Methods of inducing immune tolerance to clotting factors

Abstract

To provide methods of inducing immune tolerance in a human with hemophilia.SOLUTION: The present disclosure provides methods inducing immune tolerance in a human, comprising a step of administering to the human an effective amount of a composition or a chimeric protein comprising a clotting factor and an Fc region.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 429,516, filed on December 2, 2016; No. 62 / 466,937, filed on March 3, 2017; No. 62 / 529,866, filed on July 7, 2017; No. 62 / 558,790, filed on September 14, 2017; and No. 62 / 582,829, filed on November 7, 2017, each of which is incorporated herein by reference in its entirety.

[0002] The present disclosure generally relates to the field of therapeutic agents for hemostatic disorders.

Background Art

[0003] Hemophilia is an X - linked bleeding disorder caused by mutations and / or deletions in genes encoding coagulation proteins, particularly the mutation and / or deletion of the factor VIII (FVIII) gene that causes a deficiency in FVIII activity (hemophilia A) or the mutation and / or deletion of the factor IX gene that 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 is by replacement therapy targeting the restoration of FVIII and / or FIX activity to prevent spontaneous bleeding (see, e.g., Non - Patent Document 2).

[0004] Coagulation factor replacement therapy is the main treatment for hemophilia. However, the efficacy in these patients is greatly reduced because a significant proportion of hemophilia patients, including approximately 30% of patients with severe hemophilia A, develop inhibitors to the coagulation factor products. The immune response is a T - cell - dependent or B - cell - mediated immune response directed against the infused coagulation factor, such as FVIII replacement therapy.

[0005] People with severe hemophilia are at a higher risk of developing inhibitors, and approximately 5-8% of people with mild or moderate hemophilia A develop inhibitors. The development of coagulation factor inhibitors can be life-threatening because the antibodies can inhibit not only the infused coagulation factor concentrate but also any small amounts of coagulation factor proteins naturally produced by the body. Thus, people with mild or moderate hemophilia who have actually developed inhibitors effectively have severe hemophilia (<1% circulating factor).

[0006] Approximately 2-3% of people with hemophilia B develop inhibitors. Inhibitors in people with hemophilia B are less common than in hemophilia A, but they can be an even greater problem because about half of the patients with hemophilia B inhibitors develop anaphylactic reactions that can be life-threatening to the infused factor IX.

[0007] Therefore, there is still a need for methods to induce immune tolerance in people who have already developed an immune response to one or more coagulation factors and in people who did not respond to past immune tolerance therapies.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present disclosure provides a method of inducing immune tolerance in a human having 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, wherein the human has developed an inhibitor to the coagulation factor and is unable to respond to one or more previous immune tolerance therapies for the coagulation factor. In some embodiments, the method further comprises measuring the level of the inhibitory immune response before administration and measuring the level of the inhibitory immune response after administration. In some embodiments, the method further comprises comparing the level of the inhibitory immune response before administration to the level of the inhibitory immune response after administration.

Means for Solving the Problems

[0010] The present invention further provides a method of inducing immune tolerance in a human having hemophilia, the method comprising: (1) 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, wherein the effective amount of the chimeric protein induces immune tolerance in the human; and (2) after induction of immune tolerance, administering to the human a tapering regimen of the chimeric protein. In certain embodiments, induction of immune tolerance occurs when the titer of inhibitory antibodies in the human is less than about 0.6 Bethesda units (BU). In certain embodiments, the method further comprises (3) after the tapering regimen, administering to the human a prophylactic dose of the coagulation factor. In certain embodiments, the human has not been treated with a previous immune tolerance therapy for the coagulation factor.

[0011] In some embodiments, the human has developed an inhibitory immune response to the coagulation factor. In some embodiments, the inhibitory immune response comprises production of inhibitory antibodies to the coagulation factor. In some embodiments, the titer of inhibitory antibodies before administration is at least about 0.6 Bethesda units (BU). In some embodiments, the titer of inhibitory antibodies after administration is less than about 0.6 BU.

[0012] In some embodiments, the immune response includes a cellular immune response. In some embodiments, the cellular immune response includes the release of cytokines. In some embodiments, administration results in a reduction of cytokine levels in a human as compared to levels in the human after past treatment with a polypeptide consisting of an FVIII polypeptide. In some embodiments, the cytokine is selected from the group consisting of IL-12, IL-4, IL-17, TNF-α, and any combination thereof.

[0013] In certain embodiments, the expression of one or more tolerogenic molecules is increased after administration as compared to the expression level of the one or more tolerogenic molecules before administration. In some embodiments, the one or more tolerogenic molecules are selected from IL-10, TGF-β, IL-35, IDO-1, and any combination thereof. In other embodiments, the immune response includes clinical symptoms selected from the group consisting of an increased bleeding tendency, high coagulation factor consumption, lack of response to coagulation factor therapy, decreased efficacy of coagulation factor therapy, and shortened half-life of coagulation factors.

[0014] In some embodiments, the human was previously diagnosed as having had an inhibitory immune response to a coagulation factor at least about 1 month before administration, at least about 2 months before administration, at least about 3 months before administration, at least about 6 months before administration, at least about 12 months before administration, at least about 18 months before administration, at least about 24 months before administration, at least about 30 months before administration, at least about 36 months before administration, at least about 42 months before administration, at least about 48 months before administration, at least about 54 months before administration, at least about 60 months before administration, at least about 6 years before, at least about 7 years before, at least about 8 years before, or at least about 10 years before. In some embodiments, the period until tolerance occurs is about 1 to about 24 weeks, about 1 to about 23 weeks, about 1 to about 22 weeks, about 1 to about 21 weeks, about 2 to about 20 weeks, about 2 to about 19 weeks, about 2 to about 18 weeks, about 2 to about 17 weeks, about 3 to about 16 weeks, about 3 to about 15 weeks, about 3 to about 14 weeks, about 3 to about 13 weeks, about 4 to about 12 weeks, about 4 to about 11 weeks, about 4 to about 10 weeks, about 4 to about 9 weeks, about 5 to about 8 weeks, about 5 to about 7 weeks, about 5 to about 6 weeks, about 1 to about 12 weeks, about 1 to about 11 weeks, about 1 to about 10 weeks, about 1 to about 9 weeks, about 1 to about 8 weeks, about 1 to about 7 weeks, about 1 to about 6 weeks, about 1 to about 5 weeks, or about 1 to about 4 weeks.

[0015] In some embodiments, the coagulation factor is Factor VIII (FVIII). In some embodiments, the chimeric protein comprises FVIII-Fc. In some embodiments, the chimeric protein comprises an FVIII portion and a VWF portion, the FVIII portion comprises an 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 associate with each other.

[0016] 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), poly sialic acid, hydroxyethyl starch (HES), derivatives thereof, or any combination thereof.

[0017] In some embodiments, the effective amount of the chimeric protein comprising FVIII and an Fc region is about 20 IU / kg to about 300 IU / kg. In some embodiments, the chimeric protein comprising FVIII-Fc is administered at an administration interval of about 1 day, 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.

[0018] In certain embodiments, humans have previously mounted an FVIII-inhibitory immune response. In some embodiments, the human has a bleeding disorder selected from the group consisting of hemorrhagic coagulation disorders, hemophilic arthropathy, muscle bleeding, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fractures, bleeding in the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas fascia.

[0019] Embodiment E1. A method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region, wherein the human has generated an inhibitor to the coagulation factor and has not responded to one or more previous immune tolerance therapies for the coagulation factor.

[0020] E2. The method of E1, further comprising the step of measuring the level of inhibitory immune response before administration and the step of measuring the level of inhibitory immune response after administration.

[0021] E3. The method of E2, further comprising the step of comparing the level of inhibitory immune response before administration with the level of inhibitory immune response after administration.

[0022] E4. The method according to any one of E1 to E3, wherein the human has generated an inhibitory immune response to the coagulation factor.

[0023] E5. The method of E4, wherein the inhibitory immune response comprises the production of inhibitory antibodies to the coagulation factor.

[0024] E6. The method of E5, wherein the titer of inhibitory antibodies before administration is at least about 0.6 Bethesda units (BU).

[0025] The method according to E5 or E6, wherein the titer of the inhibitory antibody before administration is at least about 1 BU, at least about 2 BU, at least about 3 BU, at least about 4 BU, at least about 5 BU, at least about 6 BU, at least about 7 BU, at least about 10 BU, at least about 20 BU, at least about 30 BU, at least about 40 BU, at least about 50 BU, at least about 100 BU, at least about 150 BU, or at least about 200 BU.

[0026] The method according to any one of E5 to E7, wherein the titer of the inhibitory antibody before administration is at least about 5 BU.

[0027] The method according to any one of E5 to E8, wherein the titer of the inhibitory antibody after administration is less than about 0.6 BU.

[0028] The method according to any one of E5 to E9, wherein the titer of the inhibitory antibody after administration is 0 BU.

[0029] The method according to any one of E1 to E10, wherein the immune response includes a cellular immune response.

[0030] The method according to E11, wherein the cellular immune response includes the release of cytokines.

[0031] The method according to E12, wherein the cytokine level in a human is reduced upon administration as compared to the level in the human after past treatment with a polypeptide consisting of the FVIII polypeptide.

[0032] The method according to E12 or E13, wherein the cytokine is selected from the group consisting of IL-12, IL-4, IL-17, TNF-α, and any combination thereof.

[0033] The method according to any one of E1 to E14, wherein the expression of one or more tolerogenic molecules is increased after administration as compared to the expression level of the one or more tolerogenic molecules before administration.

[0034] E16. The method according to E15, wherein the one or more tolerogenic molecules are selected from IL-10, TGF-β, IL-35, IDO-1, and any combination thereof.

[0035] E17. The method according to any one of E1 to E14, wherein the immune response comprises clinical symptoms selected from the group consisting of an increased bleeding tendency, high consumption of coagulation factors, lack of response to coagulation factor therapy, reduced efficacy of coagulation factor therapy, and shortened half-life of coagulation factors.

[0036] E18. The method according to any one of E1 to E17, wherein the human has been previously diagnosed as having generated an inhibitory immune response against a coagulation factor at least about 3 months ago, at least about 6 months ago, at least about 12 months ago, at least about 18 months ago, at least about 24 months ago, at least about 30 months ago, at least about 36 months ago, at least about 42 months ago, at least about 48 months ago, at least about 54 months ago, at least about 60 months ago, at least about 6 years ago, at least about 7 years ago, at least about 8 years ago, or at least about 10 years ago.

[0037] E19. The method according to any one of E1 to E18, wherein the human has been previously diagnosed as having generated an inhibitory immune response against a coagulation factor at least about 5 years ago. The method according to any one of E1 to E18, wherein the human has been previously diagnosed as having generated an inhibitory immune response against a coagulation factor at least about 5 years ago.

[0038] E20. The method according to any one of E1 to E19, wherein the period until tolerance occurs is about 1 to about 24 weeks, about 1 to about 23 weeks, about 1 to about 22 weeks, about 1 to about 21 weeks, about 2 to about 20 weeks, about 2 to about 19 weeks, about 2 to about 18 weeks, about 2 to about 17 weeks, about 3 to about 16 weeks, about 3 to about 15 weeks, about 3 to about 14 weeks, about 3 to about 13 weeks, about 4 to about 12 weeks, about 4 to about 11 weeks, about 4 to about 10 weeks, about 4 to about 9 weeks, about 5 to about 8 weeks, about 5 to about 7 weeks, about 5 to about 6 weeks, about 1 to about 12 weeks, about 1 to about 11 weeks, about 1 to about 10 weeks, about 1 to about 9 weeks, about 1 to about 8 weeks, about 1 to about 7 weeks, about 1 to about 6 weeks, about 1 to about 5 weeks, or about 1 to about 4 weeks.

[0039] The method according to any one of E1 to E20, wherein the period until tolerance occurs is less than about 24 weeks, less than about 23 weeks, less than about 22 weeks, less than about 21 weeks, less than about 20 weeks, less than about 19 weeks, less than about 18 weeks, less than about 17 weeks, less than about 16 weeks, less than about 15 weeks, less than about 14 weeks, less than about 13 weeks, less than about 12 weeks, less than about 11 weeks, less than about 10 weeks, less than about 9 weeks, less than about 8 weeks, less than about 7 weeks, less than about 6 weeks, less than about 5 weeks, less than about 4 weeks, less than about 3 weeks, less than about 2 weeks, or less than about 1 week.

[0040] The method according to any one of E1 to E21, wherein the period until tolerance occurs is about 4 to about 12 weeks.

[0041] The method according to any one of E1 to E22, wherein the period until tolerance occurs is about 4 weeks.

[0042] The method according to any one of E1 to E23, wherein a human is undergoing interferon therapy.

[0043] The method according to any one of E1 to E24, wherein a human is undergoing antiviral therapy.

[0044] The method according to any one of E1 to E25, wherein a human has a gene polymorphism associated with an increase in TNF-α.

[0045] The method according to E26, wherein the polymorphism is TNF-308G>A.

[0046] The method according to any one of E1 to E27, wherein a human has a gene polymorphism associated with an increase in IL10.

[0047] The method according to E28, wherein the polymorphism is allele 134 of the IL10G microsatellite.

[0048] The method according to any one of E1 to E29, wherein a human has less than 150 exposure days (ED) to a coagulation factor.

[0049] E31. The method according to E30, wherein the human has ED less than 50.

[0050] E32. The method according to E31, wherein the human has ED less than 20.

[0051] E33. The method according to any one of E1 to E32, wherein the coagulation factor is factor VIII (FVIII).

[0052] E34. The method according to any one of E1 to E33, wherein the chimeric protein comprises FVIII-Fc.

[0053] E35. The chimeric protein comprises an FVIII part and a VWF part, the FVIII part comprises an FVIII polypeptide or a fragment thereof, the VWF part comprises a VWF polypeptide or a fragment thereof, the FVIII part is linked to a first Fc region, the VWF part is linked to a second Fc region, and the first Fc region and the second Fc region associate with each other. The method according to any one of E1 to E34.

[0054] E36. The method according to any one of E33 to E35, wherein the FVIII polypeptide comprises mature FVIII.

[0055] E37. The method according to any one of E33 to E35, wherein the FVIII polypeptide comprises B domain-deleted FVIII.

[0056] E38. The method according to E37, wherein the B domain-deleted FVIII comprises deletion of all or part of the B domain of FVIII.

[0057] E39. The method according to E37 or E38, wherein the B domain-deleted FVIII comprises deletion of amino acid residues 746 to 1648 of mature FVIII.

[0058] E40. The method according to any one of E33 to E39, wherein the VWF polypeptide comprises a VWF fragment containing the D’ domain and the D3 domain of VWF.

[0059] The method according to any one of E1 to E40, wherein the chimeric protein further comprises a half-life extension portion.

[0060] The method according to E41, wherein the half-life extension portion comprises albumin or a fragment thereof, an albumin-binding portion, a PAS sequence, a HAP sequence, transferrin or a fragment thereof, polyethylene glycol (PEG), poly sialic acid, hydroxyethyl starch (HES), a derivative thereof, or any combination thereof.

[0061] The method according to E41 or E42, wherein the half-life extension portion is inserted into a coagulation factor.

[0062] The method according to E41 or E42, wherein the half-life extension portion is inserted between a coagulation factor and an Fc region.

[0063] The method according to any one of E33 to E44, wherein the effective amount of the chimeric protein comprising FVIII and an Fc region is from about 20 IU / kg to about 300 IU / kg.

[0064] The effective amount of the chimeric protein comprising FVIII-Fc is from about 100 IU / kg to about 300 IU / kg, from about 100 IU / kg to about 200 IU / kg, from about 100 IU / kg to about 290 IU / kg, from about 100 IU / kg to about 280 IU / kg, from about 100 IU / kg to about 270 IU / kg, from about 100 IU / kg to about 260 IU / kg, from about 100 IU / kg to about 250 IU / kg, from about 100 IU / kg to about 240 IU / kg, from about 100 IU / kg to about 230 IU / kg, from about 100 IU / kg to about 220 IU / kg, from about 100 IU / kg to about 210 IU / kg, from about 150 IU / kg to about 300 IU / kg, from about 150 IU / kg to about 290 IU / kg, from about 150 IU / kg to about 280 IU / kg, from about 150 IU / kg to The method according to E45, wherein it is about 270 IU / kg, about 150 IU / kg to about 260 IU / kg, about 150 IU / kg to about 250 IU / kg, about 150 IU / kg to about 240 IU / kg, about 140 IU / kg to about 250 IU / kg, about 130 IU / kg to about 260 IU / kg, about 120 IU / kg to about 270 IU / kg, about 110 IU / kg to about 280 IU / kg, about 200 IU / kg to about 290 IU / kg, about 200 IU / kg to about 280 IU / kg, about 200 IU / kg to about 270 IU / kg, about 200 IU / kg to about 260 IU / kg, about 200 IU / kg to about 250 IU / kg, about 200 IU / kg to about 240 IU / kg, about 200 IU / kg to about 230 IU / kg, about 200 IU / kg to about 220 IU / kg, or about 200 IU / kg to about 210 IU / kg.

[0065] E47. The method according to E45 or E46, wherein the effective amount of the chimeric protein comprising FVIII-Fc is about 100 IU / kg, 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, about 200 IU / kg, about 225 IU / kg, about 250 IU / kg, about 275 IU / kg, or about 300 IU / kg.

[0066] E48. The method according to any one of E33 to E47, 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.

[0067] The method according to any one of E33 to E47, 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.

[0068] The method according to any one of E33 to E49, wherein the chimeric protein comprising FVIII-Fc is administered at an administration interval of about 3 days to about 5 days.

[0069] The method according to any one of E1 to E33, wherein the chimeric protein comprises an FVIII portion, a VWF portion, a first Fc region, and a second Fc region, the FVIII portion comprises an 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 associate with each other.

[0070] The method according to any one of E1 to E51, wherein a human has previously developed an FVIII inhibitory immune response.

[0071] E53. The inhibitory FVIII immune response is ADVATE (registered trademark), RECOMBINATE (registered trademark), KOGENATE FS (registered trademark), HELIXATE FS (registered trademark), XYNTHA / REFACTO AB (registered trademark), HEMOFIL-M (registered trademark), MONARC-M (registered trademark), MONOCLATE-P (registered trademark), HUMA The method according to E52, which occurs in response to an FVIII product selected from the group consisting of TE-P (registered trademark), ALPHANATE (registered trademark), KOATE-DVI (registered trademark), AFSTYLA (registered trademark), and HYATE:C (registered trademark).

[0072] E54. The method according to E52, wherein an inhibitory FVIII immune response occurs in response to a recombinant FVIII product.

[0073] E55. The method according to any one of E1 to E54, wherein a human has a bleeding condition selected from the group consisting of hemorrhagic coagulation disorders, hemophilic arthropathy, muscle bleeding, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fracture, bleeding in the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal cavity, and bleeding in the iliopsoas fascia.

[0074] E56. The method according to E55, wherein the hemorrhagic coagulation disorder is hemophilia A.

[0075] E57. The method according to any one of E33 to E56, wherein the effective amount of the chimeric protein comprising FVIII and the Fc region is from about 50 IU / kg to about 300 IU / kg.

[0076] E58. The method according to E57, wherein the effective amount of the chimeric protein comprising FVIII and the Fc region is about 50 IU / kg, about 60 IU / kg, about 70 IU / kg, about 80 IU / kg, about 90 IU / kg, about 100 IU / kg, about 110 IU / kg, about 120 IU / kg, about 130 IU / kg, about 140 IU / kg, about 150 IU / kg, about 160 IU / kg, about 170 IU / kg, about 180 IU / kg, about 190 IU / kg, about 200 IU / kg, about 225 IU / kg, about 250 IU / kg, about 275 IU / kg, or about 300 IU / kg.

[0077] E59. The method according to E57 or E58, wherein the effective amount of the chimeric protein is about 200 IU / kg and is administered daily.

[0078] The method according to E57 or E58, wherein the effective amount of the chimeric protein is about 50 IU / kg and is administered about three times a week.

[0079] E61. The method according to any one of E57 to E60, wherein the effective amount of the chimeric protein is administered in two or more doses throughout the day.

[0080] E62. The method according to any one of E57 to E61, wherein the chimeric protein is administered until immune tolerance is observed, and immune tolerance is observed when the titer of inhibitory antibodies in humans is less than about 0.6 BU.

[0081] E63. The method according to E62, wherein after immune tolerance, a tapering regimen of a chimeric protein comprising FVIII and an Fc region is administered to a human.

[0082] E64. The method according to E63, wherein the tapering regimen comprises administering a tapering dose of about 50 IU / kg to about 100 IU / kg of the chimeric protein comprising FVIII and an Fc region.

[0083] E65. The method according to E63 or E64, wherein the tapering dose is administered once a day or once every two days.

[0084] E66. The tapering dose is for at least about one week, at least about two weeks, at least about three weeks, at least about four weeks, at least about five weeks, at least about six weeks, at least about seven weeks , administered for at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, or at least about 32 weeks. The method according to any one of E63 to E65.

[0085] E67. The method according to any one of E63 to E66, wherein the tapering regimen comprises administering a tapering dose of about 50 IU / kg or about 100 IU / kg of the chimeric protein.

[0086] E68. The method according to any one of E63 to E67, wherein the tapering regimen comprises administering a tapering dose of about 50 IU / kg of the chimeric protein once daily from week 1 to week 6 after immune tolerance.

[0087] E69. The method according to any one of E63 to E67, wherein the tapering regimen comprises administering a tapering dose of about 100 IU / kg of the chimeric protein once daily from week 1 to week 6 after immune tolerance.

[0088] E70. The method according to E68 or E69, wherein the tapering regimen further comprises administering a tapering dose of about 50 IU / kg or about 100 IU / kg of the chimeric protein once every two days from week 6 to week 12 after immune tolerance.

[0089] E71. The method according to E70, wherein the tapering regimen further comprises administering a tapering dose of about 50 IU / kg or about 100 IU / kg of the chimeric protein once every two days from week 12 to week 16.

[0090] The method according to any one of E66 - E71, further comprising the step of administering a prophylactic dose of a coagulation factor after a tapered regimen.

[0091] E73. The method according to E72, wherein the prophylactic dose comprises from about 50 IU / kg to about 100 IU / kg.

[0092] E74. The method according to E72 or E73, wherein the prophylactic dose is administered about once a week, about twice a week, about three times a week, or once every about 3 - 5 days.

[0093] E75. A method for inducing immune tolerance in a human having hemophilia, (1) administering to the human an effective amount of a chimeric protein comprising a coagulation factor and an Fc region, wherein the effective amount of the chimeric protein comprising a coagulation factor and an Fc region induces immune tolerance in the human; (2) after induction of immune tolerance, administering to the human a tapered regimen of the chimeric protein.

[0094] E76. The method according to E75, wherein induction of immune tolerance occurs when the titer of inhibitory antibodies in the human is less than about 0.6 BU.

[0095] E77. The method according to E75 or E77, further comprising (3) after the tapered regimen, administering to the human a prophylactic dose of a coagulation factor.

[0096] E78. The method according to any one of E75 - E77, wherein the human has not been treated by a past immune tolerance therapy for a coagulation factor.

[0097] E79. The method according to any one of E75 - E78, further comprising the step of measuring the level of inhibitory immune response before administration and the level of inhibitory immune response after administration.

[0098] E80. The method according to E79, further comprising the step of comparing the level of inhibitory immune response before administration with the level of inhibitory immune response after administration.

[0099] E81. The method according to any one of E75 to E80, wherein a human generates an inhibitory immune response against a coagulation factor.

[0100] E82. The method according to E81, wherein the inhibitory immune response includes the production of inhibitory antibodies against a coagulation factor.

[0101] E83. The method according to E82, wherein the titer of the inhibitory antibody before administration is at least about 0.6 Bethesda unit (BU).

[0102] E84. The method according to E82 or E83, wherein the titer of the inhibitory antibody before administration is at least about 1 BU, at least about 2 BU, at least about 3 BU, at least about 4 BU, at least about 5 BU, at least about 6 BU, at least about 7 BU, at least about 10 BU, at least about 20 BU, at least about 30 BU, at least about 40 BU, at least about 50 BU, at least about 100 BU, at least about 150 BU, or at least about 200 BU.

[0103] E85. The method according to any one of E82 to E84, wherein the titer of the inhibitory antibody before administration is at least about 5 BU.

[0104] E86. The method according to any one of E82 to E85, wherein the titer of the inhibitory antibody after administration is less than about 0.6 BU.

[0105] E87. The method according to any one of E82 to E86, wherein the titer of the inhibitory antibody after administration is 0 BU.

[0106] E88. The method according to any one of E79 to E87, wherein the immune response includes a cellular immune response.

[0107] E89. The method according to E88, wherein the cellular immune response includes the release of cytokines.

[0108] The method according to E88, wherein cytokine levels in a human are reduced upon administration, as compared to levels in the human after previous treatment with a polypeptide consisting of an FVIII polypeptide.

[0109] E91. The method according to any one of E75 - E90, wherein the expression of one or more tolerogenic molecules is increased after administration, as compared to the expression level of the one or more tolerogenic molecules before administration.

[0110] E92. The method according to any one of E75 - E91, wherein the human was previously diagnosed as having generated an inhibitory immune response against a coagulation factor at least about 3 months before administration, at least about 6 months before administration, at least about 12 months before administration, at least about 18 months before administration, at least about 24 months before administration, at least about 30 months before administration, at least about 36 months before administration, at least about 42 months before administration, at least about 48 months before administration, at least about 54 months before administration, at least about 60 months before administration, at least about 6 years before, at least about 7 years before, at least about 8 years before, or at least about 10 years before.

[0111] E93. The method according to any one of E75 - E92, wherein the human was previously diagnosed as having generated an inhibitory immune response against a coagulation factor at least about 5 years before administration.

[0112] E94. The method according to any one of E75 - E93, wherein the period until tolerance occurs is about 1 - about 24 weeks, about 1 - about 23 weeks, about 1 - about 22 weeks, about 1 - about 21 weeks, about 2 - about 20 weeks, about 2 - about 19 weeks, about 2 - about 18 weeks, about 2 - about 17 weeks, about 3 - about 16 weeks, about 3 - about 15 weeks, about 3 - about 14 weeks, about 3 - about 13 weeks, about 4 - about 12 weeks, about 4 - about 11 weeks, about 4 - about 10 weeks, about 4 - about 9 weeks, about 5 - about 8 weeks, about 5 - about 7 weeks, about 5 - about 6 weeks, about 1 - about 12 weeks, about 1 - about 11 weeks, about 1 - about 10 weeks, about 1 - about 9 weeks, about 1 - about 8 weeks, about 1 - about 7 weeks, about 1 - about 6 weeks, about 1 - about 5 weeks, or about 1 - about 4 weeks.

[0113] The method according to any one of E75 to E94, wherein the period until tolerance occurs is less than about 24 weeks, less than about 23 weeks, less than about 22 weeks, less than about 21 weeks, less than about 20 weeks, less than about 19 weeks, less than about 18 weeks, less than about 17 weeks, less than about 16 weeks, less than about 15 weeks, less than about 14 weeks, less than about 13 weeks, less than about 12 weeks, less than about 11 weeks, less than about 10 weeks, less than about 9 weeks, less than about 8 weeks, less than about 7 weeks, less than about 6 weeks, less than about 5 weeks, less than about 4 weeks, less than about 3 weeks, less than about 2 weeks, or less than about 1 week.

[0114] E96. The period until tolerance occurs is about 4 to about 12 weeks. The method according to any one of E75 to E95.

[0115] E97. The period until tolerance occurs is about 4 weeks. The method according to any one of E75 to E96.

[0116] E98. The method according to any one of E75 to E97, wherein a human is undergoing interferon treatment.

[0117] E99. The method according to any one of E75 to E98, wherein a human is undergoing antiviral therapy.

[0118] E100. The method according to any one of E75 to E91, wherein a human has a gene polymorphism associated with an increase in TNF-α.

[0119] E101. The method according to E100, wherein the polymorphism is TNF-308G>A.

[0120] E102. The method according to any one of E75 to E101, wherein a human has a gene polymorphism associated with an increase in IL10.

[0121] E103. The method according to E102, wherein the polymorphism is allele 134 of the IL10G microsatellite.

[0122] The method according to any one of E75 to E103, wherein a human has an exposure days (ED) of less than 150 to a coagulation factor.

[0123] E105. The method according to E104, wherein a human has an ED of less than 50.

[0124] E106. The method according to E105, wherein a human has an ED of less than 20.

[0125] E107. The method according to any one of E75 to E106, wherein the coagulation factor is factor VIII (FVIII).

[0126] E108. The method according to any one of E75 to E107, wherein the chimeric protein comprises FVIII-Fc.

[0127] E109. The chimeric protein comprises an FVIII portion and a VWF portion, the FVIII portion comprises an 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 associate with each other. The method according to any one of E75 to E108.

[0128] E110. The method according to any one of E75 to E109, wherein the FVIII polypeptide comprises mature FVIII.

[0129] E111. The method according to any one of E75 to E109, wherein the FVIII polypeptide comprises B domain-deleted FVIII.

[0130] E112. The method according to E110, wherein the B domain-deleted FVIII comprises deletion of all or part of the B domain of FVIII.

[0131] E113. The method according to E110 or E111, wherein the B domain-deleted FVIII comprises deletion of amino acid residues 746 to 1648 of mature FVIII.

[0132] The method according to any one of E109 to E113, wherein the E114.VWF polypeptide comprises a VWF fragment comprising the D' domain and the D3 domain of VWF.

[0133] The method according to any one of E75 to E114, wherein the chimeric protein further comprises a half-life extension moiety.

[0134] The method according to E115, wherein the half-life extension 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), poly sialic acid, hydroxyethyl starch (HES), a derivative thereof, or any combination thereof.

[0135] The method according to E115 or E116, wherein the half-life extension moiety is inserted into a coagulation factor.

[0136] The method according to E115 or E116, wherein the half-life extension moiety is inserted between a coagulation factor and an Fc region.

[0137] The method according to any one of E75 to E118, wherein the effective amount of the chimeric protein comprising FVIII and an Fc region is from about 50 IU / kg to about 300 IU / kg.

[0138] The effective amount of the chimeric protein comprising FVIII-Fc is from about 100 IU / kg to about 300 IU / kg, from about 100 IU / kg to about 200 IU / kg, about 100 IU / kg The method described in E119, wherein the effective amount is from about 290 IU / kg, from about 100 IU / kg to about 280 IU / kg, from about 100 IU / kg to about 270 IU / kg, from about 100 IU / kg to about 260 IU / kg, from about 100 IU / kg to about 250 IU / kg, from about 100 IU / kg to about 240 IU / kg, from about 100 IU / kg to about 230 IU / kg, from about 100 IU / kg to about 220 IU / kg, from about 100 IU / kg to about 210 IU / kg, from about 150 IU / kg to about 300 IU / kg, from about 150 IU / kg to about 290 IU / kg, from about 150 IU / kg to about 280 IU / kg, from about 150 IU / kg to about 270 IU / kg, from about 150 IU / kg to about 260 IU / kg, from about 150 IU / kg to about 250 IU / kg, from about 150 IU / kg to about 240 IU / kg, from about 140 IU / kg to about 250 IU / kg, from about 130 IU / kg to about 260 IU / kg, from about 120 IU / kg to about 270 IU / kg, from about 110 IU / kg to about 280 IU / kg, from about 200 IU / kg to about 290 IU / kg, from about 200 IU / kg to about 280 IU / kg, from about 200 IU / kg to about 270 IU / kg, from about 200 IU / kg to about 260 IU / kg, from about 200 IU / kg to about 250 IU / kg, from about 200 IU / kg to about 240 IU / kg, from about 200 IU / kg to about 230 IU / kg, or from about 200 IU / kg to about 210 IU / kg.

[0139] E121. The method described in E119 or E120, wherein the effective amount of the chimeric protein comprising FVIII-Fc is about 50 IU / kg, about 60 IU / kg, about 70 IU / kg, about 80 IU / kg, about 90 IU / kg, about 100 IU / kg, about 110 IU / kg, about 120 IU / kg, about 130 IU / kg, about 140 IU / kg, about 150 IU / kg, about 160 IU / kg, about 170 IU / kg, about 180 IU / kg, about 190 IU / kg, about 200 IU / kg, about 225 IU / kg, about 250 IU / kg, about 275 IU / kg, or about 300 IU / kg.

[0140] E122. The method according to any one of E75 to E121, wherein the effective amount of the chimeric protein is about 200 IU / kg and is administered daily.

[0141] The method according to any one of E75 to E122, wherein the chimeric protein comprising E123.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.

[0142] The method according to any one of E75 to E121, wherein the chimeric protein comprising E124.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.

[0143] The method according to any one of E75 to E122, wherein the chimeric protein comprising E125.FVIII-Fc is administered at an administration interval of about 3 days to about 5 days.

[0144] The method according to any one of E75 to E125, wherein the chimeric protein comprises an FVIII portion, a VWF portion, a first Fc region, and a second Fc region, the FVIII portion comprises an 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 associate with each other.

[0145] The method according to any one of E75 to E126, wherein a human has previously developed an FVIII-inhibitory immune response.

[0146] E128. The method according to E127, wherein the inhibitory FVIII immune response is generated in response to an FVIII product selected from the group consisting of ADVATE (registered trademark), RECOMBINATE (registered trademark), KOGENATE FS (registered trademark), HELIXATE FS (registered trademark), XYNTHA / REFACTO AB (registered trademark), HEMOFIL-M (registered trademark), MONARC-M (registered trademark), MONOCLATE-P (registered trademark), HUMATE-P (registered trademark), ALPHANATE (registered trademark), KOATE-DVI (registered trademark), AFSTYLA (registered trademark), and HYATE:C (registered trademark).

[0147] E129. The method according to E128, wherein the inhibitory FVIII immune response is generated in response to a recombinant FVIII product.

[0148] E130. The method according to any one of E75 to E129, wherein the human has a bleeding condition selected from the group consisting of hemorrhagic coagulation disorder, hemophilic arthropathy, muscle bleeding, oral bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fracture, bleeding in the central nervous system, bleeding in the retropharyngeal space, bleeding in the retroperitoneal cavity, and bleeding in the iliopsoas fascia.

[0149] E131. The method according to E130, wherein the hemorrhagic coagulation disorder is hemophilia A.

[0150] E132. The method according to E75 to E131, wherein an effective amount of the chimeric protein is administered in a dose of two or more times per day.

[0151] E133. The method according to any one of E75 to E132, wherein the tapering regimen comprises administering a tapering dose of about 50 IU / kg to about 100 IU / kg of the chimeric protein.

[0152] E134. The method according to any one of E75 to E133, wherein the tapering dose is administered once a day, once every two days, or three times a week.

[0153] E135. The method according to any one of E75 to E134, wherein the tapered dosage is administered for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, or at least about 32 weeks.

[0154] E136. The method according to any one of E74 to E135, wherein the tapered regimen comprises administering a tapered dosage of about 50 IU / kg or about 100 IU / kg of the chimeric protein.

[0155] E137. The method according to any one of E75 to E136, wherein the tapered regimen comprises administering a tapered dosage of about 50 IU / kg of the chimeric protein once daily from the first week to the sixth week after immune tolerance.

[0156] E138. The method according to any one of E75 to E136, wherein the tapered regimen comprises administering a tapered dosage of about 100 IU / kg of the chimeric protein once daily from the first week to the sixth week after immune tolerance.

[0157] E139. The method according to E137 or E138, wherein the tapered regimen further comprises administering a tapered dosage of about 50 IU / kg or about 100 IU / kg of the chimeric protein once every two days from the sixth week to the twelfth week after immune tolerance.

[0158] The method according to E139, wherein the tapering regimen further comprises administering a tapering dose of the chimeric protein of about 50 IU / kg or about 100 IU / kg once every two days from week 12 to week 16.

[0159] The method according to any one of E77 to E140, wherein the prophylactic dose comprises from about 50 IU / kg to about 100 IU / kg.

[0160] The method according to any one of E77 to E141, wherein the prophylactic dose is administered about once a week, about twice a week, about three times a week or about three times a week.

[0161] The chimeric protein comprising a coagulation factor and an Fc region is administered to a human in less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 2 weeks, less than about 3 weeks, less than about 4 weeks, less than about 2 months, less than about 3 months, less than about 4 months, less than about 5 months, less than about 6 months, or less than about 1 year after measurement of the inhibitory immune response level in the human, according to any one of E79 to E91 and 94 to E142.

[0162] The chimeric protein comprising a coagulation factor and an Fc region is administered to a human in less than about 1 day after measurement of the inhibitory immune response level in the human, according to any one of E79 to E91 and 94 to E143.

[0163] The chimeric protein comprising a coagulation factor and an Fc region is administered to a human in less than about 12 hours after measurement of the inhibitory immune response level in the human, according to any one of E79 to E91 and 94 to E144.

[0164] The method according to any one of E1 to E145, wherein the period until tolerance occurs in a human by administration of the chimeric protein is shortened compared to the period until tolerance occurs in a human after treatment with the coagulation factor alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0165]

Figure 1

Figure 2

Figure 3-1

Figure 3-2

Figure 4

Figure 5-1

Figure 5-2

Figure 5-3

Figure 5-4

Figure 5-5

Figure 5-6

Figure 6A

Figure 6B

Figure 6C

Figure 7

Figure 8

[0166] The present disclosure provides a method for inducing immune tolerance in a human having 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, wherein the human has developed an inhibitor to the coagulation factor and has not responded to one or more past immune tolerance therapies for the coagulation factor. In some embodiments, the coagulation factor is selected from the group consisting of factor VII (FVII), factor VIIa (FVIIa), FVIII, FIX, factor X (FX), von Willebrand factor (VWF), and any combination thereof.

[0167] 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 of the It is understood to refer to a plurality of nucleotide sequences, and thus the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0168] Furthermore, "and / or" as used herein should be interpreted 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" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single) and "B" (single). Similarly, the term "and / or" as 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 (single); B (single); and C (single).

[0169] Whenever an embodiment is described herein with the language "comprising," it is understood that analogous embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0170] Unless otherwise defined, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill 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 many common dictionaries of terms used in this disclosure to one of ordinary skill in the art.

[0171] Units, prefixes, and symbols are written in the format approved by the Systeme International de Unites (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from amino to carboxy direction, left to right. The headings provided in this specification are not limitations on the various aspects of the disclosure , but can be obtained by referring to the specification as a whole. Accordingly, the terms defined below are more fully defined by referring to the specification in its entirety.

[0172] The term "about" is used herein to mean approximately, nearly, roughly, or in that region. When the term "about" is used with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. Thus, "about 10 to 20" means "about 10 to about 20". In general, the term "about" can modify the numerical values above and below the recited numerical value by, for example, a variance of up or down 10 percent (higher or lower).

[0173] As used herein, "administering" means, for example, giving a pharmaceutically acceptable composition comprising a chimeric protein as disclosed herein to a subject via a pharmaceutically acceptable route. The route of administration can be intravenous, such as intravenous injection and intravenous infusion. Further routes of administration include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The chimeric protein and the hybrid protein can be administered as part of a pharmaceutical composition comprising at least one excipient. In some embodiments, a coagulation factor and / or Fc, such as a chimeric protein, is administered to a human through gene therapy, for example, one or more polynucleotides encoding a coagulation factor and / or Fc, such as a chimeric protein, are administered to a human, and the coagulation factor and / or Fc, such as a chimeric protein, is expressed in the human.

[0174] As used herein, "treating", "treatment", or "being under treatment" refers to, for example, reducing the severity of a disease or condition; reducing the duration of a condition; improving or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition. In some embodiments, the term "treating" or "being under treatment" means reducing or eliminating an inhibitory immune response against a coagulation factor, such as FVIII.

[0175] As used herein, the term "inducing immune tolerance" means inducing in a subject a state in which the subject does not have an immune response when a specific stimulus is administered, for example, when a coagulation factor (e.g., FVIII) is administered. This state, immune tolerance, can be transient, where the subject is tolerant to the stimulus for a limited period, or can be long-term, where the subject is infinitely tolerant to the stimulus. In certain embodiments, the subject remains tolerant to the stimulus as long as the stimulus is administered to the subject. For example, in some embodiments, the subject remains tolerant to a coagulation factor as long as a chimeric protein comprising the coagulation factor and an Fc region is administered to the subject at a predetermined dosing interval. In other embodiments, the subject remains tolerant to the coagulation factor even after administration of the chimeric protein comprising the coagulation factor and an Fc region has ended.

[0176] In some embodiments, the immune response is an "inhibitory" immune response. An inhibitory immune response is an immune response that blocks or impairs the effect of the administration of a stimulus, such as a coagulation factor (e.g., FVIII). In certain embodiments, the inhibitory immune response includes the production of inhibitory antibodies against the stimulus, such as inhibitory anti-FVIII antibodies. As used herein, the term "inhibitory antibody" or "inhibitory antibodies" refers to an antibody that blocks or impairs the function of the antigen recognized by the antibody. For example, an inhibitory antibody against FVIII blocks or impairs the activity of FVIII. In some embodiments, the inhibitory antibody binds to an antigen, such as FVIII, and accelerates the clearance of the antigen from human serum. When an antibody accelerates the clearance of an antigen, the antibody reduces the half-life of the antigen.

[0177] An inhibitory immune response can be determined using clinical assays such as the Bethesda assay or the Nijmegen modification of the Bethesda assay. A level of at least 0.6 Bethesda units (BU) is inhibitory It can indicate the presence of a humoral immune response. A level of at least 5 BU can indicate the presence of high-titer inhibitors. Measurement of the in vivo recovery rate and half-life of a bolus infusion of a coagulation factor can also be used similarly. In certain embodiments, immune tolerance is observed when the titer of inhibitory antibodies in humans is less than about 5 BU, less than about 4 BU, less than about 3 BU, less than about 2 BU, less than about 1 BU, less than about 0.9 BU, less than about 0.8 BU, less than about 0.7 BU, less than about 0.6 BU, less than about 0.5 BU, less than about 0.4 BU, less than about 0.3 BU, less than about 0.2 BU, less than about 0.1 BU, or about 0 BU. In one particular embodiment, immune tolerance is observed when the titer of inhibitory antibodies in humans is less than about 0.6 BU.

[0178] In other embodiments, the immune response includes a cellular immune response. In some embodiments, the cellular immune response includes the release of cytokines. In certain embodiments, the cytokines released as part of the cellular immune response can be selected from the group consisting of IL-12, IL-4, IL-17, TNF-α, and any combination thereof.

[0179] In other embodiments, the immune response includes clinical symptoms selected from the group consisting of an increased bleeding tendency, high coagulation factor consumption, lack of response to coagulation factor therapy, decreased efficacy of coagulation factor therapy, shortened half-life of coagulation factors, and any combination thereof.

[0180] In other embodiments, immune tolerance is measured by an increase in the half-life of the clotting factor after administration to a human. In some embodiments, the half-life of the clotting factor is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1000% increased compared to the half-life of the clotting factor administered to a human before induction of immune tolerance. In certain embodiments, the half-life of the clotting factor is at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, or at least about 15 hours after induction of immune tolerance.

[0181] As used herein, the term "comparable" means, for example, that the rate or level being compared, such as due to the use of a chimeric protein, is equal to, substantially equal to, or similar to the reference rate or level. As used herein, the term "similar" means that the rate or level being compared has a difference of 10% or less or 15% or less from the reference rate or level (e.g., the rate of FXa generation by a chimeric protein consisting essentially of or consisting of two Fc portions and processed FVIII, wherein the processed FVIII is fused to one of the two Fc portions). The term "substantially equal" means having a difference of 0.01%, 0.5%, or 1% or less from the reference rate or level.

[0182] As used herein, hemostatic disorder means a genetic or acquired condition characterized by a bleeding tendency as a result of spontaneous or traumatic events due to a disorder or failure of fibrin clot formation. Examples of such disorders include hemophilia. The three main 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 disease, factor XI deficiency (PTA deficiency), factor XII deficiency, deficiency or structural abnormalities of fibrinogen, prothrombin, factor V, factor VII, factor X, or factor XIII, and Bernard-Soulier syndrome, which is a deficiency or absence of GPIb. GPIb, which is the receptor for VWF, may not function properly, resulting in a lack of primary clot formation (primary hemostasis) and an increased bleeding tendency, as well as Glanzmann-Naegeli thrombasthenia (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), the production of coagulation factors by the liver is insufficient, which can increase the bleeding risk.

[0183] As used herein, "area under the plasma concentration-time curve (AUC)" is the same as the term in the technical field of pharmacology and is based on the rate and extent of absorption of FVIII after administration. AUC is determined to infinity for a specified period such as 12, 18, 24, 36, 48, or 72 hours, or using extrapolation based on the slope of the curve. Unless otherwise specified herein, AUC is determined to infinity. The determination of AUC can be performed for a single subject or for a population of subjects to calculate the mean.

[0184] The term "coagulation promoting activity" means the ability of a coagulation factor of the present invention, such as FVIII, to participate in the blood coagulation cascade in place of the native coagulation factor, such as native FVIII. Several assays for measuring Factor VIII activity are available, including the one stage clotting assay (activated partial thromboplastin time; aPTT), thrombin generation assay (TGA), and thromboelastometry (ROTEM®).

[0185] All references to amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are incorporated herein by reference in their entirety, based on Kabat et al., 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda; MD. FcRn receptors have been isolated from several animal species, including humans. Human, rat, and mouse FcRn sequences are known (Story et al., J. Exp. Med. 180:2377 (1994), incorporated herein by reference in its entirety). Fc can include the CH2 and CH3 domains of an immunoglobulin, with or without the hinge region of the immunoglobulin. Exemplary Fc variants are provided in International Publication Nos. WO 2004 / 101740 and WO 2006 / 074199, incorporated herein by reference in their entirety.

[0186] As used herein, "hybrid" polypeptides and proteins refer to a combination of a chimeric protein and a second polypeptide. The chimeric protein and the second polypeptide in the hybrid can associate with each other via protein-protein interactions, such as charge-charge or hydrophobic interactions. The chimeric protein and the second polypeptide in the hybrid can associate with each other via disulfide bonds or other covalent bonds. The hybrids are described in International Publication No. WO 2004 / 101740 and International Publication No. WO 2006 / 074199, which are hereby incorporated by reference in their entireties. Similarly, reference is also made to U.S. Patent Nos. 7,404,956 and 7,348,004, which are hereby incorporated by reference in their entireties. The second polypeptide can be a second copy of the same chimeric protein or a non-identical chimeric protein.

[0187] As used herein, "corresponding amino acid", "corresponding site", or "equivalent amino acid" in a protein sequence is identified by alignment to maximize identity or similarity between a first protein sequence, such as an FVIII sequence, and a second protein sequence, such as a second FVIII sequence. The numbering used to identify equivalent amino acids in the second protein sequence is based on the numbering used to identify the corresponding amino acids in the first protein sequence.

[0188] As used herein, the term "insertion site" refers to the amino acid residue number in a polypeptide (typically a mature polypeptide, such as a mature FVIII polypeptide), or a fragment, variant, or derivative thereof, that is immediately upstream of a position where a heterologous moiety can be inserted. The "insertion site" is specified by a number, which is the number of the protein sequence in which the amino acid corresponding to the insertion site that is immediately N-terminal of the insertion position is specified. For example, the phrase "FVIII contains a heterologous moiety at the insertion position corresponding to amino acid 745 of a given sequence" indicates that the heterologous moiety is located between two amino acids corresponding to amino acids 745 and 746 of the sequence. However, one of ordinary skill in the art can readily identify the corresponding position in any variant of the recited protein, and the present disclosure is not limited to insertions made only in the variants specifically disclosed herein. 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 variants disclosed herein.

[0189] 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 the 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 the amino acid. Thus, as used herein, the phrase "between two amino acids of an insertion site" refers to a position where a heterologous moiety (e.g., a half-life extending moiety) is inserted between two adjacent amino acids.

[0190] As used herein, the terms "inserted," "inserting," "inserted into," or their grammatically related terms refer to the position of a heterologous moiety (e.g., a half-life extending moiety) in a fusion polypeptide as compared to a similar position in a specified protein (e.g., an FVIII protein). One of ordinary skill in the art will understand how to identify corresponding insertion positions with respect to other polypeptide sequences, e.g., other FVIII 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 polypeptides are made. For example, with reference to the fusion polypeptides provided herein, the phrase "insert a heterologous moiety immediately downstream of residue 745 of the FVIII polypeptide" means that the fusion polypeptide contains the heterologous moiety immediately downstream of the amino acid corresponding to amino acid 745 in a particular FVIII variant, e.g., in contact with the amino acids corresponding to amino acids 745 and 746 of the FVIII variant.

[0191] A "fusion" or "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature. Amino acid sequences that normally occur in different proteins can be joined together in a fusion polypeptide, or amino acid sequences that normally occur in the same protein can be placed in a new arrangement in a fusion polypeptide, e.g., a fusion of an FVIII domain and an Ig Fc domain of the invention. Fusion proteins can be made, for example, by chemical synthesis or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A fusion protein 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.

[0192] The terms "heterologous" and "heterologous moiety" mean that a polynucleotide, polypeptide, or other moiety is derived from a separate entity from the entity of the portion to which it is being compared. By way of example, a heterologous polypeptide can be synthetic or can be derived from a different species, a different cell type of an individual, or the same or different types of cells of a separate individual. In one aspect, a heterologous moiety is a polypeptide that is fused to another polypeptide to produce a fusion polypeptide or protein. In another aspect, a heterologous moiety is a non-polypeptide such as PEG that is conjugated to a polypeptide or protein.

[0193] As used herein, the terms "linked" and "fused" refer to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently attached to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly attached to or juxtaposed with the second amino acid or nucleotide sequence, or the first and second sequences can be covalently attached by an intervening sequence. The term "linked" includes not only fusions of a first amino acid sequence and a second amino acid sequence at the C-terminus or N-terminus, but also insertions of an entire first amino acid sequence (or second amino acid sequence) into any two amino acids in 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 a polypeptide and a polynucleotide chain). The term "linked" is also indicated by a hyphen (-).

[0194] As used herein, the term "associate" 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 "associate" means a covalent non-peptide bond or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In another embodiment, this means a covalent bond excluding a peptide bond. For example, the amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CH1 and CL regions are associated by disulfide bonds, and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 (positions 226 or 229 in the EU numbering system) using the Kabat 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, coordination bonds, π back-donation, double bonds, triple bonds, quadruple bonds, quintuple bonds, sextuple bonds, conjugation, hyperconjugation, aromaticity, hapticity, or antibonds. Non-limiting examples of non-covalent bonds include ionic bonds (e.g., cation-π bonds or salt bridges), 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, gold affinity, intercalation, stacking, entropy forces, or chemical polarity.

[0195] As used herein, the term "cleavage site" or "enzymatic cleavage site" refers to a site recognized by an enzyme. Certain specific 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 at such a site occurs at a clot-forming site. Exemplary such sites include, for example, sites recognized by thrombin, factor XIa, or factor Xa. Other enzymatic cleavage sites are known in the art.

[0196] As used herein, the term "processing site" or "intracellular processing site" refers to a type of enzyme 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 when transported from the lumen side of the Golgi apparatus to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides before the protein is secreted from the cell. Examples of such processing sites include, for example, sites targeted by PACE / furin (PACE is the acronym for Paired basic Amino acid Cleaving Enzyme) family endopeptidases. These enzymes are localized in 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), 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.

[0197] In constructs containing more than one processing or cleavage site, it is understood that such sites can be the same or different.

[0198] As used herein, "processable linker" refers to a linker that contains at least one intracellular processing site described elsewhere herein.

[0199] ​As used herein, "baseline" is the lowest plasma level measured for a given analyte, such as a coagulation factor (e.g., FVIII) or an antibody (e.g., anti-FVIII antibody), in a subject prior to dosing. The plasma level can be measured at two time points prior to dosing: at screening examination and immediately prior to dosing.

[0200] As used herein, "equivalent amount" means the same dose of coagulation factor activity, such as FVIII activity, expressed in international units regardless of the molecular weight of the polypeptide. For example, 1 international unit (IU) of FVIII activity approximately corresponds to the amount of FVIII in 1 milliliter of normal human plasma. Several assays are available for measuring coagulation factor activity, including the European Pharmacopoeia chromogenic substrate assay and the one-stage clotting method.

[0201] As used herein, "dose interval" means the time elapsed between multiple doses administered to a subject. Comparison of dose intervals can be done in a single subject or a population of subjects, and the mean value obtained in the population can be calculated.

[0202] As used herein, "subject" means a human individual. The subject can be a patient currently suffering from a bleeding disorder or expected to require such treatment. In some embodiments, the subject has not been previously treated with a coagulation factor (i.e., the subject is a naive subject or a naive patient). In some embodiments, the subject is a fetus, and the method includes administering the composition or chimeric protein to the mother of the fetus, and administration to the subject occurs from the mother through the placenta. In some embodiments, the subject is a pediatric or adult. In some embodiments, the subject is a pediatric less than 1 year old, less than 2 years old, less than 3 years old, less than 4 years old, less than 5 years old, less than 6 years old, less than 7 years old, less than 8 years old, less than 9 years old, less than 10 years old, less than 11 years old, or less than 12 years old. In some embodiments, the pediatric is less than 1 year old. In some embodiments, the pediatric or adult has developed a bleeding disorder, and the onset of symptoms of the bleeding disorder occurs after 1 year of age. In some embodiments, administration of the composition or chimeric protein to the subject is sufficient to prevent, inhibit, or reduce the occurrence of an immune response selected from a humoral immune response, a cellular immune response, or both a humoral and cellular immune response to a coagulation factor. In some embodiments, the subject is human and has previously had an immune response to a coagulation factor. In some embodiments, the human has not previously responded to immune tolerance therapy. In some embodiments, the previous immune tolerance therapy includes administration of a high dose of a coagulation factor. In other embodiments, the previous immune tolerance therapy includes administration of one or more immunosuppressive agents. In one embodiment, the previous immune tolerance therapy was the Malmo regimen. In another embodiment, the previous immune tolerance therapy was the Bonn protocol.

[0203] As used herein (interchangeably), "therapeutic dose", "dose", "effective amount", or "dosage" means the amount of a drug that achieves the therapeutic goal described herein. In some embodiments, "therapeutic dose" means the amount that induces immune tolerance in a subject. In certain embodiments, "therapeutic dose" means the amount that induces immune tolerance in a subject within a period until the specified tolerance occurs, e.g., within 12 weeks of administration of the first dose.

[0204] Similarly, fragments or variants of the polypeptide, and any combination thereof, are also included in the present invention. The terms "fragment" or "variant" when referring to the polypeptide used in the methods of the present disclosure include any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., the FcRn binding affinity of the FcRn binding domain or Fc variant, or the coagulation activity of FVIII). Fragments of the polypeptide include proteolytic fragments, as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere herein, and do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of the polypeptide binding domains or binding molecules used in the methods of the present disclosure include the above fragments and also include polypeptides having an amino acid sequence that has been altered by amino acid substitution, deletion, or insertion. 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 include conservative or non-conservative amino acid substitutions, deletions, or additions.

[0205] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when an amino acid in a polypeptide is replaced with another amino acid of the same side chain family, the substitution is considered to be conservative. In another embodiment, a series of amino acids can be conservatively exchanged for a structurally similar series of amino acids having a different order and / or composition of side chain family members.

[0206] The term "percent sequence identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences over a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching 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 because they are not nucleotides or amino acids. Similarly, gaps present in the reference sequence are not counted when counting nucleotides or amino acids of the target sequence, or because they are not nucleotides or amino acids of the reference sequence.

[0207] The percentage of sequence identity is calculated by determining the number of positions at which the same amino acid residue or nucleotide base is present 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. The comparison of sequences and determination of percent sequence identity between two sequences may be performed using software 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 bl2seq, which is part of the BLAST program suite available from the website of the National Center for Biotechnology Information (NCBI) of the United States government (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences and 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 and are similarly available from www.ebi.ac.uk / Tools / psa of the European Bioinformatics Institute (EBI). Information, BLAST) website (blast.ncbi.nlm.nih.gov) and is part of the BLAST program suite available from the website of the National Center for Biotechnology Information (NCBI) of the United States government (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences and 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 and are similarly available from www.ebi.ac.uk / Tools / psa of the European Bioinformatics Institute (EBI). Information, BLAST) website (blast.ncbi.nlm.nih.gov) and is part of the BLAST program suite available from the website of the National Center for Biotechnology Information (NCBI) of the United States government (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences and 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 and are similarly available from www.ebi.ac.uk / Tools / psa of the European Bioinformatics Institute (EBI).

[0208] Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that the percent sequence identity values are rounded to the nearest tenth value. For example, 80.11, 80.12, 80.13, and 80.14 are truncated to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Also note that length values are always integers.

[0209] One of ordinary skill in the art will recognize that the generation of sequence alignments for the calculation of percent sequence identity is not limited to binary sequence-sequence comparisons facilitated by primary sequence data alone. Sequence alignments can be derived from multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com / muscle / . ClustalW2 and MUSCLE are also available, for example, from EBI.

[0210] It should also be recognized 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., positions of mutations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available from www.tcoffee.org or also from EBI. Similarly, it should be recognized that the final alignment used to calculate percent sequence identity may be selected either automatically or manually.

[0211] Polynucleotide variants can include changes in the coding region, non-coding region, or both. In one embodiment, the polynucleotide variant includes changes that produce silent substitutions, additions, or deletions that do not change the properties or activities of the encoded polypeptide. In another embodiment, the nucleotide variant is produced by silent substitutions due to the degeneracy of the genetic code. In other embodiments, the variant has 5 to 10, 1 to 5, or 1 to 2 amino acids substituted, deleted, or added in any combination. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (changing the codons in human mRNA to the codons of another host, such as a bacterial host like E. coli).

[0212] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism (Genes II, edited by Lewin, B., 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. techniques or direct synthesis.

[0213] Using known methods related to protein manipulation and recombinant DNA technology, variants can be generated to improve or alter the characteristics of polypeptides. As an example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without substantially losing its biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993), which is incorporated herein by reference in its entirety, reported a variant KGF protein that still had heparin-binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma showed up to 10-fold higher activity even after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988), which is incorporated herein by reference in its entirety).

[0214] Moreover, it has been well-documented that variants often retain biological activities similar to those of the naturally occurring proteins. For example, Gayle and co-workers (J. Biol. Chem 268:22105-22111 (1993), which is incorporated herein by reference in its entirety) performed an extensive mutational analysis of 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 over the full length of the molecule. They examined every amino acid position where multiple mutations could occur. The researchers found that "most of the molecule can be changed without significantly affecting either [binding or biological activity]." (See the abstract). In fact, only 23 unique amino acid sequences out of over 3,500 nucleotide sequences examined produced proteins with significantly different activity from the wild type.

[0215] As described above, the polypeptide variant includes, for example, a modified polypeptide. Modifications include, 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 a covalent cross-link, 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 an amino acid such as arginylation to a protein, and ubiquitination. In some embodiments, FVIII is modified at any convenient position, for example, pegylated. In some embodiments, FVIII is pegylated at a surface-exposed amino acid of FVIII, such as a surface-exposed cysteine which can be an engineered cysteine. Ibid. In some embodiments, the modified FVIII, such as pegylated FVIII, is a chimeric or fusion FVIII.

[0216] 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.

[0217] The term "upstream" refers to a nucleotide sequence located 5' of a reference nucleotide sequence. "Upstream" can also refer to a peptide sequence located at the N-terminus of a reference peptide sequence.

[0218] 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 and that affects the transcription, RNA processing, stability, or translation of an associated coding region. Regulatory The region may include a promoter, a translation leader sequence, an intron, a polyadenylation recognition sequence, an RNA processing site, an effector binding site, and a stem-loop structure. When the coding region is intended for expression in eukaryotic cells, the polyadenylation signal and the transcription termination sequence are usually located 3' of the coding sequence.

[0219] A polynucleotide encoding a gene product, such as a polypeptide, may include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. Other transcription control elements other than the promoter, such as enhancers, operators, repressors, and transcription termination signals, may also be operably associated with the coding region to direct the expression of the gene product.

[0220] 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 the cytomegalovirus (immediate early promoter together with intron A), simian virus 40 (early promoter), and promoters and enhancer segments of 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 capable of controlling gene expression in eukaryotic cells. Further suitable transcription control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0221] Similarly, a variety of translation control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (in particular, internal ribosome entry sites or IRESs within the sequence, also referred to as CITE sequences).

[0222] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide.

[0223] "Vector" refers to any medium for cloning and / or transferring nucleic acid into a host cell. A vector can be a replicon that may join another nucleic acid segment to effect replication of the joined segment. "Replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous replication unit in vivo, i.e., can replicate under its own control. The term "vector" includes both viral and non-viral media for introducing nucleic acid into cells in vitro, ex vivo, or in vivo. A number of vectors are known and used in the art, including for example plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragment having complementary sticky ends to the selected vector.

[0224] The term "plasmid" refers to an extrachromosomal element often having genes in the form of circular double-stranded DNA molecules that are not part of the central metabolism of the cell. Such elements can be of any origin, and can be autonomous replication sequences, genomic integration sequences, phage or nucleotide sequences, linear, circular, or supercoiled single-stranded or double-stranded DNA or RNA, in which a plurality of nucleotide sequences are joined or recombined into a unique configuration that can introduce a promoter fragment and DNA sequence for a selected gene product into the cell along with appropriate 3' untranslated sequences.

[0225] Eukaryotic viral vectors that can be used include, but are not limited to, adenoviral vectors, retroviral vectors, adeno-associated viral vectors, and poxviruses such as vaccinia virus vectors, baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (lipofectamine), DNA-protein complexes, and biopolymers.

[0226] A "cloning vector" refers to a "replicon", which is a unit-length nucleic acid that replicates continuously and contains an origin of replication, such as a plasmid, phage, or cosmid, that may be ligated to another nucleic acid segment to effect replication of the ligated segment. A particular cloning vector can replicate in one cell type, e.g., bacteria, and can be expressed in another cell, e.g., eukaryotic cells. Cloning vectors typically contain one or more sequences that can be used to select cells containing the vector and / or one or more multiple cloning sites for insertion of the nucleic acid sequence of interest.

[0227] The term "expression vector" refers to a vehicle designed to be able to express an inserted nucleic acid sequence after insertion into a host cell. The inserted nucleic acid sequence is operably associated with the regulatory regions described above.

[0228] 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 (lysosome fusion), use of a gene gun, or a DNA vector transporter.

[0229] An "isolated" polypeptide, or fragment, variant, or derivative thereof, refers to a polypeptide that is not present in its natural environment. A specific level of purification is not required. For example, an isolated polypeptide can simply be removed from its original or natural environment. Recombinant polypeptides and proteins produced by expression in a host cell are considered isolated for the 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.

[0230] As used herein, the term "host cell" refers to a cell or population of cells that has or can have a recombinant nucleic acid. The host cell can be a prokaryotic cell (e.g., Escherichia coli), or the host cell can be a eukaryotic cell, such as a fungal cell (e.g., yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g., Sf-9), or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3).

[0231] As used herein, "steady-state volume of distribution (Vss)" has the same meaning as the term 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 plasma concentration at steady state.

[0232] II. Methods of the Invention The present disclosure is based on the discovery that a coagulation factor fused to an Fc region can be used to induce immune tolerance in a human having hemophilia who develops an inhibitor to a coagulation factor and has failed one or more previous immune tolerance therapies. Previously, FV Treatment with an III-Fc chimeric protein was thought to be able to prevent an immune response to FVIII treatment. Surprisingly, it has been discovered in the present disclosure that treatment with a coagulation factor-Fc chimeric protein can reduce a previously generated immune response in humans who did not respond to previous immunosuppressive therapies. Thus, the present disclosure provides a method for inducing immune tolerance in a human, the method comprising administering to the human an effective amount of a composition comprising a coagulation factor and Fc or a chimeric protein comprising a coagulation factor and an Fc region or a polynucleotide encoding the same.

[0233] Another aspect of the present disclosure is a method of inducing immune tolerance in a human having hemophilia, comprising: (1) administering to the human an effective amount of a composition comprising a coagulation factor and Fc or a chimeric protein comprising a coagulation factor and an Fc region, the effective amount of the composition or chimeric protein being effective to induce immune tolerance in the human; and (2) after induction of immune tolerance, administering to the human a tapering regimen of the composition or chimeric protein. In certain embodiments, induction of immune tolerance occurs when the titer of inhibitory antibodies in the human is less than about 0.6 BU. In certain embodiments, induction of immune tolerance occurs when the titer of inhibitory antibodies in the human is less than about 0.6 BU and the coagulation factor activity monitored in plasma is a 60% recovery rate. In some embodiments of the present disclosure, the method further comprises (3) after the tapering regimen, administering to the human a prophylactic dose of a coagulation factor. In certain aspects, the human has not been treated with a previous immune tolerance therapy for a coagulation factor. The composition or chimeric protein comprising a coagulation factor and an Fc region can be administered to the human at any time determined that the human has developed an inhibitor immune response, for example, after measuring the level of the inhibitor immune response in the human. In other embodiments, the composition or chimeric protein can be administered to a human who has not yet developed one or more inhibitor immune responses to prevent the occurrence of an inhibitor immune response. In some embodiments, the composition or chimeric protein is administered to a human at high risk of developing an inhibitor immune response (e.g., family history, genetic predisposition, or biomarker indication). In some embodiments, the method further comprises measuring the level of the inhibitor immune response or the likelihood of developing an inhibitor immune response prior to administration.In some embodiments, a composition or chimeric protein comprising a coagulation factor and an Fc region is administered to a human after it has been determined that the human has had an inhibitory immune response or has the potential to have an inhibitory immune response, for example, after measuring the level of the inhibitory immune response or the potential to have an inhibitory immune response in the human, within less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 2 weeks, less than about 3 weeks, less than about 4 weeks, less than about 2 months, less than about 3 months, less than about 4 months, less than about 5 months, less than about 6 months, less than about 1 year, less than about 2 years, less than about 3 years, less than about 4 years, or less than about 5 years. In certain embodiments, a composition or chimeric protein comprising a coagulation factor and an Fc region is administered to a human immediately after it has been determined that the human has had an inhibitory immune response or has the potential to have an inhibitory immune response, for example, after measuring the level of the inhibitory immune response or the potential to have an inhibitory immune response in the human. In specific embodiments, a composition or chimeric protein comprising a coagulation factor and an Fc region is administered to a human after it has been determined that the human has had an inhibitory immune response or has the potential to have an inhibitory immune response, for example, after measuring the level of the inhibitory immune response or the potential to have an inhibitory immune response in the human, within less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 30 minutes, less than about 45 minutes, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 18 hours, or less than about 24 hours. In certain embodiments, a composition or chimeric protein comprising a coagulation factor and an Fc region is administered to a human after it has been determined that the human has had an inhibitory immune response or has the potential to have an inhibitory immune response, for example, after measuring the level of the inhibitory immune response or the potential to have an inhibitory immune response in the human, within about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours. Administer to a human at intervals of about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, or about 24 hours. In certain embodiments, a composition or chimeric protein comprising a coagulation factor and an Fc region is administered to a human after it has been determined that the human has mounted an inhibitor immune response or has the potential to mount an inhibitor immune response, for example, after measuring the level of the inhibitory immune response in the human or the potential to mount an inhibitor immune response, within less than about 1 day.

[0234] Induction of the immune response can be continued until the level of the inhibitor drops below a certain level or the inhibitor becomes undetectable. In certain embodiments, the induction period may continue for at least about 24 weeks, at least about 26 weeks, at least about 28 weeks, at least about 30 weeks, at least about 32 weeks, at least about 34 weeks, at least about 36 weeks, at least about 38 weeks, at least about 40 weeks, at least about 42 weeks, at least about 44 weeks, at least about 46 weeks, at least about 48 weeks, at least about 50 weeks, at least about 52 weeks, at least about 54 weeks, at least about 56 weeks, at least about 58 weeks, at least about 60 weeks, at least about 62 weeks, at least about 64 weeks, at least about 66 weeks, at least about 68 weeks, at least about 70 weeks. In certain embodiments, the induction period is less than 60 weeks.

[0235] The inhibitory immune responses treated by the method of the present invention may include any response in a human that negatively affects one or more effects of a coagulation factor treatment. In some embodiments, the inhibitory immune response includes the production of inhibitory antibodies against a coagulation factor, such as an inhibitory anti-FVIII antibody. In certain embodiments, the method of the present disclosure further comprises measuring the titer of one or more inhibitory antibodies in a human before (e.g., at baseline) and after administration of an effective amount of a composition or chimeric protein comprising a coagulation factor and an Fc region or a polynucleotide encoding the same. In some embodiments, the titer of the inhibitory antibody before administration (e.g., at baseline) is at least about 0.6 Bethesda units (BU). In certain embodiments, the titer of the inhibitory antibody before administration (e.g., at baseline) is at least about 1 BU, at least about 2 BU, at least about 3 BU, at least about 4 BU, at least about 5 BU, at least about 6 BU, at least about 7 BU, at least about 10 BU, at least about 20 BU, at least about 30 BU, at least about 40 BU, at least about 50 BU, at least about 100 BU, at least about 150 BU, or at least about 200 BU. In one particular embodiment, the titer of the inhibitory antibody before administration (e.g., at baseline) is at least about 5 BU.

[0236] In some embodiments, the method of the present invention reduces the titer of the inhibitory antibody in a human subject as compared to the titer of the inhibitory antibody before administration. In certain embodiments, the titer of the inhibitory antibody after administration is less than about 0.6 BU. In some embodiments, the titer of the inhibitory antibody after administration is less than about 0.5 BU, less than about 0.4 BU, less than about 0.3 BU, less than about 0.2 BU, or less than about 0.1 BU. In one particular embodiment, the titer of the inhibitory antibody after administration is 0 BU. In other embodiments, the titer of the inhibitory antibody after administration is less than 5 BU, less than 4 BU, less than 3 BU, less than 2 BU, less than 1 BU, less than 0.9 BU, less than 0.8 BU, less than 0.7 BU, or less than 0.6 BU.

[0237] In some embodiments, administration increases macrophage differentiation in humans towards an M2-like phenotype as compared to macrophage differentiation in untreated controls and humans treated with coagulation factors alone. In some embodiments, the M2-like phenotype includes upregulation of the NRF2 pathway, the PPAR gamma pathway, or both the NRF2 pathway and the PPAR gamma pathway. In some embodiments, the M2-like phenotype includes upregulation of CD206 (MRC1). In some embodiments, the M2-like phenotype includes upregulation of ARG1. In some embodiments, the M2-like phenotype includes upregulation of CD206 (MRC1) and ARG1.

[0238] In some embodiments, administration results in higher expression of one or more genes in humans as compared to the expression of one or more genes in untreated subjects or subjects treated with coagulation factors only. In some embodiments, administration results in higher expression of one or more genes selected from the group consisting of Hmox1, PPAR gamma, LPL, EGR2, SLCO4A1, heme oxygenase 1 (HO-1), oxidative stress-induced growth inhibitor 1 (OSGIN1), superoxide dismutase 1 (SOD1), glutathione-disulfide reductase (GSR), glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H quinone dehydrogenase 1 (NQO1), fatty acid binding protein 5 (FABP5), B7-H3 (CD276), SLAM family member 3 (SLAMF3; lymphocyte antigen 9; LY9), SLAM family member 7 (SLAMF7), mannose receptor C-type 1 (MRC1), solute carrier family 12 member 4 (SLC12A), neuropilin 1 (NRP1), and any combination thereof. In some embodiments, administration results in higher expression of one or more genes of the NRF2 pathway. In certain embodiments, one or more genes of the NRF2 pathway are selected from the group consisting of HO-1, OSGIN1, SOD1, GSR, GCLC, GCLM, NQO1, and any combination thereof. In some embodiments, administration results in higher expression of one or more genes of the PPAR gamma pathway. In some embodiments, one or more genes of the PPAR gamma pathway are selected from the group consisting of PPAR gamma, LPL, FABP5, EGR2, and any combination thereof. In some embodiments, administration results in higher expression of one or more genes selected from the group consisting of B7-H3 (CD276), SLAMF3, SLAM7, MRC1, SLC12A, NRP1, and any combination thereof.In certain embodiments, administration results in higher expression of one or more genes as compared to the expression of one or more genes in untreated humans or humans administered coagulation factors only, where the expression is at least about 1.5-fold higher, at least about 2-fold higher, at least about 2.5-fold higher, at least about 3-fold higher, at least about 3.5-fold higher, at least about 4-fold higher, at least about 4.5-fold higher, or at least about 5-fold higher.

[0239] In some embodiments, differential expression of one or more genes is observed less than 6 hours after administration. In some embodiments, differential expression is observed less than 12 hours after administration. In some embodiments, differential expression is observed less than 18 hours after administration. In some embodiments, differential expression is observed less than 24 hours after administration.

[0240] In some embodiments, the inhibitory immune response includes a cell-mediated immune response. In certain embodiments, the cell-mediated immune response includes the release of cytokines. In some embodiments, the cytokine is any cytokine associated with an increase in the immune response. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL-6, IL-16, IL-12, IL-4, IL-17, tumor necrosis factor α (TNF-α), interferon α, interferon γ, and any combination thereof. In one embodiment, the cell-mediated immune response includes an increase in the serum level of IL-12. In another embodiment, the cell-mediated immune response includes an increase in the serum level of IL-4. In another embodiment, the cell-mediated immune response includes an increase in the serum level of IL-17. In another embodiment, the cell-mediated immune response includes an increase in the serum level of TNF-α.

[0241] Various genetic mutations have been associated with an increased risk of producing an inhibitory immune response. For example, the TNF-α-308G>A polymorphism within Hap2, which is associated with increased constitutive and induced transcriptional levels of TNF, has been associated with an increased risk of producing an inhibitory immune response. Astermark et al., Blood 108: which is hereby incorporated by reference in its entirety. See pages 3739-3745 (2006). Thus, in some embodiments, a human has a genetic polymorphism associated with an increase in TNF-α. In some embodiments, the polymorphism is the TNF-α -308G>A polymorphism. In some embodiments, a human has a polymorphism in the IL10 gene, e.g., a polymorphism associated with an increase in the secretion of IL10. In some embodiments, FVIII-Fc is administered to a subject together with allele 134 of the IL10G microsatellite in the promoter region of the IL10 gene. See Astermark et al., Hemostatis, Thrombosis, and Vascular Biology 108:3739-3745 (2006), which is incorporated herein by reference in its entirety.

[0242] In some embodiments, a human has a genetic polymorphism associated with a decrease in CTLA-4 (cytotoxic T lymphocyte antigen 4) expression. In some embodiments, a human has a mutation in DR15 (HLA-DR15) or DQB0602 MHC (major histocompatibility complex) class II molecules. Other MHC class II molecules associated with the development of an inhibitory immune response in subjects with hemophilia are A3, B7, C7, DQA0102, C2, DQA0103, DQB0603, and DR13 (see Inhibitors in Patients with Hemophilia, edited by E.C. Rodriguez-Merchan & C.A. Lee, Blackwell Science, Ltd., 2002).

[0243] In some embodiments, the methods of the present disclosure reduce the level of one or more cytokines in a subject as compared to the level of one or more cytokines in the subject after a previous treatment with a polypeptide consisting of an FVIII polypeptide. In another embodiment, the methods of the present disclosure reduce the level of one or more cytokines in a subject as compared to the level of one or more cytokines in the subject prior to administration. In other embodiments, the expression of one or more tolerogenic molecules increases after administration of the methods of the present disclosure as compared to the expression level of one or more tolerogenic molecules prior to administration. In certain embodiments, the one or more tolerogenic molecules are selected from IL-10, TGF-β, IL-35, IDO-1, and any combination thereof.

[0244] In other embodiments, the immune response includes clinical symptoms selected from the group consisting of increased bleeding tendency, high coagulation factor consumption, lack of response to coagulation factor therapy, decreased efficacy of coagulation factor therapy, shortened half-life of coagulation factors, and any combination thereof. In certain embodiments, the immune response includes clinical symptoms selected from the group consisting of increased bleeding tendency, high coagulation factor consumption, lack of response to coagulation factor therapy, decreased efficacy of coagulation factor therapy, decreased recovery of coagulation factor activity monitored in plasma, shortened half-life of coagulation factors, and any combination thereof.

[0245] In certain embodiments, a human has previously been diagnosed as having an inhibitory immune response. Such a diagnosis can be made using any method known in the art. For example, a human can be characterized as having an immune response to a coagulation factor if the human has one or more of the following: (a) a titer of inhibitory antibodies to a coagulation factor that is greater than or equal to 0.6 BU; (b) an increase in the serum level of one or more cytokines selected from the group consisting of IL-12, IL-4, IL-17, and TNF-α; (c) an increased bleeding tendency; (d) high coagulation factor consumption; (e) lack of response to coagulation factor therapy; (f) reduced efficacy of coagulation factor therapy; (g) shortened half-life of a coagulation factor, and any combination thereof. In one particular embodiment, if a human has a titer of inhibitory antibodies to a coagulation factor that is greater than or equal to 0.6 BU, the human is characterized as having an immune response to the coagulation factor.

[0246] In some embodiments, the human is at least about 1 month prior to administration, at least about 2 months prior to administration, at least about 3 months prior to administration, at least about 4 months prior to administration, at least about 5 months prior to administration, at least about 6 Months ago, at least about 7 months ago, at least about 8 months ago, at least about 9 months ago, at least about 10 months ago, at least about 11 months ago, at least about 12 months ago, at least about 13 months ago, at least about 14 months ago, at least about 15 months ago, at least about 16 months ago, at least about 17 months ago, at least about 18 months ago, at least about 19 months ago, at least about 20 months ago, at least about 21 months ago, at least about 22 months ago, at least about 23 months ago, at least about 24 months ago, at least about 27 months ago, at least about 30 months ago, at least about 33 months ago, at least about 36 months ago, at least about 39 months ago, at least about 42 months ago, at least about 45 months ago, at least about 48 months ago, at least about 51 months ago, at least about 54 months ago, at least about 57 months ago, at least about 60 months ago, at least about 6 years ago, at least about 7 years ago, at least about 8 years ago, at least about 10 years ago, at least about 15 years ago, or at least about 20 years ago, it was previously diagnosed that an inhibitory immune response to a coagulation factor had occurred. In one embodiment, the human was previously diagnosed as having had an inhibitory immune response to a coagulation factor at least about 5 years prior to administration.

[0247] In some embodiments, the methods of the present disclosure provide an improved time to tolerance as compared to standard methods of inducing immune tolerance. As used herein, the term "time to tolerance" refers to the amount of time between administration of a first dose of a composition or chimeric protein comprising a coagulation factor and an Fc region and the development of immune tolerance in a human. A decrease in the time to tolerance may have significant benefits for humans, such as, but not limited to, a reduction in the total economic burden required to achieve tolerance. In some embodiments, the time to tolerance is from about 1 to about 24 weeks, from about 1 to about 23 weeks, from about 1 to about 22 weeks, from about 1 to about 21 weeks, from about 2 to about 20 weeks, from about 2 to about 19 weeks, from about 2 to about 18 weeks, from about 2 to about 17 weeks, from about 3 to about 16 weeks, from about 3 to about 15 weeks, from about 3 to about 14 weeks, from about 3 to about 13 weeks, from about 4 to about 12 weeks, from about 4 to about 11 weeks, from about 4 to about 10 weeks, from about 4 to about 9 weeks, from about 5 to about 8 weeks, from about 5 to about 7 weeks, from about 5 to about 6 weeks, from about 1 to about 12 weeks, from about 1 to about 11 weeks, from about 1 to about 10 weeks, from about 1 to about 9 weeks, from about 1 to about 8 weeks, from about 1 to about 7 weeks, from about 1 to about 6 weeks, from about 1 to about 5 weeks, or from about 1 to about 4 weeks. In some embodiments, the time to tolerance is less than about 70 weeks, less than about 65 weeks, less than about 60 weeks, less than about 58 weeks, less than about 56 weeks, less than about 54 weeks, less than about 52 weeks, less than about 50 weeks, less than about 48 weeks, less than about 46 weeks, less than about 44 weeks, less than about 42 weeks, less than about 40 weeks, less than about 38 weeks, less than about 36 weeks, less than about 34 weeks, less than about 32 weeks, less than about 30 weeks, less than about 28 weeks, less than about 26 weeks, less than about 24 weeks, less than about 23 weeks, less than about 22 weeks, less than about 21 weeks, less than about 20 weeks, less than about 19 weeks, less than about 18 weeks, less than about 17 weeks, less than about 16 weeks, less than about 15 weeks, less than about 14 weeks, less than about 13 weeks, less than about 12 weeks, less than about 11 weeks, less than about 10 weeks, less than about 9 weeks, less than about 8 weeks, less than about 7 weeks, less than about 6 weeks, less than about 5 weeks, less than about 4 weeks, less than about 3 weeks, less than about 2 weeks, or less than about 1 week. In certain embodiments, the time to tolerance is from about 4 to about 12 weeks. In one embodiment, the time to tolerance is about 4 weeks. In another embodiment, the time to tolerance is about 12 weeks. In some embodiments, the time to tolerance is less than about 10 months. In some embodiments, the time to tolerance is less than about 9 months. In some embodiments, the time to tolerance is less than about 8 months. In some embodiments, the time to tolerance is less than about 7 months.In some embodiments, the time to tolerance is less than about 6 months. In some embodiments, the time to tolerance is less than about 5 months. In some embodiments, the time to tolerance is less than about 4 months. In some embodiments, the methods of the present disclosure result in a shorter time to tolerance in humans after treatment with a composition or chimeric protein comprising a coagulation factor and an Fc region as compared to the time to tolerance after treatment with a coagulation factor alone.

[0248] In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against a coagulation factor of less than about 0.6 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against a coagulation factor of less than about 0.5 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against a coagulation factor of less than about 0.4 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against a coagulation factor of less than about 0.3 BU . In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against a coagulation factor of less than about 0.2 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against a coagulation factor of less than about 0.1 BU. In some embodiments, the development of immune tolerance is characterized by a titer of inhibitory antibodies against a coagulation factor of about 0.0 BU. In certain embodiments, the titer of inhibitory immune antibodies is observed in two consecutive measurements, e.g., in two consecutive weeks within 4 weeks.

[0249] In some embodiments, the development of immune tolerance is characterized by an incremental recovery of greater than 66% (e.g., about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% incremental recovery). As used herein, "incremental recovery" refers to the peak FVIII level 15 - 30 minutes after infusion.

[0250] After the induction and tapering periods are completed, the subject can be placed on prophylactic treatment with the chimeric protein. Exemplary prophylactic dosing regimens may be about 50 IU / kg of the chimeric protein every 4 days or about 25 IU / kg to about 65 IU / kg of the chimeric protein at 3 - 5 day intervals. For pediatric patients under 6 years of age, about 50 IU / kg of the chimeric protein can be given twice weekly or about 25 IU / kg to about 65 IU / kg of the chimeric protein at 3 - 5 day intervals. See the package insert for ELOCTATE® available at worldwideweb.eloctate.com / _assets / pdf / ELOCTATE_pei_January2017.pdf.

[0251] In some embodiments, the human being treated using the methods of the present disclosure has received or recently received immunostimulatory therapy. For example, inhibitors have also been reported in HCV - positive hemophilia A patients treated with interferon and in HIV - positive hemophilia A patients with immune reconstitution syndrome associated with antiretroviral therapy. See Report of Expert Meeting on FVIII Products and Inhibitor Development, European Medicines Agency (February 28, 2006 - March 2, 2006). Thus, in some embodiments, the human being has received interferon therapy. In some embodiments, the human being has received antiviral therapy. In some embodiments, the human being has received antiretroviral therapy and has immune reconstitution syndrome.

[0252] In certain embodiments, the human being had less than 150 exposure days (ED) to a clotting factor, such as FVIII. In one embodiment, the human being had less than 50 ED. In another embodiment, the human being had less than 20 ED.

[0253] Some aspects of the present disclosure relate to a method of reducing the severity or occurrence of an allergic or anaphylactic reaction to a coagulation factor in a subject in need thereof, the method comprising administering to the subject a composition or chimeric protein comprising a coagulation factor and an Fc region. In some embodiments, administration of the composition or chimeric protein reduces the severity of an anaphylactic-like reaction to the coagulation factor. In some embodiments, administration of the composition or chimeric protein reduces the severity of an allergic reaction to the coagulation factor.

[0254] II.A. Chimeric Protein The methods of inducing immune tolerance disclosed herein are generally applicable to compositions or chimeric proteins comprising a coagulation factor and an Fc region, where the coagulation factor may be any known coagulation factor, fragment thereof, or variant thereof, and the Fc region may be any known Fc region, fragment thereof, or variant thereof. 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 the FVIII-Fc chimeric protein and its use is equally applicable to other chimeric proteins comprising a coagulation factor portion and an Fc portion. Any coagulation factor or any fragment thereof or any variant thereof can be used in the methods of the present disclosure. Similarly, any Fc or any fragment thereof or any variant thereof can be used in the methods of the present disclosure. In some specific examples, the coagulation factor portion of the chimeric protein is FVIII.

[0255] In some embodiments, the coagulation factor and the Fc are present on separate polypeptide chains. In some embodiments, the coagulation factor and the Fc are not linked or are covalently bound to each other.

[0256] In other embodiments, the coagulation factor may be a coagulation factor mimetic. The coagulation factor mimetic can exhibit one or more coagulation factor activities. For example, an antibody or an antigen-binding portion thereof can act like FVIII by binding to both factor IX and factor X. If the antibody or an antigen-binding portion thereof contains an Fc region, such an antibody or an 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.

[0257] In this regard, the present disclosure generally provides a method of inducing immune tolerance in a human, the method comprising administering to a subject a composition or chimeric protein comprising a coagulation factor and an Fc portion.

[0258] II.A.1. Factor VIII As used herein, throughout the present application, "Factor VIII", abbreviated as "FVIII", means an FVIII polypeptide that is functional in its normal role in coagulation, unless otherwise specified. Thus, the term FVIII includes polypeptide variants that are functional. "FVIII protein" is used interchangeably with FVIII polypeptide (or protein). Examples of FVIII function include, but are not limited to, the ability to activate coagulation, the ability to act as a cofactor for Factor IX, or the ability to convert Factor X to activated Xa after forming a tenase complex with Factor IX in the presence of Ca2+ and phospholipids. The FVIII protein may be a human, porcine, canine, rat, or murine FVIII protein. Furthermore, conserved residues that may be required for function have been identified by comparison between FVIII from humans and other species (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Patent No. 6,251,632). Full-length polypeptide and polynucleotide sequences are known, as are many functional fragments, variants, 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 / 0308230 A1, and 2014 / 0370035 A1 and International Publication No. WO2015 / 106052 A1. Examples of 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.

[0259] The FVIII moiety in a coagulation factor or chimeric protein used herein is FVI It has II activity. FVIII activity can be measured by any method known in the art. Some tests for evaluating the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen test (often by the Clauss method), platelet count, platelet function test (often by PFA-100), TCT, bleeding time, mixing test (whether the abnormality is corrected when the patient's plasma is mixed with normal plasma), coagulation factor assay, antiphospholipid antibody, D-dimer, genetic test (e.g., factor V Leiden, prothrombin mutation G20210A), diluted Russell viper venom time (dRVVT), miscellaneous platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM (registered trademark), e.g., ROTEM (registered trademark)), or euglobulin lysis time (ELT) are available.

[0260] The aPTT test is a performance indicator that measures the efficacy of both the "intrinsic" (also called the contact activation pathway) pathway and the common coagulation pathway. This test is commonly used to measure the coagulation activity of commercially available recombinant coagulation factors, such as FVIII. It is used together with the prothrombin time (PT), which measures the extrinsic pathway.

[0261] ROTEM analysis provides information on the entire dynamics of hemostasis: coagulation time, clot formation, clot stability, and lysis. The various parameters in thromboelastometry depend on the activity of the plasma coagulation system, platelet function, fibrinolysis, or many factors that affect these interactions. This assay can provide a complete view of secondary hemostasis.

[0262] The chromogenic assay mechanism is based on the principle of the blood coagulation cascade, in which activated FVIII promotes the conversion of factor X to factor Xa in the presence of activated factor IX, phospholipids, and calcium ions. Factor Xa activity is evaluated by the hydrolysis of a p-nitroanilide (pNA) substrate specific for factor Xa. The initial release rate of p-nitroaniline measured at 405 nM is directly proportional to factor Xa activity and thus directly proportional to FVIII activity in the sample.

[0263] The chromogenic assay is recommended by the FVIII and Factor IX Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostatsis (ISTH). Since 1994, the chromogenic assay has also been the reference method of the European Pharmacopoeia for the assignment of FVIII concentrate potency. Thus, in one embodiment, the chimeric protein comprising FVIII has an FVIII activity equivalent to that of a chimeric protein comprising mature FVIII or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0264] In another embodiment, the chimeric protein comprising FVIII of the present disclosure has a factor Xa generation rate equivalent to that of a chimeric protein comprising mature FVIII or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0265] To activate factor X or factor Xa, activated factor IX (factor IXa) hydrolyzes one arginine-isoleucine bond in factor X to Ca 2+, in the presence of membrane phospholipids and FVIII cofactor, factor Xa is formed. Thus, the interaction between FVIII and factor IX is important in the coagulation pathway. In certain embodiments, a chimeric protein comprising FVIII can interact with factor IXa at a rate equivalent to that of a chimeric protein comprising the mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFAC TO®, or ELOCTATE®).

[0266] Furthermore, FVIII binds to von Willebrand factor but is inactive in circulation. FVIII rapidly degrades when not bound to VWF and is released from VWF by the action of thrombin. In some embodiments, a chimeric protein comprising FVIII can bind to von Willebrand factor at a level equivalent to that of a chimeric protein comprising the mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0267] FVIII can be inactivated by activated protein C in the presence of calcium and phospholipids. Activated protein C cleaves the FVIII heavy chain after arginine 336 in the A1 domain, disrupts the factor X substrate interaction site, cleaves after arginine 562 in the A2 domain, enhances dissociation of the A2 domain, and disrupts the interaction site with factor IXa. This cleavage also cleaves the A2 domain (43 kDa) into two fragments, generating the A2-N (18 kDa) and A2-C (25 kDa) domains. Thus, activated protein C can catalyze multiple cleavage sites in the heavy chain. In one embodiment, a chimeric protein comprising FVIII is inactivated by activated protein C at a level equivalent to that of a chimeric protein comprising the mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®).

[0268] In other embodiments, the chimeric protein comprising FVIII has in vivo FVIII activity equivalent to that of a chimeric protein comprising the mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®). In certain embodiments, the chimeric protein comprising FVIII can protect HemA mice at a level equivalent to that of a chimeric protein comprising the mature FVIII sequence or BDD FVIII (e.g., ADVATE®, REFACTO®, or ELOCTATE®) in the HemA mouse tail vein transection model.

[0269] As used herein, the "B domain" of FVIII is the same as the B domain known in the art, as defined by internal amino acid sequence identity and proteolytic cleavage sites by thrombin, e.g., residues Ser741 - Arg1648 of mature human FVIII. The other human FVIII domains are the following amino acid residues compared to mature human FVIII: A1 of mature FVIII, residues Ala1 - Arg372; A2, residues Ser373 - Arg740; A3, residues Ser1690 - Ile2032; C1, residues Arg2033 - Asn2172; C2, residues Ser2173 - Tyr2332. The residue numbers of the sequences used herein without reference to sequence numbers 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, contains residues Ser1690 - Tyr2332. The remaining sequence, residues Glu1649 - Arg1689, is commonly referred to as the FVIII light chain activation peptide. The positions of the boundaries of all domains, including the B domain, for porcine, murine, 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 particular embodiment, the B domain - deleted FVIII variant contains a deletion of amino acid residues 746 - 1648 of mature FVIII.

[0270] "B domain - deleted FVIII" is described in U.S. Patent Nos. 6,316,226, 6,346, No. 5,13, No. 7,041,635, No. 5,789,203, No. 6,060,447, No. 5,595,886, No. 6,228,620, No. 5,972,885, No. 6,048,720, No. 5,543,502, No. 5,610,278, No. 5,171,844, No. 5,112,950, No. 4,868,112, and No. 6,458,563 and International Publication No. WO2015106052A1 (PCT / US2015 / 010738), which may be complete or have partial deletions. In some embodiments, the B-domain deleted FVIII sequences used in the methods of the present disclosure include any one of the deletions disclosed in column 4, rows 4 - 5, row 28, and Examples 1 - 5 of U.S. Patent No. 6,316,226 (also in U.S. Patent No. 6,346,513). In another embodiment, the factor VIII with B-domain deletion is the S743 / Q1638 B-domain deleted factor VIII (SQ BDD FVIII) (e.g., factor VIII having a deletion of amino acids 744 - 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 has the deletions disclosed in column 2, rows 26 - 51, and Examples 5 - 8 of U.S. Patent No. 5,789,203 (U.S. Patent No. 6,060,447, U.S. Patent No. 5,595,886, and U.S. Patent No. 6,228,620).In some embodiments, the B-domain deleted Factor VIII has deletions as described in column 1, lines 25 to column 2, line 40 of U.S. Patent No. 5,972,885; column 6, lines 1 to 22 and Example 1 of U.S. Patent No. 6,048,720; column 2, lines 17 to 46 of U.S. Patent No. 5,543,502; column 4, line 22 to column 5, line 36 of U.S. Patent No. 5,171,844; column 2, lines 55 to 68, Figure 2, and Example 1 of U.S. Patent No. 5,112,950; column 2, lines 2 to column 19, line 21 and Table 2 of U.S. Patent No. 4,868,112; column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39 of U.S. Patent No. 7,041,635; or column 4, lines 25 to 53 of U.S. Patent No. 6,458,563. In some embodiments, the B-domain deleted FVIII has multiple deletions of the B-domain as described in WO91 / 09122, but still contains the amino-terminal sequence of the B-domain that is essential for the in vivo proteolytic processing of the primary translation product into two polypeptide chains. In some embodiments, the B-domain deleted FVIII is constructed using a deletion of amino acids 747 to 1638, i.e., substantially complete deletion of the B-domain. Hoeben R.C. et al., J. Biol. Chem. 265(13):7318 - 7323 (1990). The B-domain deleted Factor VIII may also contain a deletion of amino acids 771 to 1666 or amino acids 868 to 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 deletions of amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, pp. 5939-5942), 797-1562 (Eaton et al., Biochemistry (1986) 25:8343-8347), 741-1646 (Kaufman (PCT Publication No. WO87 / 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 particular embodiment, the B domain deleted FVIII comprises a deletion of amino acid residues 746-1648 of mature FVIII. In another embodiment, the B domain deleted FVIII comprises a deletion of amino acid residues 745-1648 of mature FVIII.

[0271] In other embodiments, the BDD FVIII contains 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. It comprises an FVIII polypeptide. Examples of B domain fragments include the 226 amino acids or 163 amino acids of the B domain disclosed in Miao, H.Z. et al., Blood 103(a):3412-3419 (2004), Kasuda, A et al., J. Thromb. Haemost. 6:1352-1359 (2008), and Pipe, S.W. et al., J. Thromb. Haemost. 9:2235-2242 (2011) (i.e., the first 226 amino acids or 163 amino acids of the B domain are retained). In yet other embodiments, the BDD FVIII further comprises a point mutation (from Phe to Ser) at residue 309 to improve the expression of the BDD FVIII protein. In yet other embodiments, the BDD FVIII comprises an FVIII polypeptide that contains a portion of the B domain but does not contain one or more furin cleavage sites (e.g., Arg1313 and Arg1648). See Pipe, S.W. et al., J. Thromb. Haemost. 9:2235-2242 (2011). In some embodiments, the BDD FVIII comprises a single-chain FVIII containing a deletion in amino acids 765-1652 corresponding to mature full-length FVIII (also known as rVIII-SingleChain and AFSTYLA®). See U.S. Patent No. 7,041,635. Each of said deletions can be made in any FVIII sequence.

[0272] As considered above and below, many functional FVIII variants are known. Further, 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 arise more from their location within the three-dimensional structure of FVIII rather than the nature of the substitution (Cutler et al., Hum. Mutat. 19:274-278 (2002), which is hereby incorporated by reference in its entirety). Further, comparison between FVIII from humans and other species has identified conserved residues that may be required for function (Cameron et al., Thromb. Haemost. 79:317-322 (1998), which is hereby incorporated by reference in its entirety; U.S. Patent No. 6,251,632).

[0273] In some embodiments, the effective amount of the chimeric protein comprising FVIII and the Fc region is equivalent to the effective amount of FVIII without the Fc region. In certain embodiments, the effective amount is from about 20 IU / kg to about 400 IU / kg. In certain embodiments, the effective amount is from about 20 IU / kg to about 300 IU / kg. In some embodiments, the effective amount is from about 50 IU / kg to about 300 IU / kg. In some embodiments, the effective amount is from about 50 IU / kg to about 200 IU / kg. In some embodiments, the effective amount is from about 100 IU / kg to about 300 IU / kg, from about 100 IU / kg to about 200 IU / kg, from about 100 IU / kg to about 290 IU / kg, from about 100 IU / kg to about 280 IU / kg, from about 100 IU / kg to about 270 IU / kg, from about 100 IU / kg to about 260 IU / kg, from about 100 IU / kg to about 250 IU / kg, from about 100 IU / kg to about 240 IU / kg, from about 100 IU / kg to about 230 IU / kg, from about 100 IU / kg to about 220 IU / kg, from about 100 IU / kg to about 210 IU / kg, from about 150 IU / kg to about 300 IU / kg, from about 150 IU / kg to about 290 IU / kg, from about 150 IU / kg to about 280 IU / kg, from about 150 IU / kg to about 270 IU / kg, from about 150 IU / kg to about 260 IU / kg, from about 150 IU / kg to about 250 IU / kg, from about 150 IU / kg to about 240 IU / kg, from about 140 IU / kg to about 250 IU / kg, from about 130 IU / kg to about 260 IU / kg, from about 120 IU / kg to about 270 IU / kg, from about 110 IU / kg to about 280 IU / kg. In one particular embodiment, the effective amount is from about 200 IU / kg to about 300 IU / kg. In another embodiment, the effective amount is from about 200 IU / kg to about 290 IU / kg. In other embodiments, the effective amount is from about 200 IU / kg to about 280 IU / kg, from about 200 IU / kg to about 270 IU / kg, from about 200 IU / kg to about 260 IU / kg, from about 200 IU / kg to about 250 IU / kg, from about 200 IU / kg to about 240 IU / kg, from about 200 IU / kg to about 230 IU / kg, from about 200 IU / kg to about 220 IU / kg, or from about 200 IU / kg to about 210 IU / kg.

[0274] In some embodiments, the effective amount is about 50 IU / kg, about 60 IU / kg, about 70 IU / kg, about 80 IU / kg, about 90 IU / kg, about 100 IU / kg, 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, about 200 IU / kg, about 225 IU / kg, about 250 IU / kg, about 275 IU / kg, or about 200 IU / kg. In one particular embodiment, the effective amount is about 150 IU / kg / kg. In another embodiment, the effective amount is about 200 IU / kg. In another embodiment, the effective amount is about 250 IU / kg. In another embodiment, the effective amount is about 50 IU / kg. In another embodiment, the effective amount is about 100 IU / kg.

[0275] When administering a chimeric protein comprising FVIII and an Fc region or fragment thereof, the dosing interval may be at least about 1.5-fold longer than the dosing interval required for an equivalent amount of a clotting factor that does not contain the Fc domain. The dosing interval may be at least about 1.5 to 6-fold longer, 1.5-fold to 5-fold longer, 1.5-fold to 4-fold longer, 1.5-fold to 3-fold longer, or 1.5-fold to 2-fold longer than the dosing interval required for an equivalent amount of FVIII that does not contain the Fc domain.

[0276] In some embodiments, an effective dose of the chimeric protein comprising FVIII and an Fc region is administered to a human at a dosing interval of about 1 day, 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 the 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.

[0277] In some embodiments, a composition or chimeric protein comprising FVIII and an Fc region is administered at dosing intervals 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, a composition or chimeric protein comprising FVIII and an Fc region is administered at dosing intervals of 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 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, about 15 to about 20 days. In certain embodiments, a composition or chimeric protein comprising FVIII and an Fc region is administered at a dosing interval of about 2 to about 6 days. In another embodiment, a composition or chimeric protein comprising FVIII and an Fc region is administered at a dosing interval of about 3 to about 5 days.

[0278] In one embodiment, the effective amount is 25 to 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 to 5, 3 to 6, 3 to 7, 3, 4, 5, 6, 7, or 8 days or more days, or three times a week, or three times a week or less. In another embodiment, the effective amount is 65 IU / kg and the dosing interval is once a week or once every 6 to 7 days. The dosage can be administered repeatedly as long as they are needed (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 dosage is about 25 to 65 IU / kg and the dosing interval is once every 3 to 5 days.

[0279] In one embodiment, the effective amount is about 200 IU / kg and the effective amount is administered daily. In another embodiment, the effective amount is about 50 IU / kg and the effective amount is administered about three times a week.

[0280] In certain embodiments, the effective amount or effective dosage is administered as a single dose. In some embodiments, the effective amount or effective dosage is administered in two or more doses throughout the day.

[0281] In some embodiments, a composition or chimeric protein comprising FVIII and an Fc region is administered to a human at a dose of about 200 IU / kg once a day until tolerance is observed. In some embodiments, the tolerance period extends from about 4 weeks to about 36 months. In some embodiments, the tolerance period extends for about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, or about 36 months.

[0282] In certain embodiments, once immune tolerance is achieved, the human is taken through a taper period. As used herein, the terms "taper period" and "taper regimen" are used interchangeably to refer to a dosing regimen in which one or more tapered doses are administered. In some embodiments, the taper period includes administration of from about 20 IU / kg to about 400 IU / kg. In certain embodiments, the taper period includes administration of from about 20 IU / kg to about 300 IU / kg. In some embodiments, the taper period includes administration of from about 50 IU / kg to about 300 IU / kg. In some embodiments, the taper period includes administration of from about 50 IU / kg to about 100 IU / kg. In some embodiments, the taper period includes administration of from about 100 IU / kg to about 300 IU / kg, from about 100 IU / kg to about 200 IU / kg, from about 100 IU / kg to about 290 IU / kg, from about 100 IU / kg to about 280 IU / kg, from about 100 IU / kg to about 270 IU / kg, from about 100 IU / kg to about 260 IU / kg, from about 100 IU / kg to about 250 IU / kg, from about 100 IU / kg to about 240 IU / kg, from about 100 IU / kg to about 230 IU / kg, from about 100 IU / kg to about 220 IU / kg, from about 100 IU / kg to about 210 IU / kg, from about 150 IU / kg to about 300 IU / kg, from about 150 IU / kg to about 290 IU / kg, from about 150 IU / kg to about 280 IU / kg, from about 150 IU / kg to about 270 IU / kg, from about 150 IU / kg to about 260 IU / kg, from about 150 IU / kg to about 250 IU / kg, from about 150 IU / kg to about 240 IU / kg, from about 140 IU / kg to about 250 IU / kg, from about 130 IU / kg to about 260 IU / kg, from about 120 IU / kg to about 270 IU / kg, from about 110 IU / kg to about 280 IU / kg. In one particular embodiment, the taper period is about It includes administration at 200 IU / kg to approximately 300 IU / kg. In another embodiment, the tapering period includes administration at about 200 IU / kg to about 290 IU / kg. In other embodiments, the tapering period includes administration at about 200 IU / kg to about 280 IU / kg, about 200 IU / kg to about 270 IU / kg, about 200 IU / kg to about 260 IU / kg, about 200 IU / kg to about 250 IU / kg, about 200 IU / kg to about 240 IU / kg, about 200 IU / kg to about 230 IU / kg, about 200 IU / kg to about 220 IU / kg, or about 200 IU / kg to about 210 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 50 IU / kg to about 100 IU / kg. In one particular embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 50 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 150 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 125 IU / kg. In another particular embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 100 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 90 U / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 80 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 75 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 70 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 60 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 40 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 30 IU / kg. In another embodiment, the tapering regimen includes administering a tapering dose of a composition or chimeric protein at about 25 IU / kg.In another embodiment, the tapering regimen comprises administering a tapering dose of the composition or chimeric protein of about 20 IU / kg. In another embodiment, the tapering regimen comprises administering a tapering dose of the composition or chimeric protein of about 10 IU / kg.

[0283] In some embodiments, the tapering period comprises daily administration of the composition or chimeric protein. In other embodiments, the tapering period comprises administration of the composition or chimeric protein once every about 2 days, once every about 3 days, once every about 4 days, once every about 5 days, once every about 6 days, once every about 7 days, once every about 8 days, once every about 9 days, once every about 10 days, once every about 11 days, once every about 12 days, once every about 13 days, or once every about 14 days.

[0284] In certain embodiments, the tapering dose is administered once a day, once every other day, or three times a week. In some embodiments, the tapering dose is administered over at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, or at least about 32 weeks. In a particular embodiment, the tapering dose is administered over about 16 weeks or less.

[0285] In certain embodiments, the dose of the composition or chimeric protein gradually decreases during the tapering period and the dosing interval remains the same. In other embodiments, the dosing interval increases during the tapering period and the dose of the composition or chimeric protein remains the same. In some embodiments, the composition Alternatively, the dosage of the chimeric protein gradually decreases during the tapering period, and the dosing interval gradually increases.

[0286] In one particular embodiment, the tapering period includes administration of about 200 IU / kg of the chimeric coagulation factor every other day, followed by further decreases in dosage and dosing interval. In other embodiments, the dosage of the chimeric protein required per day can be divided into two dosages, three dosages, or more dosages. For example, about 200 IU / kg of the chimeric protein can be divided into about 100 IU / kg twice a day, about 70 IU / kg three times a day, or about 50 IU / kg four times a day.

[0287] In some embodiments, the tapering period extends from about 1 month to about 6 months. In certain embodiments, the tapering period extends for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months. In one particular embodiment, the tapering period extends for about 4 months.

[0288] In certain embodiments, the tapering regimen includes administering a tapering dosage of about 100 IU / kg of the chimeric protein once a day from week 1 to week 6 after immune tolerance. In certain embodiments, the tapering regimen further includes administering a tapering dosage of about 100 IU / kg of the chimeric protein once every other day from week 6 to week 12 after immune tolerance. In certain embodiments, the tapering regimen further includes administering a tapering dosage of about 50 IU / kg of the chimeric protein once every other day from week 12 to week 16.

[0289] In some embodiments, there is a follow-up period after the tapering period. In some embodiments, the follow-up period includes prophylactic treatment with the composition or chimeric protein. In some embodiments, the follow-up period includes prophylactic treatment with a coagulation factor. The coagulation factor used during the follow-up period can be selected from the coagulation factors used during the tolerance induction and tapering periods, including, or not including, the Fc region and any variants thereof. Coagulation factors include, but are not limited to, natural coagulation factors, any variants described herein (e.g., the B domain-deleted variant of FVIII), and any chimeric coagulation factors described herein (e.g., FVIII-Fc, FVIII-albumin, etc.). In certain embodiments, the prophylactic treatment includes, for example, administration of an approved prophylactic dose of recombinant FVIIIFc. In some embodiments, the prophylactic treatment includes 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 days, 3-6 days, 3-7 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days or more, or three times a week, or three times a week or less. In another embodiment, the prophylactic treatment includes 65 IU / kg, and the dosing interval is once a week or once every 6-7 days. In another embodiment, the prophylactic treatment includes administering a dose of 50 IU / kg of a coagulation factor. In another embodiment, the prophylactic treatment includes administering a dose of 50 IU / kg of a coagulation factor, and the dosing interval is approximately three times a week. In one particular embodiment, the prophylactic treatment includes approximately 25-65 IU / kg, and the dosing interval is once every 3-5 days. In certain embodiments, the follow-up period extends for about 8 months.

[0290] In one particular embodiment, the chimeric protein, e.g., FVIIIFc, is administered at about 200 IU / kg / day until immune tolerance is observed, e.g., until the titer of inhibitory antibodies in humans is less than about 0.6 BU; then, after immune tolerance, a tapered dose of the chimeric protein, e.g., FVIIIFc, of about 100 IU / kg is administered once daily from week 1 to week 6, a tapered dose of the chimeric protein, e.g., FVIIIFc, of about 100 IU / kg is administered once every other day from week 6 to week 12, and a tapered dose of the chimeric protein, e.g., FVIIIFc, of about 50 IU / kg is administered once every other day from week 12 to week 16, and a tapered regimen, including administering a tapered dose of the chimeric protein, e.g., FVIIIFc, is administered after immune tolerance; then, after the tapered regimen, a prophylactic dose of a coagulation factor of about 50 IU / kg is administered about three times a week. A tapered regimen, including administering a tapered dose of the chimeric protein, e.g., FVIIIFc, is administered after immune tolerance; then, after the tapered regimen, a prophylactic dose of a coagulation factor of about 50 IU / kg is administered about three times a week.

[0291] Compositions or chimeric proteins comprising FVIII 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.

[0292] As used herein, the term parenteral includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, intraspinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intraarticular, 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 can be formulated as a suppository with traditional binders and carriers such as triglycerides.

[0293] In one example, the pharmaceutical formulation is a liquid formulation, e.g., a buffered, isotonic aqueous solution. In another example, the pharmaceutical composition has a pH that is physiological or near physiological conditions. In other examples, the aqueous formulation has an osmotic pressure and salt concentration that are physiological or near physiological conditions. It may contain sodium chloride and / or sodium acetate.

[0294] In some embodiments, the chimeric protein comprising FVIII and the Fc region used in the method of the present invention is formulated in a pharmaceutical composition comprising: (a) the chimeric protein; (b) one or more stabilizers selected from sucrose, trehalose, raffinose, arginine, or mixtures 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) the chimeric protein at 50 IU / ml to 2500 IU / ml; (b) sucrose at 10 mg / ml to 25 mg / ml; (c) sodium chloride (NaCl) at 8.8 mg / ml to 14.6 mg / ml; (d) L-histidine at 0.75 mg / ml to 2.25 mg / ml; (e) calcium chloride dihydrate at 0.75 mg / ml to 1.5 mg / ml; and (f) polysorbate 20 or polysorbate 80 at 0.08 mg / ml to 0.25 mg / ml. In some examples, the pharmaceutical composition used in the methods of the present disclosure is lyophilized.

[0295] In some embodiments, the pharmaceutical composition is free of immune cells. In some embodiments, the pharmaceutical composition is cell-free.

[0296] In certain embodiments, the human being treated using the methods of the present disclosure has previously had an FVIII inhibitory immune response. In some embodiments, the previously occurring FVIII inhibitory response occurred in response to recombinant FVIII. In some embodiments, the previously occurring FVIII inhibitory response occurred in response to an FVIII product selected from the group consisting of ADVATE®, RECOMBINATE®, KOGENATE FS®, HELIXATE FS®, XYNTHA / REFACTO AB®, HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®, ALPHANATE®, KOATE-DVI®, AFSTYLA®, and HYATE:C®.

[0297] In some embodiments, once tolerance is reached in response to a decrease in the titer of the inhibitory antibody, the serum level of the coagulation factor is maintained at about 100 IU / dL to about 200 IU / dL. In some embodiments, if the serum level of the coagulation factor is higher than or equal to 200 IU / dL, the effective amount of the composition or chimeric protein is decreased to about 175 IU / kg / day. A In certain embodiments, if the serum level of the coagulation factor is higher than or equal to 200 IU / dL, the effective amount of the composition or chimeric protein is decreased to about 150 IU / kg / day. In certain embodiments, if the serum level of the coagulation factor is higher than or equal to 200 IU / dL, the effective amount of the composition or chimeric protein is decreased to about 125 IU / kg / day. In certain embodiments, if the serum level of the coagulation factor is higher than or equal to 200 IU / dL, the effective amount of the composition or chimeric protein is decreased to about 100 IU / kg / day. In certain embodiments, if the serum level of the coagulation factor is higher than or equal to 200 IU / dL, the effective amount of the composition or chimeric protein is decreased to about 75 IU / kg / day. In certain embodiments, if the serum level of the coagulation factor is higher than or equal to 200 IU / dL, the effective amount of the composition or chimeric protein is decreased to about 50 IU / kg / day. In certain embodiments, if the serum level of the coagulation factor is higher than or equal to 200 IU / dL, the effective amount of the composition or chimeric protein is decreased to about 25 IU / kg / day.

[0298] Certain aspects of the present disclosure relate to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising about 200 IU / kg of a coagulation factor and an Fc region. In certain embodiments, the composition or chimeric protein is administered every other day. In other embodiments, the composition or chimeric protein is administered daily.

[0299] In other aspects, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 202 IU / kg. In certain embodiments, the composition or chimeric protein is administered daily.

[0300] In other aspects, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 150 IU / kg. In certain embodiments, the composition or chimeric protein is administered daily.

[0301] In other aspects, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 130 IU / kg. In certain embodiments, the composition or chimeric protein is administered daily.

[0302] In other aspects, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 115 IU / kg. In certain embodiments, the composition or chimeric protein is administered every other day.

[0303] In other aspects, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 100 IU / kg. In certain embodiments, the composition or chimeric protein is administered daily. In other embodiments, the composition or chimeric protein is administered three times a week.

[0304] In another aspect, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 102 IU / kg. In certain embodiments, the composition or chimeric protein is administered every other day.

[0305] In another aspect, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 96 IU / kg. In certain embodiments, the composition or chimeric protein is administered daily.

[0306] In another aspect, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 85 IU / kg. In certain embodiments, the composition or chimeric protein is administered daily.

[0307] In another aspect, the present disclosure relates to a method of inducing immune tolerance in a human having hemophilia, the method comprising administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 50 IU / kg. In certain embodiments, the composition or chimeric protein is administered three times a week.

[0308] Certain aspects of the present disclosure relate to a method of inducing immune tolerance in a human having hemophilia, the method comprising: (1) administering to the human a composition or chimeric protein comprising a coagulation factor and an Fc region at about 200 IU / kg, the composition or chimeric protein being one that induces immune tolerance in humans; and (2) after induction of immune tolerance, administering to the human a tapering regimen of the composition or chimeric protein.

[0309] II.A.2 Fc In some embodiments, the compositions, chimeric proteins, and / or coagulation factors of the present disclosure include an Fc domain or a portion thereof that binds to an Fc receptor (FcR; e.g., FcRn). In some embodiments, the Fc domain is fused to a coagulation factor, for example, as part of a chimeric protein that includes the coagulation factor and an Fc region. In other embodiments, the Fc domain is fused to a polypeptide other than a coagulation factor, where the composition includes (1) a coagulation factor and (2) a chimeric protein that includes the Fc domain and an additional polypeptide. 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 the molecule fused to the Fc domain or a portion thereof.

[0310] As used herein, the term “Fc domain” of the “Fc region” as used herein means a functional FcR (e.g., FcRn) binding partner, unless otherwise specified. The Fc domain is the portion of the polypeptide that corresponds to the Fc domain of a native Ig, i.e., formed by the dimeric binding of the Fc domains of each of its two heavy chains. The native Fc domain forms a homodimer with another Fc domain. In contrast, as used herein, the terms “genetically fused Fc region” or “single-chain Fc region” (scFv region) refer to a synthetic dimeric Fc region that includes an Fc domain genetically linked (i.e., encoded in a single continuous gene sequence) within a single polypeptide chain.

[0311] In one embodiment, the “Fc region” refers to the portion of a single IgG heavy chain that begins at the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in IgG, assuming the first residue of the heavy chain constant region is 114) and ends at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain.

[0312] The Fc region of the Ig constant region may include CH2, CH3, and CH4 domains, as well as the hinge region, depending on the Ig isotype. Chimeric proteins containing the Fc region of Ig 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 hereby incorporated by reference in their entirety).

[0313] FcRn receptors have been isolated from several mammalian species, including humans. The sequences of human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al., 1994, J. Exp. Med. 180:2377). The FcRn receptor binds to IgG at a relatively low pH (but not other Ig classes such as IgA, IgM, IgD, and IgE), actively transports IgG intracellularly in the luminal direction, and then releases IgG at the relatively high pH found in the interstitial fluid. It is expressed in adult epithelial tissues, including the lung and intestinal epithelium (Israel et al., 1997, Immunology 92:69), renal proximal tubular epithelium (Kobayashi et al., 2002, Am. J. Physiol. Renal Physiol. 282:F358), and nasal epithelium, vaginal surface, and biliary tract surface (U.S. Patent Nos. 6,485,726, 6,030,613, 6,086,875; WO03 / 077834; US2003-0235536A1).

[0314] Fc regions useful in the present invention include molecules that can specifically bind to FcRs, including whole IgG, Fc fragments of IgG, and other fragments containing the complete binding region of FcR. For example, the region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al., 1994, Nature 372:379). The main contact region between Fc and FcRn is close to the junction of the CH2 and CH3 domains. All Fc-FcRn are within a single Ig heavy chain. The Fc region includes whole IgG, Fc fragments of IgG, and other fragments of IgG containing the complete binding region of FcRn. The main contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. All references to amino acid numbers of Ig or Ig fragments, or regions, are based on Kabat et al., 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

[0315] Specific binding refers to two molecules that form a relatively stable complex under physiological conditions. Specific binding is characterized by high affinity and low to moderate avidity, such that it is typically distinguished from non-specific binding, which has low affinity and usually moderate to high avidity. Typically, binding is considered specific if the affinity constant KA is 10 6 M -1 or higher, or 10 8 M -1 or higher. If desired, non-specific binding can be reduced by changing the binding conditions without substantially affecting specific binding. One of ordinary skill in the art can optimize appropriate binding conditions such as the concentration of the molecules, the ionic strength of the solution, the temperature, the time of binding, the concentration of blocking agents (e.g., serum albumin, milk casein), etc., using routine techniques.

[0316] In certain embodiments, the chimeric protein of the invention comprises one or more truncated Fc regions that, nevertheless, are sufficient to confer Fc binding properties to the Fc region. For example, the portion of the Fc region that binds to FcRn (i.e., the FcRn binding portion) comprises approximately amino acids 282-438 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 region of the invention may comprise or consist of the FcRn binding portion.

[0317] The FcR binding portion may be derived from the heavy chain of any isotype including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcR binding portion derived from an antibody of human isotype IgG1 is used. In another embodiment, an FcR binding portion derived from an antibody of human isotype IgG4 is used.

[0318] In another embodiment, the "Fc region" includes an amino acid sequence of the Fc domain or derived from the Fc domain. In certain embodiments, the Fc region includes the hinge (e.g., upper, middle, and / or lower hinge region) domain (approximately amino acids 216-230 of the antibody Fc region according to EU numbering), the CH2 domain (approximately amino acids 231-340 of the antibody Fc region according to EU numbering), the CH3 domain (approximately amino acids 341-438 of the antibody Fc region according to EU numbering), the CH4 domain, or at least one of its variants, portions, or fragments. In other embodiments, the Fc region includes the complete Fc domain (i.e., the hinge domain, the CH2 domain, and the CH3 domain). In some embodiments, the Fc region consists essentially of or consists of a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to both a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In yet other embodiments, the Fc region lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). In certain embodiments, the Fc region comprises or consists of amino acids corresponding to EU numbers 221-447.

[0319] The Fc region, denoted 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 be derived from the 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. Further, the polypeptide of the Fc domain 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 another embodiment, the human isotype IgG1 is used.

[0320] In certain embodiments, the Fc variant confers a change in at least one effector function provided by the Fc region containing the wild-type Fc domain (e.g., an improvement or decrease in the binding of the Fc region to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII), a complete protein (e.g., C1q), or another Fc-binding partner (e.g., DC-SIGN), or an improvement or decrease in the ability of the Fc region to induce antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides engineered cysteine residues.

[0321] The Fc region of the present invention may use Fc variants recognized in the art that are known to confer changes (e.g., enhancements or decreases) in effector function and / or FcR or FcRn binding. Specifically, the binding molecules of the present invention are described, for example, in International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, 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 / 0231329, 2007 / 0237765, 2007 / 0237766, 2007 / 0237767, 2007 / 0243188, 2007 / 0248603, 2007 / 0286859, 2008 / 0057056; or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091 may include a change (e.g., substitution) at one or more amino acid positions. 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 of the disclosed amino acid positions. In another embodiment, different changes (e.g., different substitutions at one or more amino acid positions disclosed in the art) can be made at one or more of the disclosed amino acid positions.

[0322] The Fc region can be modified according to well-known procedures such as site-directed mutagenesis to obtain a modified Fc fragment or a portion thereof to which FcγRIIB and / or DC-SIGN binds. Such modifications include modifications distant from the FcγRIIB and / or DC-SIGN contact sites as well as modifications within the contact sites that preserve or even enhance binding to 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, 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, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, 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 FcγRIIB and / or DC-SIGN, where, for example, P238A represents wild-type proline substituted by alanine at position number 238. By way of example, certain embodiments include the N297A mutation, excluding the highly conserved N-glycosylation site. In addition to alanine, other amino acids can be substituted for the wild-type amino acids at the positions specified above. Mutations can be introduced singly into the Fc to generate more than 100 Fc regions different from native Fc. Further, 2 One, three, or more combinations of these individual mutations can be introduced together to generate hundreds more Fc regions. Further, one of the Fc regions of the constructs of the invention can be mutated and the other Fc regions of the construct either left completely unmutated or both mutated using different mutations.

[0323] Certain of the above mutations can confer new functions on the Fc region or FcRn binding partners. For example, one embodiment includes N297A, excluding the highly conserved N-glycosylation site. The effect of this mutation is to enhance the circulating half-life of the Fc region by reducing immunogenicity and to render the Fc region unable to bind to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA without impairing its affinity for FcRn (Routledge et al., 1995, Transplantation 60:847; Friend et al., 1999, Transplantation 68:1632; Shields et al., 1995, J. Biol. Chem. 276:6591). As a further example of a new function resulting from the above mutations, the affinity for FcRn can, in some instances, be increased beyond that of the wild-type. This increase in affinity may reflect an increase in the "on" rate, a decrease in the "off" rate, or both an increase in the "on" rate and a decrease in the "off" rate. Examples of mutations thought to confer an increase in affinity for FcRn include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al., 2001, J. Biol. Chem. 276:6591).

[0324] Furthermore, at least three human Fc gamma receptors are thought to recognize binding sites on IgG within the lower hinge region, generally amino acids 234-237. Thus, another example of a new function and potential reduction in immunogenicity could result from mutations in this region, such as by replacing amino acids 233-236 "ELLG" of human IgG1 with the corresponding sequence "PVA" from IgG2 (including a one amino acid deletion). FcγRI, FcγRII, and FcγRIII, which mediate various effector functions, have been shown not to bind IgG1 when such mutations are introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al., 1999, Eur. J. Immunol. 29:2613.

[0325] In one embodiment, the Fc domain or a portion thereof is a polypeptide that includes SEQ ID NO: 3 of U.S. Patent No. 5,739,277 and optionally further includes a sequence selected from SEQ ID NOs: 11, 1, 2, and 31 of U.S. Patent No. 5,739,277.

[0326] In certain embodiments, the Fc domain or a portion thereof is hemiglycosylated. For example, a chimeric protein that includes two Fc regions may contain a first, glycosylated Fc region (e.g., a glycosylated CH2 region) and a second, non-glycosylated Fc region (e.g., a non-glycosylated CH2 region). In one embodiment, a linker can be placed between the glycosylated Fc region and the non-glycosylated Fc region. In another embodiment, the Fc region is fully glycosylated, i.e., all of the Fc region is glycosylated. In other embodiments, the Fc region may be non-glycosylated, i.e., none of the Fc moieties are glycosylated.

[0327] In certain embodiments, the chimeric proteins of the invention include amino acid substitutions to the Fc domain or a portion thereof (e.g., an Fc variant) that alter the antigen-independent effector functions of the Fc domain, particularly the circulating half-life of the protein.

[0328] Such proteins, when compared to proteins lacking these substitutions, bind to FcRs Indicates an increase or decrease in the binding that occurs, and thus, a corresponding increase or decrease in the half-life in serum, respectively. Fc variants with improved affinity for FcR are expected to have a longer serum half-life, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered polypeptide is desirable, for example, in treating chronic diseases or disorders (see, e.g., U.S. Patent Nos. 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with decreased FcR binding affinity are expected to have a shorter half-life, and such molecules are also useful, for example, when a shortened circulation time is advantageous, for example, for diagnostic imaging in vivo, or when the starting polypeptide has toxic side effects when present in circulation over a long period of time, for administration to mammals. Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and are thus useful in treating diseases or disorders in pregnant women. Further, other applications where a decrease in FcRn binding affinity is desirable include applications where localization in the brain, kidney, and / or liver is desirable. In one exemplary embodiment, the chimeric protein of the present invention exhibits a decrease in transport through the epithelium of the renal glomerulus from the vasculature. In another embodiment, the chimeric protein of the present invention exhibits a decrease in transport through the blood-brain barrier (BBB) from the brain to the vascular lumen. In one embodiment, the protein with altered FcR binding comprises at least one Fc region (e.g., one or two Fc regions) having one or more amino acid substitutions within the "FcR binding loop" of the Ig constant region. The FcR binding loop, in one embodiment, comprises amino acid residues 280-299 (according to EU numbering) of the wild-type, full-length Fc region. In other embodiments, the Ig constant region or a portion thereof in the chimeric protein of the present invention with altered FcR binding affinity comprises at least one Fc region having one or more amino acid substitutions within a 15 Å FcR "contact zone".As used herein, the term "contact zone" of FcRn of 15 Å includes the residues at the following positions of the wild-type, full-length Fc portion: 243-261, 275-280, 282-293, 302-319, 336-348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering). In other embodiments, the Fc domain or a portion thereof of the present invention with altered FcR binding affinity comprises at least one Fc region having one or more amino acid substitutions at amino acid positions corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions with altered FcR binding activity are disclosed in International PCT Publication No. WO05 / 047327, which is incorporated herein by reference.

[0329] The Fc region used in the present invention may also include amino acid substitutions recognized in the art that alter the glycosylation of the chimeric protein. For example, the Fc region of a chimeric protein linked to the FVIII protein may include an Fc region having a mutation that results in a decrease in glycosylation (e.g., N- or O-linked glycosylation), or may include a change in the glycoform of the wild-type Fc portion (e.g., hypofucosylated or afucosylated glycan).

[0330] In one embodiment, the unprocessed chimeric protein of the present invention may include a genetically fused Fc region (i.e., scFc region) having two or more of its constituent Ig constant regions or portions thereof independently selected from the Ig constant regions described herein or portions thereof. 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 present invention may contain the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In still other embodiments, the Fc regions of the proteins of the present invention may differ in sequence at one or more amino acid positions. For example, 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.

[0331] In some embodiments, the chimeric protein used in the methods of the present disclosure includes more than one polypeptide chain. In some embodiments, the chimeric protein includes two polypeptide chains. In certain embodiments, the first polypeptide chain includes a coagulation factor and a first Fc region, and the second polypeptide chain includes a second Fc region. In certain embodiments, the first Fc region and the second Fc region are joined by a covalent bond. In one embodiment, the first Fc region and the second Fc region are joined by a peptide bond. In another embodiment, the first Fc region and the second Fc region are joined by a disulfide bond.

[0332] In one particular embodiment, the chimeric protein comprises a Factor VIII portion and a von Willebrand factor (VWF) portion, wherein the FVIII portion comprises an 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.

[0333] The Fc region or a portion thereof for producing the chimeric protein used in the methods of the present disclosure can be obtained from several different sources. In some embodiments, the Fc region or a portion thereof is derived from a human Ig. However, it is understood that the Fc region or a portion thereof may also be derived from the 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. Further, the Fc region or a portion thereof may be derived from any Ig class including IgM, IgG, IgD, IgA, and IgE, as well as any Ig isotype including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgG1 is used.

[0334] A variety of Fc region gene sequences (e.g., human Fc gene sequences) are available in a publicly accessible deposit form. Fc sequences having a particular effector function (or lacking a particular effector function) or having particular modifications that reduce immunogenicity can be selected. Many sequences of antibodies and antibody-encoding genes are publicly available, and suitable Fc region sequences can be derived from these sequences using techniques recognized in the art. The genetic material obtained using any of the foregoing methods can then be altered or synthesized to obtain the chimeric proteins used in the methods of the present disclosure. Further, it is understood that the scope of the present invention encompasses alleles, variants, and mutations of the constant region DNA sequences.

[0335] Sequences of 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 a sequence of an Fc region or a portion thereof derived from an antibody, mRNA can be isolated from a hybridoma, spleen, or lymphocytes, reverse transcribed into DNA, and the antibody gene can be amplified by PCR. PCR amplification methods are described 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 A" pplications」, 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 more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, the library can be screened with larger homologous probes such as consensus primers or mouse constant region probes. Several primer sets suitable for amplification of antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan, 1994, Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al., 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 to Newman et al., filed January 25, 1995, which is incorporated herein by reference.

[0336] II.B. Half-life extension moiety In some embodiments, the chimeric protein used in the method of the present disclosure further comprises one or more half-life extension moieties. The half-life of a coagulation factor can be determined by any method known to those skilled in the art, such as an FVIII activity assay (chromogenic assay or one-stage clotting aPTT assay) for detecting plasma FVIII activity or an FVIII ELISA for detecting plasma FVIII antigen levels. In certain embodiments, the half-life of the coagulation activity of the coagulation factor is determined by the one-stage clotting method. In more specific embodiments, the half-life of the coagulation activity of the coagulation factor is determined in HemA mice or factor VIII and von Willebrand factor double knockout (DKO) mice.

[0337] In certain aspects, the heterologous moiety that increases the half-life of the coagulation factor of the present invention includes, but is not limited to, heterologous polypeptides such as albumin, the Fc region of immunoglobulin, XTEN sequence, the C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, PAS sequence, HAP sequence, transferrin, albumin-binding moiety, or any fragment, derivative, variant, or combination of these peptides. In other related aspects, the half-life extension moiety may include a binding site for non-polypeptide moieties such as polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, or any derivative, variant, or combination of these moieties. In certain embodiments, the half-life extension moiety includes albumin or a fragment thereof, an albumin-binding moiety, PAS sequence, HAP sequence, transferrin or a fragment thereof, or any combination thereof. In some embodiments, the half-life extension moiety does not include XTEN. In other embodiments, the half-life extension moiety includes XTEN.

[0338] In other embodiments, the chimeric proteins of the 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 bound to a coagulation factor, Fc, or other moiety conjugated to a coagulation factor or Fc. Non-limiting examples of polymers may be poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, or poly(acryloyl morpholine). For example, a further form of FVIII conjugated to a polymer is disclosed in U.S. Patent No. 7,199,223, which is hereby incorporated by reference in its entirety.

[0339] In certain embodiments, the chimeric proteins of the invention may include one, two, three, or more half-life extending moieties, which may be the same or different molecules, respectively.

[0340] In some embodiments, the half-life extending moiety is fused to the N-terminus or C-terminus of the chimeric protein. In some embodiments, the half-life extending moiety is fused to the N-terminus or C-terminus of the coagulation factor. In some embodiments, the half-life extending moiety is fused to the N-terminus or C-terminus of Fc. In certain embodiments, the half-life extending moiety is inserted within the coagulation factor of the chimeric protein.

[0341] In some embodiments, the chimeric protein includes FVIII or a portion thereof, and the half-life extending moiety is inserted within FVIII at one or more positions disclosed in U.S. Patent Application Publication No. 2015-0158929 A1 and / or International Publication No. WO2015106052 A1, which are hereby incorporated by reference in their entireties. In one particular embodiment, the half-life extending moiety is inserted within the B domain (or a fragment thereof) of FVIII. In one particular embodiment, the half-life extending moiety is inserted within FVIII immediately downstream of amino acid residue 745 of mature FVIII.

[0342] II.B.1. Albumin In certain embodiments, the chimeric proteins used in the methods of the disclosure include at least one albumin polypeptide or 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 for endogenous and exogenous ligands. As used herein, the term "albumin" includes full-length albumin or functional fragments, variants, derivatives, or analogs thereof. 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 hereby incorporated by reference in their entirety.

[0343] The albumin-binding polypeptide (ABP) may include, but is not limited to, a bacterial albumin-binding domain, an albumin-binding peptide, or an albumin-binding antibody fragment that can bind to albumin. Domain 3 derived from streptococcal protein G, 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. Examples of albumin-binding peptides are 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 hereby incorporated by reference in their entirety.

[0344] In certain embodiments, the chimeric proteins used in the methods of the present disclosure include at least one binding site for a non-polypeptide small molecule, a variant thereof that can bind to albumin, or a derivative. For example, the chimeric protein may include one or more organic albumin-binding moieties. Examples of such albumin-binding moieties are 2-(3-maleimidopropanamido)-6-(4 -(4-iodophenyl)butanamido)hexanoate (the "Albu" tag), as disclosed by Trussel et al., Bioconjugate Chem. 20:2286-2292 (2009).

[0345] II.B.2.XTEN In certain embodiments, the chimeric proteins used in the methods of the present disclosure include at least one XTEN polypeptide or a fragment, variant, or derivative thereof. As used herein, an "XTEN sequence" refers to an extended-length polypeptide having a substantially non-repetitive sequence that is not naturally occurring and that primarily comprises small hydrophilic amino acids, along with a sequence having little or no structure under physiological conditions or no secondary or tertiary structure. Similar to chimeric protein partners, XTEN can function as a carrier that provides certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties when fused to or inserted into a coagulation factor of a chimeric protein, for example. Such desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility properties.

[0346] In the methods of the present disclosure, XTEN sequences fused to or inserted within a coagulation factor of a chimeric protein useful in the methods 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 increase in hydrodynamic (or Stokes) radius. In certain embodiments, the XTEN sequence can increase pharmacokinetic properties such as a longer half-life (e.g., in vivo half-life) or an increase in the area under the curve (AUC) such that the chimeric protein remains in vivo and has procoagulant activity over a longer period compared to a chimeric protein that does not contain XTEN.

[0347] Examples of XTEN sequences that can be inserted into the recombinant FVIII protein of the present invention are disclosed, 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. WO2010091122 A1, WO2010144502 A2, WO2010144508 A1, WO2011028228 A1, WO2011028229 A1, WO2011028344 A2, or WO2015106052 A1, each of which is incorporated herein by reference in its entirety.

[0348] II.B.3.VWF or a fragment thereof In certain embodiments, the chimeric proteins used in the methods of the present disclosure include at least one VWF polypeptide or fragment, variant, or derivative thereof. VWF (also known as F8VWF) is a large, multimeric glycoprotein present in plasma and is constitutively produced in endothelial cells (Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2,813 amino acid protein. All monomers contain several specific domains with specific functions, a D’ / D3 domain (which binds to factor VIII), an A1 domain (which binds to platelet GPIb-receptor, heparin, and / or perhaps collagen), an A3 domain (which binds to collagen), a C1 domain (where the RGD domain binds to this when platelet integrin αIIbβ3 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)).

[0349] In one embodiment, the VWF polypeptide is a VWF fragment. As used herein, the term “VWF fragment” includes, but is not limited to, functional VWF fragments containing the D’ domain and the D3 domain that can inhibit the binding of endogenous VWF to FVIII. Exemplary. In one embodiment, the chimeric protein used in the methods of the present disclosure includes a coagulation factor, an Fc region, and a VWF fragment, where the coagulation factor includes FVIII and the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment inhibits the interaction between the FVIII protein and endogenous VWF by blocking the VWF binding site on the FVIII protein. The VWF fragment includes derivatives, variants, mutants, or analogs that retain these activities of VWF. In certain embodiments, the VWF fragment includes the D’ domain and the D3 domain of VWF.

[0350] The 2,813 monomer 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 as NM_000552.3.

[0351] In certain embodiments, the VWF proteins useful herein can be further modified to improve their interaction with FVIII, e.g., to improve the binding affinity for FVIII. In other embodiments, the VWF proteins useful for the present invention may have other modifications, e.g., the protein can be pegylated, glycosylated, hexyled, 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 / 0023959 A1, US2015 / 0266943 A1, 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 a fragment thereof is fused to albumin or co-administered with albumin or a polypeptide comprising albumin.

[0352] II.B.4.CTP In certain aspects, the chimeric proteins used in the methods of the present disclosure comprise at least one C-terminal peptide (CTP) or a fragment, variant, or derivative thereof of the β subunit of human chorionic gonadotropin. CTP peptides are known to increase the half-life of the protein. See, for example, U.S. Patent No. 5,712,122, which is hereby incorporated by reference in its entirety. Non-limiting CTP peptides are disclosed in U.S. Patent Application Publication No. US2009 / 0087411 A1, which is hereby incorporated by reference.

[0353] II.B.5.PAS In certain embodiments, the chimeric proteins used in the methods of the present disclosure include at least one PAS peptide or fragment, variant, or derivative thereof. As used herein, a PAS peptide or PAS sequence refers to an amino acid sequence that consists primarily of alanine and serine residues, or consists primarily of 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 that can be used as part of a heterologous moiety in a chimeric protein. The 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 components" means that amino acids other than alanine, serine, and proline can be added to the PAS sequence to a certain extent, for example, up to about 12%, i.e., up to about 12 out of 100 amino acids of the PAS sequence, up to about 10%, up to about 9%, up to about 8%, up to about 6%, about 5%, about 4%, about 3%, i.e., about 2%, or about 1% of the amino acids. Amino acids different from 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 an increase in in vivo and / or in vitro stability against the recombinant proteins of the present invention and having a coagulation promoting activity.

[0354] Non-limiting examples of PAS peptides are disclosed, for example, in U.S. Patent Application Publication No. 2010 / 0292130 A1; PCT Application Publication No. WO2008 / 155134 A1; and European Patent No. EP2173890.

[0355] II.B.6.HAP In certain embodiments, the chimeric proteins used in the methods of the present disclosure include at least one homoamino acid polymer (HAP) peptide or a fragment, variant, or derivative thereof. The HAP peptide may be at least 50 amino acids in length, at least 100 amino acids in length, 120 amino acids in length, 140 amino acids in length, 160 amino acids in length, 180 amino acids in length, 200 amino acids in length, 250 amino acids in length, 300 amino acids in length, 350 amino acids in length, 400 amino acids in length, 450 amino acids in length, or 500 amino acids in length, and may include a repeat sequence of glycine. 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 (Gly 4 Ser) n or S(Gly 4 Ser) 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, for example, Schlapschy M et al., Protein Eng. Design Selection, 20:273-284 (2007).

[0356] II.B.7. Transferrin In certain embodiments, the chimeric proteins used in the methods of the present disclosure include at least one transferrin peptide or fragment, variant, or derivative thereof. Any transferrin can be fused to the chimeric proteins used in the methods of the present disclosure. By way of example, wild-type human Tf (Tf) is a ~75 kDa (not accounting for glycosylation) 679 amino acid protein with two major domains, N (≈330 amino acids) and C (≈340 amino acids), 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 hereby incorporated by reference in their entirety).

[0357] Transferrin transports iron via transferrin receptor (TfR)-mediated endocytosis. After the iron is released into the endosomal compartment 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 exceeding 14 - 17 days (Li et al., Trends Pharmacol. Sci. 23:206 - 209 (2002)). Transferrin fusion proteins have been studied for half-life extension, targeted delivery for cancer therapy, oral delivery, and sustained activation of proinsulin (Brandsma et al., Biotechnol. Adv., 29:230 - 238 (2011); Bai et al., Proc. Natl. Acad. Sci. USA 102:7292 - 7296 (2005); Kim et al., J.Pha rmacol. Exp. Ther., 334:682 - 692 (2010); Wang et al., J. Controlled Release 155:386 - 392 (2011)).

[0358] II.B.8.PEG In certain embodiments, the chimeric proteins used in the methods of the present disclosure include at least one binding site or fragment, variant, or derivative thereof for a non-polypeptide heterologous moiety. For example, the chimeric proteins used in the methods of the present disclosure may include one or more polyethylene glycol (PEG) moieties attached to one or more amino acid residues in a coagulation factor and / or an Fc region.

[0359] Protein PEGylation may refer to a conjugate formed between a protein and at least one polyethylene glycol (PEG) molecule. PEGs are commercially available in a variety of molecular weights and average molecular weight ranges. Exemplary 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 merely as examples and are not meant to be limiting in any way.

[0360] The chimeric proteins used in the methods of the present disclosure can be PEGylated to include mono- or poly- (e.g., 2 - 4) PEG moieties. PEGylation can be carried out by any PEGylation reaction known in the art. Methods for preparing PEGylated protein products generally include (i) reacting a polypeptide with a polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions such that the peptide of the invention becomes attached to one or more PEG groups; and (ii) obtaining the reaction product. Generally, the optimal reaction conditions for the reaction will be determined individually based on known parameters and the desired result.

[0361] There are several PEG conjugation methods available to those skilled in the art, see, for example, Malik F et al., Exp. Hematol. 20:1028-1035 (1992); Francis, Focus on Growth Factors 3(2):4-10 (1992); European Patent Publications EP0401384, EP0154316, and EP0401384; and International Patent Application Publications WO92 / 16221 and WO95 / 34326. By way of non-limiting example, the FVIII variant may contain cysteine substitutions and the cysteine can be further conjugated to a PEG polymer. See Mei et al., Blood 116:270-279 (2010) and U.S. Patent No. 7,632,921, which are hereby incorporated by reference in their entirety.

[0362] II.B.9.HES In certain embodiments, the chimeric proteins used in the methods of the disclosure include at least one hydroxyethyl starch (HES) polymer. HES is a derivative of naturally occurring amylopectin and is degraded in vivo by alpha-amylase. HES exhibits significant biological properties and is used in clinics as a blood volume expander and in hemodilution therapy. See, for example, Sommermeyer et al., Krankenhauspharmazie 8:271-278 (1987); and Weidler et al., Arzneim.-Forschung / Drug Res. 41:494-498 (1991).

[0363] HES is mainly characterized by its molecular weight distribution and degree of substitution. HES has an average molecular weight (weight average) of 1 to 300 kD, 2 to 200 kD, 3 to 100 kD, or 4 to 70 kD. Hydroxyethyl starch may further exhibit a degree of molar substitution of 0.1 to 3, 0.1 to 2, 0.1 to 0.9, or 0.1 to 0.8, and a C2:C6 substitution ratio in the range of 2 to 20 with respect to the hydroxyethyl group. HES having an average molecular weight of about 130 kD is VOLUVEN® from Fresenius. VOLUVEN® is an artificial colloid used, for example, for volume exchange in the treatment and prevention of decreased blood volume and in therapeutic indications. There are several HES conjugation methods available to those skilled in the art, for example, the same PEG conjugation method as described above.

[0364] II.B.10.PSA In certain embodiments, the chimeric proteins used in the methods of the present disclosure comprise 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 cells in mammals. See, for example, Roth J. et al. (1993) Polysialic Acid: From Microbes to Man, eds. Roth J., Rutishauser U., Troy F.A. (Birkhäuser Verlag, Basel, Switzerland), pp. 335 - 348. PSA can be produced at 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 fractionation of natural bacterial-derived polymers. In certain embodiments, activated PSA can also be conjugated to cysteine amino acid residues within a coagulation factor, for example, on FVIII, or within the Fc region. See, for example, U.S. Patent No. 5,846,951.

[0365] II.B.11.Clearance receptor In certain embodiments, the half-life of the chimeric protein used in the methods of the disclosure can be extended when the coagulation factor of the chimeric protein comprises at least one fragment of FVIII and an FVIII clearance receptor, or an FVIII-binding fragment, variant, or derivative thereof. Insertion of a soluble clearance receptor, such as 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., its in vivo half-life. 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 include, for example, LDLR (low density lipoprotein receptor), VLDLR (very low density lipoprotein receptor), and megalin (LRP-2), or fragments thereof. See, for example, Bovenschen et al., Blood 106:906-912 (2005); Bovenschen, Blood 116:5439-5440 (2010); Martinelli et al., Blood 116:5688-5697 (2010).

[0366] III. Polynucleotides, Vectors, and Host Cells In some embodiments, the disclosure provides a method of immune tolerance in a human, the method comprising administering to the human an effective amount of a polynucleotide or set of polynucleotides encoding a coagulation factor and / or an Fc region, e.g., a chimeric protein comprising a coagulation factor and an Fc region, wherein the human has not responded to one or more previous immune tolerance therapies. In some embodiments, the polynucleotide or set of polynucleotides is in an expression vector or set of expression vectors. In certain embodiments, the expression vector or set of expression vectors is in one or more host cells. There.

[0367] In the methods of the present disclosure, the polynucleotide encoding a coagulation factor and / or an Fc region, e.g., 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, the single nucleotide sequence encodes a chimeric protein comprising a coagulation factor (e.g., FVIII 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) 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.

[0368] In some embodiments, the polynucleotide is codon-optimized.

[0369] As used herein, an expression vector refers to any nucleic acid construct containing the essential elements for transcription and translation of an inserted coding sequence, or for replication and translation in the case of an RNA viral vector, when introduced into a suitable host cell. Expression vectors may include plasmids, phagemids, viruses, and derivatives thereof.

[0370] The gene expression control sequences used herein are any regulatory nucleotide sequences, such as promoter sequences or promoter-enhancer combinations, that facilitate efficient transcription and translation of the coding nucleic acid to which they are operably linked. The gene expression control sequences may be mammalian or viral promoters, such as, for example, constitutive or inducible promoters. Examples of constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeat (LTR) of Moloney leukemia virus, and promoters derived from other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters useful as the gene expression sequences of the present invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducer. 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.

[0371] For the purposes of the present invention, several expression vector systems can be used. These expression vectors are typically replicable in a host cell as episomes or as an integral part of the host chromosomal DNA. Expression vectors may contain expression control sequences including, but not limited to, a promoter (e.g., a native or heterologous promoter), an enhancer, a signal sequence, a splice signal, an enhancer element, and a transcription termination sequence. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Expression vectors may also contain DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), cytomegalovirus (CMV), or SV40 virus Other uses include the use of polycistronic systems having an internal ribosome binding site.

[0372] Generally, expression vectors contain a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to enable the detection of cells transfected with the desired DNA sequence (e.g., Itakura et al., U.S. Patent No. 4,704,362). By introducing one or more markers that enable the selection of transfected host cells, cells in which the DNA has been integrated into its chromosome can be selected. Markers can provide prototrophy for auxotrophic hosts, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can be ligated directly to the DNA sequence to be expressed or introduced into the same cell by co-transformation.

[0373] 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 a cytomegalovirus promoter / enhancer, a mouse beta-globin major promoter, an SV40 origin of replication, a bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exons 1 and 2, a dihydrofolate reductase gene, and a leader sequence. This vector has been found to result in very high levels of antibody expression upon integration of the variable and constant region genes, transfection into cells, and then selection in G418-containing medium and methotrexate amplification. The vector systems are also taught in U.S. Patent Nos. 5,736,137 and 5,658,570, which are hereby incorporated by reference in their entireties. This system provides high expression levels, for example, expression levels exceeding 30 pg / cell / day. Other exemplary vector systems are disclosed, for example, in U.S. Patent No. 6,413,777.

[0374] In other embodiments, the polypeptides of the invention are expressed using a polycistronic construct. In these expression systems, multiple gene products of interest, such as multiple polypeptides of a multimeric binding protein, can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (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 hereby incorporated by reference.

[0375] 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 accomplished by a variety of techniques well known to those of skill 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, and the like.

[0376] Although the invention has been described in detail herein, 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 limit the invention.

Example

[0377] Hemophilia A ("Factor VIII [FVIII] deficiency") is a rare bleeding disorder and the most common form of hemophilia. The most serious treatment complication in patients with hemophilia A is the development of inhibitory IgG antibodies against FVIII. Inhibitors result in rapid clearance and a marked decrease or absence of efficacy of infused FVIII. FVIII inhibitor bypass agents are used to treat acute bleeding in patients with inhibitors, but eradication of the inhibitor is the goal of long-term management.

[0378] Immune tolerance induction ("ITI") therapy using frequent administration of high-dose FVIII has been shown to be the only strategy to achieve antigen-specific tolerance. ITI is usually attempted to remove high-responsive FVIII inhibitors (≥ 5 BU titer). Several studies have examined the efficacy of various FVIII products in achieving ITI success using various doses and injection frequencies, and international consensus recommendations have been issued to support ITI approaches in clinical practice. In the international ITI study, patients in the high-dose group ("HD") (200 IU / Kg / day) achieved negative titers and normal recovery significantly more rapidly than patients in the low-dose group ("LD"). Hay and DiMichele, Blood 119(6):1335-44 (2012). HD patients also experienced significantly less bleeding than LD patients, and for this reason, the Data Safety Monitoring Board ("DSMB") recommended terminating the trial as they identified bleeding as a safety issue. The high dose of 200 IU / Kg / day is the recommended dose in "high-risk" patients, defined as those with a peak historical titer > 200 BU, pre-ITI titer > 10 BU and / or those with more than 5 years since inhibitor diagnosis. There is increasing interest in examining the use of extended half-life ("EHL") rFVIIIFc in ITI. rFVIIIFc was approved in the United States in 2014 under the name ELOCTATE®, and in Europe in 2015 under the name ELOCTA®.

[0379] rFVIIIFc is produced in a human cell line ("HE293") as a recombinant B domain deleted ("BDD") factor VIII fused to the Fc domain of human IgG. The protein produced by HEK has post-translational modifications similar to natural human proteins, in contrast to proteins produced in cell lines derived from other species such as hamster (e.g., CHO cells). In such proteins, non-human glycans resulting from post-translational modifications (such as N-glycolylneuraminic acid, NGNA and galactose-alpha-1,3-galactose, alpha-Gal, etc.) can potentially be immunogenic. Neither NGNA nor alpha-Gal is detected in rFVIIIFc. A mouse model has shown that rFVIIIFc induces a regulatory T cell response against FVIII (see Batsuli, Hemophilia (2016), 22 (Suppl. 5), pp. 31-35), which has led some investigators to suggest that rFVIIIFc could provide a more effective ITI than rFVIII, specifically, a shortening of ITI.

[0380] Objective The primary objective of this study is to describe the time to tolerance using rFVIIIFc in patients after ITI treatment. This study also aims to describe the outcome of ITI treatment; describe the recurrence rate over a defined period after successful ITI performed with rFVIIIFc; describe breakthrough bleeding during ITI and during the period after successful ITI performed with rFVIIIFc; describe the safety and tolerability of rFVIIIFc when used for ITI; describe specific quality of life (QoL) issues; and demonstrate rFVIIIFc consumption.

Example

[0381] This study aims to describe the use of rFVIIIFc for immune tolerance induction (ITI) in patients with severe hemophilia A who have failed previous ITI therapies. Specifically, the primary objective of this study is to describe the outcome of ITI procedures performed with rFVIIIFc in patients who have failed previous attempts at tolerance induction, such as the use of immunosuppressive agents after ITI treatment. The second objectives and their evaluation items are: (1) to describe the time to ITI success along with the evaluation items, the time to tolerance induction of ITI performed with rFVIIIFc in patients who have failed previous attempts at tolerance induction, such as the use of immunosuppressive agents; (2) to describe the recurrence rate after the success of ITI performed with rFVIIIFc along with the evaluation items for the occurrence of recurrence; (3) to describe the bleeding complications during ITI and during the period after the success of ITI performed with rFVIIIFc; (4) to describe the safety and tolerability of rFVIIIFc when used for ITI along with the evaluation items for adverse events and / or injection site reactions; and (5) to describe the consumption of rFVIIIFc in ITI performed with Elocta® along with the evaluation items for rFVIIIFc.

Example

[0382] In this study, male subjects of all ages with severe hemophilia A and high-titer inhibitors (historical peak ≥ 5 Bethesda units [BU] / mL) receive recombinant coagulation factor FVIII Fc fusion protein (rFVIIIFc) for the first immune tolerance induction (ITI) therapy to eradicate and neutralize anti-coagulation factor VIII (FVIII) alloantibodies.

[0383] Participants started at baseline visit and received rFVIIIFc at a dose of 200 international units (IU) / kilogram (kg) as a once-daily injection or divided into several injections per day at the discretion of the investigator for up to 48 weeks during the ITI period. Participants who met the criteria for successful immune tolerance induction (ITI) entered the taper period and received rFVIIIFc (as an intravenous injection powder) at a dose adjusted according to the investigator's judgment (50 or 100 IU / kg), once daily from week 1 to week 6 and then once every other day until week 16.

[0384] The primary outcome measure of this study is to describe the time to tolerance induction with rFVIIIFc within a 12-month time frame. Tolerance induction is defined as an inhibitor titer <0.6 VU / ml, an FVIII recovery rate >66%, and a t 1 / 2 half-life of more than 7 hours.

[0385] The secondary outcome measure is the number of participants in whom immune tolerance induction (ITI) was successful. Success of ITI is defined as a negative titer for inhibitors <0.6 BU / mL by the Nijmegen modified Bethesda assay; an FVIII incremental recovery (IR) of more than 1.3 international units / deciliter (IU / dL) per kg in two consecutive determinations, representing 66% of the expected IR of 2 IU / dL per kg; and a half-life (t 1 / 2 ) of more than 7 hours. Success of ITI is monitored over a 48-week time frame.

[0386] Another secondary outcome measure is the number of participants who experience recurrence. The percentage of participants in whom ITI was successful and who meet the recurrence criteria (defined as an inhibitor titer exceeding 0.6 BU / mL or an abnormal recovery after tolerance) is evaluated. Recurrence is monitored over a 48-week time frame.

[0387] Another secondary outcome measure is the number of bleeding episodes. A bleeding episode was considered the same if any signs of bleeding at the same site or injection occurred within 72 hours or less and started from the first sign of bleeding and ended within 72 hours after the last treatment for bleeding. Bleeding episodes were monitored over a time frame of up to 104 weeks.

[0388] Another secondary outcome measure is the number of participants who showed adverse events (AEs) that occurred during the treatment and serious adverse events (SAEs) that occurred during the treatment. An AE is any undesirable medical occurrence that does not necessarily have a causal relationship with this treatment. An SAE is, at any dose: resulting in death; in the investigator's opinion, placing the participant at immediate risk of death (life-threatening event); requiring hospitalization of an inpatient or prolonging an existing hospitalization; resulting in persistent or significant physical impairment / disability; resulting in congenital anomalies / birth defects, any undesirable medical occurrence; in the investigator's opinion, placing the participant at risk, or an intervention may be required to prevent one of the other outcomes listed in the definition, any other medically important event. AEs and SAEs were measured over a time frame of approximately 2 years.

[0389] Another secondary outcome measure is the number of days away from work or school. The number of days absent from school or work was descriptively summarized over a time frame of up to 104 weeks.

[0390] Another secondary outcome measure is the number of hospital days. The number of hospital days was descriptively summarized and monitored over a time frame of up to 104 weeks.

[0391] Another secondary outcome measure is adherence to the treatment regimen, defined as the percentage of the administered dose relative to the planned dose, and was monitored over a time frame of up to 104 weeks.

[0392] Another secondary outcome measure is the consumption of rFVIIIFc. The consumption is evaluated based on the amount of the test treatment administered and is monitored over a time frame up to 104 weeks.

[0393] This study is directed to male participants of any age diagnosed with severe hemophilia A (confirmed from medical records). Subjects were diagnosed with high-titer inhibitors (historical peak of greater than or equal to 5 biological units per milliliter [BU / mL] by medical records) and subjects have been previously treated with any plasma-derived or recombinant conventional or extended half-life FVIII. Exclusion criteria include, in addition to hemophilia A, any other coagulation disorder; any previous ITI therapy; any history of hypersensitivity or anaphylaxis associated with administration of recombinant coagulation factor VIII Fc (rFVIIIFc); any renal dysfunction (serum creatinine greater than 2.0 milligrams per deciliter [mg / dL]) as evaluated by the local lab; and / or subjects having serum alanine aminotransferase or aspartate aminotransferase greater than 5 times the upper limit of normal (ULN) as evaluated by the local lab.

Example

[0394] A non-interventional retrospective chart review of ITI with rFVIIIFc in patients with severe hemophilia A and high-titer inhibitors (HTI; ≥5 BU) was conducted at 10 sites in the United States and Canada between July 1, 2014 and June 1, 2017. All-age male patients with severe hemophilia A with HTI who initiated treatment with rFVIIIFc for ITI as primary or rescue therapy were included regardless of response.

[0395] After regulatory approval, unspecified clinical information was collected via an electronic survey. Patients treated for the first time with ITI were considered to be at high risk of ITI failure according to the previously listed criteria. A negative Bethesda titer was defined as ≤ 0.6 BU / mL. Tolerance was defined as a negative Bethesda titer and normal FVIII recovery (≥ 66%) and half-life (≥ 6 hours). The primary objective of this study was to report the clinical characteristics and outcomes of ITI using rFVIIIFc. Results were summarized using descriptive statistics; inferential statistical analysis was not performed.

[0396] Results Study population Nineteen patients were identified. Of these, 7 had received ITI for the first time and 12 had received rescue ITI (Tables 1 and 2). The median age at the start of rFVIIIFc ITI was 1.3 years (range: 0.8 - 4.3 years) for first ITI and 6.4 years (range: 1.6 - 12.6 years) for rescue ITI patients.

[0397] First ITI patients had a median peak historical inhibitor (pre-ITI) titer of 151 BU (range: 11 - 1126 BU); the median inhibitor titer at the start of rFVIIIFc ITI was 52 BU (range: 3 - 1126 BU). At the start of ITI, 6 of the 7 first ITI patients had titers > 10 BU; 4 of these 6 had titers > 50 BU. The median time from inhibitor diagnosis to the start of rFVIIIFc ITI was 4.4 weeks (range: 0 - 41 weeks).

[0398] For rescue ITI patients, the mean number of previous ITI courses with other FVIII products was 2.6 (range: 1 - 5), and the median time from inhibitor diagnosis to the start of rFVIIIFc ITI was 5.5 years (range: 0.8 - 12 years). FVIII genotypes for 18 of the 19 patients are shown in Tables 1 and 2.

[0399] Outcomes of first ITI patients At the time of data collection, 4 out of 7 patients who were receiving the initial ITI were tolerized and transitioned to prophylaxis with rFVIIIFc. Three of these 4 patients achieved negative Bethesda titers and normal FVIII recovery and half-life; as such, they met the standard definition of tolerization at 5, 7, and 9 months. The fourth patient was at 13 months after completion of rFVIIIFc ITI and was considered tolerized by the treating physician at 14.8 months based on negative inhibitor titer and transition to prophylaxis at the time of data collection when he continued to have a negative inhibitor on rFVIIIFc prophylaxis. Normal half-life was also reported at that time.

[0400]

Table 1

[0401] Among the 4 patients, the median time to achieve a negative Bethesda titer was 27.7 weeks (range: 4.1 - 64 weeks). The ITI regimen for 3 of the 4 tolerized patients consisted of daily rFVIIIFc (85 - 200 IU / kg) compared to thrice-weekly dosing (50 IU / kg) for the fourth patient (Table 1). The median time to report of tolerization was 33.9 weeks (7.8 months; range: 21 - 64 weeks) for all 4 patients. For the 3 patients treated with daily rFVIIIFc (85 - 200 IU / kg), tolerization took 29 weeks (6.7 months; range: 20.6 - 38 weeks), while the fourth patient treated thrice-weekly at 50 IU / kg tolerized at 64 weeks (14.8 months).

[0402] Of the remaining patients (n = 3), 2 had a decrease in Bethesda titer (a decrease from 32 BU to 18 BU and from 378 BU to 23 BU, respectively, 18 and 58 weeks after ITI). At the time of this evaluation, 1 patient had an increase in Bethesda titer (an increase from 3 BU to 16 BU 15 weeks after ITI); this patient had a low compliance with ITI, either performed or discontinued ITI and interrupted rFVIIIFc according to the report of the treating physician (Table 1). All 7 first-time ITI patients continued rFVIIIFc ITI or prophylaxis.

[0403] Outcomes of Rescue ITI Patients Seven of the 12 patients (Table 2) receiving rescue ITI achieved a negative Bethesda for rFVIIIFc ITI first. The median time to achieve a negative titer was 14.1 weeks (range: 3 - 67.6 weeks). Three of these 7 patients remained Bethesda negative and continued rFVIIIFc ITI or switched to prophylaxis without rFVIIIFc. The other 4 patients who achieved a negative titer first later developed a titer exceeding 0.6 BU Two of these continued rFVIIIFc ITI and 2 switched to ITI with other factor (Table 2).

[0404]

Table 2

[0405] Of the 7 patients who achieved a negative Bethesda, 3 also achieved recovery of normal FVIII at 3, 14, and 65 weeks, and the 4th patient reached a normal FVIII half-life at 27 weeks. Recovery and half-life were not available for others (Table 2). Of the remaining 5 patients, 1 had a decrease in Bethesda titer (a decrease from 36 BU to 22 BU 10 weeks later), and the Bethesda titers of 4 patients remained unchanged or increased during ITI (Table 2). Of these 5 patients, 4 continued rFVIIIFc ITI and 1 was excluded from ITI and placed on bypass therapy only.

[0406] Outcome of dosing, use of bypass agents, and current treatment status The patient population evaluated in this trial received a wide range / wide timing of doses (Tables 1 and 2). For the high doses administered daily, there was a tendency towards a rapid negative inhibitor titer. Five out of five patients (one primary ITI and four rescue ITIs) who received a daily rFVIIIFc dose of 130 IU / kg or more achieved a negative baseline titer at a median of 28 weeks. Eighteen out of 19 patients used bypass agents concomitantly with rFVIIIFc ITI; 14 were mainly for prophylaxis (9 used aPCC and 5 used rFVIIa), and 4 were treated with rFVIIa on demand. Overall, 16 out of 19 patients were still on rFVIIIFc (prophylaxis or ITI) at the time of data collection (Tables 1 and 2).

[0407] Overall, 16 out of 19 patients were still on rFVIIIFc (prophylaxis or ITI) at the time of data collection (Tables 1 and 2).

[0408] Safety No adverse events such as thromboembolism were reported. Six surgeries were performed, and in all of them, there was no interruption of rFVIIIFc ITI (knee synovectomy, intracranial neurosurgical removal, and 4 port catheter exchanges). Bypass therapy was used in all. Inhibitor titers during surgery were not collected for this trial.

[0409] Conclusion In summary, these results indicate that ITI with rFVIIIFc is possible and can lead to inhibitor eradication and ITI success in many patients receiving primary ITI (who are at high risk of ITI failure) and in some patients receiving rescue ITI. Furthermore, rFVIIIFc ITI demonstrated a rapid decline in Bethesda titer and a rapid time to tolerance in the majority of patients receiving primary ITI, despite its risk profile. For rescue ITI, it was more difficult to draw conclusions as many of these patients were still receiving ITI with rFVIIIFc at the time of data collection. However, some patients receiving rescue treatment were thought to have induced a therapeutic benefit in that they achieved Bethesda negativity or demonstrated a significant decrease in inhibitor titer. This was particularly true when higher rFVIIIFc doses (≥130 IU / kg) were administered daily.

Example

[0410] The main complication of replacement therapy with factor 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 the affected individual. 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 rFVIII Fc fusion protein with an extended half-life (rFVIIIFc) is an effective and well-tolerated therapy for preventing and controlling bleeding episodes. The Fc region of this molecule has been shown 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 As suggested by 22(5): pp. e462 - e464 (2016), it can not only cause an increase in the rFVIII half - life, but also promote antigen - specific tolerance.

[0411] Method Using peripheral blood - derived human APCs or THP - 1 monocytes, the effects of rFVIIIFc on FcγR binding, internalization, signal transduction and cytokine production, as well as gene expression changes, and subsequent interactions in vitro and effects on T cells were examined (Figure 1).

[0412] Results A decrease in the cell - surface expression of FcγR indicates internalization during rFVIIIFc treatment (Figure 2A ~2C). Monocyte - derived macrophages and dendritic cells were treated with horseradish peroxidase immune complex (HRP - IC) as a positive control, human immunoglobulin G1 (IgG1) as a negative control, and equimolar concentrations (200 nM) of recombinant factor VIII (rFVIII) or rFVIII Fc fusion protein (rFVIIIFc) for 24 hours. The cell - surface expression of Fcγ receptors (FcγR) CD16 (Figure 2A), CD32 (Figure 2B), and CD64 (Figure 2C) was measured by flow cytometry (n = 3; ** P≤0.01, *** P≤0.005, the significance of HRP - IC compared to other treatments is not shown). Treatment with rFVIIIFc was correlated with a decrease in the cell - surface expression of CD16 (Figure 2A), CD32 (Figure 2B), and CD64 (Figure 2C) compared to the cell - surface after treatment with rFVIII.

[0413] rFVIIIFc engages with FcγR and induces signal transduction in monocytes and macrophages without subsequent inflammatory cytokine production (Figure 3A - 3C). The THP - 1 monocyte 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 3A). Syk phosphorylation was measured in cell lysates using the MSD platform (n = 3 - 7,* P ≤ 0.05). Syk phosphorylation was measured after treating macrophages with rFVIIIFc(WT), an rFVIIIFc mutant that cannot bind to the neonatal Fc receptor (FcRn mutant), or an rFVIIIFc mutant that cannot bind to FcγR (FcγR mutant) (n = 4, * P ≤ 0.05) (Figure 3B). Inflammatory cytokine production in macrophages treated for 24 hours was measured by MSD ELISA (n = 4, not significant) (Figure 3C).

[0414] rFVIIIFc phosphorylates molecules involved in immune regulation rather than molecules that play a role in activation and inflammatory cytokine production (Table 3 and Figure 4). Phosphorylated proteins in lysates derived from monocyte-derived macrophages treated with rFVIIIFc for 15 minutes were interrogated using a proteome profiler phosphokinase and phosphoimmunoreceptor array. A list of phosphorylated molecules in macrophages treated with rFVIIIFc identified by the proteome profiler array is shown in Table 3. Phosphorylation of phosphatases responsible for inhibitory signaling was measured using the MSD platform (n = 3; ** P ≤ 0.01, *** P ≤ 0.005) (Figure 4).

[0415]

Table 3

[0416] rFVIIIFc induces a gene expression pattern characteristic of tolerogenic macrophages (Figure 5A - 5G). Exploratory RNA sequencing was performed on genes significantly downregulated (Figure 5A) For genes that were upregulated significantly (Figure 5B), pathway analysis was performed on genes upregulated by rFVIIIFc (Table 4) to examine molecular pathways showing selectivity among these cells, which were performed on monocytes-derived macrophages treated with IgG1, rFVIII, or rFVIIIFc for 6 hours (n = 3) and compared with cells treated with rFVIII. Various genes in the NRF2 and PPAR-gamma pathways, as well as various other immunomodulatory factors, were found to be upregulated (Figure 5H). Selected genes in the NRF2 and lipid metabolism pathways were verified by Q-PCR (n = 8; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005) (Figures 5C - 5G). Furthermore, macrophages educated by rFVIIIFc were found to exhibit a characteristic M2-like phenotype (Figures 5I - 5M). In particular, macrophages treated with rFVIIIFc had higher relative CD206 expression than cells treated with rFVIII at 6 hours (Figure 5I) and 24 hours (Figure 5J), and macrophages treated with rFVIIIFc had higher relative ARG1 expression than cells treated with rFVIII at 24 hours (Figure 5M).

[0417]

Table 4

[0418] Antigen-presenting cells treated with rFVIIIFc affect regulatory T cell differentiation that requires APC-T cell cell contact (Figs. 6A-6C). Peripheral blood monocyte-derived macrophages were treated with IgG1, rFVIII, or rFVIIIFc and then placed in co-cultures with naive CD4-positive T cells isolated from peripheral blood from the same donor. After 6 days in co-culture (Fig. 6A), the percentage of regulatory T cells (CD4+CD25+FoxP3+) was quantified using flow cytometry (n = 4) (Fig. 6B). The percentage of regulatory T cells was also quantified when naive T cells were cultured in conditioned media from APCs pretreated with IgG1, rFVIII, or rFVIIIFc (n = 4) (Fig. 6C).

[0419] Conclusion rFVIIIFc is thought to bind via Fcγ receptors on APCs, inducing internalization and signaling. This signaling is not converted to inflammatory cytokine production and does not activate APCs (data not shown). Immunoregulatory signaling events are initiated upon rFVIIIFc treatment. These events are thought to drive macrophage differentiation towards an M2-like phenotype characterized by upregulation of the NRF2 and PPARγ pathways (Fig. 5H) as well as upregulation of CD206 and arginase 1 molecules. Various other immunoregulatory factors also showed increased expression, but at least soluble guanylate cyclase 1 beta subunit (2GUCY1B2), protoporphyrinogen oxidase (PPOX), and suppressor of cytokine signaling 3 (SOCS3) showed decreased expression in rFVIIIFc-treated cells (Fig. 5H). These macrophages can perform previously reported beneficial immunological effects such as regulatory T cell differentiation, FVIII tolerance, and anti-FVIII inhibitor reduction (Fig. 7).

Example

[0420] Initial preclinical and clinical data have shown that rFVIIIFc enables a relatively short time to negative inhibitor titers when used for ITI treatment, presumably due to immunomodulatory effects attributable to the Fc domain of the molecule. To obtain more robust clinical data, a standardized protocol was developed. Here, the study design is presented.

[0421] The ReITIrate (NCT03103542), a predictive, interventional, multi-site, open-label trial, aims to enroll 20 severe HA inhibitor patients of all ages who have previously failed ITI attempts. The primary objective of this study is to characterize the outcome of ITI performed with rFVIIIFc within a 60-week time frame. The primary endpoint is ITI success; secondary endpoints evaluated during ITI treatment include time to ITI success, occurrence of relapse, number of bleeding episodes, rFVIIIFc consumption, school or work absenteeism days, hospitalizations, and compliance. The ITI treatment includes rFVIIIFc 200 IU / kg / day (divided into once or twice daily doses) for up to 60 weeks. After tolerance is achieved, a 16-week taper period and a 32-week follow-up with prophylactic rFVIIIFc are conducted. Success criteria include a negative inhibitor titer (<0.6 Bethesda units), incremental recovery exceeding 66% of prediction, and a terminal half-life of more than 7 hours.

[0422] Figure 8 shows the proposed effect of rFIXFc on macrophages. rFVIIIFc is thought to bind via Fcγ receptors on APCs, inducing internalization and signaling. This signaling is not converted to inflammatory cytokine production and does not activate APCs. Rather, immunomodulatory signaling events are initiated upon rFVIIIFc treatment. These events are thought to drive macrophage differentiation towards a "Mox / M2-like" phenotype characterized by upregulation of the NRF2 and PPARγ pathways.

[0423] The foregoing description of specific embodiments fully discloses the general nature of the present invention such that others can, by applying knowledge within the skill of the art, readily modify and / or adapt such specific embodiments without undue experimentation and without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based upon the teachings and guidance presented herein. It is to be understood that the terminology or phraseology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by those of ordinary skill in the art considering the teachings and guidance.

[0424] Other embodiments of the invention will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the invention disclosed herein. The specification and examples are considered exemplary only, and the true scope and spirit of the invention is intended to be indicated by the following claims.

[0425] All publications, patents, and patent applications disclosed herein are hereby 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. A composition for inducing immune tolerance in a human under the age of 12 with hemophilia who has developed an inhibitor against a coagulation factor, comprising a chimeric protein comprising a coagulation factor and an Fc region, the chimeric protein comprising any one of the following (1) to (3): (1) administering to the human 85 IU / kg to 300 IU / kg of a chimeric protein daily for a period of time sufficient to induce immune tolerance, wherein said amount of chimeric protein induces immune tolerance in the human; (2) after induction of immune tolerance, administering to the human a tapering regimen of the chimeric protein; (3) following the tapering regimen, administering to the human a prophylactic dose of the chimeric protein; and administering the same according to the dosing regimen of wherein the human having hemophilia has not responded to one or more prior immune tolerance therapies.

2. A composition for inducing immune tolerance in a human with hemophilia who has developed an inhibitor against a coagulation factor, comprising a chimeric protein comprising a coagulation factor and an Fc region, the chimeric protein comprising any one of the following (1) to (3): (1) administering to the human 85 IU / kg to 300 IU / kg of a chimeric protein daily for a period of time sufficient to induce immune tolerance, wherein said amount of chimeric protein induces immune tolerance in the human; (2) after induction of immune tolerance, administering to the human a tapering regimen of the chimeric protein; (3) following the tapering regimen, administering to the human a prophylactic dose of the chimeric protein; and administering the same according to the dosing regimen of wherein the human having hemophilia has not responded to one or more prior immune tolerance therapies and has an inhibitory antibody titer of at least about 10 Bethesda Units (BU) prior to administration.

3. A composition for inducing immune tolerance in a human aged 12 years or older with hemophilia who has developed an inhibitor against a coagulation factor, comprising a chimeric protein comprising a coagulation factor and an Fc region, the chimeric protein comprising any one of the following (1) to (3): (1) administering to the human 85 IU / kg to 300 IU / kg of a chimeric protein daily for a period of time sufficient to induce immune tolerance, wherein said amount of chimeric protein induces immune tolerance in the human; (2) after induction of immune tolerance, administering to the human a tapering regimen of the chimeric protein; (3) following the tapering regimen, administering to the human a prophylactic dose of the chimeric protein; and administering the same according to the dosing regimen of wherein said human having hemophilia has not responded to one or more prior immune tolerance therapies.

4. A composition for inducing immune tolerance in a human with hemophilia who has developed an inhibitor against a coagulation factor, comprising a chimeric protein comprising a coagulation factor and an Fc region, the chimeric protein comprising any one of the following (1) to (3): (1) administering to the human 85 IU / kg to 300 IU / kg of a chimeric protein daily for a period of time sufficient to induce immune tolerance, wherein said amount of chimeric protein induces immune tolerance in the human; (2) after induction of immune tolerance, administering to the human a tapering regimen of the chimeric protein; (3) following the tapering regimen, administering to the human a prophylactic dose of the chimeric protein; and administering the same according to the dosing regimen of wherein the human having hemophilia has not responded to one or more previous immune tolerance therapies, immune tolerance is observed when the titer of inhibitory antibodies in the human is less than about 0.6 BU, and the time period until tolerance occurs is less than about 24 weeks.

5. The composition according to any one of claims 1 to 4, wherein the chimeric protein comprising a coagulation factor and an Fc region comprises factor VIII-Fc or factor IX-Fc.

6. The composition of any one of claims 1 to 5, wherein the tapering regimen comprises administering tapering doses of about 50 IU / kg to about 100 IU / kg of a chimeric protein comprising a coagulation factor and an Fc region.

7. The composition of any one of claims 1 to 6, wherein the tapering dose is administered once a day, once every two days, or three times a week.

8. 8. The composition of any one of claims 1-7, wherein the tapering dose is administered for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 11 weeks, at least about 12 weeks, at least about 13 weeks, at least about 14 weeks, at least about 15 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 21 weeks, at least about 22 weeks, at least about 23 weeks, at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, or at least about 32 weeks.

9. The tapering regimen comprises administering tapering doses of about 50 IU / kg of the chimeric protein once daily from week 1 to week 6 after immune tolerance, or administering tapering doses of about 100 IU / kg of the chimeric protein once daily from week 1 to week 6 after immune tolerance.

9. The composition according to any one of claims 1 to 8.

10. The composition of claim 9, wherein the tapering regimen further comprises administering tapering doses of about 50 IU / kg or about 100 IU / kg of the chimeric protein once every two days from week 6 to week 12 after immune tolerance, and / or administering tapering doses of about 50 IU / kg or about 100 IU / kg of the chimeric protein once every two days from week 12 to week 16.

11. The composition of any one of claims 1 to 10, wherein the previous immune tolerance therapy comprises administration of one or more immunosuppressants.

12. The composition of any one of claims 1 to 11, wherein the previous immune tolerance therapy comprises the Malmo regimen or the Bonn protocol.

13. The composition of any one of claims 1 to 12, wherein the human has been diagnosed with developing an inhibitor to FVIII at least about 24 months prior to administration of step (1).

14. The composition of any one of claims 1 to 13, wherein the human has been diagnosed with developing an inhibitor to FVIII at least about 5 years prior to administration of step (1).

15. 15. The composition of any one of claims 1 to 14, wherein the inhibitors to the coagulation factors were developed in response to a recombinant or plasma-derived FVIII product selected from the group consisting of ADVATE®, RECOMBINATE®, KOGENATEFS®, HELIXATEFS®, AFSTYLA®, XYNTHA / REFACTOAB®, HEMOFIL-M®, MONARC-M®, MONOCLATE-P®, HUMATE-P®, ALPHANATE®, KOATE-DVI®, and HYATE:C®.

16. The composition of any one of claims 1 to 15, wherein the Fc region is fused to the C-terminus of the coagulation factor.

17. The method of any one of claims 1 to 16, further comprising administering a prophylactic dose of the chimeric protein after the tapering regimen.

18. The composition of any one of claims 5 to 17, wherein the FVIII comprises a complete or partial B domain deleted FVIII.

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