56), AE144_4A (sequence number 57), AE144_5A (sequence number 58), AE144_6B (sequence number 59), AG144_A (sequence number 60), AG144_B (sequence number 61), AG144_C (sequence number 62), AG144_F (sequence number 63), AE864 (sequence number 10). The amino acid sequence is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of (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 (sequence number 9), AE72 (sequence number 10), AE144_2A (sequence number 55), AE144_3B (sequence number 56), AE144_4A (sequence number 57), AE144_5A (sequence number 58), AE144_6B (sequence number 59), AG144_A (sequence number 60), AG144_B (sequence number 61), AG144_C (sequence number 62), AG144_F (sequence number 63), AE864 (sequence number 15), AE576 (sequence number 16), AE288 (sequence number 8), AE288_2 (sequence number 54), AE144 (sequence number 11), AG864 (sequence number 17), AG576 (sequence number 18), AG288 (sequence number 19), AG144 (sequence number 14), and any combination thereof. In some embodiments, the XTEN sequence is AE288. The amino acid sequences of certain XTEN sequences in this 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 components consist of less than 100% 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 a sequence consisting of a sequence motif from Table 7 or an XTEN sequence from Table 8, the other amino acid residues of the XTEN are selected from any of the other 14 native L-amino acids, but are preferentially selected from hydrophilic amino acids so 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. The XTEN amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P) are These components are either scattered throughout the XTEN sequence, located within or between sequence motifs, or concentrated in one or more short stretches of the XTEN sequence to form linkers, for example, between XTEN and FVIII or VWF components. In such cases where the XTEN components include amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), it is preferable that less than 2% of the amino acids or less than 1% of the amino acids are hydrophobic residues, such that the resulting sequence generally lacks secondary structure, and does not have more than 2% alpha helices or more than 2% beta sheets, for example, as determined by the methods disclosed herein. Hydrophobic residues that are less favorable for the construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. In addition, XTEN sequences can be designed to contain less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the following amino acids: cysteine ​​(to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine, and glutamine (to avoid deamidation), or to contain none of them. 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 in which less than 5% of residues contribute to alpha-helices and beta-sheets when measured by the Chou-Fasman algorithm, and have at least 90%, or at least about 95%, or more random coil formation when measured by the GOR algorithm.

[0261] In further embodiments, the XTEN sequences used in the Disclosure affect the physical or chemical properties of the chimeric polypeptides of the Disclosure, such as pharmacokinetics. The XTEN sequences used in the Disclosure may exhibit one or more of the following advantageous properties: conformational flexibility, enhanced water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius. In some embodiments, the XTEN sequences ligated to the FVIII protein of the Disclosure increase pharmacokinetic properties such as a longer terminal phase half-life or an increased area under the curve (AUC), thereby causing the chimeric polypeptides described herein to remain in vivo for an increased period compared to wild-type FVIII. In further embodiments, the XTEN sequences used in the Disclosure increase pharmacokinetic properties such as a longer terminal phase half-life or an increased area under the curve (AUC), thereby causing the FVIII protein to remain 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 the XTEN sequence. 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, refractive index measurement, and UV / visible light spectroscopy. Additional methods include Amau et al., Prot Expr and This is disclosed in Purif 48, 1-13 (2006).

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

[0264] III.C.2. Albumin Albumin or a portion thereof may 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, responsible for a significant proportion of serum osmotic pressure, and also functions as a carrier for 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 Publications 2008 / 0194481A1, 2008 / 0004206A1, 2008 / 0161243A1, 2008 / 0261877A1, or 2008 / 0153751A1, or PCT Application Publications 2008 / 033413A2, 2009 / 058322A1, or 2007 / 021494A2, which are incorporated herein by reference in their entirety.

[0265] Albumin-binding polypeptides (ABPs) may, but are not limited to, bacterial albumin-binding domains, albumin-binding peptides, or albumin-binding antibody fragments capable of binding to albumin. 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 fragments of which are incorporated herein by reference.

[0266] In certain embodiments, the chimeric polypeptide used in the methods of the present disclosure comprises at least one binding site of a non-polypeptide small molecule, a variant thereof, or a derivative capable of binding to albumin. For example, the chimeric polypeptide may comprise one or more organicalbumin-binding moieties. An example of such an albumin-binding moiety is 2-(3-maleimidopropanamide)-6-(4-(4-iodophenyl)butanamide)hexanoate ("Albu" tag) 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 the VWF fused to albumin binds to the FVIII polypeptide. In certain embodiments, the D'D3 domain of the VWF fused to albumin binds to the FVIII polypeptide by an interaction stronger than the innate non-covalent interaction between wild-type FVIII and VWF. In certain embodiments, the D'D3 domain of the VWF and / or the FVIII polypeptide contains one or more mutations that increase the affinity between the D'D3 domain of the VWF and the FVIII polypeptide. In certain embodiments, the D'D3 domain of the VWF and / or the FVIII polypeptide contains one or more mutations that enable the formation of a disulfide bond between the D'D3 domain of the 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-restrictive 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 means an amino acid sequence comprising 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 comprising, essentially consisting of, or composed of alanine, serine, and proline, which can be used as part of a heterologous portion in a chimeric polypeptide. Amino acid polymers can also form a random coil conformation if residues other than alanine, serine, and proline are added as trace components in the PAS sequence. "Trace components" means that amino acids other than alanine, serine, and proline can be added to the PAS sequence to some extent, for example, up to about 12%, i.e., about 12 out of 100 amino acids in 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%. 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 can form a random coil conformation, thereby mediating increased in vivo and / or in vitro stability for the recombinant protein of this disclosure and possessing procoagulant activity.

[0270] Non-restrictive examples of PAS peptides are disclosed, for example, in U.S. Patent Publication 2010 / 0292130A1; PCT Application Publication WO2008 / 155134A1; and European Patent Publication 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 repeating 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 portion fused or linked to the HAP sequence. Non-restrictive 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. Example For example, see 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 with 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 (not considering glycosylation) with two major domains, N (approximately 330 amino acids) and C (approximately 340 amino acids), which appears to originate from gene duplication. See GenBank accessions NM001063, XM002793, M12530, XM039845, XM039847, and S95936, the whole of which is incorporated herein by reference (www.ncbi.nlm.nih.gov).

[0273] Transferrin transports iron via transferrin receptor (TfR)-mediated endocytosis. After iron is released into the endosomal compartment 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 has a long half-life of over 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 treatment, 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, the chimeric polypeptide used in the methods of the present disclosure comprises at least one binding site of a non-polypeptide heterogeneous moiety, or a fragment, variant, or derivative thereof. For example, the chimeric polypeptide used in the methods of the present disclosure may comprise one or more polyethylene glycol (PEG) moieties bound to one or more amino acid residues in the coagulation factor and / or Fc region.

[0275] Protein PEGylation 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 PEGs include, but are not limited to, approximately 200, 300, 400, 600, 1000, 1300-1600, 1450, 2000, 3000, 3000-3750, 3350, 3000-7000, 3500-4500, 5000-7000, 7000-9000, 8000, 10000, 8500-11500, 16000-24000, 35000, 40000, 60000, and 80000 Daltons. These average molecular weights are provided merely as examples and are by no means restrictive.

[0276] The chimeric polypeptide used in the methods of this disclosure can be PEGylated to include mono or poly(e.g., 2 to 4) PEG moieties. PEGylation is known in the art. This can be carried out by any PEGylation reaction. 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 that the peptide of the Disclosure is bound to one or more PEG groups; and (ii) obtaining a reaction product. Generally, the optimal reaction conditions for the reaction are determined on a case-by-case basis based on known parameters and the desired results.

[0277] Numerous PEG-conjugation methods are 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 Publications EP0401384, EP0154316, and EP0401384; and International Patent Application Publications WO92 / 16221 and WO95 / 34326. As a non-limiting example, the FVIII variant may contain cysteine ​​substitution, and cysteine ​​may be further conjugated to the PEG polymer. The whole is incorporated herein by reference, Mei et al. See Blood 116:270-279 (2010) and U.S. Patent No. 7,632,921.

[0278] In certain embodiments, the chimeric polypeptide comprises a single-stranded FVIII polypeptide covalently linked to a VWF fragment containing the D' and D3 domains of a VWF, wherein the FVIII polypeptide contains a deletion of all or part of the B domain, the FVIII polypeptide is PEGylated, and the VWF fragment is directly or indirectly fused 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-stranded FVIII polypeptide covalently linked to a VWF fragment containing the D' and D3 domains of a VWF, wherein the FVIII polypeptide contains a deletion of all or part of the B domain, the VWF fragment is PEGylated, and the VWF fragment is directly or indirectly fused to the N-terminus of the FVIII polypeptide (e.g., scFVIII-D'D3-PEG). In other embodiments, the chimeric polypeptide comprises a single-stranded FVIII polypeptide covalently linked to a VWF fragment containing the D' and D3 domains of the VWF, wherein the FVIII polypeptide comprises a deletion of all or part of the B domain, and the FVIII polypeptide and VWF fragment are PEGylated, with the VWF fragment directly or indirectly fused 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 methods of the present disclosure comprises at least one hydroxyethyl starch (HES) polymer. HES is a naturally occurring derivative of amylopectin and is degraded by alpha-amylase in the body. HES exhibits advantageous bioproperties and is used as a blood fluid 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: See 494-498 (1991).

[0280] HES is primarily characterized by its molecular weight distribution and degree of substitution. HES has average molecular weights (weight average) of 1–300 kD, 2–200 kD, 3–100 kD, or 4–70 kD. Hydroxyethyl starch can also exhibit molar substitution degrees of 0.1–3, 0.1–2, 0.1–0.9, or 0.1–0.8, and C2:C6 substitution ratios ranging from 2–20 with respect to the hydroxyethyl group. HES with an average molecular weight of approximately 130 kD, This is Fresenius's VOLUVEN®. VOLUVEN® is an artificial colloid used in replacement fluids, for example, for therapeutic indications such as the treatment and prevention of hypovolemia. Numerous HES-binding methods are available to those skilled in the art, such as the same PEG-binding method 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 in certain cells of humans and mammals. 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 in various degrees of polymerization from n=about 80 or more sialic acid residues to n=2 by limited acid hydrolysis, digestion by neuraminidase, or fractionation of the naturally occurring bacterial form of the polymer. Several methods of PSA conjugation exist available to those skilled in the art, e.g., the same PEG conjugation method as described above. In certain embodiments, activated PSA can also be conjugated to cysteine ​​amino acid residues within coagulation factors, e.g., on FVIII, or within the Fc region. For example, see U.S. Patent No. 5846951.

[0282] III.C.10. Clearance Receptors In certain embodiments, the half-life of the chimeric polypeptide used in the methods of the present disclosure can be extended if the coagulation factor of the chimeric polypeptide includes an FVIII polypeptide and at least one fragment of an FVIII clearance receptor, or an FVIII-binding fragment, variant, or derivative thereof. Insertion of a soluble form of a clearance receptor, such as the low-density lipoprotein-associated protein receptor LRP1 or a fragment thereof, can block the binding of FVIII to the clearance receptor, thereby extending its half-life, for example, its in vivo half-life. LRP1 is a 600 kDa membrane-bound protein involved in receptor-mediated clearance of a variety of proteins, including FVIII. See, for example, Lenting et al., Haemophilia 16:6-16 (2010). Other suitable FVIII clearance receptors include, for example, LDLR (low-density lipoprotein receptor), VLDLR (very low-density lipoprotein receptor), and megalin (LRP-2), or fragments thereof. For example, see 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, A1 domain, A2 domain, A3 domain, B domain, C1 domain, C2 domain, C-terminus, or two or more combinations thereof, or between two domains of the FVIII polypeptide, such as the A1 domain and the a1 acidic region, 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, 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 include 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, and the N-terminus. The group is selected from the C-terminus, A1 and A2 domains, A1 and A3 domains, A1 and B domains, A1 and C1 domains, A1 and C2 domains, A1 domain and C-terminus, A2 and A3 domains, A2 and B domains, A2 and C1 domains, A2 and C2 domains, A2 domain and C-terminus, A3 and B domains, A3 and C1 domains, A3 and C2 domains, A3 domain and C-terminus, B and C1 domains, B and C2 domains, B domain and C-terminus, C1 and C2 domains, C1 and C-terminus, C2 domain and C-terminus, and two or more combinations thereof.

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

[0285] The FVIII polypeptides useful in this 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 they 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 other embodiments, one or more XTENs are inserted into the B domain of FVIII. In one example, the XTEN is inserted between amino acids 740 and 1640 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 740 and 1640 is omitted if necessary. In another example, the XTEN is inserted between amino acids 741 and 1690 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 740 and 1690 is omitted if necessary. In yet another example, the XTEN is inserted between amino acids 741 and 1648 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 741 and 1648 is omitted if necessary. In yet another example, the XTEN is inserted between amino acids 743 and 1638 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 743 and 1638 is omitted if necessary. In yet another example, the XTEN is inserted between amino acids 745 and 1656 corresponding to SEQ ID NO: 65, and the FVIII sequence between amino acids 745 and 1656 is omitted if necessary. In some cases, 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 omitted if necessary. In a particular case, 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 omitted if necessary. In yet another case, 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 omitted if necessary. In some other cases, 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 omitted if necessary. In yet another case, 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 omitted if necessary. 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 deletion FVIII polypeptide.In certain embodiments, the amino acid sequence of AE288 (SEQ ID NO: 8) 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%, and at least about 97%. An XTEN sequence containing an amino acid sequence having approximately 98%, at least approximately 99%, or approximately 100% sequence identity is inserted between amino acids 745 and 746 of mature FVIII, and mature FVIII has deletions of amino acid residues 746-1648. In certain embodiments, XTEN is inserted immediately downstream of amino acid 745 of B-domain deletion FVIII (SEQ ID NO: 68) in 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 in vivo by various proteases when administered to a subject, for example, at a coagulation site. In some embodiments, the cleavable linker enables cleavage from...

Claims

[Claim 1] The invention described in the specification.

Images