Methods of treating hemophilic arthropathy using chimeric coagulation factors

Abstract

To provide a method for treating reversible hemophilic arthropathy of a joint in a human having hemophilia.SOLUTION: Uses of a chimeric protein or composition comprising a coagulation factor and an Fc region are provided. The coagulation factor is selected from the group consisting of Factor VII (FVII), Factor VIIa (FVIIa), Factor VIII (FVIII), Factor IX (FIX), Factor X (FX), von Willebrand Factor (VWF), an antigen-binding portion thereof that specifically binds to FIX and FX, or any combination thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 429,509, filed December 2, 2016, 62 / 529,896, filed July 7, 2017, 62 / 550,488, filed August 25, 2017, and 62 / 558,793, filed September 14, 2017, each of which is incorporated by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to the field of therapeutic agents for the treatment of hemostatic disorders. [Background technology]

[0003] Hemophilia is an X-linked bleeding disorder caused by mutations and / or deletions in genes encoding coagulation proteins, particularly the factor VIII (FVIII) gene, which causes a deficiency in FVIII activity (hemophilia A), or mutations and / or deletions in the factor IX gene, which causes a deficiency in FIX activity (hemophilia B) (see, e.g., Non-Patent Document 1). The disease is characterized by spontaneous bleeding and excessive bleeding after trauma. Treatment of hemophilia relies on alternative therapies that target the restoration of FVIII and / or FIX activity to prevent spontaneous bleeding (see, e.g., Non-Patent Document 2).

[0004] Over time, repeated bleeding into muscles and joints, often beginning in early childhood, leads to hemophilic arthropathy and joint damage. Hemophilic arthropathy is a common and severe complication associated with hemophilia, often causing pain, deformity, and disability. The most common patients affected by hemophilic arthropathy are young males between the ages of 3 and 15. The joint most likely to be affected is the knee, but hemophilic arthropathy can also be present in the elbow, ankle, shoulder, and vertebrae. Hemophilic arthropathy is known to be irreversible. For this reason, currently available treatments for hemophilic arthropathy are limited. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Peyvandi, F. et al., Haemophilia 12:82-89 (2006) [Non-patent document 2] Mannucci, PM et al., N. Engl. J. Med. 344:1773-1779 (2001) Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need to develop new methods for treating hemophilic arthropathy. [Means for solving the problem]

[0007] One aspect of the present disclosure provides a method for treating reversible hemophilic arthropathy of a joint in a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region or a composition comprising the chimeric protein. In some embodiments, the reversible hemophilic arthropathy comprises synovitis. In certain embodiments, the reversible hemophilic arthropathy comprises microhemorrhage or asymptomatic bleeding.

[0008] Another aspect of the present disclosure is a method of treating synovitis in a human with hemophilia, comprising administering to the patient a chimeric protein comprising a clotting factor and an Fc region or a composition comprising a clotting factor and an Fc region. and administering to a human an effective amount of the compound of formula (I) or (II). In some embodiments, the synovitis is associated with hemophilic arthropathy.

[0009] Another embodiment of the present disclosure discloses a method for reducing the occurrence of vascular remodeling in the joints of a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region. Another embodiment of the present disclosure provides a preventive treatment for vascular remodeling in the joints of a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region or a composition comprising a coagulation factor and an Fc region.

[0010] Another aspect of the present disclosure discloses a method for improving periarticular soft tissue in a human with hemophilia, the method comprising administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region or a composition comprising a coagulation factor and an Fc region.

[0011] In some embodiments, administration improves the Joint Health Score (HJHS) in a human. In some embodiments, administration reduces joint pain in a human.

[0012] In certain embodiments, the Fc region specifically binds to low-affinity immunoglobulin gamma Fc region receptor II-b (FcγRIIB). In some embodiments, the Fc region specifically binds to dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin (DC-SIGN).

[0013] In some aspects, the method further includes identifying a human in need of treatment. In some embodiments, the identifying includes using an imaging system. In certain embodiments, the imaging system includes radiography, magnetic resonance imaging, ultrasound imaging, power Doppler ultrasonography, or any combination thereof. In some embodiments, the human expresses one or more biomarkers associated with joint inflammation.

[0014] In some aspects, the coagulation factor is selected from the group consisting of factor VII (FVII), factor VIIa (FVIIa), factor VIII (FVIII), factor IX (FIX), factor X (FX), von Willebrand factor (VWF), antigen-binding portions thereof that specifically bind to FIX and FX, or any combination thereof. In some embodiments, the chimeric protein comprises FVIII-Fc. In other embodiments, the chimeric protein comprises FIX-Fc. In one embodiment, the chimeric protein comprises a factor VIII portion and a VWF portion, wherein the FVIII portion comprises a FVIII polypeptide or a fragment thereof, the VWF portion comprises a VWF polypeptide or a fragment thereof, the FVIII portion is linked to a first Fc region, the VWF portion is linked to a second Fc region, and the first Fc region and the second Fc region are associated with each other.

[0015] In some embodiments, the chimeric protein further comprises a half-life extending moiety. In certain embodiments, the half-life extending moiety comprises albumin or a fragment thereof, an albumin binding moiety, a PAS sequence, a HAP sequence, transferrin or a fragment thereof, polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), a derivative thereof, or any combination thereof.

[0016] In some aspects, an effective amount of a composition comprising FVIII and an Fc region, e.g., a chimeric protein, is about 20 IU / kg to about 300 IU / kg. In some embodiments, a chimeric protein comprising FVIII-Fc is administered for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 58 days, about 59 days, about 60 days, about 61 days, about 62 days, about 63 days, about 64 days, about 65 days, about 66 days, about 67 days, about 68 days, about 69 days, about 70 days, about 71 days, about 72 days, about 73 days, about 74 days, about 75 days, about 76 days, about The doses are administered at intervals of 0 days, about 21 days, about 22 days, about 23 days, or about 24 days.

[0017] In other aspects, the effective amount of the chimeric protein comprising FIX-Fc is about 20 IU / kg to about 100 IU / kg. In some embodiments, the chimeric protein comprising FIX-Fc is administered at an interval of about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0018] In one specific embodiment, the chimeric protein comprises a FVIII portion, a VWF portion, a first Fc region, and a second Fc region; the FVIII portion comprises a FVIII polypeptide or a fragment thereof; the VWF portion comprises a VWF polypeptide or a fragment thereof; the FVIII portion is linked to the first Fc region; the VWF portion is linked to the second Fc region; and the first Fc region and the second Fc region are associated with each other. Embodiment

[0019] E1. A method for treating reversible hemophilic arthropathy of the joints in a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a clotting factor and an Fc region.

[0020] E2. The method of E1, wherein the reversible hemophilic arthropathy comprises synovitis.

[0021] E3. The method of E1 or E2, wherein the reversible hemophilic arthropathy comprises microhemorrhages.

[0022] E4. A method of treating synovitis in a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a clotting factor and an Fc region.

[0023] E5. The method of E4, wherein the synovitis is associated with hemophilic arthropathy.

[0024] E6. A method for preventing or reducing the occurrence of vascular remodeling in the joints of a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region.

[0025] E7. A method for improving periarticular soft tissue in a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a clotting factor and an Fc region.

[0026] E8. The method of any one of E1-E7, wherein administration improves human joint health score (HJHS).

[0027] E9. Joint health score is the sum of the total joint score and the global gait score. The method according to E8,

[0028] E10. The method of E9, wherein a total joint score is measured based on swelling, duration of swelling, muscle atrophy, joint friction rub, loss of flexion, loss of extension, joint pain, and muscle strength.

[0029] E11. The method of E9, wherein the total gait score is measured based on walking, stairs, running, or hopping on one leg.

[0030] E12. Any one of E1 to E11, wherein administration reduces joint pain in humans. The method described below.

[0031] E13. The method of any one of E1-E12, wherein the joints are selected from the group consisting of one or both elbows, one or both knees, one or both ankles, one or both shoulders, one or both hips, one or both wrists, one or more joints of the hand, one or more joints of the foot, and any combination thereof.

[0032] E14. The method of any one of E1 to E13, wherein the joint is the elbow.

[0033] E15. The method of any one of E1 to E13, wherein the joint is the knee.

[0034] E16. The method of any one of E1 to E13, wherein the joint is the ankle.

[0035] E17. The method of any one of E1 to E16, wherein the Fc region specifically binds to the low affinity immunoglobulin gamma Fc region receptor II-b (FcγRIIB).

[0036] E18. The method of any one of E1 to E17, wherein the Fc region specifically binds to dendritic cell-specific intercellular adhesion molecule-3-binding nonintegrin (DC-SIGN).

[0037] E19. The method of any one of E1-E18, further comprising identifying a human in need of treatment.

[0038] E20. The method of E19, wherein identifying includes using an imaging system.

[0039] E21. The method of E20, wherein the imaging system comprises radiography, magnetic resonance imaging, ultrasound imaging, power Doppler ultrasonography, or any combination thereof.

[0040] E22. The method of E21, wherein the human expresses one or more biomarkers associated with joint inflammation.

[0041] E23. The method of any one of E1-E22, wherein the coagulation factor is selected from the group consisting of factor VII (FVII), factor VIIa (FVIIa), factor VIII (FVIII), factor IX (FIX), factor X (FX), von Willebrand factor (VWF), an antigen-binding portion thereof that specifically binds to FIX and FX, or any combination thereof.

[0042] E24. The method of any one of E1 to E23, wherein the chimeric protein comprises FVIII-Fc.

[0043] E25. The method of any one of E1 to E23, wherein the chimeric protein comprises FIX-Fc.

[0044] E26. The method of any one of E1-E24, wherein the chimeric protein comprises a Factor VIII portion and a VWF portion, wherein the FVIII portion comprises a FVIII polypeptide or a fragment thereof, wherein the VWF portion comprises a VWF polypeptide or a fragment thereof, wherein the FVIII portion is linked to a first Fc region, wherein the VWF portion is linked to a second Fc region, and wherein the first Fc region and the second Fc region are associated with each other.

[0045] E27. The FVIII polypeptide comprises full-length mature FVIII, E23, E24, and the method of any one of E26.

[0046] E28. The method of any one of E23, E24 and E26, wherein the FVIII polypeptide comprises a B-domain deleted FVIII.

[0047] E29. The method of E28, wherein the B-domain deleted FVIII comprises a deletion of all or part of the B-domain of FVIII.

[0048] E30. The method of E28 or E29, wherein the B-domain deleted FVIII comprises a deletion of amino acid residues 746 to 1648 of mature FVIII.

[0049] E31. The method of any one of E23, E24, and E25 to E30, wherein the VWF polypeptide comprises a VWF fragment comprising the D' and D3 domains of VWF.

[0050] E32. The method of any one of E1-E31, wherein the chimeric protein further comprises a half-life extending moiety.

[0051] E33. The method of E32, wherein the half-life extending moiety comprises albumin or a fragment thereof, an albumin binding moiety, a PAS sequence, a HAP sequence, transferrin or a fragment thereof, polyethylene glycol (PEG), polysialic acid, hydroxyethyl starch (HES), a derivative thereof, or any combination thereof.

[0052] E34. The method of E32 or E33, wherein a half-life extending moiety is inserted into the clotting factor.

[0053] E35. The method of E32 or E33, wherein the half-life extending moiety is inserted between the clotting factor and the Fc region.

[0054] E36. The method according to any one of E24 and E26 to E35, wherein the effective amount of the chimeric protein comprising FVIII and an Fc region is about 20 IU / kg to about 300 IU / kg.

[0055] E37. The effective amount of a chimeric protein containing FVIII-Fc is about 20 IU / kg to about 275 IU / kg, about 20 IU / kg to about 250 IU / kg, about 20 IU / kg to about 200 IU / kg, about 20 IU / kg to about 175 IU / kg, about 20 IU / kg to about 150 IU / kg, about 20 IU / kg to about 125 IU / kg, Approximately 20IU / kg to approximately 100IU / kg, approximately 20IU / kg to approximately 90IU / kg, approximately 20IU / kg to approximately 80IU / kg, approximately 20IU / kg to approximately 70IU / kg , about 20IU / kg to about 60IU / kg, about 20IU / kg to about 50IU / kg, about 20IU / kg to about 40IU / kg, about 20IU / kg to about 30IU / kg , about 30IU / kg to about 100IU / kg, about 40IU / kg to about 100IU / kg, about 50IU / kg to about 100IU / kg, about 60IU / kg to about 100I U / kg, about 70IU / kg~about 100IU / kg, about 80IU / kg~about 100IU / kg, about 90IU / kg~about 100IU / kg, about 100IU / kg~ The method of claim E36, wherein the dose is about 200 IU / kg, about 150 IU / kg to about 200 IU / kg, about 200 IU / kg to about 300 IU / kg, about 225 IU / kg to about 300 IU / kg, about 250 IU / kg to about 300 IU / kg, about 275 IU / kg to about 300 IU / kg, or about 25 IU / kg to about 75 IU / kg.

[0056] E38. The method according to E36 or E37, wherein the effective amount of the chimeric protein comprising FVIII-Fc is about 25 IU / kg to about 65 IU / kg.

[0057] E39. The method of any one of E24 and E26 to E38, wherein the chimeric protein comprising FVIII-Fc is administered at an administration interval of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, or about 24 days.

[0058] E40. The method of any one of E24 and E26 to E38, wherein the chimeric protein comprising FVIII-Fc is administered at an administration interval of about 1 to about 14 days, about 1 to about 13 days, about 1 to about 12 days, about 1 to about 11 days, about 1 to about 10 days, about 1 to about 9 days, about 1 to about 8 days, about 1 to about 7 days, about 1 to about 6 days, about 1 to about 5 days, about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about 14 days, about 3 to about 14 days, about 4 to about 14 days, about 5 to about 14 days, about 6 to about 14 days, about 7 to about 14 days, about 8 to about 14 days, about 9 to about 14 days, about 10 to about 14 days, about 11 to about 14 days, about 12 to about 14 days, about 13 to about 14 days, or about 5 to about 10 days.

[0059] E41. The method according to any one of E24 and E26 to E40, wherein the chimeric protein comprising FVIII-Fc is administered at an administration interval of about 3 days to about 5 days.

[0060] E42. The method according to E25, wherein the effective amount of the chimeric protein comprising FIX-Fc is about 20 IU / kg to about 100 IU / kg.

[0061] E43. An effective amount of a chimeric protein containing FIX-Fc is about 20 IU / kg to about 100 IU / kg, about 30 IU / kg to about 100 IU / kg, about 40 IU / kg to about 100 IU / kg, about 50 IU / kg to about 100 IU / kg, about 60 IU / kg to about 100 IU / kg, about 70 IU / kg to about 100 IU / kg, about 80 IU / kg to about 100 IU / kg, about 90 IU / kg to about 100 IU / kg, about 100 IU / kg to about 100 IU / kg, about 110 IU / kg to about 100 IU / kg, about 120 IU / kg to about 100 IU / kg, about 130 IU / kg to about 100 IU / kg, about 140 IU / kg to about 100 IU / kg, about 150 IU / kg to about 100 IU / kg, about 160 IU / kg to about 100 IU / kg, about 170 IU / kg to about 100 IU / kg, about 180 IU / kg to about 100 IU / kg, about 190 IU / kg to about 100 IU / kg, about 200 IU / kg to about 100 IU / kg, about 210 IU / kg to about 100 IU / kg, about 220 IU / kg to about 100 IU / kg, about 230 IU / kg to about 100 IU / kg, about 240 IU / kg to about 100 IU / kg, about 250 IU / kg to about 100 IU / kg, about 260 IU / kg to about 100 IU / kg, The method according to E25 to E42, wherein the dose is 0 IU / kg to about 100 IU / kg, about 20 IU / kg to about 90 IU / kg, about 20 IU / kg to about 80 IU / kg, about 20 IU / kg to about 70 IU / kg, about 20 IU / kg to about 60 IU / kg, about 20 IU / kg to about 50 IU / kg, about 20 IU / kg to about 40 IU / kg, or about 20 IU / kg to about 30 IU / kg.

[0062] E44. The method according to any one of E25 and E42 to E44, wherein the effective amount of the chimeric protein comprising FIX-Fc is about 50 IU / kg to 100 IU / kg.

[0063] E45. The method of any one of E25 and E42 to E44, wherein the chimeric protein comprising FIX-Fc is administered at an administration interval of about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0064] A chimeric protein comprising E46.FIX-Fc is expressed for about 1 to about 21 days, about 1 to about 20 days, about 1 to about 19 days, about 1 to about 18 days, about 1 to about 17 days, about 1 to about 16 days, about 1 to about 15 days, about 1 to about 14 days, about 1 to about 13 days, about 1 to about 12 days, about 1 to about 11 days, about 1 to about 10 days, about 1 to about 9 days, about 1 to about 8 days, about 1 to about 7 days, about 1 to about 6 days, about 1 to about 5 days, or about 1 to about 4 days. about 1 to about 3 days, about 1 to about 2 days, about 2 to about 21 days, about 3 to about 21 days, about 4 to about 21 days, about 5 to about 21 days, about 6 to about 21 days, about 7 to about 21 days, about 8 to about 21 days, about 9 to about 21 days, about 10 to about 21 days, about 11 to about 21 days, about 12 to about 21 days, about 13 to about 21 days, about 14 to about 21 days, about 15 to about 21 days, about 16 to about 21 days, about 17 to about 21 days, about 18 to about 21 days, about 1 The method according to any one of E25 and E42 to E45, wherein the administration is performed at an administration interval of 9 to about 21 days, about 20 to about 21 days, about 5 to about 10 days, about 10 to about 15 days, or about 15 to about 20 days.

[0065] E47. The method of any one of E25 and E42 to E46, wherein the chimeric protein comprising FIX-Fc is administered at an administration interval of about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0066] E48. The method of any one of E1-E32, wherein the chimeric protein comprises a FVIII portion, a VWF portion, a first Fc region, and a second Fc region, wherein the FVIII portion comprises a FVIII polypeptide or a fragment thereof, the VWF portion comprises a VWF polypeptide or a fragment thereof, the FVIII portion is linked to the first Fc region, the VWF portion is linked to the second Fc region, and the first Fc region and the second Fc region are associated with each other.

[0067] E49. The method of any one of E1-E47, wherein the Fc region of the chimeric protein promotes localization of the chimeric protein to the joint.

[0068] E50. The method of any one of E1 to E49, wherein the human is under 6 years of age.

[0069] E51. The method of any one of E1-E49, wherein the human is between 6 and under 12 years of age.

[0070] E52. The method of any one of E1-E49, wherein the human is 12 years of age or older.

[0071] E53. The method of any one of E1-E52, wherein the coagulation factor is distributed in tissues outside the plasma compartment as well as within the plasma compartment. [Brief explanation of the drawings]

[0072] [Figure 1-1] Figures 1A-1F show the median (IQR) annualized bleeding rate (ABR) before (Figures 1A, 1C, and 1E) and during (Figures 1B, 1D, and 1F) studies from the FVIII-Fc study (Figures 1A and 1B) and the children's FVIII-Fc study (Figures 1C-1F) along with target joints at baseline. Figures 1C-1D show the combined data from the children's FVIII-Fc study, while Figures 1E-1F show the same data stratified based on the subjects' age (<6 years and 6-12 years). [Figure 1-2] Continued from Figure 1-1. [Figure 2] Figures 2A-2B show the mean change in total modified Hemophilia Joint Health Score (mHJHS; y-axis) from FVIII-Fc study baseline to expansion study year 2 (Figure 2A; x-axis) and expansion study year 3 (Figure 2B; x-axis). Figure 2B distinguishes between the presence (yes; triangles) and absence (no; circles) of target joints at baseline. [Figure 3-1] Figures 3A-3C show the mean change in total mHJHS (y-axis) from FVIII-Fc study baseline to extension year 2 (Figure 3A; x-axis) and extension year 3 (Figure 3B), and from the FVIII-Fc study for children to extension year 2 for subjects receiving pre-study prophylaxis and for subjects receiving pre-study hemostatic (on-demand) treatment (Figure 3C; x-axis). [Figure 3-2] Continued from Figure 3-1. [Figure 4] FIG. 1 shows the mean change in total mHJHS (y-axis) from FVIII-Fc study baseline to year 2 (x-axis) for subjects with target joints at FVIII-Fc study baseline (squares) and subjects without target joints at FVIII-Fc study baseline (diamonds). [Figure 5] FIG. 1 shows the mean change in total mHJHS (y-axis) from FVIII-Fc study baseline to year 2 (x-axis) for subjects in the lowest quartile of disability in mHJHS score at baseline of FVIII-Fc study (Q1; ≧1-10) (diamonds), the second lowest quartile of disability in mHJHS score at baseline of FVIII-Fc study (Q2; ≧10-22) (squares), the second highest quartile of disability in mHJHS score at baseline of FVIII-Fc study (Q3; ≧22-34) (triangles), and the highest quartile of disability in mHJHS score at baseline of FVIII-Fc study (Q4; ≧34-37) (diamonds). [Figure 6] FIG. 1 shows the mean change in total mHJHS (y-axis) from FVIII-Fc study baseline to year 2 (x-axis) for subjects with target joints at FVIII-Fc study baseline. [Figure 7]FIG. 1 shows the mean change in total mHJHS (y-axis) from FVIII-Fc study baseline to year 2 (x-axis) for weight-bearing joints (diamonds) and non-weight-bearing target joints (squares). [Figure 8] FIG. 1 shows the mean change in total mHJHS (y-axis) from FVIII-Fc study baseline to year 2 (x-axis) for swelling (diamonds), range of motion (squares), and muscle strength (triangles). [Figure 9] Figure 1 shows the mean (SEM) change (y-axis) in mHJHS in FVIII-Fc study patients for joint instability (dark gray squares), swelling (black triangles), muscle atrophy (light gray squares), joint pain (light gray diamonds), joint crepitus (black squares), and muscle strength (light gray circles). Total baseline (BL) scores for each mHJHS measurement are shown. [Figure 10A] Figure 10 shows pre-study (Figure 10A) and on-study median (Figure 10B) (IQR) annualized bleeding rate (ABR) for subjects from the rFIXFc study. Figure 10B further shows the overall ABR (dark circles), the overall target joint ABR (gray diamonds), and the spontaneous target joint ABR (gray triangles). WP = weekly prophylaxis; IP = individualized interval prophylaxis; and MP = modified prophylaxis (Figures 10A-10B). [Figure 10B] Figure 10 shows pre-study (Figure 10A) and on-study median (Figure 10B) (IQR) annualized bleeding rate (ABR) for subjects from the rFIXFc study. Figure 10B further shows the overall ABR (dark circles), the overall target joint ABR (gray diamonds), and the spontaneous target joint ABR (gray triangles). WP = weekly prophylaxis; IP = individualized interval prophylaxis; and MP = modified prophylaxis (Figures 10A-10B). [Figure 11]1 is a graphical representation showing the number of evaluable target joints (ankle, knee, elbow, hip, wrist, and shoulder) that resolved and did not resolve in subjects (n=37) with at least 12 months of continuous follow-up who had not undergone joint surgery within 12 months since the start of follow-up. The number (n) of each target joint is overlaid on the associated data, for a total of 93 target joints evaluated. The percentage of resolved (100%) and non-resolved (0%) target joints is shown below the x-axis. [Figure 12-1] Figures 12A-12C are single-photon emission computed tomography (SPECT) images of mice administered I-SIB-labeled FIX (Figure 12A), I-SIB-labeled FIXFc (Figure 12B), or I-SIB-labeled glycoPEGylated FIX (Figure 12C). Figures 12D-12G show a direct comparison of mice administered I-SIB-labeled FIX, I-SIB-labeled FIXFc, or I-SIB-labeled glycoPEGylated FIX at various time points. Heat maps show the relative concentration (%ID / g) of I-SIB labeling in each mouse (Figures 12A-12G). [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 12-4] Continued from Figure 12-3. [Figure 13] 13A-13B are graphs showing the intensity of localization of I-SIB labeling in the mice shown in Figures 12A-12G after administration of I-SIB-labeled FIX, I-SIB-labeled FIXFc, or I-SIB-labeled GlycoPEGylated FIX over time in the knee (Figure 13A) and shoulder (Figure 13B). Data were collected for both the right and left knees and shoulders and combined to generate the data shown (Figures 13A and 13B, respectively). [Figure 14]Figures 14A-B are graphs showing the relative activity of labeled FIX and labeled FIXFc compared to unlabeled FIX and FIXFc, as measured by chromogenic assay or one-stage clotting assay, and Figure 14B is a graph showing the pharmacokinetics of labeled and unlabeled FIX molecules in HemB mice. [Figure 15-1] 15A-15F show the range of motion of the elbow (flexion: FIG. 15A; and extension: FIG. 15B), knee (flexion: FIG. 15C; and extension: FIG. 15D), and ankle (plantar flexion: FIG. 15E; and dorsiflexion: FIG. 15F) joints, along with the modified Hemophilia Joint Health Score (HJHS) and superimposed degree of flexion / extension. [Figure 15-2] Continued from Figure 15-1. [Figure 15-3] Continued from Figure 15-2. [Figure 16] Figure 16 is a flow chart outlining the methods used to investigate the effects of rFVIIIFc on FcγR binding, internalization, signaling and cytokine production, and gene expression changes, as well as subsequent interactions and effects on T cells in vitro. [Figure 17] Figures 17A-17C are graphical representations of the relative surface expression levels of the Fcγ receptors CD16 (Figure 17A), CD32 (Figure 17B), and CD64 (Figure 17C) on macrophages and dendritic cells after treatment with horseradish peroxidase immune complexes (HRP-IC; positive control), IgG1, recombinant FVIII (rFVIII), or rFVIII Fc fusion protein (rFVIIIFc). Asterisks (*) indicate the degree of significance (n=3; *=P≦0.05, **=P≦0.01, ***=P≦0.005, not indicating significance of HRP-IC compared to other treatments). [Figure 18-1]Figures 18A-18C are graphical representations showing relative signaling following treatment with rFVIII or rFVIIIFc. Figure 18A shows signaling, as measured by Syk phosphorylation, in THP-1 monocyte line ("THP-1"), monocytes, peripheral blood monocyte-derived macrophages ("macrophages"), and peripheral blood monocyte-derived dendritic cells treated with HRP-IC, IgG1, rFVIII, or rFVIIIFc for 15 minutes. Figure 18B shows relative Syk phosphorylation in macrophages treated with rFVIIIFc ("WT"), an rFVIIIFc variant unable to bind to the neonatal Fc receptor ("FcRn variant"), or an rFVIIIFc variant unable to bind to FcγR ("FcgR variant"). Figure 18C shows the relative production of the inflammatory cytokines interleukin-1b (IL-1b), IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNFa) in macrophages 24 hours after treatment with HRP-IC, IgG1, rFVIII, or rFVIIIFc. [Figure 18-2] Continuation of Figure 18-1. [Figure 19] Figure 19 shows the relative phosphorylation status of Src homology region 2 domain-containing phosphatase-1 (SHP1), pSHP2, phosphatidylinositol-3,4,5-triphosphate 5-phosphatase 1 (SHIP1), and pSHIP2 at 1, 5, and 30 minutes after treatment with rFVIII or rFVIIIFc. Asterisks (*) indicate the degree of significance (n=3; **P≦0.01, ***P≦0.005). [Figure 20-1]Figures 20A-20M are graphical representations of gene expression patterns in tolerogenic macrophages after treatment with rFVIII or rFVIIIFc. Figures 20A-20B are Venn diagrams showing the distribution of significantly down-regulated genes (Figure 20A) and significantly up-regulated genes (Figure 20B) in monocyte-derived macrophages (n=3) treated with IgG1, rFVIIIc for 6 hours. Figures 20C-20G are graphs showing the relative expression of various NRF2 and lipid metabolism pathway genes, such as heme oxygenase 1 (Hmox1; Figure 20C), peroxisome proliferator-activated receptor gamma (PPARγ; Figure 20D), lipoprotein lipase (LPL; Figure 20E), early growth response protein 2 (EGR2; Figure 20F), and solute-carrying organic anion transporter family member 4A1 (SLCO4A1; Figure 20G), CD206, and arginase 1 (ARG1; Figure 20L), at 6 hours (Figure 20I) and 12 hours (Figure 20J) after treatment with rFVIII or rFVIIIFc, as measured by quantitative PCR. Asterisks (*) indicate the degree of significance (n = 8; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005; Figures 20C-20G). Figures 20K and 20M are graphs showing the number of cells collected by flow cytometry that express CD206. Furthermore, macrophages educated with rFVIIIFc were found to exhibit a characteristic M2-like phenotype (Figures 20I-20M). Notably, macrophages treated with rFVIIIFc had higher relative CD206 (mannose receptor type C-1; also known as MRC1) expression than cells treated with rFVIII after 6 hours (Figure 20I) and 24 hours (Figure 20J), and macrophages treated with rFVIIIFc had higher relative ARG1 expression than cells treated with rFVIII after 24 hours (Figure 20M). [Figure 20-2] Continuation of Figure 20-1. [Figure 20-3] Continued from Figure 20-2. [Figure 20-4] Continued from Figure 20-3. [Figure 20-5] Continued from Figure 20-4. [Figure 20-6] Continued from Figure 20-5. [Figure 21-1] Figure 21A is a flow chart showing the method used to determine the effect of rFVIIIFc treatment on T cell differentiation. Figure 21B is a graphical representation of the percentage of regulatory T cells 6 days after macrophages or dendritic cells were treated with IgG1 (control), rFVIII, or rFVIIIFc for 24 hours and then placed in co-culture with naive CD4-positive T cells. Figure 21C is a graphical representation showing the percentage of regulatory T cells after culture of naive CD4-positive T cells in conditioned medium of macrophages or dendritic cells pretreated with IgG1, rFVIII, or rFVIIIFc. [Figure 21-2] Continuation of Figure 21-1. [Figure 22] FIG. 22 shows a proposed mechanism of rFVIIIFc-regulatory T cell differentiation. [Figure 23] FIG. 23 shows the proposed effect of rFIXFc on macrophages. DETAILED DESCRIPTION OF THE INVENTION

[0073] The present disclosure provides a method for treating reversible hemophilic arthropathy in a human with hemophilia, comprising administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region or a composition comprising a coagulation factor and an Fc region. The chimeric protein disclosed herein can also be used to treat synovitis, microhemorrhage, inflammation of one or more joints, vascular remodeling, or any combination thereof, in the joints of a human with hemophilia. In certain embodiments, the method of the present invention improves periarticular soft tissue in a human with hemophilia.

[0074] I. Definition The terms "a" or "an" entity refer to one or more of that entity; for example, "a nucleotide sequence" refers to one or more It is understood to refer to a plurality of nucleotide sequences, and as such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0075] Furthermore, "and / or" as used herein should be construed as a specific disclosure of each of the two specified features or components in the presence or absence of the other features or components. Thus, the term "and / or" when used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0076] Whenever an embodiment is described herein with the language "comprising," it is understood that similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0077] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0078] Units, prefixes, and symbols are written in the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, but may be obtained by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification in its entirety.

[0079] The term "about" is used herein to mean approximately, roughly, roughly, or in the region thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. Thus, "about 10 to 20" means "about 10 to about 20." In general, the term "about" can modify numerical values ​​above and below the stated numerical value by, for example, a variance above or below 10 percent (higher or lower).

[0080] As used herein, "administering" refers to providing a pharmaceutically acceptable composition, such as the chimeric protein disclosed herein, to a subject via a pharmaceutically acceptable route. The administration route can be intravenous, such as intravenous injection and intravenous infusion. Additional administration routes include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The chimeric and hybrid proteins can be administered as part of a pharmaceutical composition containing at least one excipient.

[0081] In some embodiments, the composition, e.g., the chimeric protein, is administered to a human through gene therapy, e.g., one or more polynucleotides encoding a clotting factor and / or an Fc region are administered to a human and the clotting factor and / or Fc region are expressed in the human.

[0082] As used herein, "treat," "treatment," or "treating" refers to, for example, reducing the severity of a disease or condition; reducing the duration of a disease; alleviating or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject with a disease or condition without necessarily curing the disease or condition, but not including the prevention or prophylaxis of hemophilic arthropathy or its symptoms. In some embodiments, the term "treat" or "treatment" refers to improving a subject's Hemophilia Joint Health Score (HJHS) or modified HJHS (mHJHS). In some embodiments, the total HJHS or mHJHS is improved. In some embodiments, the individual scores of one or more target joints are improved. In some embodiments, the term "treat" or "treatment" refers to improving a subject's quality of life (QoL). In certain embodiments, the QoL score analyzes a patient's propensity for sports and leisure, physical health, commitment to hemophilia, family planning, feelings about the future (for hemophilia), partnership and sexuality, treatment, mindset (for a person), work and schoolwork, or any combination thereof. In another embodiment, the term "treating" or "treatment" refers to reducing the effects and / or severity of one or more microhemorrhages. In another embodiment, the term "treating" or "treatment" refers to reducing swelling and / or inflammation and / or pain in one or more target joints. In another embodiment, the term "treating" or "treatment" refers to reducing vascular remodeling in one or more target joints.

[0083] As used herein, "prevent" or "preventing" refers to reducing or lessening the occurrence or severity of a particular outcome. In some embodiments, prevention of an outcome is achieved through prophylactic treatment.

[0084] The term "comparable" as used herein means, for example, that the rate or level being compared resulting from the use of a chimeric polypeptide is equal to, substantially equal to, or similar to, the reference rate or level. The term "similar" as used herein means that the rate or level being compared differs by no more than 10% or no more than 15% from the reference rate or level (e.g., the FXa production rate by a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and processed FVIII, where the processed FVIII is fused to the Fc of one of the two Fc moieties). The term "substantially equal" means that the rate or level being compared differs by no more than 0.01%, 0.5%, or 1% from the reference rate or level.

[0085] As used herein, hemostatic disorder refers to a hereditary or acquired condition characterized by a tendency to bleed spontaneously or as a result of trauma due to impaired or hypoplastic fibrin clot formation. Examples of such disorders include hemophilia. The three major types are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency, or "Christmas disease"), and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, for example, von Willebrand's disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiency or structural abnormality of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, and Bernard-Soulier syndrome, which is a deficiency or defect of GPIb. The receptor for VWF, GPIb, may not function properly, resulting in a lack of primary clot formation (primary hemostasis) and an increased tendency to bleed, as well as Glanzmann-Naegeli thrombasthenia. In liver failure (acute and chronic), the liver produces insufficient clotting factors, which can increase the risk of bleeding.

[0086] As used herein, the "area under the plasma concentration versus time curve (AUC)" is the same as in the art of pharmacology and is based on the rate and extent of absorption of FVIII after administration. The AUC is determined for a specified period, such as 12, 18, 24, 36, 48, or 72 hours, or to infinity using extrapolation based on the slope of the curve. Unless otherwise specified, AUC is determined at infinity. AUC determinations can be performed on a single subject or can be performed on a population of subjects to calculate an average.

[0087] The term "procoagulant activity" refers to the ability of a coagulation factor of the present invention, e.g., a FVIII or FIX protein, to participate in the coagulation cascade in blood in place of a native coagulation factor, e.g., native FVIII or FIX. For example, the recombinant FIX protein of the present invention has procoagulant activity because it can convert factor X (FX) to activated factor X (FXa) in the presence of factor VIII (FVIII), as tested, for example, in a chromogenic assay. In another embodiment, FIX activity is the ability to generate a tenase complex. In another embodiment, FIX activity is the ability to generate thrombin (or a clot).

[0088] All references to the amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are based on Kabat et al., 1991, Sequences of Proteins of Immunological Interest, USDapartment of Public Health, Bethesda, MD, which are incorporated herein by reference in their entireties. FcRn receptors have been isolated from several animal species, including humans. Human FcRn, rat FcRn, and mouse FcRn sequences are known (Story et al., J. Exp. Med. 180:2377 (1994)), which are incorporated herein by reference in their entireties). Fc can include the CH2 and CH3 domains of immunoglobulins, with or without the hinge region of the immunoglobulin. Exemplary Fc variants are provided in WO 2004 / 101740 and WO 2006 / 074199, which are incorporated herein by reference in their entireties.

[0089] As used herein, "hybrid" polypeptides and proteins refer to a combination of a chimeric polypeptide and a second polypeptide. The chimeric polypeptide and the second polypeptide in the hybrid can associate with each other through protein-protein interactions, such as charge-charge or hydrophobic interactions. The chimeric polypeptide and the second polypeptide in the hybrid can associate with each other through disulfide bonds or other covalent bonds. Hybrids are described in International Publication Nos. 2004 / 101740 and 2006 / 074199, each of which is incorporated herein by reference in its entirety. See also U.S. Patent Nos. 7,404,956 and 7,348,004, each of which is incorporated herein by reference in its entirety. The second polypeptide can be a second copy of the same chimeric polypeptide or a non-identical chimeric polypeptide.

[0090] As used herein, an "amino acid corresponding to," "site corresponding to," or "equivalent amino acid" in a protein sequence is identified by alignment to maximize identity or similarity between a first protein sequence, e.g., a FVIII or FIX sequence, and a second protein sequence, e.g., a second FVIII sequence or a second FIX. The number used to identify the equivalent amino acid in the second protein sequence is based on the number used to identify the corresponding amino acid in the first protein sequence.

[0091] The term "insertion site" as used herein refers to the number of amino acid residues in a polypeptide (typically a mature polypeptide, e.g., a mature FVIII polypeptide or a mature FIX polypeptide), or a fragment, variant, or derivative thereof, that is immediately upstream of a position at which a heterologous moiety can be inserted. An "insertion site" is specified as a number, and the number corresponds to the amino acid in the protein that is specified, immediately N-terminal to the insertion position. The insertion positions are numbers in the protein sequence. For example, the phrase "an EGF2 domain comprises a heterologous moiety at an insertion position corresponding to amino acid 105 of a given sequence" indicates that the heterologous moiety is located between the two amino acids corresponding to amino acids 105 and 106 of the sequence. However, one of skill in the art can readily identify corresponding positions in any variant of the designated protein, and the present disclosure is not limited to insertions made only in the variants specifically disclosed in this disclosure. Rather, the insertions disclosed herein can be made in any related variant or fragment thereof that has activity at a position corresponding to the position of the variant disclosed herein.

[0092] As used herein, the phrase "immediately downstream of an amino acid" refers to the position immediately to the right of the terminal carboxyl group of an amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to the position immediately to the right of the terminal amine group of an amino acid. Thus, as used herein, the phrase "between the two amino acids at the insertion site" refers to the position where a heterologous moiety (e.g., a half-life extending moiety) is inserted between two adjacent amino acids.

[0093] As used herein, the terms "inserted," "inserted into," "inserted into," or grammatically related terms refer to the location of a heterologous moiety (e.g., a half-life extending moiety) in a fusion polypeptide relative to the analogous location in a specified protein (e.g., a FVIII protein or a FIX protein). One of skill in the art will understand how to identify corresponding insertion positions for other polypeptide sequences, e.g., other FVIII and FIX variants. As used herein, the terms refer to features of the recombinant polypeptides disclosed herein and do not indicate, imply, or infer any method or process by which the fusion polypeptide is made. For example, with reference to a fusion polypeptide provided herein, the phrase "a heterologous moiety is inserted into the EGF2 domain immediately downstream of residue 105 of the FIX polypeptide" means that the fusion polypeptide contains a heterologous moiety immediately downstream of the amino acid corresponding to amino acid 105 in the particular FIX variant, e.g., adjacent to amino acids corresponding to amino acids 105 and 106 of the FIX variant.

[0094] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. Amino acid sequences normally present in different proteins can be joined together in a fusion polypeptide, or amino acid sequences normally present in the same protein can be placed in a new arrangement in a fusion polypeptide, such as a fusion of an Ig Fc domain with a FVIII or FIX domain of the present invention. Fusion proteins are created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Fusion proteins can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond.

[0095] The terms "heterologous" and "heterologous moiety" mean that a polynucleotide, polypeptide, or other moiety is derived from a separate entity from the moiety of the entity to which it is compared. By way of example, a heterologous polypeptide can be synthetic or derived from a different species, a different cell type of an individual, or the same or different cell type of a separate individual. In one embodiment, a heterologous moiety is a polypeptide fused to another polypeptide to produce a fusion polypeptide or protein. In another embodiment, a heterologous moiety is a non-polypeptide, such as PEG conjugated to a polypeptide or protein.

[0096] The terms "linked" and "fused" as used herein refer to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently linked, respectively, to a second amino acid sequence or nucleotide sequence. The amino acid or nucleotide sequences can be directly linked or juxtaposed, or the first and second sequences can be covalently linked by an intervening sequence. The term "linked" includes not only C- or N-terminal fusion of the first and second amino acid sequences, but also insertion of the entire first amino acid sequence (or second amino acid sequence) into any two amino acids of the second amino acid sequence (or first amino acid sequence, respectively). In one embodiment, the first amino acid sequence is linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or polypeptide (e.g., in the case of a polypeptide chain), or a nucleotide or nucleotide chain (in the case of a nucleotide chain), or any chemical moiety (in the case of both polypeptides and polynucleotide chains). The term "linked" can also be indicated by a hyphen (-).

[0097] As used herein, the term "associated" refers to a covalent or non-covalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term "associated" refers to a covalent, non-peptide bond or a non-covalent bond. This association can be indicated by a colon (:). In another embodiment, it refers to a covalent bond excluding a peptide bond. For example, the amino acid cysteine ​​contains a thiol group that can form a disulfide bond or crosslink with a thiol group on a second cysteine ​​residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by a disulfide bond, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229 in the EU numbering system). Examples of covalent bonds include, but are not limited to, peptide bonds, metal bonds, hydrogen bonds, disulfide bonds, sigma bonds, pi bonds, delta bonds, glycosidic bonds, agonist bonds, bent bonds, coordinate bonds, π back-donation bonds, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation π bonds or salt bonds), metal bonds, hydrogen bonds (e.g., dihydrogen bonds, dihydrogen complexes, low-barrier hydrogen bonds, or symmetric hydrogen bonds), van der Waals forces, London dispersion forces, mechanical bonds, halogen bonds, aurophilicity, intercalation, stacking, entropic forces, or chemical polarity.

[0098] As used herein, the term "cleavage site" or "enzyme cleavage site" refers to a site recognized by an enzyme. Certain enzymatic cleavage sites include intracellular processing sites. In one embodiment, a polypeptide has an enzymatic cleavage site that is cleaved by an enzyme activated during the coagulation cascade, such that cleavage of such a site occurs at the site of clot formation. Exemplary such sites include sites recognized by, for example, thrombin, factor XIa, or factor Xa. Other enzymatic cleavage sites are known in the art.

[0099] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzymatic cleavage site in a polypeptide that is the target of an enzyme that functions after translation of the polypeptide. In one embodiment, such an enzyme functions during transport from the lumenal side of the Golgi apparatus to the trans-Golgi compartment. The intracellular processing enzyme cleaves the polypeptide before the protein is secreted from the cell. Examples of such processing sites include, for example, PACE / furin (PACE is a Paired Basic Amino Acid These include sites targeted by the "furin" family endopeptidases, an acronym for Acid Cleaving Enzyme. These enzymes are localized to the Golgi membrane and cleave proteins on the carboxy-terminal side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein, "furin" family enzymes include, for example, PCSK1 (also known as PC1 / PC3), PCSK2 (also known as PC2), and PCSK3 (also known as PC4). 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.

[0100] In constructs that include more than one processing or cleavage site, it is understood that such sites may be the same or different.

[0101] As used herein, a "processable linker" refers to a linker that includes at least one intracellular processing site as described elsewhere herein.

[0102] As used herein, "baseline" refers to the lowest plasma level measured for a given analyte, e.g., a clotting factor (e.g., FVIII or FIX), in a subject prior to administration of a dose. Plasma levels can be measured at two time points prior to administration: at a screening visit and immediately prior to administration. Alternatively, (a) the baseline for subjects whose pre-treatment clotting factor activity is <1% and who have no detectable clotting factor antigen but who have a nonsense genotype can be defined as 0%, (b) the baseline for subjects whose pre-treatment clotting factor activity is <1% and who have detectable clotting factor antigen can be set at 0.5%, and (c) the baseline for subjects whose pre-treatment clotting factor activity is 1-2% can be set at 0.5%. min (the lowest activity across PK studies), and (d) subjects whose pre-treatment clotting factor activity is ≧2% may have a baseline set at 2%.

[0103] As used herein, "equivalent amount" refers to the same dose of clotting factor activity, e.g., FVIII activity or FIX activity, expressed in international units that are independent of the molecular weight of the polypeptide. For example, one international unit (IU) of FVIII activity corresponds approximately to the amount of FVIII in one milliliter of normal human plasma. Several assays are available for measuring clotting factor activity, including the European Pharmacopoeia chromogenic substrate assay and the one-stage clotting assay.

[0104] As used herein, "dosing interval" refers to the time that elapses between doses administered to a subject. Comparisons of dosing intervals can be made for a single subject or a population of subjects, and average values ​​obtained for the population can be calculated.

[0105] As used herein, "subject" refers to a human individual. The subject may be a patient currently suffering from a bleeding disorder or anticipated to require such treatment. In some embodiments, the subject has not previously been treated with a clotting factor (i.e., the subject is a previously treatment-naive subject or a previously treatment-naive patient). In some embodiments, the subject is a fetus, and the method includes administering a composition, e.g., a chimeric polypeptide, to the mother of the fetus, where administration to the subject occurs from the mother across the placenta. In some embodiments, the subject is a child or an adult. In some embodiments, the subject is a child under 1 year old, under 2 years old, under 3 years old, under 4 years old, under 5 years old, under 6 years old, under 7 years old, under 8 years old, under 9 years old, under 10 years old, under 11 years old, or under 12 years old. In some embodiments, the child is under 1 year old. In certain embodiments, the subject is under 6 years old. In other embodiments, the subject is between 6 and 12 years old. In other embodiments, the subject is 12 years old or older. In some embodiments, the child or adult suffers from a bleeding disorder, and onset of symptoms of the bleeding disorder occurs after age 1. In some embodiments, administration of a composition, e.g., a chimeric polypeptide, to a subject is sufficient to prevent, inhibit, or reduce development of an immune response selected from a humoral immune response, a cellular immune response, or both a humoral immune response and a cellular immune response against a clotting factor.

[0106] As used herein (interchangeably), a "therapeutic dose," "dose," "effective amount," or "administration" refers to a dose that achieves a therapeutic goal as described herein. In some embodiments, a "therapeutic dose" refers to a dose that improves the HJHS, mHJHS, or QoL score compared to the HJHS, mHJHS, or QoL score before treatment. In some embodiments, a "therapeutic dose" refers to a dose that reduces swelling, inflammation, and / or pain in one or more joints of a subject compared to the level of swelling, inflammation, and / or pain in the joints before treatment. In some embodiments, a "therapeutic dose" refers to a dose that reduces the effect and / or severity of one or more microhemorrhages compared to the effect and / or severity of microhemorrhages before treatment. In another embodiment, a "therapeutic dose" refers to a dose that reduces vascular remodeling in one or more target joints compared to the vascular remodeling before treatment.

[0107] Similarly, fragments or variants of polypeptides, and any combination thereof, are also encompassed by the present invention. The term "fragment" or "variant" when referring to a polypeptide used in the methods of the present disclosure includes any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., the clotting activity of an Fc variant or a FVIII or FIX variant). Polypeptide fragments include proteolytic fragments as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere herein, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Variants of polypeptide binding domains or binding molecules used in the methods of the present disclosure include the above-described fragments, as well as polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides can contain conservative or non-conservative amino acid substitutions, deletions, or additions. One particular FIX variant disclosed herein is the R338L FIX (Padua) variant. See, e.g., Simioni, P. et al., "X-Linked Thrombophilia with a Mutant Factor IX (Factor IX Padua)," NEJM 361:1671-75 (October 2009), which is incorporated by reference in its entirety.

[0108] "Conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain.The art defines a family of amino acid residues with similar side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).In this way, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another embodiment, a series of amino acids can be conservatively exchanged for a series of structurally similar amino acids that differ in the order and / or composition of side chain family members.

[0109] The term "percent sequence identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions that the sequences share over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target and reference sequences. Gaps present in the target sequence are not counted, since they are not nucleotides or amino acids. Similarly, gaps present in the reference sequence are not counted, since they are not nucleotides or amino acids in the target sequence. is not a nucleotide or amino acid in the reference sequence and is therefore not counted.

[0110] The percentage of sequence identity is calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of percent sequence identity between two sequences may be performed using software that is readily available both for online use and for download. Suitable software programs for alignment of protein and nucleotide sequences are available from a variety of sources. One suitable program for determining percent sequence identity is the nucleotide sequence identity program provided by the U.S. government's National Center for Biotechnology Information. One suitable program is bl2seq, which is part of the BLAST suite of programs available from the National Institute for Bioinformatics and Systems Sciences (BLAST) website (blast.ncbi.nlm.nih.gov). Bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs, also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0111] Different regions within a single polynucleotide or polypeptide target sequence that are aligned with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Note also that length values ​​are always integers.

[0112] Those skilled in the art will recognize that the generation of sequence alignment for calculating percent sequence identity is not limited to binary sequence-sequence comparison, which is driven only by primary sequence data.Sequence alignment can be derived from multiple sequence alignment.One suitable program for generating multiple sequence alignment is ClustalW2, available from www.clustal.org.Another suitable program is MUSCLE, available from www.drive5.com / muscle / .ClustalW2 and MUSCLE can also be obtained from, for example, EBI.

[0113] It should also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, also available from www.tcoffee.org or EBI. Similarly, it should be appreciated that the final alignment used to calculate percent sequence identity may be curated either automatically or manually.

[0114] Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In one embodiment, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activity of the encoded polypeptide. In another embodiment, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. In other embodiments, variants may contain 5-10, 1-5, or 20 nucleotides. Or one to two amino acids are substituted, deleted, or added in any combination. Polynucleotide variants can be produced for a variety of reasons, such as to optimize codon expression for a particular host (changing codons in human mRNA to those of another host, e.g., a bacterial host such as E. coli).

[0115] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on an organism's chromosome (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at the polynucleotide and / or polypeptide level and are included in the present disclosure. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or direct synthesis.

[0116] Known methods of protein engineering and recombinant DNA technology can be used to generate variants to improve or alter the characteristics of a polypeptide. For example, one or more amino acids can be deleted from the N- or C-terminus of a secreted protein without substantial loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993), incorporated herein by reference in its entirety, reported variant KGF proteins that retained heparin-binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma exhibited up to 10-fold increased activity after deletion of 8 to 10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated herein by reference in its entirety).

[0117] Moreover, ample evidence demonstrates that variants often retain similar biological activity to naturally occurring proteins. For example, Gayle and colleagues (J. Biol. Chem. 268:22105-22111 (1993), incorporated herein by reference in its entirety) conducted an extensive mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants with an average of 2.5 amino acid changes per variant throughout the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The researchers found that "most of the molecule could be altered with little effect on either [binding or biological activity]" (see abstract). In fact, only 23 unique amino acid sequences out of over 3,500 nucleotide sequences examined produced proteins with activity significantly different from the wild-type.

[0118] As described above, polypeptide variants include, for example, modified polypeptides, including, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010)), which is incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. In some embodiments, FVIII is modified, e.g., PEGylated, at any convenient position. In some embodiments, FVIII is PEGylated at a surface-exposed amino acid of FVIII, e.g., a surface-exposed cysteine, which may be an engineered cysteine. Ibid. In some embodiments, the modified FVIII, e.g., PEGylated FVIII, is a chimeric or fusion FVIII.

[0119] The term "downstream" refers to a nucleotide sequence located 3' of a reference nucleotide sequence. "Downstream" can also refer to a peptide sequence located at the C-terminus of a reference peptide sequence.

[0120] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. "Upstream" can also refer to a peptide sequence located at the N-terminus of a reference peptide sequence.

[0121] As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region that influences the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences are usually located 3' of the coding sequence.

[0122] A polynucleotide encoding a gene product, e.g., a polypeptide, can include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. Other transcription control elements besides a promoter, e.g., enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct expression of the gene product.

[0123] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as promoter and enhancer segments from cytomegalovirus (immediate-early promoter with intron A), Simian Virus 40 (early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include regions derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences that can control gene expression in eukaryotic cells. Further suitable transcription control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0124] Similarly, a variety of translational control elements are known to those skilled in the art, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also called CITE sequences).

[0125] The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product, eg, RNA or a polypeptide.

[0126] A "vector" refers to any vehicle for cloning and / or transferring a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be attached so as to bring about replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and non-viral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors are known and used in the art, including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. A polynucleotide is inserted into a suitable vector by ligating an appropriate polynucleotide fragment with complementary cohesive termini into the vector of choice. This can be done.

[0127] The term "plasmid" refers to an extrachromosomal element that often contains genes that are not part of the cell's central metabolism and are usually in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled single- or double-stranded DNA or RNA from any source, in which multiple nucleotide sequences are combined or recombined into a unique configuration that allows the introduction of a promoter fragment and DNA sequence for a selected gene product, along with appropriate 3' untranslated sequences, into a cell.

[0128] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and poxviruses, such as vaccinia virus vectors, baculovirus vectors, or herpes virus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

[0129] "Cloning vector" refers to a "replicon," a unit length of nucleic acid that replicates continuously and contains an origin of replication, such as a plasmid, phage, or cosmid, to which another nucleic acid segment may be attached so as to bring about replication of the attached segment. A particular cloning vector is capable of replicating in one cell type, e.g., bacteria, and expressing in another cell, e.g., a eukaryotic cell. Cloning vectors typically contain one or more sequences that can be used to select cells that contain the vector and / or one or more multiple cloning sites for inserting a nucleic acid sequence of interest.

[0130] The term "expression vector" refers to a vehicle designed to enable expression of an inserted nucleic acid sequence after insertion into a host cell, the inserted nucleic acid sequence being placed in operative association with the regulatory regions described above.

[0131] Vectors are introduced into host cells by methods well known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, lipofection (lysosomal fusion), use of a gene gun, or DNA vector transporters.

[0132] An "isolated" polypeptide, or a fragment, variant, or derivative thereof, refers to a polypeptide that is not present in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0133] As used herein, the term "host cell" refers to a cell or population of cells that contains or is capable of containing a recombinant nucleic acid. Host cells can be prokaryotic cells (e.g., E. coli), or they can be eukaryotic cells, such as fungal cells (e.g., yeast cells such as Saccharomyces cerevisiae, Pichia pastori, or Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g., Sf-9), or mammalian cells (e.g., HEK 293F, CHO, COS-7, NIH-3T3).

[0134] "Volume of distribution at steady state (Vss)" as used herein has the same meaning as the term is used in pharmacology and is the apparent space (volume) into which a drug is distributed. Vss = amount of drug in the body divided by the steady state plasma concentration.

[0135] II. The Methods of the Invention This disclosure is based on the discovery that hemophilic arthropathy can be reversed using clotting factors fused to Fc regions. Hemophilic arthropathy was previously known to be irreversible once it developed, as only MRI could visualize soft tissue changes. Currently known treatment options for individuals with hemophilic arthropathy include surgery to remove the enlarged synovial membrane or sclerosing agents. Sclerosing agents, either radioactive or chemical, can prevent further deterioration of cartilage and bone; however, they cannot reverse hemophilic arthropathy. In knee and hip arthropathy, they have been successful in reducing pain and loss of mobility, despite the failure of other attempts to control synovial hypertrophy. Hilgartner M., Current Opinion in Pediatrics: February 2002, V14, No. 1, pp. 46-49.

[0136] Thus, the present disclosure provides a method for treating hemophilic arthropathy of a joint in a human with hemophilia, comprising administering to the human an effective amount of a composition comprising a coagulation factor and an Fc region, e.g., a chimeric protein, or a polynucleotide encoding the chimeric protein. Treatment of hemophilic arthropathy can partially or completely reverse hemophilic arthropathy (e.g., one or more symptoms of hemophilic arthropathy). Thus, in one embodiment, the present disclosure provides a method for treating a human with hemophilia who has already developed hemophilic arthropathy, e.g., synovitis.

[0137] Generally, hemophilic arthropathy refers to a joint disease that occurs in human subjects with hemophilia as a result of repeated bleeding into the target joints over a long period of time. Spontaneous bleeding into one or more joints is common in hemophilia patients. Joints that have several consecutive bleeding episodes within six months are often called "target joints," and these joints often progress to hemophilic arthropathy.

[0138] Hemophilic arthropathy can present with a variety of symptoms, including, but not limited to, synovial hypertrophy, chronic inflammation (including synovitis), fibrosis, hemosideria, subarticular cyst formation, pain, reduced range of motion, muscle atrophy, joint ankylosis, osteoporosis, cartilage degeneration with joint space collapse, and any combination thereof. Hemophilic arthropathy includes various stages: (i) Stage I, which involves soft tissue swelling but no skeletal abnormalities; (ii) Stage II, which involves epiphyseal overgrowth and osteoporosis but maintains joint integrity. There are no bone cysts or narrowing of the articular cartilage space. Radiographic Stage II is similar to the clinical stage of subacute hemophilic arthropathy; and (iii) Stage III, which involves minimal to moderate joint space narrowing with subchondral cysts. Widening of the intercondylar notch of the knee and the trochlear notch of the ulna may also be present. In the knee, sharpening of the patellar margin may also be present. (iv) Stage IV involves destruction of the articular cartilage with severe narrowing of the joint space. Other bone changes seen in Stage III are more pronounced. And (v) Stage V involves complete loss of the joint space with fibrous ankylosis of the joint. There is marked incongruity of the joint structures with severe irregular hypertrophy of the epiphysis.

[0139] The present disclosure provides that treatment with a composition, e.g., a chimeric protein, comprising a coagulation factor and an Fc region can reduce and / or alleviate the symptoms of reversible hemophilic arthropathy. In some embodiments, the composition, e.g., the chimeric protein, treats one or more stages of hemophilic arthropathy, e.g., stages I, II, and / or III. As used herein, "reversible" hemophilic arthropathy is a manifestation of hemophilic arthropathy that can be partially or completely reversed to its original healthy state after treatment. In contrast, "irreversible" hemophilic arthropathy is a manifestation of hemophilic arthropathy that is persistent and does not improve after treatment. Thus, the methods of the invention can improve, reduce, or alleviate (or partially or completely reverse) one or more symptoms of hemophilic arthropathy, such as synovial hyperplasia, chronic inflammation (including synovitis), hemosideria, subarticular cyst formation, pain, reduced range of motion, swelling, vascular remodeling, or any combination thereof.

[0140] The hemophilic arthropathy (reversible) treated using the method of the present disclosure can affect any joint in the body.In some embodiments, the joint is a weight-bearing joint, such as one or both knees, one or both ankles, one or both hips, one or more foot joints, and any combination thereof.In another embodiment, the joint is a non-weight-bearing joint, such as one or both elbows, one or both shoulders, one or both wrists, one or more hand joints, or any combination thereof.In another embodiment, the joint is a knee.

[0141] In some embodiments, the reversible hemophilic arthropathy includes synovitis. Synovitis refers to inflammation of the synovial membrane around a joint. In some embodiments, synovitis is inflammation of any joint in the body. In some embodiments, synovitis manifests as joint swelling, and the disclosed methods reduce joint swelling. In some embodiments, synovitis manifests as joint pain, and the disclosed methods reduce joint pain. In other embodiments, synovitis manifests as a decrease in joint range of motion, and the disclosed methods increase joint range of motion.

[0142] In other embodiments, reversible hemophilic arthropathy includes microhemorrhages in joints. In some embodiments, reversible hemophilic arthropathy is the result of microhemorrhages. Microhemorrhages refer to very small amounts of bleeding in one or more joints, which, if repeated, can result in hemophilic arthropathy. In some embodiments, reversible hemophilic arthropathy includes acute joint hemorrhages. In some embodiments, reversible hemophilic arthropathy is the result of acute joint hemorrhages. Acute joint hemorrhages refer to more substantial bleeding episodes in one or more joints.

[0143] In certain embodiments, the reversible hemophilic arthropathy comprises inflammation of one or more joints. In some embodiments, the inflammation is present in any joint of the body. In some embodiments, the inflammation manifests as joint swelling, and the methods of the present disclosure reduce joint swelling. In some embodiments, the inflammation manifests as joint pain, and the methods of the present disclosure reduce joint pain. In other embodiments, the inflammation manifests as a decrease in joint range of motion, and the methods of the present disclosure increase joint range of motion. In certain embodiments, the method further provides measuring inflammation of one or more joints before administration of an effective amount of a composition or chimeric protein comprising a coagulation factor and an Fc region. In some embodiments, the method further comprises measuring inflammation of one or more joints after administration of an effective amount of a composition or chimeric protein comprising a coagulation factor and an Fc region.

[0144] In other embodiments, hemophilic arthropathy is evidenced by the expression of one or more biomarkers associated with joint inflammation and / or joint damage.In some embodiments, one or more biomarkers indicating increased joint inflammation and / or joint damage are upregulated in humans.In some embodiments, one or more biomarkers indicating increased joint inflammation and / or joint damage are downregulated in humans.In some embodiments, the human in need of treatment is identified based on the expression of one or more biomarkers associated with increased responsiveness to treatment using the methods of the present disclosure.

[0145] In some embodiments, the methods of the present invention increase the localization of a coagulation factor to one or more target joints. In certain embodiments, a composition or chimeric protein comprising a coagulation factor and an Fc region is localized to a target joint to a greater extent than the coagulation factor alone after administration. In certain embodiments, a composition or chimeric protein comprising a coagulation factor and an Fc region remains localized to one or more target joints for a longer period of time than the coagulation factor alone after administration.

[0146] In yet other embodiments, the methods of the present invention further include identifying a subject exhibiting one or more markers of reversible hemophilic arthropathy and then administering a composition or chimeric protein comprising a coagulation factor and an Fc region.

[0147] In some embodiments, the methods of the present invention prevent or reduce the occurrence of vascular remodeling in the joints of a person with hemophilia. A common factor associated with hemophilic arthropathy is remodeling of the vasculature around joints, particularly target joints. Vascular remodeling can be characterized by increased angiogenesis and increased occurrence of microbleeds in the joint. In some embodiments, the present disclosure provides a method for preventing or reducing the occurrence of vascular remodeling in the joints of a person with hemophilia, comprising administering to the person an effective amount of a composition or chimeric protein comprising a clotting factor and an Fc region. In other embodiments, the present disclosure provides a method for reversing existing vascular remodeling associated with hemophilic arthropathy in the joints of a person with hemophilia, comprising administering to the person an effective amount of a composition or chimeric protein comprising a clotting factor and an Fc region. In some embodiments, vascular remodeling is present in any joint of the body. In certain embodiments, vascular remodeling is present in the target joint. In other embodiments, vascular remodeling is present in a joint other than the target joint. In other embodiments, vascular remodeling is present in weight-bearing joints, such as joints selected from the group consisting of one or both knees, one or both ankles, one or both hips, one or more foot joints, and any combination thereof. In another embodiment, vascular remodeling is present in non-weight-bearing joints, such as joints selected from the group consisting of one or both elbows, one or both shoulders, one or both wrists, one or more hand joints, or any combination thereof. In another embodiment, vascular remodeling is present in the knee. In some embodiments, vascular remodeling is present in muscle. In some embodiments, vascular remodeling is present in the spleen and / or liver.

[0148] In certain embodiments, the method of the present disclosure improves soft tissue around joints in people with hemophilia. Another common pathology of hemophilic arthropathy is overgrowth of soft tissue in joints. In some embodiments, the soft tissue improved by the method of the present disclosure is present in any joint of the body. In certain embodiments, the soft tissue improved by the method of the present disclosure is present in a target joint. In other embodiments, the soft tissue improved by the method of the present disclosure is present in a joint other than the target joint.

[0149] In some embodiments, the methods of the present disclosure reduce the severity of one or more symptoms associated with soft tissue hypergrowth in joints. In some embodiments, soft tissue hypergrowth in joints manifests as joint swelling, and the methods of the present disclosure reduce joint swelling. In some embodiments, soft tissue hypergrowth in joints manifests as joint pain, and the methods of the present disclosure reduce joint pain. In other embodiments, soft tissue hypergrowth in joints manifests as a decrease in joint range of motion, and the methods of the present disclosure increase joint range of motion.

[0150] The methods disclosed herein can be practiced on subjects who have been treated and who show a reduction in hemophilic arthropathy to prevent further development of hemophilic arthropathy in one or more joints. In some embodiments, the methods of the disclosure are applied to subjects to treat existing hemophilic arthropathy in one or more joints and to prevent the development of further hemophilic arthropathy in the same or different joints.

[0151] In some embodiments, the methods of the present disclosure allow for distribution of coagulation factors to tissues outside the plasma compartment as well as within the plasma compartment.

[0152] The methods of the present disclosure improve joint health of one or more joints in humans with hemophilia. Joint health can be measured using any measurement method known in the art. In some embodiments, joint health is measured using the Hemophilia Joint Health Score (HJHS) system (Feldman et al., "Hemophilia Joint Health Score"). See the "HJHS Joint Health Score (HJHS) 2.1," available at http: / / www.wfh.org / en / page.aspx?pid=885 (last accessed November 18, 2016, incorporated herein by reference in its entirety). The HJHS measures joint health in terms of structural regions and function (i.e., impairment) of the joints most commonly affected by bleeding in hemophilia: knees, ankles, and elbows. It was primarily designed for children with hemophilia aged 4-18 years who have mild joint impairment (e.g., treated prophylactically), but can be applied to any population. In some embodiments, the HJHS measures swelling, duration (of swelling), muscle atrophy, joint crepitus, loss of flexion, loss of extension, joint pain, and muscle strength for each elbow, knee, and ankle in a person with hemophilia. In some embodiments, each parameter and assigned a numerical score. In a particular embodiment, the standard HJHS, version 2.1, is used to measure joint health. In some embodiments, swelling is scored from 0 to 3, with 0 being no swelling and 3 being severe swelling. In some embodiments, swelling duration is scored from 0 to 1, with 0 being no swelling or swelling for less than 6 months and 1 being swelling for more than or equal to 6 months. In some embodiments, muscle atrophy is scored from 0 to 2, with 0 being no atrophy and 2 being severe atrophy. In some embodiments, joint crepitus is scored from 0 to 2, with 0 being no joint crepitus and 2 being severe joint crepitus. In some embodiments, loss of flexion is scored from 0 to 3, with 0 being loss of less than 5° of flexion and 3 being loss of more than 20° of flexion. In some embodiments, loss of extension is scored from 0 to 3, with 0 being loss of less than 5° of extension and 3 being loss of more than 20° of extension.In some embodiments, joint pain is scored from 0 to 2, with 0 being no pain throughout active range of motion and 2 being pain throughout active range of motion. In some embodiments, total gait is scored as follows: 0 reflects that all skills are within normal range; 1, 2, and 3 reflect that skills 1, 2, and 3 are not within normal range, respectively; and 4 reflects that no skill is within normal range. In certain embodiments, total gait score is measured based on walking, stair climbing, running, and / or single-leg hopping. In some embodiments, the scores are combined to create a total score. In other embodiments, the individual scores of one or more joints are evaluated as an indicator of the health of one or more joints.

[0153] In other embodiments, a modified HJHS system is used to measure joint health. In some embodiments, the mHJHS differs from the standard HJHS, version 2.1, by collapsing joint pain and gait response options into fewer categories, adding an instability assessment, and providing a lower total score (range, 0-116; 0 indicates normal joint function, 116 indicates severe disease) than the standard HJHS (range, 0-124). Scores within 2 weeks after bleeding were excluded. Scores for joints undergoing surgical intervention were imputed by using an extension of the last observation. To assess annual change, subjects in the rFVIIIFc extension study who had mHJHS data at four time points (baseline in the pivotal phase 3 clinical trial of rFVIIIFc, baseline in the rFVIIIFc extension study, year 1 in the rFVIIIFc extension study, and year 2 in the rFVIIIFc extension study) were included in this post-hoc analysis. Changes in mHJHS scores (negative values ​​indicate improvement) from baseline in the pivotal phase 3 clinical trial of rFVIIIFc to year 2 in the extension study of rFVIIIFc were summarized using descriptive statistics. Changes in HJHS scores were summarized for (1) total score (range, 0–116; by prestudy regimen (prophylactic vs. episodic); by severity of functional impairment based on the initial mHJHS; and by the presence of target joints at baseline; (2) target joints (range, 0–19: sum of all questions pertaining to a single target joint); (3) weight-bearing (e.g., ankle and knee) and non-weight-bearing (e.g., elbow) joints (range, 0–38: sum of bilateral joints at a single location); and (4) individual components (range of motion (range, 0–36: combination of questions "loss of extension [ankle dorsiflexion]" and "loss of flexion [ankle plantarflexion]" for all joints); swelling (range, 0–24: combination of questions "swelling" and "duration of swelling" for all joints); and muscle strength (range, 0–6: sum of all joints)).

[0154] In some embodiments, joint scoring is performed separately for six joints (left ankle-LA, right ankle-RA, left elbow-LE, right elbow-RE, left knee-LK, right knee-RK). In some embodiments, swelling is scored as follows: 0=none; 1=mild; 2=moderate; 3=severe. In some embodiments, swelling duration is scored as follows: 0=no swelling or less than 6 months; 1=more than 6 months. In some embodiments, muscle atrophy is scored as follows: 0=none; 1=mild; 2=severe. In some embodiments, joint friction is scored as follows: 0=none; 1=present. In some embodiments, loss of flexion, including loss of ankle plantar flexion, is scored as follows: 0=none; 1=mild; 2=moderate; 3=severe. In some embodiments, loss of extension, including loss of ankle dorsiflexion, is scored as follows: 0=none; 1=mild; 2=moderate; 3=severe. In some embodiments, instability is scored as follows: 0 = absent; 1 = significant pathological joint laxity. In some embodiments, joint pain is scored as follows: 0 = no pain in either range of motion or end range of motion; 1 = present. In some embodiments, muscle strength is scored as follows: 0 = normal (holding position against gravity and maximum resistance); 1 = minimal reduction (holding position against gravity and moderate resistance, but not maximum resistance); 2 = mild reduction (holding position against gravity or minimum resistance); 3 = moderate reduction (joint can move without gravity); 4 = severe reduction (slight or no muscle contraction). In certain embodiments, the following applies to scoring loss of flexion and loss of extension at the knee and elbow: none = approximately 0-5°; mild = approximately 5-10°; moderate = approximately 11-20°; and severe = approximately >20°.

[0155] In some embodiments, walking is scored once (range 0-2), where 0 = no problem walking or climbing stairs; 1 = no problem walking, but stairs are difficult; and 2 = difficulty walking and stairs.

[0156] In certain embodiments, joint scoring is performed separately for six joints (left ankle - LA, right ankle - RA, left elbow - LE, right elbow - RE, left knee - LK, right knee - RK) according to the following categories and scales (range 0-19 for each joint and 0-114 for all six joints): swelling (0=none; 1=mild; 2=moderate; 3=severe); duration of swelling (0=no swelling or less than 6 months; 1=more than 6 months); muscle atrophy (0=none; 1=mild; 2=severe); joint friction (0=none; 1=present); loss of flexion, including loss of ankle plantar flexion (0=none; 1=mild; 2=moderate; 3=severe); loss of extension, including loss of ankle dorsiflexion (0=none; 1=mild; 2=moderate; 3=severe); instability ( 0 = absent; 1 = significantly pathological joint laxity); joint pain (0 = no pain in either range of motion or end range of motion; 1 = present); muscle strength (0 = normal (holding position against gravity and maximum resistance); 1 = minimal reduction (holding position against gravity and moderate resistance, but not at maximum resistance); 2 = mild reduction (holding position against gravity or minimum resistance); 3 = moderate reduction (joint can move without gravity); 4 = severe reduction (slight or no muscle contraction)); for scoring loss of flexion and loss of extension at the knee and elbow, the following applies: none = approximately 0-5°; mild = approximately 5-10°; moderate = approximately 11-20°; and severe = approximately >20°.

[0157] In some embodiments, the present disclosure provides a method for treating reversible hemophilic arthropathy of a joint in a human with hemophilia, comprising administering to the human an effective amount of a composition or chimeric protein comprising a coagulation factor and an Fc region, wherein the administration improves the human's HJHS score compared to the HJHS score before administration. In some embodiments, the HJHS score is a total HJHS score that includes the sum of all measured joint scores plus the total walking score. In some embodiments, the HJHS score is a total HJHS score that includes the sum of all measured joint scores but does not include the total walking score. In other embodiments, the HJHS score is a score for one or both elbows, one or both knees, one or both ankles, or any combination thereof. In a specific embodiment, the HJHS score is an elbow score. In another embodiment, the HJHS score is a knee score. In another embodiment, the HJHS score is an ankle score. In another embodiment, the HJHS score reflects total walking.

[0158] In some embodiments, the present disclosure provides a method for treating reversible hemophilic arthropathy of a joint in a human with hemophilia, comprising administering to the human an effective amount of a composition or chimeric protein comprising a clotting factor and an Fc region, wherein the administration reduces joint pain in the human. In some embodiments, the joint pain is reduced compared to joint pain before administration. In certain embodiments, the method further comprises measuring joint pain in one or more joints before administering an effective amount of the composition or chimeric protein comprising a clotting factor and an Fc region. In some embodiments, the method further comprises measuring joint pain in one or more joints after administering an effective amount of the composition or chimeric protein comprising a clotting factor and an Fc region.

[0159] In certain embodiments, the effect of the method of the present invention is observed in one or more joints of a human.In some embodiments, the effect of the method of the present invention is observed in at least one joint.In some embodiments, the effect of the method of the present invention is observed in at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 joints.

[0160] In some embodiments, the method of the present disclosure further includes identifying a human in need of treatment, for example, identifying a subject with hemophilic arthropathy. Hemophilic arthropathy can be detected and / or monitored using any method known in the art. In some embodiments, an imaging system is used to detect and / or monitor hemophilic arthropathy in a subject. In some embodiments, the imaging system includes any imaging system known in the art for use in characterizing joints. For example, in certain embodiments, the imaging system includes radiography, magnetic resonance imaging, ultrasound imaging, power Doppler ultrasonography, or any combination thereof.

[0161] In some embodiments, the subject has been previously treated with a clotting factor protein that is not fused to an Fc portion. The clotting factor may be a full-length or mature clotting factor. In some embodiments, such a clotting factor may be ADVATE®, RECOMBINATE®, KOGENATE FS®, HELIXATE FS®, XYNTHA® / REFACTO AB®, HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®, ALPHANATE®, KOATE-DVI®, AFSTYLA®, HYATE:C®, or IDELVION®.

[0162] II.A. Chimeric Proteins The method for treating reversible hemophilic arthropathy disclosed herein is a generally applicable chimeric protein or composition comprising a coagulation factor and an Fc region, wherein the coagulation factor can be any known coagulation factor, fragment, or variant thereof, and the Fc region can be any known Fc region, fragment, or variant thereof. In some embodiments, the composition comprises a chimeric protein comprising a coagulation factor and an Fc region. In some embodiments, the coagulation factor is selected from the group consisting of factor VII (FVII), factor VIIa (FVIIa), factor VIII (FVIII), factor IX (FIX), factor X (FX), von Willebrand factor (VWF), or any combination thereof. Thus, the present disclosure regarding FVIIIFc and FIXFc chimeric polypeptides and their uses is equally applicable to other chimeric polypeptides comprising a coagulation factor portion and an Fc portion. Any coagulation factor, or any fragment or variant thereof, can be used in the disclosed method.

[0163] Without being bound by any theory, it is believed that Fc regions fused to coagulation factors are useful in treating reversible hemophilic arthropathy. Inflammation in hemophilia occurs during bleeding into joints. TNF-α, an inflammatory cytokine involved in hemophilic arthropathy, induces NF-κB signaling, thereby upregulating FcRn expression in human monocytes (Liu et al., J Immunol, 2007, 179(5):2999-3011). Thus, FcRn is upregulated at sites of inflammation and, as part of regulating the inflammatory process, can localize Fc-containing proteins to the site of inflammation by binding to Fc receptors. Fc regions fused to coagulation factors can also interact with inhibitory Fc receptors, which can cause downregulation of immune and inflammatory pathways. For example, rFVIIIFc can block Fc neonatal receptors (FcRn) and activate Fcγ receptors (FcγR), resulting in altered levels of pro- and anti-inflammatory molecules. Thus, in some embodiments, the Fc region of the chimeric protein facilitates localization of the chimeric protein to joints, e.g., sites of inflammation and / or injury and / or damage.

[0164] In other embodiments, the coagulation factor may be a coagulation factor mimic. The coagulation factor mimic can exhibit one or more coagulation factor activities. For example, an antibody or antigen-binding portion thereof can act like FVIII by binding to both factor IX and factor X. If the antibody or antigen-binding portion thereof contains an Fc region, such an antibody or antigen-binding portion thereof can be used for the methods of the present invention. In another embodiment, the coagulation factor is a peptide having FVIII activity.

[0165] In this regard, the present disclosure generally provides a method of treating reversible hemophilic arthropathy in a subject in need thereof, the method comprising administering to the subject a chimeric protein comprising a coagulation factor portion and an Fc portion.

[0166] II.A.1. Factor VIII As used herein, "factor VIII," abbreviated throughout this application as "FVIII," refers to a FVIII polypeptide that is functional in its normal role in coagulation, unless otherwise specified. Thus, the term FVIII includes functional polypeptide variants. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of FVIII functions include, but are not limited to, the ability to activate coagulation, act as a cofactor for factor IX, or convert factor X to activated Xa after forming a tenase complex with factor IX in the presence of Ca and phospholipids. The FVIII protein may be human, porcine, canine, rat, or mouse FVIII protein. Furthermore, comparisons between FVIII derived from humans and other species reveal differences in function. Conserved residues that may be required have been identified (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat. No. 6,251,632). Full-length polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants, and modified forms. Various FVIII amino acid and nucleotide sequences are disclosed, for example, in U.S. Patent Application Publication Nos. 2015 / 0158929 A1, 2014 / 0308280 A1, and 2014 / 0370035 A1 and International Publication No. WO2015 / 106052 A1, each of which is incorporated herein by reference in its entirety. FVIII polypeptides include, for example, full-length FVIII, full-length FVIII-N-terminal Met, mature FVIII (-signal sequence), mature FVIII with an additional Met at the N-terminus, and / or FVIII with a complete or partial deletion of the B domain. FVIII variants include deletions of the B domain, whether partial or complete deletions.

[0167] In some embodiments, the FVIII of the chimeric protein or composition of the present disclosure comprises a B-domain-deleted FVIII. As used herein, the "B domain" of FVIII is the same as a B domain known in the art, defined by internal amino acid sequence identity and the site of proteolytic cleavage by thrombin, e.g., residues Ser741 to Arg1648 of mature human FVIII. Other human FVIII domains are defined by the following amino acid residues relative to mature human FVIII: A1, residues Ala1 to Arg372; A2, residues Ser373 to Arg740; A3, residues Ser1690 to Ile2032; C1, residues Arg2033 to Asn2172; and C2, residues Ser2173 to Tyr2332 of mature FVIII. Residue numbers in sequences used herein without reference to a SEQ ID NO correspond to the FVIII sequence without the signal peptide sequence (19 amino acids), unless otherwise indicated. The A3-C1-C2 sequence, also known as the FVIII heavy chain, includes residues Ser1690 to Tyr2332. The remaining sequence, residues Glu1649 to Arg1689, is commonly referred to as the FVIII light chain activation peptide. The locations of all domain boundaries, including the B domain, for porcine, mouse, and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted ("B-domain deleted FVIII" or "BDD FVIII"). An example of BDD FVIII is REFACTO® (recombinant BDD FVIII). In one specific embodiment, the B-domain deleted FVIII variant contains a deletion of amino acid residues 746 to 1648 of mature FVIII.

[0168] The "B domain deleted FVIII" may have a complete or partial deletion as disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563 and International Publication No. WO2015106052A1 (PCT / US2015 / 010738). In some embodiments, the B-domain deleted FVIII sequence used in the methods of the disclosure comprises any one of the deletions disclosed in column 4, lines 4-5, lines 28 and Examples 1-5 of U.S. Patent No. 6,316,226 (also U.S. Patent No. 6,346,513). In another embodiment, the B-domain deleted factor VIII is S743 / Q1638 B-domain deleted factor VIII (SQ BDD FVIII) (e.g., factor VIII having a deletion of amino acids 744 to 1637, e.g., factor VIII having amino acids 1-743 and amino acids 1638-2332 of mature FVIII). In some embodiments, the B domain deleted FVIII used in the methods of the present disclosure is a B domain deleted FVIII as disclosed in column 2, lines 26-51 and Examples 5-8 of U.S. Pat. No. 5,789,203 (U.S. Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620). In some embodiments, the B-domain deleted factor VIII has a deletion as described in U.S. Pat. No. 5,972,885, column 1, line 25 to column 2, line 40; U.S. Pat. No. 6,048,720, column 6, lines 1-22 and Example 1; U.S. Pat. No. 5,543,502, column 2, lines 17-46; U.S. Pat. No. 5,171,844, column 4, lines 22 to column 5, line 36; U.S. Pat. No. 5,112,950, column 2, lines 55-68, No. 4,868,112; column 2, lines 2-19, line 21, and Table 2 of U.S. Pat. No. 4,868,112; column 2, lines 1-3, line 19, column 3, lines 40-4, line 67, column 7, lines 43-8, line 26, and column 11, lines 5-13, line 39 of U.S. Pat. No. 7,041,635; or column 4, lines 25-53 of U.S. Pat. No. 6,458,563. In some embodiments, the B-domain-deleted FVIII has a deletion of much of the B domain, as described in WO 91 / 09122, but still contains the amino-terminal sequence of the B domain that is essential for in vivo proteolytic processing of the primary translation product into two polypeptide chains. In some embodiments, B-domain deleted FVIII is constructed using a deletion of amino acids 747-1638, i.e., a substantially complete deletion of the B domain. Hoeben RC et al., J. Biol. Chem. 265(13):7318-7323 (1990). B-domain deleted factor VIII may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P. et al., Protein Eng. 2(4):301-6 (1988).Additional B domain deletions that are part of the present invention include amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. USA (1986) 83, pp. 5939-5942), 797-1562 (Eaton et al., Biochemistry (1986) 25:8343-8347), 741-1646 (Kaufman (PCT Publication No. WO 87 / 04187)), 747-1560 (Sarver et al., DNA (1987) 6:553-564), 741-1648 (Pasek (PCT Publication 88 / 00831)), or 816-1598 or 741-1648 (Lagner (Behring Inst.Mitt.(1988)82:16-25; EP295597). In one specific embodiment, the B domain deleted FVIII comprises a deletion of amino acid residues 746 to 1648 of mature FVIII. In another embodiment, the B domain deleted FVIII comprises a deletion of amino acid residues 745 to 1648 of mature FVIII.

[0169] In other embodiments, the BDD FVIII comprises a FVIII polypeptide containing a fragment of the B domain that retains one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or, optionally, 943, corresponding to the amino acid sequence of the full-length FVIII sequence. Examples of B domain fragments include 226 or 163 amino acids of the B domain disclosed in Miao, HZ et al., Blood 103(a):3412-3419 (2004), Kasuda, A et al., J. Thromb. Haemost. 6:1352-1359 (2008), and Pipe, SW et al., J. Thromb. Haemost. 9:2235-2242 (2011) (i.e., the first 226 or 163 amino acids of the B domain are retained). In yet other embodiments, the BDD FVIII further comprises a point mutation at residue 309 (Phe to Ser) to improve expression of the BDD FVIII protein. In yet other embodiments, the BDD FVIII comprises a FVIII polypeptide containing a portion of the B domain but not one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, SW et al., J. Thromb. Haemost. 9:2235-2242 (2011). In some embodiments, the BDD FVIII includes single-chain FVIII (also known as rVIII-SingleChain and AFSTYLA®) containing a deletion between amino acids 765 and 1652 corresponding to mature, full-length FVIII. See U.S. Patent No. 7,041,635. Each of the deletions can be made in any FVIII sequence.

[0170] As discussed above and below, many functional FVIII variants are known. Furthermore, hundreds of non-functional mutations in FVIII have been identified in hemophilia patients, and it has been determined that the effects of these mutations on FVIII function are largely due to their location within the three-dimensional structure of FVIII, rather than the nature of the substitution (Cutler et al., Hum. Mutat. 19:274-8 (2002), which is incorporated herein by reference in its entirety). Furthermore, comparisons between human and other species-derived FVIII have identified conserved residues that may be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998), which is incorporated herein by reference in its entirety; U.S. Patent No. 6,251,632).

[0171] In some embodiments, an effective amount of a composition or chimeric protein comprising FVIII and an Fc region is equivalent to an effective amount of FVIII without an Fc region. In certain embodiments, the effective amount is about 10 IU / kg to about 300 IU / kg. In some embodiments, the effective amount is about 20 IU / kg to about 300 IU / kg. In some embodiments, an effective amount is about 20 IU / kg to about 250 IU / kg, about 20 IU / kg to about 200 IU / kg, about 20 IU / kg to about 190 IU / kg, about 20 IU / kg to about 180 IU / kg, about 20 IU / kg to about 170 IU / kg, about 20 IU / kg to about 160 IU / kg, about 20 IU / kg to about 150 IU / kg, about 20 IU / kg to about 140 IU / kg, about 20 IU / kg to about 130 IU / kg, or about 20 IU / kg to about 120 IU / kg. , about 20 IU / kg to about 110 IU / kg, about 20 IU / kg to about 100 IU / kg, about 20 IU / kg to about 90 IU / kg, about 20 IU / kg to about 80 IU / kg, about 20 IU / kg to about 70 IU / kg, about 20 IU / kg to about 60 IU / kg, about 25 IU / kg to about 100 IU / kg, about 25 IU / kg to about 90 IU / kg, about 25 IU / kg to about 80 IU / kg, about 25 IU / kg to about 70 IU / kg, or about 25 IU / kg to about 65 IU / kg. In one specific embodiment, the effective amount is about 20 IU / kg to about 100 IU / kg. In another embodiment, the effective amount is about 25 IU / kg to about 65 IU / kg. In other embodiments, the effective amount is about 20 IU / kg to about 100 IU / kg, about 30 IU / kg to about 100 IU / kg, about 40 IU / kg to about 100 IU / kg, about 50 IU / kg to about 100 IU / kg, about 60 IU / kg to about 100 IU / kg, about 70 IU / kg to about 100 IU / kg, about 80 IU / kg to about 100 IU / kg, about 90 IU / kg to about 100 IU / kg, about 100 IU / kg to about 100 IU / kg, about 110 IU / kg to about 100 IU / kg, about 120 IU / kg to about 100 IU / kg, about 130 IU / kg to about 100 IU / kg, about 140 IU / kg to about 100 IU / kg, about 150 IU / kg to about 100 IU / kg, about 160 IU / kg to about 100 IU / kg, about 170 IU / kg to about 100 IU / kg, about 180 IU / kg to about 100 IU / kg, about 190 IU / kg to about 100 IU / kg, about 200 IU / kg to about 100 IU / kg, about 210 IU / kg to about 100 IU / kg, about 220 IU / kg to about 100 IU / kg, about 230 IU / kg to about 100 IU / kg, about 240 IU / kg to about 100 IU / kg, about 250 IU / kg to about 100 IU / kg, about 260 IU / kg to about 100 IU / kg, about 270 IU / kg to about The dose is 0 IU / kg to about 100 IU / kg, about 20 IU / kg to about 90 IU / kg, about 20 IU / kg to about 80 IU / kg, about 20 IU / kg to about 70 IU / kg, about 20 IU / kg to about 60 IU / kg, about 20 IU / kg to about 50 IU / kg, about 20 IU / kg to about 40 IU / kg, or about 20 IU / kg to about 30 IU / kg.

[0172] In some embodiments, an effective amount is 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 105 IU / kg, about 110 IU / kg, or about 115 IU / kg. , about 120 IU / kg, about 125 IU / kg, about 130 IU / kg, about 135 IU / kg, about 140 IU / kg, about 145 IU / kg, about 150 IU / kg, about 155 IU / kg, about 160 IU / kg, about 165 IU / kg, about 170 IU / kg, about 175 IU / kg, about 180 IU / kg, about 185 IU / kg, about 190 IU / kg, about 195 IU / kg, about 200 IU / kg, about 225 IU / kg, about 250 IU / kg, about 275 IU / kg, or about 300 IU / kg. In one particular embodiment, the effective amount is about 50 IU / kg. In another embodiment, the effective amount is about 100 IU / kg. In another embodiment, the effective amount is about 200 IU / kg.

[0173] When administering a composition or chimeric protein comprising FVIII and an Fc region or a fragment thereof, the dosing interval may be at least about 1.5 times longer than the dosing interval required for an equivalent amount of a coagulation factor that does not contain an Fc domain. The dosing interval may be at least about 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than the dosing interval required for an equivalent amount of FVIII that does not contain an Fc domain.

[0174] In some embodiments, an effective dose of a composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, or about 24 days. In some embodiments, an effective dose of a composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 45 days, or about 60 days.

[0175] In some embodiments, the composition or chimeric protein comprising FVIII and an Fc region is administered at a dosing interval of about 1 to about 14 days, about 1 to about 13 days, about 1 to about 12 days, about 1 to about 11 days, about 1 to about 10 days, about 1 to about 9 days, about 1 to about 8 days, about 1 to about 7 days, about 1 to about 6 days, about 1 to about 5 days, about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about 14 days, about 3 to about 14 days, about 4 to about 14 days, about 5 to about 14 days, about 6 to about 14 days, about 7 to about 14 days, about 8 to about 14 days, about 9 to about 14 days, about 10 to about 14 days, about 11 to about 14 days, about 12 to about 14 days, about 13 to about 14 days, or about 5 to about 10 days. In other embodiments, the composition or chimeric protein comprising FVIII and an Fc region is expressed for about 1 to about 21 days, about 1 to about 20 days, about 1 to about 19 days, about 1 to about 18 days, about 1 to about 17 days, about 1 to about 16 days, about 1 to about 15 days, about 1 to about 14 days, about 1 to about 13 days, about 1 to about 12 days, about 1 to about 11 days, about 1 to about 10 days, about 1 to about 9 days, about 1 to about 8 days, about 1 to about 7 days, about 1 to about 6 days, about 1 to about 5 days, about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about In a specific embodiment, the composition or chimeric protein comprising FVIII and an Fc region is administered at a dosing interval of about 2 to about 21 days, about 3 to about 21 days, about 4 to about 21 days, about 5 to about 21 days, about 6 to about 21 days, about 7 to about 21 days, about 8 to about 21 days, about 9 to about 21 days, about 10 to about 21 days, about 11 to about 21 days, about 12 to about 21 days, about 13 to about 21 days, about 14 to about 21 days, about 15 to about 21 days, about 16 to about 21 days, about 17 to about 21 days, about 18 to about 21 days, about 19 to about 21 days, about 20 to about 21 days, about 5 to about 10 days, about 10 to about 15 days, or about 15 to about 20 days. In another embodiment, the chimeric protein comprising FVIII and an Fc region is administered at a dosing interval of about 3 to about 5 days.

[0176] In one embodiment, the effective amount is 25-65 IU / kg (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 62, 64, or 65 IU / kg), and the dosing interval is once every 3-5, 3-6, 3-7, 3, 4, 5, 6, 7, or 8 days or more, or three times per week, or less than three times per week. In another embodiment, the effective amount is 65 IU / kg, and the dosing interval is once per week, or once every 6-7 days. Doses can be administered repeatedly for as long as necessary (e.g., at least 10, 20, 28, 30, 40, 50, 52, or 57 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years). In one particular embodiment, the effective dose is about 25-65 IU / kg, and the dosing interval is once every 3-5 days.

[0177] A composition or chimeric protein comprising FVIII and an Fc region can be administered, for example, topically (e.g., They can be formulated for any suitable mode of administration, including oral, buccal, nasal, vaginal, rectal or parenteral administration (e.g., transdermal or ocular).

[0178] The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial, and intraperitoneal injection, as well as any similar injection or infusion technique. The composition may also be, for example, a suspension, emulsion, sustained-release formulation, cream, gel, or powder. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0179] In one example, the pharmaceutical formulation is a liquid formulation, for example, a buffered isotonic aqueous solution. In another example, the pharmaceutical composition has a pH that is physiological or close to physiological conditions. In another example, the aqueous formulation has an osmolality and salt concentration that is physiological or close to physiological conditions. It may contain sodium chloride and / or sodium acetate.

[0180] In some embodiments, the chimeric protein comprising FVIII and an Fc region used in the methods of the present invention is formulated in a pharmaceutical composition comprising: (a) the chimeric polypeptide; (b) one or more stabilizers selected from sucrose, trehalose, raffinose, arginine, or a mixture thereof; (c) sodium chloride (NaCl); (d) L-histidine; (e) calcium chloride; and (f) polysorbate 20 or polysorbate 80. In certain embodiments, the pharmaceutical composition comprises (a) 50 IU / ml to 2500 IU / ml of the chimeric polypeptide; (b) 10 mg / ml to 25 mg / ml of sucrose; (c) 8.8 mg / ml to 14.6 mg / ml of sodium chloride (NaCl); (d) 0.75 mg / ml to 2.25 mg / ml of L-histidine; (e) 0.75 mg / ml to 1.5 mg / ml of calcium chloride dihydrate; and (f) 0.08 mg / ml to 0.25 mg / ml of polysorbate 20 or polysorbate 80. In some examples, the pharmaceutical composition used in the methods of the present disclosure is lyophilized.

[0181] In some embodiments, the pharmaceutical composition does not comprise immune cells. In some embodiments, the pharmaceutical composition does not comprise cells.

[0182] II.A.2. Factor IX Human factor IX (FIX) is a serine protease that is a key component of the intrinsic pathway of the blood coagulation cascade. As used herein, "factor IX" or "FIX" refers to coagulation factor proteins and species and their sequence variants, including, but not limited to, the 461 single-chain amino acid sequence of the human FIX precursor polypeptide ("prepro"), the 415 single-chain amino acid sequence of mature human FIX, and the R338L FIX (Padua) variant. FIX includes any form of the FIX molecule that possesses the typical characteristics of blood coagulation. As used herein, "Factor IX" and "FIX" are intended to encompass polypeptides comprising the domain Gla (the region containing gamma-carboxyglutamic acid residues), EGF1 and EGF2 (regions containing sequences homologous to human epidermal growth factor), the activation peptide ("AP" formed by residues R136-R180 of mature FIX), and the C-terminal protease domain ("Pro"), or synonyms of these domains known in the art, or may be truncated fragments or sequence variants that retain at least some of the biological activity of the native protein.

[0183] FIX or sequence variants have been cloned as described in U.S. Pat. Nos. 4,770,999 and 7,700,734, and cDNA encoding human factor IX has been isolated, characterized, and cloned into expression vectors (e.g., Choo et al., Nature, 299:178-180 (1982); Fair et al., Blood, 64:194-204 (1984); and Kurachi et al., Proc. Natl. Acad. Sci., 1999:141-142 (1985)). (See, e.g., J. Acad. Sci. USA 79:6461-6464 (1982)). One particular variant of FIX, the R339L FIX (Padua) variant, characterized by Simioni et al., 2009, contains a gain-of-function mutation that correlates with an approximately eight-fold increase in activity of the Padua variant compared to native FIX. A FIX variant may also include any FIX polypeptide with one or more consecutive amino acid substitutions that do not affect the FIX activity of the FIX polypeptide.

[0184] In some embodiments, FIX comprises Coagulation Factor IX (recombinant), Albumin Fusion Protein (also known as rIX-FP and IDELVION®).

[0185] The FIX polypeptide is 55 kDa and is synthesized as a prepropolypeptide chain consisting of three regions: a 28-amino acid signal peptide (amino acids 1–28), an 18-amino acid propeptide (amino acids 29–46) required for gamma-carboxylation of glutamic acid residues, and the 415-amino acid mature factor IX. The propeptide is an 18-amino acid sequence N-terminal to the gamma-carboxyglutamic acid domain. After binding to vitamin K-dependent gamma-carboxylase, the propeptide is cleaved from the FIX precursor polypeptide by an endogenous protease, most likely PACE (paired basic amino acid cleaving enzyme), also known as furin or PCSK3. Without gamma-carboxylation, the Gla domain cannot adopt the correct conformation required to bind calcium and anchor the protein to negatively charged phospholipid surfaces, thereby rendering factor IX nonfunctional. Even though it is carboxylated, the Gla domain also depends on cleavage of the propeptide for proper function, because the retained propeptide inhibits the conformational changes in the Gla domain necessary for optimal binding to calcium and phospholipids. In humans, the resulting mature factor IX is secreted into the bloodstream from liver cells as an inactive zymogen, a single-chain protein of 415 amino acid residues containing approximately 17% carbohydrate by weight (Schmidt, AE et al. (2003) Trends Cardiovasc Med 13:39).

[0186] Mature FIX consists of several domains, from N to C terminus: the GLA domain, the EGF1 domain, the EGF2 domain, the activation peptide (AP) domain, and the protease (or catalytic) domain. A short linker connects the EGF2 domain and the AP domain. FIX contains two activation peptides, R145-A146 and R180-V181, respectively. After activation, single-chain FIX becomes a two-chain molecule, with the two chains linked by a disulfide bond. Coagulation factors can be engineered by replacing their activation peptides, which alters activation specificity. In mammals, mature FIX is expected to be activated by activated factor XI to yield factor IXa. The protease domain provides the catalytic activity of FIX during activation of FIX to FIXa. Activated factor VIII (FVIIIa) is a specific cofactor for full expression of FIXa activity.

[0187] In other embodiments, the FIX polypeptide comprises the Thr148 allelic form of plasma-derived factor IX and has structural and functional characteristics similar to endogenous factor IX.

[0188] Numerous functional FIX variants are known. WO 02 / 040544 A3 discloses, on page 4, lines 9-30 and on page 15, lines 6-31, mutants that exhibit increased resistance to inhibition by heparin. WO 03 / 020764 A2 discloses, in Tables 2 and 3 (pages 14-24) and on page 12, lines 1-27, FIX mutants with reduced T-cell immunogenicity. WO 2007 / 149406 A2 discloses, on page 4, lines 1-19, line 11, FIX mutants with increased protein stability. and functional FIX molecule variants exhibiting increased activity, increased in vivo and in vitro half-lives, and increased resistance to proteases. WO 2007 / 149406 A2 also discloses chimeric and other variant FIX molecules on page 19, line 12 to page 20, line 9. WO 08 / 118507 A2 discloses FIX variants exhibiting increased coagulation activity on page 5, line 14 to page 6, line 5. WO 09 / 051717 A2 discloses FIX variants with an increased number of N-linked and / or O-linked glycosylation sites, resulting in increased half-life and / or recovery, on page 9, line 11 to page 20, line 2. International Publication No. WO 09 / 137254 A2 also discloses factor IX variants with an increased number of glycosylation sites on page 2, paragraphs

[0006] to

[0011] , and on pages 16,

[0044] to

[0057] . International Publication No. WO 09 / 130198 A2 discloses functional FIX molecule variants with an increased number of glycosylation sites, resulting in an increased half-life, on page 4, line 26 to page 12, line 6. International Publication No. WO 09 / 140015 A2 discloses functional FIX variants with an increased number of Cys residues, which can be used for polymer (e.g., PEG) conjugation, on page 11, paragraphs

[0043] to

[0053] . The FIX polypeptides described in International Application No. PCT / US2011 / 043569, filed July 11, 2011, and published January 12, 2012 as WO2012 / 006624, are also incorporated herein by reference in their entirety.

[0189] Additionally, hundreds of non-functional mutations in FIX have been identified in hemophilia subjects, many of which are disclosed in Table 5 on pages 11-14 of WO 09 / 137254 A2. Such non-functional mutations are not included in the present invention, but provide further guidance as to which mutations are likely to result in a more or less functional FIX polypeptide.

[0190] Factor IX clotting activity is expressed as international units (IU). 1 IU of FIX activity corresponds approximately to the amount of FIX in 1 milliliter of normal human plasma. Several assays are available for measuring factor IX activity, such as the one-stage clotting assay (activated partial thromboplastin time; aPTT), thrombin generation time (TGA), and rotational thromboelastometry (ROTEM®). The present invention contemplates sequences that have homology to the FIX sequence, naturally occurring sequence fragments, such as those derived from humans, non-human primates, mammals (such as livestock), and non-naturally occurring sequence variants that retain at least some of the biological activity or function of FIX, and / or are useful for preventing, treating, mediating, or ameliorating coagulation factor-related diseases, deficiencies, disorders, or conditions (e.g., bleeding episodes associated with coagulation factor deficiencies, trauma, surgery). Sequences with homology to human FIX can be found by standard homology search techniques, such as NCBI BLAST.

[0191] In some embodiments, an effective amount of a composition or chimeric protein comprising FIX and an Fc region is equivalent to an effective amount of FIX without an Fc region. In certain embodiments, the effective amount is about 0.1 IU / kg to about 500 IU / kg. In some embodiments, the effective amount is about 10 IU / kg to about 400 IU / kg. In some embodiments, the effective amount is about 20 IU / kg to about 300 IU / kg. In some embodiments, the effective amount is about 20 IU / kg to about 275 IU / kg, about 20 IU / kg to about 250 IU / kg, about 20 IU / kg to about 225 IU / kg, about 20 IU / kg to about 200 IU / kg, about 20 IU / kg to about 175 IU / kg, about 20 IU / kg to about 150 IU / kg, about 20 IU / kg to about 100 IU / kg, or about 2 0IU / kg ~ approx. 90IU / kg, approx. 20IU / kg ~ approx. 80IU / kg, approx. 20IU / kg ~ approx. 70IU / kg, approx. 20IU / kg ~ approx. 60IU / kg, approx. 20IU / kg ~ approx. 50IU / kg, approx. 20IU / kg ~ approx. 40IU / kg, approx. 20IU / kg ~ approx. 30IU / kg, approx. 30IU / kg ~ approx. 100IU / kg, approx. 40IU / k In one specific embodiment, the effective dose is about 20 IU / kg to about 100 IU / kg.

[0192] In some embodiments, an effective amount is 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, or about 105 IU / kg. , about 110 IU / kg, about 115 IU / kg, about 120 IU / kg, about 125 IU / kg, about 130 IU / kg, about 135 IU / kg, about 140 IU / kg, about 145 IU / kg, about 150 IU / kg, about 155 IU / kg, about 160 IU / kg, about 165 IU / kg, about 170 IU / kg, about 175 IU / kg, about 180 IU / kg, about 185 IU / kg, about 190 IU / kg, about 195 IU / kg, or about 200 IU / kg. In one particular embodiment, the effective amount is about 50 IU / kg. In another embodiment, the effective amount is about 100 IU / kg.

[0193] When administering a composition or chimeric protein comprising FIX and an Fc region or fragment thereof, the dosing interval may be at least about 1.5 times longer than the dosing interval required for an equivalent dose of the FIX without the Fc domain. The dosing interval may be at least about 1.5-6 times, 1.5-5 times, 1.5-4 times, 1.5-3 times, or 1.5-2 times longer than the dosing interval required for an equivalent dose of the FIX without the Fc domain. In some embodiments, the dosing interval is at least about 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, or 6 times longer than the dosing interval required for an equivalent dose of the FIX without the Fc domain. The dosing interval may be about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or longer. The interval between doses may be at least about 1.5 to 5 days, 1.5 days, 2 days, 3 days, 4 days, or 5 days, or longer. For on-demand treatment, the chimeric polypeptide or hybrid is administered at intervals of about once every 24 to 36, 24 to 48, 24 to 72, 24 to 96, 24 to 120, 24 to 144, 24 to 168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours, or longer.

[0194] In some embodiments, an effective dose of a composition or chimeric protein comprising FIX and an Fc region is administered to a human at an interval of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days. In some embodiments, an effective dose of a composition or chimeric protein comprising FVIII and an Fc region is administered to a human at an interval of about 25, 26, 27, 28, 29, 30, 45, or 60 days. In certain embodiments, an effective dose of a composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In one particular embodiment, an effective dose of a composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of about 2 days (e.g., about 48 hours). In another embodiment, an effective dose of a composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of about 2 days (e.g., about 48 hours). An effective dose of the chimeric protein is administered to a human at a dosing interval of about 7 days. In another embodiment, an effective dose of the composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of about 10 days. In some embodiments, an effective dose of the composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of every 6 to 10 hours. In certain embodiments, an effective dose of the composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a daily dosing interval.

[0195] In some embodiments, the composition or chimeric protein comprising FVIII and an Fc region is expressed for about 1 to about 21 days, about 1 to about 20 days, about 1 to about 19 days, about 1 to about 18 days, about 1 to about 17 days, about 1 to about 16 days, about 1 to about 15 days, about 1 to about 14 days, about 1 to about 13 days, about 1 to about 12 days, about 1 to about 11 days, about 1 to about 10 days, about 1 to about 9 days, about 1 to about 8 days, about 1 to about 7 days, about 1 to about 6 days, about 1 to about 5 days, about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about The administration interval is about 21 days, about 3 to about 21 days, about 4 to about 21 days, about 5 to about 21 days, about 6 to about 21 days, about 7 to about 21 days, about 8 to about 21 days, about 9 to about 21 days, about 10 to about 21 days, about 11 to about 21 days, about 12 to about 21 days, about 13 to about 21 days, about 14 to about 21 days, about 15 to about 21 days, about 16 to about 21 days, about 17 to about 21 days, about 18 to about 21 days, about 19 to about 21 days, about 20 to about 21 days, about 5 to about 10 days, about 10 to about 15 days, or about 15 to about 20 days.

[0196] In one embodiment, an effective dose is one that can maintain circulating FIX of 1 to 50 IU / dL (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 IU / dL). In another embodiment, an effective dose is 50 IU / kg and the administration interval is once weekly. In another embodiment, an effective dose is 100 IU / kg and the administration interval is once every 10 days. Doses can be administered repeatedly for as long as necessary (e.g., at least 10, 20, 28, 30, 40, 50, 52, or 57 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years).

[0197] A composition or chimeric protein comprising FIX and an Fc region can be formulated for any suitable mode of administration, including, for example, topical (e.g., transdermal or intraocular), oral, buccal, nasal, vaginal, rectal, or parenteral administration.

[0198] The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial, and intraperitoneal injection, as well as any similar injection or infusion technique. The composition may be, for example, a suspension, emulsion, sustained-release formulation, cream, gel, or powder. The composition may be formulated as a suppository, using traditional binders and carriers such as triglycerides.

[0199] In one example, the pharmaceutical formulation is a liquid formulation, such as a buffered, isotonic, aqueous solution. In another example, the pharmaceutical composition has a physiological or near-physiological pH. In another example, the aqueous formulation has a physiological or near-physiological osmolality and salt concentration. It may contain sodium chloride and / or sodium acetate.

[0200] In some embodiments, the chimeric protein comprising FIX and an Fc region used in the methods of the present invention is formulated in a pharmaceutical composition comprising: (a) the chimeric polypeptide; (b) a carbohydrate mixture comprising sucrose and mannitol; (c) sodium chloride (NaCl); (d) L-histidine; and (e) polysorbate 20 or polysorbate 80. In certain embodiments, the pharmaceutical composition comprises: (a) about 25 IU / ml to about 700 IU / ml of Factor IX polypeptide; (b) about 10 mg / ml to about 20 mg / ml of sucrose. (c) about 20 mg / ml to about 40 mg / ml of mannitol; (d) about 3 mg / ml to about 4 mg / ml of NaCl; (e) about 3 mg / ml to about 6 mg / ml of L-histidine; (f) about 0.08 mg / ml to about 0.2 mg / ml of polysorbate 20 or polysorbate 80; or (g) any combination thereof. In some examples, the pharmaceutical compositions used in the methods of the present disclosure are lyophilized.

[0201] In some embodiments, the pharmaceutical composition does not comprise immune cells. In some embodiments, the pharmaceutical composition does not comprise cells.

[0202] II.A.3 Fc The compositions or chimeric proteins of the present disclosure comprise an Fc domain or a portion thereof that binds to FcRn, FcγRIIB, and / or FC-SIGN. In some embodiments, the Fc domain is fused to a clotting factor, e.g., as part of a chimeric protein comprising the clotting factor and an Fc region. In other embodiments, the Fc domain is fused to a polypeptide other than a clotting factor, wherein the composition comprises (1) a clotting factor and (2) a chimeric protein comprising an Fc domain and an additional polypeptide. In some embodiments, the Fc domain is coadministered with the clotting factor. The Fc domain or a portion thereof can improve the pharmacokinetic or pharmacodynamic properties of the chimeric protein. In certain embodiments, the Fc domain or a portion thereof extends the half-life of a molecule fused to the Fc domain or a portion thereof. In some embodiments, the Fc region of the chimeric protein facilitates localization of the chimeric protein to joints.

[0203] As used herein, the term "Fc domain" or "Fc region" refers to a functional portion of a polypeptide corresponding to the Fc domain of a native Ig, formed by the dimeric association of the Fc domains of each of its two heavy chains. A native Fc domain forms a homodimer with another Fc domain. In contrast, as used herein, the term "genetically fused Fc region" or "single-chain Fc region" (scFv region) refers to a synthetic dimeric Fc region comprising Fc domains genetically linked (i.e., encoded in a single contiguous gene sequence) within a single polypeptide chain.

[0204] In one embodiment, "Fc region" refers to the portion of a single IgG heavy chain beginning at the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in an IgG, if the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc domain includes at least the hinge, CH2, and CH3 domains.

[0205] The Fc region of an Ig constant region may contain CH2, CH3, and CH4 domains, as well as a hinge region, depending on the Ig isotype. Chimeric proteins containing an Ig Fc region confer several desirable properties on the chimeric protein, including increased stability, increased serum half-life (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) (these documents are incorporated herein by reference in their entirety).

[0206] In some embodiments, the Fc region specifically binds to FcRn. 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). FcRn receptors bind IgG (but not other Ig classes, such as IgA, IgM, IgD, and IgE) at relatively low pH and bind to the lumen. It actively transports IgG intercellularly toward the serosal membrane and then releases it at the relatively high pH found in interstitial fluid. It is expressed in adult epithelial tissues, including lung and intestinal epithelium (Israel et al., 1997, Immunology 92:69), renal proximal tubular epithelium (Kobayashi et al., 2002, Am. J. Physiol. Renal Physiol. 282:F358), and nasal epithelium, vaginal surface, and biliary surface (U.S. Patent Nos. 6,485,726, 6,030,613, 6,086,875; WO 03 / 077834; US 2003-0235536 A1).

[0207] Fc regions useful in the present invention include molecules capable of specifically binding to FcRn, FcγRIIB, and / or DC-SIGN, including whole IgG, Fc fragments of IgG, and other fragments containing the complete binding region of the FcRn receptor. The regions of the Fc portion of IgG that bind to FcRn, FcγRIIB, and / or DC-SIGN have been described.

[0208] In one specific embodiment, the Fc region specifically binds to the low-affinity immunoglobulin gamma Fc region receptor II-b (FcγRIIB). FcγRIIB is an inhibitory Fc receptor that controls aspects of the inflammatory response. In particular, activation of FcγRIIB inhibits activation signals that lead to inflammation. Thus, activation of FcγRIIB essentially inhibits inflammation.

[0209] In another embodiment, the Fc region specifically binds to dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). DC-SIGN, also known as CD209, is a C-type lectin receptor expressed by many bone marrow-derived cells, including certain monocytes, dendritic cells, and macrophages. Studies in mice have shown that activation of DC-SIGN can inhibit inflammation. Nimerjahn, Chapter 5, Molecular and Cellular Pathways See Involved in the Anti-inflammatory Activity of IgG, Molecular Mechanisms of Antibody Activity, New York, NY 2013:113-138.

[0210] Specific binding refers to two molecules forming a complex that is relatively stable under physiological conditions. Specific binding is characterized by a high affinity and a low-to-moderate capacity, as distinguished from nonspecific binding, which usually has a low affinity with a moderate-to-high capacity. Typically, binding occurs with an affinity constant KA of 10 or greater. 6 M -1 Higher or 108 M -1 If the binding activity is higher, it is considered specific. If necessary, the binding conditions can be changed to reduce non-specific binding without substantially affecting specific binding. Those skilled in the art can use routine techniques to optimize appropriate binding conditions, such as molecule concentration, ionic strength of the solution, temperature, binding time, and concentration of blocking agent (e.g., serum albumin, milk casein).

[0211] In certain embodiments, chimeric proteins of the present invention comprise one or more truncated Fc regions that are sufficient to nonetheless confer FcRn, FcγRIIB, and / or DC-SIGN binding properties to the Fc region. For example, the portion of the Fc region that binds to FcRn, FcγRIIB, and / or DC-SIGN (i.e., the FcRn, FcγRIIB, and / or DC-SIGN binding portion) comprises approximately amino acids 282-438 of IgG1 according to EU numbering (the major 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 regions of the present invention comprise FcRn, FcγRIIB, and / or DC-SIGN binding portions. The FcRn, FcγRIIB, and / or DC-SIGN binding moiety may comprise or consist of a cγRIIB and / or DC-SIGN binding moiety. The FcRn, FcγRIIB, and / or DC-SIGN binding moiety may be derived from a heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, an FcRn, FcγRIIB, and / or DC-SIGN binding moiety derived from an antibody of human isotype IgG1 is used. In another embodiment, an FcRn, FcγRIIB, and / or DC-SIGN binding moiety derived from an antibody of human isotype IgG4 is used.

[0212] In another embodiment, an "Fc region" comprises an amino acid sequence of or derived from an Fc domain. In certain embodiments, an Fc region comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region) domain (approximately amino acids 216-230 of an antibody Fc region according to EU numbering), a CH2 domain (approximately amino acids 231-340 of an antibody Fc region according to EU numbering), a CH3 domain (approximately amino acids 341-438 of an antibody Fc region according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, an Fc region comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, the Fc region comprises, consists essentially of, or consists of a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), a hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), or a CH2 domain (or portion thereof) fused to both a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In still other embodiments, the Fc region lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). In certain embodiments, the Fc region comprises or consists of amino acids corresponding to EU numbers 221-447.

[0213] The Fc region, designated herein as F, F1, or F2, can be obtained from several different sources. In one embodiment, the Fc region of the polypeptide is derived from human Ig. However, it is understood that the Fc region may also be derived from Ig of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, or guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Furthermore, the Fc domain 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 another embodiment, the human isotype IgG1 is used.

[0214] In certain embodiments, Fc variants provide an alteration in at least one effector function conferred by an Fc region comprising a wild-type Fc domain (e.g., an improved or decreased ability of the Fc region to bind to an Fc receptor (e.g., reduced binding to FcγRI or FcγRIII or improved binding to FcRn or FcγRII), a complement protein (e.g., C1q), or other Fc binding partner (e.g., improved binding to DC-SIGN), or to induce antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, Fc variants provide engineered cysteine ​​residues.

[0215] The Fc regions of the present invention may employ art-recognized Fc variants known to confer altered (e.g., enhanced or diminished) effector function and / or FcR binding. Specifically, binding molecules of the present invention may employ Fc variants described in, for example, International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 9 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 07456 9A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2 , WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2; U.S. Patent Application Publication Nos. 2007 / 0231329, 2007 / 02 37765, 2007 / 0237766, 2007 / 0237767, 2007 / 0243188, 20070248603, 20070286859, 20080057056; or U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; The amino acid sequence may include a change (e.g., a substitution) at one or more amino acid positions disclosed in Nos. 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 one embodiment, a specific change (e.g., a specific substitution of one or more amino acids disclosed in the art) can be made at one or more disclosed amino acid positions. In another embodiment, different changes (e.g., different substitutions of one or more amino acid positions disclosed in the art) can be made at one or more disclosed amino acid positions.

[0216] The Fc region can be modified according to well-recognized procedures, such as site-directed mutagenesis, to obtain modified Fc fragments or portions thereof that bind FcRn, FcγRIIB, and / or DC-SIGN. Such modifications include modifications away from the FcRn, FcγRIIB, and / or DC-SIGN contact sites as well as modifications within the contact sites that preserve or even enhance binding to FcRn, FcγRIIB, and / or DC-SIGN. For example, human IgG1 The following single amino acid residues in Fc (Fcγ1): P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q3 42A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380 A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A can be substituted without significant loss of Fc binding affinity for FcRn, FcγRIIB, and / or DC-SIGN, where, for example, P238A represents the wild-type proline substituted with alanine at position 238.

[0217] By way of example, certain embodiments include the N297A mutation, which removes the highly conserved N-glycosylation site. In addition to alanine, other amino acids may be substituted at the positions identified above relative to the wild-type amino acid. The amino acids can be substituted with other amino acids. Mutations can be introduced singly into the Fc to generate over 100 Fc regions that differ from the native Fc. Furthermore, combinations of two, three, or more of these individual mutations can be introduced together to generate hundreds more Fc regions. Furthermore, one of the Fc regions of the constructs of the invention can be mutated and the other Fc region of the construct can be left unmutated, or both can be mutated, but with different mutations.

[0218] In one embodiment, the Fc domain or portion thereof is a polypeptide comprising SEQ ID NO: 3 of US Pat. No. 5,739,277, and optionally further comprising a sequence selected from SEQ ID NOs: 11, 1, 2 and 31 of US Pat. No. 5,739,277.

[0219] In certain embodiments, the Fc domain, or a portion thereof, is hemiglycosylated. For example, a chimeric protein comprising two Fc regions may contain a first glycosylated Fc region (e.g., a glycosylated CH2 region) and a second aglycosylated Fc region (e.g., an aglycosylated CH2 region). In one embodiment, a linker can be placed between the glycosylated Fc region and the aglycosylated Fc region. In another embodiment, the Fc region is fully glycosylated, i.e., the entire Fc region is glycosylated. In other embodiments, the Fc region may be aglycosylated, i.e., none of the Fc portions are glycosylated.

[0220] In certain embodiments, the chimeric proteins of the present invention comprise amino acid substitutions to the Fc domain or portions thereof (e.g., Fc variants) that alter the antigen-independent effector functions of the Fc domain, in particular the circulating half-life of the protein.

[0221] The Fc region used in the present invention may also contain art-recognized amino acid substitutions that alter the glycosylation of the chimeric protein. For example, the Fc region of a chimeric protein linked to a FVIII protein or FXI protein may contain an Fc region with a mutation that results in reduced glycosylation (e.g., N- or O-linked glycosylation), or may contain an altered glycoform of the wild-type Fc portion (e.g., low-fucose or fucose-free glycans).

[0222] In one embodiment, an unprocessed chimeric protein of the invention may comprise a genetically fused Fc region (i.e., an scFc region) having two or more of its component Ig constant regions or portions thereof, independently selected from the Ig constant regions or portions thereof described herein. In one embodiment, the Fc regions of the dimeric Fc region are the same. In another embodiment, at least two Fc regions are different. For example, the Fc regions of the proteins of the invention may comprise the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet other embodiments, the Fc regions of the proteins of the invention may differ in sequence at one or more amino acid positions. For example, the at least two Fc regions 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.

[0223] In some embodiments, the chimeric protein used in the methods of the present disclosure comprises more than one polypeptide chain. In some embodiments, the chimeric protein comprises two polypeptide chains. In certain embodiments, the first polypeptide chain comprises a coagulation factor and a first Fc region, and the second polypeptide chain comprises a second Fc region. Certain embodiments In one embodiment, the first Fc region and the second Fc region are covalently linked. In one embodiment, the first Fc region and the second Fc region are linked by a peptide bond. In another embodiment, the first Fc region and the second Fc region are linked by a disulfide bond.

[0224] In one specific embodiment, the chimeric protein comprises a factor VIII portion and a von Willebrand factor (VWF) portion, wherein the FVIII portion comprises a FVIII polypeptide or a fragment thereof, the VWF portion comprises a VWF polypeptide or a fragment thereof, the FVIII portion is linked to a first Fc region, the VWF portion is linked to a second Fc region, and the first Fc region and the second Fc region bind to each other. In certain embodiments, the VWF portion comprises the D' and D3 domains of VWF. In one embodiment, the first polypeptide, the second polypeptide, or both the first and second polypeptides further comprise one or more half-life extending moieties.

[0225] The Fc region or a portion thereof for producing the chimeric protein used in the method of the present disclosure can be obtained from several different sources. In some embodiments, the Fc region or a portion thereof is derived from human Ig. However, it is understood that the Fc region or a portion thereof may also be derived from Ig of another mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, or guinea pigs) or non-human primates (e.g., chimpanzees, macaques). Furthermore, the Fc region or a 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 one embodiment, human isotype IgG1 is used.

[0226] Various Fc region gene sequences (e.g., human Fc gene sequences) are available in the form of publicly accessible deposits. Fc sequences can be selected that have specific effector functions (or lack specific effector functions) or that have specific modifications that reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Fc region sequences can be derived from these sequences using art-recognized techniques. The genetic material obtained using any of the above methods can then be altered or synthesized to obtain chimeric proteins for use in the methods of the present disclosure. Furthermore, it is understood that the scope of the present invention encompasses alleles, variants, and mutations of constant region DNA sequences.

[0227] The sequence of an Fc 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 an Fc region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymphocytes, reverse transcribed into DNA, and the antibody gene can be amplified by PCR. PCR amplification methods are described in detail in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 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 by consensus constant region primers or by more specific primers based on the published DNA and amino acid sequences of heavy and light chains. As discussed above, PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, libraries can be screened with consensus primers or larger homologous probes, such as mouse constant region probes. Several primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primer sets based on the N-terminal sequence of purified antibodies). rapid amplification of cDNA ends (Ruberti, F. et al., 1994, J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al., 1989, Biochem. Biophys. Res. Commun. 160:1250). Cloning of antibody sequences is further described in U.S. Patent No. 5,658,570, filed January 25, 1995, to Newman et al., which is incorporated herein by reference.

[0228] II.B. Half-Life Extending Moieties In some embodiments, the chimeric protein used in the methods of the present disclosure further comprises one or more half-life extending moieties. The half-life of a coagulation factor can be determined by any method known to those skilled in the art, such as a FVIII activity assay (colorimetric assay or one-stage clotting aPTT assay) to detect plasma FVIII activity or a FVIII / FIX ELISA to detect plasma FVIII / FIX antigen levels. In certain embodiments, the half-life of the coagulation activity of a coagulation factor is determined by a one-stage coagulation assay. In more specific embodiments, the half-life of the coagulation activity of a coagulation factor is determined in HemA mice or FVIII and von Willebrand factor double knockout (DKO) mice.

[0229] In certain aspects, heterologous moieties that increase the half-life of the coagulation factors of the present invention include heterologous polypeptides such as, but not limited to, albumin, immunoglobulin Fc regions, XTEN sequences, the C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, PAS sequences, HAP sequences, transferrin, albumin-binding moieties, or any fragments, derivatives, variants, or combinations of these peptides. In other related aspects, the half-life extending moiety may comprise binding sites for non-polypeptide moieties such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivatives, variants, or combinations of these moieties. In certain embodiments, the half-life extending moiety comprises albumin or a fragment thereof, an albumin-binding moiety, a PAS sequence, a HAP sequence, transferrin or a fragment thereof, or any combination thereof. In some embodiments, the half-life extending moiety does not comprise XTEN. In other embodiments, the half-life extending moiety comprises XTEN.

[0230] In other embodiments, the chimeric proteins of the present invention are conjugated to one or more polymers. The polymers may be water-soluble or water-insoluble. The polymers can be covalently or non-covalently attached to the clotting factor, Fc, or other moieties conjugated to the clotting factor or Fc. Non-limiting examples of polymers may be poly(alkylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, or poly(acryloylmorpholine). For example, additional forms of polymer-conjugated FVIII are disclosed in U.S. Patent No. 7,199,223, the entire disclosure of which is incorporated by reference.

[0231] In certain embodiments, the chimeric proteins of the present invention may comprise one, two, three or more half-life extending moieties, each of which may be the same or different molecules.

[0232] In some embodiments, the half-life extending moiety is fused to the N-terminus or C-terminus of the chimeric polypeptide. In some embodiments, the half-life extending moiety is fused to the N-terminus or C-terminus of the clotting factor. In some embodiments, the half-life extending moiety is fused to the N-terminus or C-terminus of the Fc. In certain embodiments, the half-life extending moiety is inserted within the clotting factor of the chimeric protein.

[0233] In some embodiments, the chimeric protein comprises FVIII or a portion thereof and has a half-life The extender is inserted into FVIII at one or more positions disclosed in U.S. Patent Application Publication No. 2015-0158929 A1 and / or International Publication No. WO2015106052 A1, the entire contents of which are incorporated by reference herein. In one particular embodiment, the half-life extender is inserted into the B domain (or a fragment thereof) of FVIII. In one particular embodiment, the half-life extender is inserted into FVIII immediately downstream of amino acid residue 745 of mature FVIII.

[0234] In other embodiments, the chimeric protein comprises FIX or a portion thereof, wherein the half-life extending moiety is inserted into FIX at one or more positions disclosed in International Application No. PCT / US16 / 045401. In one specific embodiment, the half-life extending moiety is inserted into FIX at an insertion site immediately downstream of an amino acid residue selected from the group consisting of amino acid 103, amino acid 105, amino acid 142, amino acid 149, amino acid 162, amino acid 166, amino acid 174, amino acid 224, amino acid 226, amino acid 228, and amino acid 413 of mature Padua FIX. In one embodiment, the chimeric protein comprises FIX and an Fc region, wherein FIX comprises a half-life extending moiety inserted into FIX within the activation peptide (AP) domain of FIX. In one specific embodiment, the chimeric protein comprises FIX and an Fc region, wherein FIX comprises a half-life extending moiety inserted into FIX immediately downstream of amino acid residue 166 of mature Padua FIX.

[0235] II.B.1. Albumin In certain embodiments, the chimeric protein 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), a 609 amino acid protein in its full-length form, accounts for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. As used herein, the term "albumin" includes full-length albumin or its functional fragment, variant, derivative, or analog. Examples of albumin or fragments or variants thereof are disclosed in U.S. Patent Publication Nos. 2008 / 0194481 A1, 2008 / 0004206 A1, 2008 / 0161243 A1, 2008 / 0261877 A1, or 2008 / 0153751 A1 or PCT Application Publication Nos. 2008 / 033413 A2, 2009 / 058322 A1, or 2007 / 021494 A2, which are incorporated by reference in their entireties.

[0236] Albumin-binding polypeptides (ABPs) may include, 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, as disclosed by 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), which are incorporated herein by reference in their entireties.

[0237] In certain embodiments, the chimeric protein used in the methods of the present disclosure comprises at least one binding site for a non-polypeptide small molecule, variant, or derivative thereof capable of binding to albumin. For example, the chimeric protein may comprise one or more organic albumin-binding moieties. Examples of such albumin-binding moieties include Tr 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate ("Albu" tag), as disclosed by Ussel et al., Bioconjugate Chem. 20:2286-2292 (2009).

[0238] II.B.2.XTEN In certain embodiments, the chimeric protein used in the methods of the present disclosure comprises at least one XTEN polypeptide or a fragment, variant, or derivative thereof. As used herein, "XTEN sequence" refers to an extended-length polypeptide having a non-naturally occurring, substantially non-repetitive sequence that primarily contains small, hydrophilic amino acids, with a sequence that has little or no secondary or tertiary structure under physiological conditions. Similar to a chimeric protein partner, XTEN can act as a carrier that provides certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties, for example, when fused to or inserted into a clotting factor of a chimeric protein. Such desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility properties.

[0239] The XTEN sequence fused to or inserted into a coagulation factor of a chimeric protein useful in the methods of the present disclosure can provide the chimeric protein with one or more of the following significant properties: conformational flexibility, enhanced aqueous solubility, a high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or an increased hydrodynamic (or Stokes) radius. In certain embodiments, the XTEN sequence can enhance pharmacokinetic properties, such as a longer half-life (e.g., in vivo half-life) or an increased area under the curve (AUC), such that the chimeric protein persists in vivo and has procoagulant activity for a longer period of time compared to a chimeric protein that does not contain the XTEN.

[0240] Examples of XTEN sequences that can be inserted into the recombinant FVIII proteins of the present invention are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1, or 2011 / 0172146 A1, or International Patent Application Publication Nos. Nos. WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, WO2011028344A2, or WO2015106052A1, each of which is incorporated herein by reference in its entirety.

[0241] II.B.3. VWF or a fragment thereof In certain embodiments, the chimeric protein used in the methods of the present disclosure comprises at least one VWF polypeptide or a fragment, variant, or derivative thereof. VWF (also known as F8VWF) is a large, multimeric glycoprotein present in plasma and constitutively produced in the endothelium (Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2813 amino acid protein. All monomers contain several specific domains with specific functions: the D' / D3 domain (which binds factor VIII), the A1 domain (which binds platelet GPIb-receptors, heparin, and / or possibly collagen), the A3 domain (which binds collagen), the C1 domain (the RGD domain binds to platelet integrin αIIbβ3 when it is activated), and a "cysteine ​​knot" domain at the C-terminus of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ), and β-human chorionic gonadotropin (βHCG)).

[0242] In one embodiment, the VWF polypeptide is a VWF fragment. The term "VWF fragment" includes, but is not limited to, a functional VWF fragment comprising the D' and D3 domains that can inhibit the binding of endogenous VWF to FVIII. In one embodiment, the chimeric protein used in the disclosed method comprises a coagulation factor, an Fc region, and a VWF fragment, wherein the coagulation factor comprises FVIII and the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment inhibits the interaction of the FVIII protein with endogenous VWF by blocking the VWF-binding site on the FVIII protein. VWF fragments include derivatives, variants, mutants, or analogs that retain these activities of VWF. In certain embodiments, the VWF fragment comprises the D' and D3 domains of VWF.

[0243] The 2813 monomeric amino acid sequence of human VWF is reported in Genbank under accession number NP_000543.2. The nucleotide sequence encoding human VWF is reported in Genbank under accession number NM_000552.3.

[0244] In certain embodiments, the VWF protein useful herein can be further modified to improve its interaction with FVIII, for example, to improve binding affinity to FVIII. In other embodiments, the VWF protein useful for the present invention can have other modifications, for example, the protein can be pegylated, glycosylated, hesylated, or polysialylated. Exemplary VWF sequences useful in the methods of the present disclosure are provided, for example, in U.S. Patent Application Publication Nos. US2015 / 0023959A1, US2015 / 0266943A1, and US2015 / 0158929. In certain embodiments, the VWF protein or a fragment thereof is fused to or co-administered with an FcRn binding partner. In some embodiments, the VWF protein or a fragment thereof is fused to Fc or co-administered with Fc or a polypeptide comprising Fc. In some embodiments, the VWF protein or fragment thereof is fused to albumin or co-administered with albumin or a polypeptide comprising albumin.

[0245] II.B.4.CTP In certain embodiments, the chimeric protein 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, the entire contents of which are incorporated herein by reference. Non-limiting examples of CTP peptides are disclosed in U.S. Patent Application Publication No. US2009 / 0087411 A1, the entire contents of which are incorporated herein by reference.

[0246] II.B.5.PAS In certain embodiments, the chimeric protein used in the methods of the present disclosure comprises at least one PAS peptide or a fragment, variant, or derivative thereof. As used herein, a PAS peptide or PAS sequence refers to an amino acid sequence that contains primarily alanine and serine residues, or primarily alanine, serine, and proline residues, and that forms a random coil conformation under physiological conditions. Thus, a PAS sequence is a component, amino acid polymer, or sequence cassette that contains, consists essentially of, or consists of alanine, serine, and proline, and can be used as part of a heterologous moiety in a chimeric protein. An amino acid polymer can also form a random coil conformation when residues other than alanine, serine, and proline are added as minor components in the PAS sequence. "Minor constituents" refer to amino acids other than alanine, serine, and proline that are present to a certain extent, e.g., up to about 12%, i.e., up to about 12 of 100 amino acids in a PAS sequence, up to about 10%, up to about 9%, up to about 8%, about 6%, about 5%, about This means that up to about 4%, about 3%, i.e., about 2%, or about 1% of the amino acids can be added to the PAS sequence. Amino acids other than alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Under physiological conditions, the PAS peptide forms a random coil conformation, thereby mediating increased in vivo and / or in vitro stability for the recombinant protein of the present invention and having procoagulant activity.

[0247] Non-limiting examples of PAS peptides are disclosed, for example, in US Patent Application Publication No. 2010 / 0292130 A1; PCT Application Publication No. WO2008 / 155134 A1; and European Patent No. EP2173890.

[0248] II.B.6.HAP In certain embodiments, the chimeric protein used in the method of the present disclosure comprises at least one homoamino acid polymer (HAP) peptide or a fragment, variant, or derivative thereof. The HAP peptide may comprise a repeat sequence of glycine having a length of at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids. The HAP sequence can extend the half-life of the moiety fused or linked to the HAP sequence. Non-limiting examples of HAPPY sequences include, but are not limited to, (Gly) n (Gly4Ser) n or S(Gly4Ser) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one embodiment, 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 another embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. See, e.g., Schlapschy M et al., Protein Eng. Design Selection, 20:273-284 (2007).

[0249] II.B.7. Transferrin In certain embodiments, the chimeric protein used in the methods of the present disclosure comprises at least one transferrin peptide or a fragment, variant, or derivative thereof. Any transferrin can be fused to the chimeric protein used in the methods of the present disclosure. For example, wild-type human Tf (Tf) is a 679-amino acid protein of approximately 75 kDa (excluding glycosylation), with two major domains, N (approximately 330 amino acids) and C (approximately 340 amino acids), which is thought to result from gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM039847, and S95936 (www.ncbi.nlm.nih.gov), all of which are incorporated herein by reference in their entirety.

[0250] Transferrin transports iron via transferrin receptor (TfR)-mediated endocytosis. After iron is released into endosomal compartments and the Tf-TfR complex recycles to the cell surface, Tf is released back into the extracellular space for the next cycle of iron transport. Tf has a long half-life of more than 14-17 days (Li et al., Trends Pharmacol. Sci. 23:206-209 (2002)). Transferrin fusion proteins have been investigated for half-life extension, targeted delivery for cancer therapy, oral delivery, and sustained activation of proinsulin (Brandsma et al., Biotechnol. Adv. v., 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)).

[0251] II.B.8.PEG In certain embodiments, a chimeric protein used in the methods of the present disclosure comprises at least one binding site for a non-polypeptide heterologous moiety, or a fragment, variant, or derivative thereof. For example, a chimeric protein used in the methods of the present disclosure may comprise one or more polyethylene glycol (PEG) moieties attached to one or more amino acid residues in a coagulation factor and / or Fc region.

[0252] PEGylation of a protein may refer to a conjugate formed between a protein and at least one polyethylene glycol (PEG) molecule. PEG is commercially available in a variety of molecular weights and average molecular weight ranges. Typical examples of PEG average molecular weight ranges include, but are not limited to, about 200, about 300, about 400, about 600, about 1000, about 1300-1600, about 1450, about 2000, about 3000, about 3000-3750, about 3350, about 3000-7000, about 3500-4500, about 5000-7000, about 7000-9000, about 8000, about 10000, about 8500-11500, about 16000-24000, about 35000, about 40000, about 60000, and about 80000 daltons. These average molecular weights are provided as examples only and are not meant to be limiting in any way.

[0253] The chimeric proteins used in the methods of the present disclosure can be PEGylated to contain mono- or poly- (e.g., 2-4) PEG moieties. PEGylation can be carried out by any PEGylation reaction known in the art. Methods for preparing PEGylated protein products will generally include: (i) reacting a polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions such that the peptide of the present invention becomes bound to one or more PEG groups; and (ii) obtaining the reaction product. Generally, optimal reaction conditions for the reaction will be determined on a case-by-case basis based on known parameters and the desired results.

[0254] There are several PEG conjugation methods available to those skilled in the art, see, for example, Malik F et al., Exp. Hematol. 20:1028-35 (1992); Francis, Focus on Growth Factors 3(2):4-10 (1992); European Patent Publication Nos. EP0401384, EP0154316, and EP0401384; and International Patent Application Publication Nos. WO92 / 16221 and WO95 / 34326. As a non-limiting example, FVIII variants may contain cysteine ​​substitutions, which can be further conjugated to PEG polymers. See Mei et al., Blood 116:270-279 (2010) and U.S. Patent No. 7,632,921, the entire contents of which are incorporated herein by reference.

[0255] II.B.9.HES In certain embodiments, the chimeric protein used in the methods of the present disclosure comprises at least one hydroxyethyl starch (HES) polymer. HES is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body. HES exhibits significant biological properties and is used in clinics as a blood volume replacement agent and in hemodilution therapy. See, for example, Sommermeyer et al., Krankenhauspharmazie 8:271-278 (1987); and Weidler et al., See Arzneim.-Forschung / Drug Res. 41:494-498 (1991).

[0256] HES is primarily characterized by its molecular weight distribution and degree of substitution. HES has an average molecular weight (weight average) of 1-300 kD, 2-200 kD, 3-100 kD, or 4-70 kD. Hydroxyethyl starch may further exhibit a molar substitution of 0.1-3, 0.1-2, 0.1-0.9, or 0.1-0.8, and a C2:C6 substitution ratio with respect to the hydroxyethyl group ranging from 2 to 20. An HES with an average molecular weight of approximately 130 kD is VOLUVEN® from Fresenius. VOLUVEN® is an artificial colloid used for volume exchange in therapeutic applications, such as the treatment and prevention of hypovolemia. Several HES conjugation methods are available to those skilled in the art, including the same PEG conjugation method described above.

[0257] II.B.10.PSA In certain embodiments, the chimeric protein used in the methods of the present disclosure comprises at least one polysialic acid (PSA) polymer. PSA is a naturally occurring, unbranched polymer of sialic acid produced by certain bacterial strains and in certain mammalian cells. See, for example, Roth J. et al. (1993) Polysialic Acid: From Microbes to Man (eds.), Roth J., Rutishauser U., Troy FA (Birkhauser Verlag, Basel, Switzerland), pp. 335-348. PSA can be produced with various degrees of polymerization, from n=about 80 or more sialic acid residues to n=2, by limited acid hydrolysis or digestion with neuraminidase, or by fractionation of naturally occurring bacterially derived forms of the polymer. In certain embodiments, activated PSA can also be conjugated to cysteine ​​amino acid residues within coagulation factors, such as FVIII or FIX, or within the Fc region. See, for example, U.S. Patent No. 5,846,951.

[0258] II.B.11. Clearance receptors In certain embodiments, the half-life of a chimeric protein used in the methods of the present disclosure can be extended when the coagulation factor of the chimeric protein comprises FVIII and at least one fragment of an FVIII clearance receptor, or a FVIII-binding fragment, variant, or derivative thereof. Insertion of a soluble clearance receptor, such as the low-density lipoprotein-related protein receptor LRP1, or a fragment thereof, can block the binding of FVIII to the clearance receptor, thereby extending its half-life, e.g., in vivo. LRP1 is a 600-kDa integral membrane protein involved in receptor-mediated clearance of various proteins, including FVIII. See, for example, Lenting et al., Haemophilia 16:6-16 (2010). Other suitable FVIII clearance receptors are, for example, LDLR (low-density lipoprotein receptor), VLDLR (very-low-density lipoprotein receptor), and megalin (LRP-2), or fragments thereof. See, e.g., Bovenschen et al., Blood 106:906-912 (2005); Bovenschen, Blood 116:5439-5440 (2010); Martinelli et al., Blood 116:5688-5697 (2010).

[0259] III. Polynucleotides, Vectors, and Host Cells In some aspects, the present disclosure provides methods of treating reversible hemophilic arthropathy of a joint in a human with hemophilia, the method comprising administering to the human an effective amount of a polynucleotide or set of polynucleotides encoding a clotting factor and / or an Fc region, e.g., encoding a chimeric protein comprising a clotting factor and an Fc region. In some embodiments, the polynucleotide or set of polynucleotides is an expression vector or In certain embodiments, the expression vector or set of expression vectors is in one or more host cells.

[0260] The polynucleotides used in the methods of the present disclosure encoding a coagulation factor and / or an Fc region, for example, encoding a chimeric protein comprising a coagulation factor and an Fc region, may be a single nucleotide sequence, two nucleotide sequences, three nucleotide sequences, or more. In one embodiment, a single nucleotide sequence encodes a chimeric protein comprising a coagulation factor (e.g., a FVIII or FIX polypeptide) and an Fc region. In another embodiment, the polynucleotide comprises two nucleotide sequences: a first nucleotide sequence encoding a coagulation factor (e.g., FVIII) and a second nucleotide sequence encoding an Fc region. In another embodiment, the polynucleotide comprises two nucleotides: a first nucleotide sequence encoding a coagulation factor (e.g., FVIII or FIX) and an Fc region and a second nucleotide sequence encoding a second Fc region. In certain embodiments, the encoded Fc domains form a covalent bond after expression.

[0261] In some embodiments, the polynucleotide is codon optimized.

[0262] As used herein, an expression vector refers to any nucleic acid construct that contains the necessary elements for transcription and translation of an inserted coding sequence when introduced into a suitable host cell, or the necessary elements for replication and translation in the case of an RNA viral vector. Expression vectors may include plasmids, phagemids, viruses, and their derivatives.

[0263] As used herein, a gene expression control sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, that facilitates efficient transcription and translation of an operably linked encoding nucleic acid. A gene expression control sequence may be, for example, a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, promoters derived from cytomegalovirus (CMV), simian viruses (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeat (LTR), and other retroviruses, as well as the herpes simplex virus thymidine kinase promoter. Other constitutive promoters are known to those skilled in the art. Promoters useful as gene expression sequences of the present invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

[0264] For the purposes of the present invention, several expression vector systems can be used. These expression vectors are typically replicable in host cells either as episomes or as an integral part of the host chromosomal DNA. Expression vectors may contain expression control sequences, including, but not limited to, promoters (e.g., naturally associated or heterologous promoters), enhancers, signal sequences, splice signals, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Expression vectors also include those derived from bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOML). DNA elements derived from animal viruses such as SV40, cytomegalovirus (CMV), or SV40 viruses can also be utilized. Others involve the use of polycistronic systems with internal ribosome binding sites.

[0265] Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to permit detection of cells transfected with the desired DNA sequence (e.g., Itakura et al., U.S. Pat. No. 4,704,362). Cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for selection of transfected host cells. Markers can provide prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selection marker gene can be directly linked to the DNA sequence to be expressed, or introduced into the same cell by cotransformation.

[0266] An example of a vector useful for optimized expression of the chimeric proteins used in the methods of the present disclosure is NEOSPLA (U.S. Patent No. 6,159,730). This vector contains the cytomegalovirus promoter / enhancer, mouse beta globin major promoter, SV40 origin of replication, bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, dihydrofolate reductase gene and leader sequence. This vector has been shown to result in very high levels of antibody expression upon incorporation of variable and constant region genes, transfection into cells, and subsequent selection in G418-containing medium and methotrexate amplification. Vector systems are also taught in U.S. Patent Nos. 5,736,137 and 5,658,570, each of which is incorporated herein by reference in its entirety. This system provides high expression levels, e.g., greater than 30 pg / cell / day. Other exemplary vector systems are disclosed, for example, in U.S. Patent No. 6,413,777.

[0267] In other embodiments, the polypeptides of the present invention are expressed using polycistronic constructs. These expression systems allow multiple gene products of interest, such as multiple polypeptides of a multimeric binding protein, to be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is also incorporated herein.

[0268] More generally, once a vector or DNA sequence encoding a polypeptide has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be achieved by various techniques well known to those skilled in the art, as discussed above. The transformed cells are grown under conditions appropriate for the production of the chimeric protein, and assayed for chimeric protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, etc.

[0269] Having now described the invention in detail, the same will be more clearly understood by reference to the following examples, which are included herein for purposes of illustration only and are not intended to be limiting of the invention. [Example]

[0270] Long-term efficacy of rFVIIIFc prophylaxis in target joints and in children, adolescents, and adults with severe hemophilia A For people with hemophilia, frequent bleeding into the same joints (target joints) can contribute to hemophilic arthropathy (chronic joint disease). rFVIIIFc was developed to extend the half-life of factor VIII (FVIII) compared to conventional FVIII products (Peters et al., J. Thromb. Haemost. 11(1):132-41 (2013)). The completed rFVIIIFc pivotal phase 3 trial (ClinicalTrials.gov Identifier: NCT01181128; Mahlangu et al., Blood 123(3):317-25 (2014)) and the children's rFVIIIFc pivotal phase 3 trial (NCT01458106; Young et al., J. Thromb. Haemost. 13(6):967-77 (2015)) established the safety and efficacy of rFVIIIFc among adults / adolescents (patients 12 years of age or older) and children (patients younger than 12 years of age) with severe hemophilia A, respectively. The long-term safety and efficacy of rFVIIIFc are being evaluated in the ongoing rFVIIIFc extension trial (NCT01454739; Nolan et al., Haemophilia 22(1):72-80 (2016)).

[0271] The objective of this study is to report cumulative data on the sustainable efficacy and quality of life of rFVIIIFc in subjects with target joints at the time of enrollment in the rFVIIIFc pivotal phase 3 study and the pediatric rFVIIIFc pivotal phase 3 study as of the second rFVIIIFc expansion study interim data cut (December 8, 2014).

[0272] method Patients with ≥1 target joint (a major joint with ≥3 bleeding episodes in 6 months (World Federation of Subjects with Hemophilia, Guidelines for the Management of Hemophilia 2nd ed., Blackwell Publishing: Montreal, Canada (2012) were evaluated. The rFVIIIFc expansion study had four treatment arms (Table 1).

[0273] [Table 1]

[0274] Outcomes for subjects with target joints were analyzed post hoc over the cumulative period of the parent study up to the second rFVIIIFc extension study interim data cut. Outcomes included ABR, number and resolution of target joint bleeding episodes, and prophylactic dose and frequency of administration. Analysis of resolution in target joints was performed for subjects with a continuous follow-up time of 12 months or more. This was performed on subjects who had not undergone major surgery (i.e., replacement or removal) of the target joint since the beginning of the follow-up period. A target joint was considered clinically resolved if there were two or fewer spontaneous bleeding episodes in the target joint over a consecutive 12-month period (Blanchette et al., J Thromb Haemost. 12(11):1935-39 (2014)).

[0275] QOL measures were assessed by the Haem-A-QOL index in prevention subjects who were 17 years of age or older, had one or more resolved target joints during the study, and had Haem-A-QOL scores at both baseline in the rFVIIIFc pivotal phase 3 study and at year 2 in the rFVIIIFc expansion study. Among subjects from the pediatric rFVIIIFc pivotal phase 3 study, QOL was measured by the Canadian Hemophilia Outcomes-Kids Life Assessment Tool (CHO-KLAT).

[0276] result Study population Of the 113 subjects from the rFVIIIFc pivotal phase 3 trial who had target joints at baseline, 111 subjects with pre-study prophylaxis or hemostatic regimens and on-study data had 287 target joints at baseline (median age, 31.0 years; interquartile range (IQR), 24.0-44.0 years; Table 2). Thirteen subjects from the pediatric rFVIIIFc pivotal phase 3 trial had 15 target joints at baseline and had pre-study and on-study data (median age, 6.0 years; IQR, 5.0-8.0 years; Table 2).

[0277] [Table 2]

[0278] Number of bleeding episodes The median (IQR) on-study total ABR with rFVIIIFc prophylaxis was lower than the bleeding rate on pre-study prophylaxis for adults / adolescents and children aged 12 years or younger (Figures 1A-1D). Pediatric rFVIIIFc pivotal phase 3 study data were further stratified by patient age, and median pre-study and on-study ABRs are shown in Figures 1E and 1F. Before the study, patients younger than 6 years had lower median (IQR) ABRs than patients aged 6 to 12 years (Figure 1E). During the study, patients younger than 6 years had higher on-study ABRs, total target joint ABRs, and target joint spontaneous ABRs than patients aged 6 to 12 years (Figure 1F).

[0279] During the rFVIIIFc pivotal phase 3 trial, 46.3% of subjects on individualized prophylaxis, 40.7% on weekly prophylaxis, and 21.4% on modified prophylaxis had a target joint bleed episode, whereas 53.8% of subjects on individualized prophylaxis in the pediatric rFVIIIFc pivotal phase 3 trial did not have a target joint bleed episode.

[0280] Resolution in clinical target joints Among subjects on prophylaxis who had target joints at baseline and 12-month follow-up, 100% (93 / 93) of subjects in the rFVIIIFc pivotal phase 3 study and 100% (7 / 7) of subjects in the pediatric rFVIIIFc pivotal phase 3 study had one or more resolved target joints (i.e., ≤2 spontaneous bleeding episodes in any consecutive 12-month period). and resolution of all bleeds in 98.3% (231 / 235) and 100% (9 / 9) of target joints (based on all bleeds) in subjects in the rFVIIIFc pivotal phase 3 trial and the pediatric rFVIIIFc pivotal phase 3 trial, respectively.

[0281] Preventive factor consumption The median (IQR) weekly prophylactic factor consumption among prophylactic subjects with target joints at baseline in the rFVIIIFc pivotal phase 3 trial (n=105) and the pediatric rFVIIIFc pivotal phase 3 trial (n=13) was 76.0 (68.0-90.9) IU / kg and 83.5 (79.9-111.6) IU / kg, respectively.

[0282] Consumption was similar to subjects in the parent studies who were on prophylaxis before both the rFVIIIFc pivotal phase 3 trial / pediatric rFVIIIFc pivotal phase 3 trial and during the rFVIIIFc pivotal phase 3 trial / pediatric rFVIIIFc pivotal phase 3 trial and the rFVIIIFc extension trial and had available pre- and on-study dosing data (rFVIIIFc pivotal phase 3 trial, n=79, 75.0 [70.0-113.8] IU / kg; pediatric rFVIIIFc pivotal phase 3 trial, n=54, 95.0 [75.0-113.0] IU / kg).

[0283] For both patient populations, the median (IQR) dosing interval was also similar between prophylaxis subjects with target joints at baseline (rFVIIIFc pivotal phase 3 trial, n = 105, 3.8 [3.5-5.6] days; pediatric rFVIIIFc pivotal phase 3 trial, n = 13, 3.5 [3.5-3.5] days) and prophylaxis subjects in the parent trials who had available pre- and in-study dosing data (rFVIIIFc pivotal phase 3 trial, n = 79, 3.5 [3.0-5.0] days; pediatric rFVIIIFc pivotal phase 3 trial, n = 54, 3.5 [3.5-3.5] days).

[0284] Pediatric rFVIIIFc pivotal phase 3 trial data were further stratified by patient age. The mean weekly consumption in patients younger than 6 years was 89.6 (75.3-97.5; n=6) IU / kg with a dosing interval of 3.5 (3.5-3.5) days. For patients aged 6-12 years, the mean weekly consumption was 82.2 (79.4-113.2) IU / kg with a dosing interval of 3.5 (3.0-3.6) days.

[0285] Quality of life QOL improved by 18% in Year 2 of the rFVIIIFc expansion study among adults / adolescents (n=48) compared with rFVIIIFc pivotal phase 3 baseline (Table 3). Among pediatric rFVIIIFc pivotal phase 3 study subjects who had self-reported CHO-KLAT scores at pediatric rFVIIIFc pivotal phase 3 baseline and Year 1 of the rFVIIIFc expansion study (n=6), the mean (standard deviation [SD]) baseline score was 85.5 (12.1); in Year 1 of the rFVIIIFc expansion study, the CHO-KLAT score improved by 28% (a mean [SD] improvement of 24.1 [15.3]).

[0286] [Table 3]

[0287] conclusion Efficacy data from the rFVIIIFc pivotal phase 3 trial and the pediatric rFVIIIFc pivotal phase 3 trial and the ongoing rFVIIIFc expansion trial demonstrate sustained low annualized target joint bleed rates (ABRs) and effective target joint resolution in children, adolescents, and adults with severe hemophilia A on long-term rFVIIIFc prophylaxis. Weekly prophylactic factor consumption in this analysis of subjects with target joints at baseline was consistent with that in the full population of the previously published rFVIIIFc pivotal phase 3 trial and the pediatric rFVIIIFc pivotal phase 3 trial. Improvements in quality of life (QOL) were seen in subjects who demonstrated target joint resolution with rFVIIIFc prophylaxis without changes in prophylactic factor consumption or dosing interval. [Example]

[0288] Long-term modified Hemophilia Joint Health Score (mHJHS) outcomes with recombinant factor VIII Fc fusion protein (rFVIIIFc) prophylaxis in subjects with severe hemophilia A Hemophilic arthropathy remains a challenge in the management of hemophilia (Knobe et al., J Comorbidity. 2011;1(1):51-59; Simpson et al., Expert Rev Hematol. 2012;5(4):459-68). The Hemophilia Joint Health Score (HJHS) is a first-line tool that can be used to detect the development of hemophilic arthropathy (Oymak et al., J Pediatr Hematol Oncol. 2015;37(2):e80-5). Improvement in skeletal muscle outcomes is an important measure of the effectiveness of prophylactic treatment for hemophilia A (Blanchette et al., Haemop hilia.2004;10(Suppl.S4):97-104). The long-term safety and efficacy of rFVIIIFc among adults / adolescents and children who have completed the rFVIIIFc pivotal phase 3 trial (Mahlangu et al., Blood.2014;123(3):317-325) and the pediatric rFVIIIFc pivotal phase 3 trial (Young et al., J Thromb Haemost.2015;13(6):967-977), respectively, have been established using interim data from an ongoing rFVIIIFc expansion study (Nolan et al., Haemophilia.2016;22(1):72-80). Determining the long-term effects of rFVIIIFc on skeletal muscle outcomes will require further studies.

[0289] The objective of this study is to report long-term joint health data from the rFVIIIFc pivotal phase 3 trial and the rFVIIIFc extension trial using the modified Hemophilia Joint Health Score (mHJHS).

[0290] Study participants and design The analysis population included adults / adolescents (ages 12 years and older) who completed the rFVIIIFc pivotal phase 3 trial and enrolled in the 2-year rFVIIIFc extended trial. Patients may have received pre-study prophylaxis or hemostatic treatment. Joint health was assessed using the mHJHS at rFVIIIFc pivotal phase 3 trial screening (post-protocol amendment) and baseline, and then annually for the rFVIIIFc extended trial.

[0291] The mHJHS differs from the standard HJHS in that it condenses response options for joint pain and ambulation into fewer categories, adds an assessment for instability, and results in a lower total score (range, 0–116; 0 indicates normal joint function and 116 indicates severe disease) compared with the standard HJHS (range, 0–124) (see Table 4). Scores prior to bleeding within 2 weeks were excluded. Scores for joints that underwent surgical intervention were imputed using last observation carried forward. To assess year-to-year change, subjects in the rFVIIIFc expansion study who had mHJHS data at four time points (rFVIIIFc pivotal phase 3 study baseline, rFVIIIFc expansion study baseline, rFVIIIFc expansion study year 1, and rFVIIIFc expansion study year 2) were included in this post hoc analysis. Changes in mHJHS scores (negative values ​​indicate improvement) from rFVIIIFc pivotal phase 3 study baseline to year 2 of the rFVIIIFc extension study were summarized using descriptive statistics. Changes in mHJHS scores from baseline to follow-up visits in the rFVIIIFc pivotal phase 3 trial were summarized for (1) total score (range, 0–116; by pretrial regimen (prophylaxis and hemostasis); severity of functional impairment based on the initial mHJHS; and presence of target joints at baseline); (2) target joints (range, 0–19: sum of all questions regarding a single target joint); (3) weight-bearing (e.g., ankle and knee) and non-weight-bearing (e.g., elbow) joints (range, 0–38: sum of both joints in a single position); and (4) individual components: range of motion (range, 0–36: combination of "loss of extension [ankle dorsiflexion]" and "loss of flexion [ankle plantarflexion]" questions for all joints; swelling (range, 0–24: combination of "swelling" and "swelling duration" questions for all joints); and muscle strength (range, 0–6: sum of all joints).

[0292] [Table 4]

[0293] Differences between the rFVIIIFc pivotal phase 3 study baseline and the rFVIIIFc extension study year 2 were analyzed using paired t-tests. Because this analysis was ad hoc, P values ​​were not used to infer statistical significance. For subgroup analyses, only descriptive statistics are provided.

[0294] result Baseline characteristics Baseline characteristics were similar between the population of completers included in this analysis (n = 47) and the patient population in which mHJHS was collected at rFVIIIFc pivotal phase 3 study baseline and enrolled in the rFVIIIFc expansion study (n = 74) (Table 5 ).

[0295] [Table 5]

[0296] Long-Term Joint Health A continuous improvement in mHJHS scores was observed from baseline in the rFVIIIFc pivotal phase 3 study to year 2 in the rFVIIIFc extension study, with a mean reduction in total score from 23.4 to 19.3 (Figure 2A). Of the 74 subjects, 24 were evaluated in year 3 of the extension study. These improvements in mHJHS scores were observed in year 3 of the extension study, regardless of the presence of target joints at baseline in the rFVIIIFc pivotal phase 3 study (Figure 2B). The mean follow-up period was 2.8 years (range, 2.5–3.3). Continuous improvement was observed regardless of the patient's prestudy treatment (Figure 3) or the presence of target joints at baseline in the rFVIIIFc pivotal phase 3 study (Figure 4). For these patients evaluated in year 3 of the extension study, the mean mHJHS total score for adults / adolescents across all time points was 25.0 (standard error of the mean [SEM], 2.9) at baseline. The mean (SEM) change from baseline was -2.0 (1.2) at expansion baseline, -3.8 (1.5) at expansion year 1, -4.5 (1.6) at expansion year 2, and -5.1 (1.5) at expansion year 3 (Figure 3B). The changes from baseline to expansion year 3 were statistically significant (P < 0.002). The pediatric rFVIIIFc pivotal phase 3 analysis population (n = 24) also demonstrated a statistically significant mean (SEM) improvement from baseline to expansion year 2 (-1.2 [0.56]; P < 0.05) (Figure 3C). Subjects with the highest quartile of disability in the mHJHS score at rFVIIIFc pivotal phase 3 baseline demonstrated the greatest improvement in mHJHS total score from baseline to rFVIIIFc expansion year 2 (Figure 5). A sustained improvement in mHJHS target joint scores was observed from baseline in the rFVIIIFc pivotal phase 3 study to year 2 of the rFVIIIFc expansion study, ranging from a mean of 7.0 at baseline to 1.0 at year 2 of the rFVIIIFc expansion study. The mean score improved to a mean of 5.3 at year 2 of the extension study (Figure 6). The mHJHS joint scores for weight-bearing joints decreased from a mean of 8.0 to a mean of 6.8 from the rFVIIIFc pivotal phase 3 study baseline to year 2 of the rFVIIIFc extension study, and for non-weight-bearing joints, they decreased from a mean of 6.7 to a mean of 4.8 (Figure 7). To calculate weight-bearing (ankle and knee) and non-weight-bearing (elbow) joint scores, scores were first derived as the sum of the per-joint scores for the left and right joint pairs (ankle, knee, or elbow). The weight-bearing joint score was then calculated as the mean of the scores for the ankle and knee, and the non-weight-bearing joint score was the same as the score for the elbow. Furthermore, specific improvements in swelling, range of motion, and muscle strength were also observed from the rFVIIIFc pivotal phase 3 study baseline to year 2 of the rFVIIIFc extension study, which were the most significant contributors to the change in the mHJHS total score (Figure 8).

[0297] Statistically significant mean improvements from baseline in the rFVIIIFc pivotal phase 3 study were observed for both weight-bearing and non-weight-bearing joints at year 3 (-1.1 [SEM, 0.5; P = 0.036] and -3.0 [SEM, 0.8; P = 0.001], respectively). Individual components of the mHJHS that showed a 20% or greater decrease from baseline to year 3 were swelling (-47%), muscle atrophy (-26%), and joint crepitus (-20%) (P < 0.05 for all three components), as well as joint instability (-89%), joint pain (-31%), and muscle strength (-26%) (Figure 9). [Example]

[0298] Targeted joint outcomes of prophylaxis with rFIXFc in adults and adolescents with hemophilia B In patients with severe hemophilia B, repeated bleeding into joints without adequate treatment can lead to serious chronic joint disease, pain, and reduced quality of life (Djambas Khayat, J Blood Med. 7:275-82 (2016)). Prophylactic treatment with recombinant factor IX Fc fusion protein (rFIXFc) resulted in fewer annualized bleeding episodes (ABR) and fewer spontaneous / traumatic bleeding events in adolescents and adults with severe hemophilia B (Kavakli et al., Haemophilia 22(3):381-88 (2016); Powell et al., N Engl J Med. 369(24):2313-23 (2013); Powell et al., Br J Haematol. 168(1):124-34 (2015); Powell et al., Br J Haematol. 168(1):113-23 (2015)). In addition to preventing joint damage and reducing bleeding events, prophylactic treatment with both conventional and long-acting rFIX may result in reduced work or school interruptions, fewer hospitalizations, less frequent monitoring, and improved quality of life (Kavakli et al., Haemophilia 22(3):381-88 (2016); Wyrwich et al., Haemophilia 22(6):866-72 (2016)). Prophylactic treatment initiated early in life reduces bleeding and prevents joint damage. Even when initiated later in life, once joint damage has occurred, prophylactic treatment can still significantly reduce the number of bleeding episodes, including bleeding into joints (Fischer et al., Haemophilia 20(Suppl 4):106-13 (2014)). Adults / adolescents with severe hemophilia B who completed the rFIXFc pivotal phase 3 trial (NCT01027364) were able to enroll in a long-term extension study evaluating the safety and efficacy of rFIXFc (NCT01425723; Pasi et al., Thromb Haemost. 117(3):508-18 (2017)). Long-term outcomes from subjects with target joints at enrollment in the rFIXFc pivotal phase 3 trial through the long-term extension study are reported here.

[0299] The purpose of this study is to obtain long-term data from the rFIXFc pivotal Phase 3 study from subjects with target joints at enrollment through the second interim data cut of the extension study (September 11, 2015). The goal is to provide the best possible outcomes.

[0300] method Study design and population Subjects with severe hemophilia B (endogenous FIX ≤2 IU / dL) completing the rFIXFc pivotal Phase 3 study were enrolled in one of four treatment arms in the expansion study: (1) weekly prophylaxis (WP; 20–100 IU / kg every 7 days); (2) individualized interval prophylaxis (IP; 100 IU / kg every 8–16 days); (3) modified prophylaxis (MP; investigators can individualize dosing for subjects not achieving optimal dosing with IP or WP); and (4) emergency treatment (ET; on-demand dosing based on the type and severity of the bleeding episode). Subjects could switch treatment arms at enrollment in the expansion study and at any time during the study. Subjects who switched treatment arms were included in the analysis of each treatment arm for the time spent on that treatment regimen, so individual subjects were counted in more than one treatment arm in the analysis. Subjects with one or more target joints (large joints with three or more bleeding episodes in a three-month period) at the time of pivotal Phase 3 study enrollment were evaluated.

[0301] Outcome measures and statistical analyses Outcomes were analyzed over the cumulative period from the rFIXFc pivotal Phase 3 trial through the second B-YOND interim data cut (September 11, 2015). Analysis of target joint resolution was performed. Target joint resolution was defined as two or fewer spontaneous bleeds in the target joint over a consecutive 12-month period (Blanchette et al., J Thromb Haemost. 12(11):1935-39 (2014)).

[0302] result Baseline characteristics for subjects with target joints are shown in Table 6. Of the 117 rFIXFc pivotal Phase 3 study subjects with on-study data, 60 had a total of 166 target joints at baseline. These subjects received rFIXFc for a cumulative median (interquartile range [IQR]) duration of 3.4 (1.4-4.2) years.

[0303] [Table 6]

[0304] Prophylactic administration The mean weekly doses and dosing intervals are summarized in Table 7.

[0305] [Table 7]

[0306] Number of bleeding episodes Pre-study and on-study bleeding data are shown in Figures 10A and 10B, respectively. Subjects receiving rFIXFc prophylaxis whose target joints did not rebleed during the study were as follows: WP: 15 of 40 (37.5%); IP: 1 of 12 (8.3%); MP: 4 of 12 (33.3%); ET: 0 of 14 (0%). Target joint bleeding at baseline In subjects who had rFIXFc prophylaxis, the on-study total and target joint ABRs were lower than the number of bleeds with pre-study treatment (FIGS. 10A and 10B).

[0307] [Table 8]

[0308] Resolution at target joints Overall, 100% (93 / 93) of target joints (out of 37) resolved as indicated by two or fewer spontaneous bleeds over a 12-month period (Figure 11).

[0309] conclusion In adults / adolescents with severe hemophilia B, long-term rFIXFc prophylaxis resulted in target joint resolution and lower target joint ABR in 100% of subjects with evaluable target joints at baseline across all treatment groups. Physicians should consider the beneficial and significantly improved long-term outcomes for patients with target joints achieved with rFIXFc when designing treatment prophylaxis plans. [Example]

[0310] In vivo single photon emission computed tomography (SPECT) in HemB mice 125 Biodistribution of I-labeled FIXFc, FIX, and GlycoPEGylated FIX In vivo mouse studies were performed to assess the biodistribution of FIX protein at multiple time points after administration by in vivo single photon emission computed tomography (SPECT). 125 The FIX protein was labeled at lysine residues using an I-labeled SIB linker. The labeled FIX protein was administered to 7- to 12-week-old HemB mice in a single therapeutically relevant dose. 125 I-SIB-FIX was administered as a single dose of 1 mg / kg (n=3); 125 I-SIB-FIXFc was administered as a single dose of 2 mg / kg (n=3), 125 I-SIB-FIX-PEG was administered as a single dose of 1 mg / kg (n=3).

[0311] The localization of the labeled FIX protein was imaged at 0.5, 2.5, 20, 48, 92, 120, 168, and / or 216 h (Figures 10A-10C). Specific localization to the joint regions (e.g., right / left knee and right / left shoulder), heart (left ventricle), and liver was analyzed using region-of-interest (ROI) placement. The ROIs for the left ventricle and liver were fixed volume objects placed within the exact organ. The ROI for the knee joint was defined by placing a cylinder around the bone from approximately halfway down the femur to halfway down the tibia / fibula. The shoulder ROI was defined by placing a uniform sphere at the center of the shoulder, then placing a bone threshold within the sphere and expanding the ROI.

[0312] Labeled FIXFc was found to distribute to the periarticular region at 30 min (Figure 12D) and 2.5 h (Figure 12E) after administration and persisted for 48–216 h after administration (Figures 12F–12G), corresponding to a half-life and five-fold half-life, respectively. FIXFc localization to the knee (Figure 13A) and shoulder (Figure 13B) was more pronounced than that of FIX and GlycoPEG-FIX in the same joint regions.

[0313] 125 I-SIB labeling did not affect the FIX activity of the labeled FIX protein (Figure 14A), nor did labeling affect the pharmacokinetic properties of the FIX protein compared to unlabeled FIX protein after administration to HemB mice (Figure 14B). [Example]

[0314] Baseline joint assessment Six joints (left ankle - LA, right ankle - RA, left elbow - LE, right elbow - RE, left knee - LK, right knee - RK) are scored on a scale of 0 to 19 according to the following criteria: swelling, duration, muscle atrophy, joint friction, loss of flexion, loss of extension, instability, joint pain, and muscle strength. Gait is scored on a scale of 0 to 2 based on walking and stair climbing. The total score is the sum of the scores from all six joints plus the gait score (ranging from 0 to 116, with 0 being normal and 116 being the most severe disease).

[0315] Screening visit The elbows, knees, and ankles on each side of the body are evaluated for joint disease at screening. Joint function should be assessed in the absence of active bleeding episodes. A joint score of 0 reflects a normal state.

[0316] Scoring Details 1. Joint scoring is performed separately for the six joints (LA, RA, LE, RE, LK, RK) according to these categories and scales (range 0-19 for each joint and 0-114 for all six joints): swelling (0=none; 1=mild; 2=moderate; 3=severe); duration of swelling (0=no swelling or less than 6 months; 1=more than 6 months); muscle atrophy (0=none; 1=mild; 2=severe); friction rubs on movement (0=none; 1=present); loss of flexion, such as loss of ankle plantar flexion (0=none; 1=mild; 2=moderate; 3=severe); instability (0=none; 1=significant pathological joint laxity); joint pain (0= muscle strength (0 = normal (holds position against gravity and maximum resistance); 1 = minimal decrease (holds position against gravity and moderate resistance, but not at maximum resistance); 2 = mild decrease (holds position against gravity or minimum resistance); 3 = moderate decrease (joint can move when gravity is removed); 4 = severe decrease (slight or no muscle contraction). To score flexion and extension loss at the knee and elbow, the following applies: none = approximately 0-5°; mild = approximately 5-10°; moderate = approximately 11-20°; and severe = more than approximately 20°.

[0317] 2. Gait is scored once (range 0-2) as 0 = no difficulty walking or climbing stairs; 1 = no difficulty walking but difficulty climbing stairs; and 2 = difficulty walking and climbing stairs.

[0318] This modified Hemophilia Joint Health Score (HJHS) is based on the scoring system used in a joint scoring reliability study in boys with hemophilia (Hilliard, Funk et al., Hemophilia 2002, 14, 1999, which is incorporated herein by reference in its entirety). ia 12(5):518-525 (2006)). It has been used as a tool to assess skeletal muscle outcomes in a cohort of 20 boys aged 4-17 years (Saulyte Trakymiene, Ingerslev et al., Haemophilia 16(3):479-486 (2010), incorporated herein by reference in its entirety). Modifications were made to this scoring system to adapt it to the adult hemophilia population and following comments in a recent validation study by the International Hemophilia Prevention Study Group (Feldman, Funk et al., Arthritis Care Res (Hoboken), 2010, incorporated herein by reference in its entirety). [Example]

[0319] The major complication of factor-based replacement therapy in hemophilia A is the formation of inhibitors (neutralizing anti-factor VIII antibodies) in approximately 30% of patients with severe hemophilia A. The development of inhibitors affects the efficacy of treatment as well as the quality of life of affected individuals. Further understanding of how the immune system responds to recombinant factor VIII (rFVIII) is an ongoing effort in hemophilia research to efficiently eradicate inhibitors. The half-life extended rFVIII Fc fusion protein (rFVIIIFc) is an effective and well-tolerated therapy for preventing and controlling bleeding episodes. The Fc region of this molecule has been shown to inhibit immune tolerance in preclinical animal models (Krishnamoorthy S. et al., Cell Immunol. 301:30-39 (2016)) and in case reports of immune tolerance induction (Groomes CL et al., Pediatr Blood Cancer 63(5):922-24 (2016); Malec LM et al., Haemophilia 22(6):e552-e554 (2016); Ragni MV et al., Haemophilia 22(5):e462-e464 (2016)), it not only contributes to an increase in rFVIII half-life but can also promote antigen-specific tolerance.

[0320] method Using peripheral blood-derived human APCs or THP-1 monocytes, we investigated the effects of rFVIIIFc on FcγR binding, internalization, signaling and cytokine production, as well as gene expression changes, and subsequent interactions and effects on T cells in vitro (Figure 16).

[0321] result Decreased cell surface expression of FcγR indicates internalization upon rFVIIIFc treatment (Figures 17A-17C). Monocyte-derived macrophages and dendritic cells were treated for 24 hours with equimolar concentrations (200 nM) of recombinant factor VIII (rFVIII) or rFVIII Fc fusion protein (rFVIIIFc), including horseradish peroxidase immunoconjugate (HRP-IC) as a positive control and human immunoglobulin G1 (IgG1) as a negative control. Cell surface expression of Fcγ receptors (FcγR) CD16 (Figure 17A), CD32 (Figure 17B), and CD64 (Figure 17C) was measured by flow cytometry (n=3; ** P ≤ 0.01; *** *P<0.005, significance of HRP-IC versus other treatments not shown. Treatment with rFVIIIFc correlated with decreased cell surface expression of CD16 (Figure 17A), CD32 (Figure 17B), and CD64 (Figure 17C) compared with the cell surface after treatment with rFVIII.

[0322] rFVIIIFc engages FcγR and induces signaling in monocytes and macrophages without subsequent inflammatory cytokine production (Figures 18A-18C). THP-1 monocytic cell line, monocytes, peripheral blood monocyte-derived macrophages, and peripheral blood monocyte-derived dendritic cells were treated with HRP-IC, IgG1, rFVIII, or rFVIIIFc for 15 minutes (Figure 18A). Syk phosphorylation was measured in cell lysates using the MSD platform (n=3-7). * P ≤ 0.05). Syk phosphorylation was significantly reduced by rFVIIIFc (WT ), an rFVIIIFc mutant unable to bind to neonatal Fc receptors (FcRn mutants), or an rFVIIIFc mutant unable to bind to FcγR (FcγR mutants) (n=4, * *P≦0.05) (FIG. 18B). Inflammatory cytokine production in macrophages treated for 24 hours was measured by MSD ELISA (n=4, significance not shown) (FIG. 18C).

[0323] rFVIIIFc phosphorylates molecules involved in immune regulation rather than molecules that play a role in activation and inflammatory cytokine production (Table 9 and Figure 19). Phosphorylated proteins in lysates from monocyte-derived macrophages treated with rFVIIIFc for 15 minutes were queried using Proteome Profiler phosphokinase and phosphoimmunoreceptor arrays. A list of phosphorylated molecules in rFVIIIFc-treated macrophages identified by Proteome Profiler arrays is shown in Table 9. Phosphorylation of phosphatases responsible for inhibitory signaling was measured using the MSD platform (n=3; ** P ≤ 0.01; *** P ≤ 0.005) (Figure 19).

[0324] [Table 9]

[0325] rFVIIIFc induces gene expression patterns characteristic of tolerogenic macrophages (Figures 20A-20G). Exploratory RNA sequencing was performed on monocyte-derived macrophages treated with IgG1, rFVIII, or rFVIIIFc for 6 hours (n=3) for significantly downregulated genes (Figure 20A) and significantly upregulated genes (Figure 20B). To investigate the molecular pathways that showed selectivity in these cells compared with rFVIII-treated cells, pathway analysis was performed on genes upregulated by rFVIIIFc (Table 9). Various genes in the NRF2 and PPAR-gamma pathways, as well as various other immune regulators, were found to be upregulated (Figure 20H). Selected genes in the NRF2 and lipid metabolism pathways were validated by Q-PCR (n=8; * P ≤ 0.05; ** P ≤ 0.01; *** P < 0.005) (Figures 20C-20G). Furthermore, macrophages educated by rFVIIIFc were found to exhibit a characteristic M2-like phenotype (Figures 20I-20M). Notably, macrophages treated with rFVIIIFc had higher relative CD206 expression than cells treated with rFVIII after 6 hours (Figure 20I) and 24 hours (Figure 20J), and macrophages treated with rFVIIIFc had higher relative ARG1 expression than cells treated with rFVIII after 24 hours (Figure 20M).

[0326] [Table 10]

[0327] Antigen-presenting cells treated with rFVIIIFc affect regulatory T cell differentiation, which requires APC-T cell cell contact (Figures 21A-21C). Peripheral blood monocyte-derived macrophages were treated with IgG1, rFVIII, or rFVIIIFc and then cocultured with naive CD4+ T cells isolated from peripheral blood from the same donor. After 6 days in coculture (Figure 21A), the percentage of regulatory T cells (CD4+CD25+FoxP3+) was quantified using flow cytometry (n=4) (Figure 21B). The percentage of regulatory T cells was also quantified when naive T cells were cultured in conditioned medium from APCs pretreated with IgG1, rFVIII, or rFVIIIFc (n=4) (Figure 21C).

[0328] conclusion rFVIIIFc appears to bind via Fcγ receptors on APCs, inducing their internalization and signaling. This signaling does not translate into inflammatory cytokine production or activate APCs (data not shown). Upon rFVIIIFc treatment, immunoregulatory signaling events are initiated. These events appear to drive macrophage differentiation toward an M2-like phenotype, characterized by upregulation of the NRF2 and PPARγ pathways (Figure 20H) and upregulation of CD206 and arginase 1 molecules. Various other immunoregulatory factors also showed increased expression, although at least guanylate cyclase 1 soluble subunit beta (2GUCY1B2), protoporphyrinogen oxidase (PPOX), and suppressor of cytokine signaling 3 (SOCS3) showed decreased expression in rFVIIIFc-treated cells (Figure 20H). These macrophages are capable of carrying out previously reported beneficial immunological effects such as regulatory T cell differentiation, FVIII tolerization, and anti-FVIII inhibitor reduction (Figures 22 and 23). [Example]

[0329] The pharmacokinetic (PK) profile of factor IX (FIX) is consistent with a two-compartment model in which the majority of FIX is distributed to the extravascular space outside the plasma compartment. Using SPECT imaging, we previously demonstrated that the biodistribution profiles of rFIXFc and rFIX in hemophilia B are consistent with the hypothesis that FIX can distribute to tissues outside the plasma compartment, but that most of the glycoPEGylated rFIX remains in the plasma compartment due to modifications imposed by the PEG moiety. In this study, we evaluated the distribution of rFIX and rFIXFc in nonhuman primates (NHPs).

[0330] rFIXFc and rFIX were purified using 124I-SIB (succinimidyl iodobenzoate) Cynomolgus monkeys were administered a single IV bolus injection of approximately 2 mCi microdose of 124I-SIB-rFIXFc (2 mg / kg) or 124I-SIB-rFIX (1 mg / kg). Reconstructed whole-body PET / CT scans were used to generate maximum intensity projection (MIP) images, and in vivo biodistribution was determined for region-of-interest (ROI) analysis.

[0331] PET imaging in NHPs demonstrated initial distribution of 124I-SIB-rFIXFc and 124I-SIB-rFIX in the blood pool, heart, liver, and kidney. At later time points, both rFIXFc and rFIX showed distribution to the shoulder joint and other bony joints (wrist, ankle, and jaw), with rFIXFc showing significantly higher distribution to these regions even at time points when FIX plasma levels were below detection levels. Both in vivo and ex vivo data demonstrated blood pool clearance of rFIXFc and rFIX. TCA precipitation data indicated that >95% of the radioactivity was associated with FIX.

[0332] The biodistribution profiles of rFIXFc and rFIX in NHPs were similar to those observed in hemophilia B mice, consistent with the hypothesis that FIX can distribute to tissues outside the plasma compartment. The significantly higher distribution of rFIXFc to the joint region compared with rFIX could potentially reflect retention in these tissues due to Fc-mediated mechanisms or improved PK. While the role of extravascular distribution in preventing bleeding and overall protection of joint health remains an area of ​​investigation, this study is consistent with observations in mice that showed differences in the biodistribution of FIX variants, suggesting that plasma levels are not equivalent or have the same meaning across FIX variants.

[0333] The foregoing description of specific embodiments is intended to fully illustrate the general nature of the present invention, such that others, by applying knowledge within the skill of the art, may readily modify and / or adapt such specific embodiments to various applications without undue experimentation and without departing from the general concept of the present invention. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, and should be interpreted by one of ordinary skill in the art in light of the teaching and guidance.

[0334] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0335] All publications, patents, and patent applications disclosed in this specification are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. Use of a chimeric protein or composition comprising a coagulation factor and an Fc region in a method for treating reversible hemophilic arthropathy of the joints in humans with hemophilia.

2. The use of claim 1 , wherein the reversible hemophilic arthropathy comprises synovitis, microhemorrhages, or both.

3. Use of a chimeric protein or composition comprising a coagulation factor and an Fc region in a method for reducing the occurrence of or prophylactically treating vascular remodeling in the joints of humans with hemophilia.

4. 1. Use of a chimeric protein or composition comprising a coagulation factor and an Fc region in a method for improving periarticular soft tissue in humans with hemophilia.

5. The use according to any one of claims 1 to 4, wherein the administration improves the Joint Health Score (HJHS) in humans.

6. 6. The use of any one of claims 1 to 5, wherein administration reduces joint pain in humans.

7. 7. The use according to any one of claims 1 to 6, wherein the joints are selected from the group consisting of one or both elbows, one or both knees, one or both ankles, one or both shoulders, one or both hips, one or both wrists, one or more joints of the hand, one or more joints of the foot, and any combination thereof.

8. The use according to any one of claims 1 to 7, wherein the Fc region specifically binds to the low affinity immunoglobulin gamma Fc region receptor II-b (FcγRIIB).

9. 8. The use of any one of claims 1 to 7, further comprising identifying the human in need of treatment using an imaging system selected from the group consisting of radiography, magnetic resonance imaging, ultrasound contrast imaging, power Doppler ultrasonography, or any combination thereof.

10. 10. The use according to any one of claims 1 to 9, wherein the coagulation factor is selected from the group consisting of factor VII (FVII), factor VIIa (FVIIa), factor VIII (FVIII), factor IX (FIX), factor X (FX), von Willebrand factor (VWF), antigen-binding portions thereof that specifically bind to FIX and FX, or any combination thereof.

11. The use according to any one of claims 5 to 10, wherein the chimeric protein comprises FVIII-Fc or FIX-Fc.

12. The use according to claim 11, wherein the effective amount of the chimeric protein comprising FVIII-Fc is about 20 IU / kg to about 300 IU / kg.

13. The use of claim 11 or 12, wherein the chimeric protein comprising FVIII-Fc is administered at an administration interval of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, or about 24 days.

14. The use according to claim 11, wherein the effective amount of the chimeric protein comprising FIX-Fc is about 20 IU / kg to about 100 IU / kg.

15. 15. The use of claim 11 or 14, wherein the chimeric protein comprising FIX-Fc is administered at an administration interval of about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

16. The use according to any one of claims 1 to 15, wherein the coagulation factor is distributed in tissues outside the plasma compartment as well as within the plasma compartment.

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