FACTOR VIII-Fc CHIMERIC AND HYBRID POLYPEPTIDES, AND METHODS OF USE THEREOF

Abstract

To provide factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof.SOLUTION: The present invention provides methods of administering Factor VIII; methods of administering chimeric and hybrid polypeptides comprising Factor VIII; chimeric and hybrid polypeptides comprising Factor VIII; polynucleotides encoding such chimeric and hybrid polypeptides; cells comprising such polynucleotides; and methods of producing such chimeric and hybrid polypeptides using such cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention generally relates to the field of methods for treating hemostatic disorders.

Background Art

[0002] Hemophilia A is an X-linked bleeding disorder caused by mutations and / or deletions in the factor VIII (FVIII) gene resulting in a deficiency of FVIII activity (Peyvandi et al. 2006). This disease is characterized by spontaneous bleeding and excessive bleeding after trauma. Over a long period, bleeding occurs repeatedly in muscles and joints (often starting in childhood), resulting in hemophilic arthropathy and irreversible joint damage. This damage is progressive and can lead to severely limited joint mobility, muscle atrophy, and chronic pain (Non-Patent Document 1, which is incorporated herein by reference in its entirety).

[0003] The A2 domain is required for the coagulation-promoting activity of the factor VIII molecule. Studies have shown that porcine factor VIII has a coagulation-promoting activity six times higher than that of human factor VIII (Non-Patent Document 2), and the difference in coagulation activity between human factor VIII and porcine factor VIII appears to be based on differences in the amino acid sequences between one or more residues in the human A2 domain and the porcine A2 domain (Non-Patent Document 3) (which is incorporated herein by reference in its entirety).

[0004] The treatment of hemophilia A aims to prevent spontaneous bleeding by restoring FVIII activity to 1-5% of normal levels through replacement therapy (Non-Patent Document 4, which is incorporated herein by reference in its entirety). There are plasma-derived and recombinant FVIII products available for treating bleeding symptoms on demand or for preventing bleeding symptoms that occur prophylactically. Based on the half-lives of these products, frequent intravenous administration is required in treatment regimens. Such frequent administration is painful and inconvenient.

[0005] Reduction of mortality, prevention of joint damage, and improvement of quality of life are important achievements resulting from the development of plasma-derived recombinant FVIII. Prolonged prevention of bleeding will be another important advance in the treatment of patients with hemophilia A. However, to date, no product enabling long-term protection has been developed. Accordingly, there remains a need for an improved treatment for hemophilia resulting from factor VIII deficiency that is more tolerable and effective than current therapies.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0007] The present invention relates to a method of administering factor VIII; a method of administering a chimeric polypeptide comprising factor VIII and a hybrid of such a chimeric polypeptide; factor VIII Chimeric polypeptides comprising the same and hybrids of such chimeric polypeptides; polynucleotides encoding such chimeric and hybrid polypeptides; cells comprising such polynucleotides; and methods of producing such chimeric and hybrid polypeptides using such cells are provided.

[0008] The present invention provides a method of administering factor VIII to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a chimeric factor VIII polypeptide, such as a chimeric factor VIII-Fc polypeptide, at an administration interval that is at least about 1.5-fold longer than the administration interval required for an equivalent amount of the factor VIII that does not contain a non-factor VIII moiety (a polypeptide consisting of the factor VIII moiety), for example, the factor VIII that does not contain an Fc moiety.

[0009] The administration interval may be at least about 1.5 to 6-fold longer, 1.5 to 5-fold longer, 1.5 to 4-fold longer, 1.5 to 3-fold longer, or 1.5 to 2-fold longer than the administration interval required for an equivalent amount of the factor VIII that does not contain a non-factor VIII moiety (a polypeptide consisting of the factor VIII moiety), such as an Fc moiety. The administration interval may be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6-fold longer than the administration interval required for an equivalent amount of the factor VIII that does not contain a non-factor VIII moiety (a polypeptide consisting of the factor VIII moiety). The administration interval may be every 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more.

[0010] The administration interval may be at least about 1.5 to 5, 1.5, 2, 3, 4, or 5 days or more.

[0011] The present invention further provides a method of administering factor VIII to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a chimeric factor VIII polypeptide, such as a chimeric factor VIII-Fc polypeptide, to obtain an area under the plasma concentration-time curve (AUC) that is at least about 1.25-fold greater than the AUC obtained with an equivalent amount of said factor VIII (a polypeptide consisting of said factor VIII moiety) that does not contain a non-factor VIII moiety such as an Fc moiety.

[0012] The present invention further provides a method of administering factor VIII to a subject in need thereof, the method comprising administering a therapeutically effective amount of a polypeptide comprising factor VIII and Fc at an administration interval of about every 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or more.

[0013] The methods of the present invention can be practiced on subjects in need of prophylactic treatment or on-demand treatment.

[0014] On-demand treatment includes treatment of bleeding symptoms, hemarthrosis, muscle bleeding, oral bleeding, major bleeding, major bleeding into muscle, major oral bleeding, trauma, head trauma (trauma capitis), gastrointestinal bleeding, intracranial major bleeding, intra-abdominal major bleeding, intra-thoracic major bleeding, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the iliopsoas sheath. The subject may be in need of treatment for surgical prophylaxis, intraoperative management, or surgery. Such surgeries include, for example, minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hernia incision, synovectomy, total knee arthroplasty, craniotomy, bone fixation, trauma surgery, intracranial surgery, intra-abdominal surgery, intra-thoracic surgery, or joint replacement surgery.

[0015] For on-demand treatment, the administration interval of said chimeric polypeptide is approximately 24 to 36, 24 to 48, 24 to 72, 24, 25, 26, 27, 28, 29, 30, 31, 32, Once every 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or more.

[0016] The therapeutic amount that can be used in the method of the present invention is about 10 to about 100 IU / kg, and more specifically, about 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 IU / kg, and more specifically, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / kg.

[0017] The therapeutic amount that can be used in the method of the present invention is about 10 to about 150 IU / kg, and more specifically, about 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150 IU / kg, and more specifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150 IU / kg.

[0018] The subject in the method of the present invention may be a human subject or a non-human mammal. Examples of non-human mammals include mice, dogs, primates, monkeys, cats, horses, cows, pigs, and other domestic animals and small animals. The determination of the dosing interval and AUC can be carried out in a single subject or in a population of subjects.

[0019] Factor VIII (or the Factor VIII portion of the chimeric polypeptide) can be human Factor VIII or non-human Factor VIII, such as porcine, murine or canine Factor VIII. Factor VIII (or the Factor VIII portion of the chimeric polypeptide) can have a complete or partial deletion of the B domain.

[0020] Factor VIII (or the Factor VIII portion of a chimeric polypeptide) can be at least 90% or 95% identical to the Factor VIII amino acid sequence shown in Table 2 that does not include the signal sequence (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). Factor VIII (or the Factor VIII portion of a chimeric polypeptide) can be identical to the Factor VIII amino acid sequence shown in Table 2 that does not include the signal sequence (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12).

[0021] Factor VIII (or the Factor VIII portion of a chimeric polypeptide) can be at least 90% or 95% identical to the Factor VIII amino acid sequence shown in Table 2 that includes the signal sequence (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). Factor VIII (or the Factor VIII portion of a chimeric polypeptide) can be identical to the Factor VIII amino acid sequence shown in Table 2 that includes the signal sequence (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12).

[0022] The Fc portion (or the Fc portion of a chimeric polypeptide) can be at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12). F The c portion (or the Fc portion of the chimeric polypeptide) may be identical to the Fc amino acid sequences shown in Table 2 (amino acids 1439 - 1665 of SEQ ID NO: 2; amino acids 2333 - 2559 of SEQ ID NO: 6; amino acids 741 - 967 of SEQ ID NO: 8; amino acids 746 - 972 of SEQ ID NO: 10; amino acids 685 - 924 of SEQ ID NO: 12).

[0023] The chimeric polypeptide may comprise a sequence that is at least 90% or 95% identical to Factor VIII and the Fc amino acid sequence (amino acids 1 - 1665 of SEQ ID NO: 2) shown in Table 2A(i) without a signal sequence, or Factor VIII and the Fc amino acid sequence (amino acids - 19 - 1665 of SEQ ID NO: 2) shown in Table 2A(i) with a signal sequence. The chimeric polypeptide may comprise a sequence that is identical to Factor VIII and the Fc amino acid sequence (amino acids 1 - 1665 of SEQ ID NO: 2) shown in Table 2A(i) without a signal sequence, or Factor VIII and the Fc amino acid sequence (amino acids - 19 - 1665 of SEQ ID NO: 2) shown in Table 2A(i) with a signal sequence.

[0024] The chimeric polypeptide may be in the form of a hybrid comprising a second polypeptide bound to the chimeric polypeptide, where the second polypeptide may contain Fc or may consist essentially of Fc.

[0025] The second polypeptide may comprise a sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 1 to 227 of SEQ ID NO: 4), or may comprise a sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with the signal sequence (amino acids -20 to 227 of SEQ ID NO: 4), or may consist essentially of this sequence. The second polypeptide may be identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 1 to 227 of SEQ ID NO: 4), or may comprise a sequence that is identical to the amino acid sequence shown in Table 2A(ii) with the signal sequence (amino acids -20 to 227 of SEQ ID NO: 4), or may consist essentially of this sequence.

[0026] The chimeric polypeptide or hybrid can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0027] The present invention further provides the chimeric and hybrid polypeptides themselves, polynucleotides encoding them, cultured human embryonic cells containing the polynucleotides, and methods for producing such chimeric and hybrid polypeptides, and polypeptides produced by such methods. In this embodiment, for example, the following items are provided. (Item 1) A method for administering factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric polypeptide comprising a factor VIII portion and a second portion at a dosing interval that is at least about 1.5 times longer than the dosing interval required for an equivalent amount of polypeptide consisting of the factor VIII portion. (Item 2) The method according to item 1, wherein the chimeric polypeptide comprises an Fc portion. (Item 3) The method according to item 1 or 2, wherein the dosing interval is at least about 1.5 to 6 times, 1.5 to 5 times, 1.5 to 4 times, 1.5 to 3 times, or 1.5 to 2 times longer than the dosing interval required for an equivalent amount of polypeptide consisting of the Factor VIII moiety. (Item 4) The dosing interval is required for an equivalent amount of polypeptide consisting of the Factor VIII moiety The method according to item 3, wherein the dosing interval is at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times longer than the dosing interval required for an equivalent amount of polypeptide consisting of the Factor VIII moiety. (Item 5) The method according to any one of items 1 to 4, wherein the dosing interval of the chimeric polypeptide is at intervals of about 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. (Item 6) The method according to any one of items 1 to 5, wherein the subject requires prophylactic treatment. (Item 7) The method according to any one of items 1 to 4, wherein the subject requires on-demand treatment. (Item 8) The method according to item 7, wherein the subject requires treatment for the appearance of bleeding symptoms. (Item 9) The method according to item 8, wherein the subject requires treatment related to joint bleeding, muscle bleeding, oral bleed, bleeding, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the iliopsoas sheath. (Item 10) The method according to item 7, wherein the subject requires treatment related to surgical prophylaxis, intraoperative management, or surgery. (Item 11) The method according to item 10, wherein the surgery is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hernia repair, synovectomy, total knee arthroplasty, craniotomy, bone fixation, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement. (Item 12) The method according to any one of items 7 to 11, wherein the dosing interval of the chimeric polypeptide is about once every 24 to 36, 24 to 48, 24 to 72, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours or longer. (Item 13) The method according to any one of items 1 to 12, wherein the therapeutic dose is 10 to 100 IU / kg. (Item 14) The method according to item 12, wherein the therapeutic dose is 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100 IU / kg. (Item 15) The method according to item 13, wherein the therapeutic dose is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 IU / kg. (Item 16) The method according to any one of items 1 to 15, wherein the subject is human. (Item 17) The method according to any one of items 1 to 16, wherein the Factor VIII is human Factor VIII. (Item 18) The Factor VIII according to any one of items 1 to 17, which has a complete or partial deletion of the B domain in any of the methods described above. (Item 19) The method according to item 17, wherein the Factor VIII portion of the chimeric polypeptide has no signal sequence and is at least 90% or 95% identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). (Item 20) The method according to item 19, wherein the Factor VIII portion of the chimeric polypeptide has no signal sequence and is identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). (Item 21) The method according to item 17, wherein the Factor VIII portion of the chimeric polypeptide has a signal sequence and is at least 90% or 95% identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 22) The method according to item 21, wherein the Factor VIII portion of the chimeric polypeptide has a signal sequence and is identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 23) The method according to item 17 or 18, wherein the second portion of the chimeric polypeptide is at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12). (Item 24) The method according to item 23, wherein the second portion of the chimeric polypeptide is identical to the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12). (Item 25) The method according to any one of items 1 to 24, wherein the chimeric polypeptide is in the form of a hybrid comprising a second polypeptide bound to the chimeric polypeptide, and the second polypeptide consists essentially of Fc. (Item 26) The method according to item 25, wherein the chimeric polypeptide has no signal sequence and is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids 1 to 1665 of SEQ ID NO: 2), or has a signal sequence and is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids -19 to 1665 of SEQ ID NO: 2). (Item 27) The method according to item 26, wherein the chimeric polypeptide has no signal sequence and is identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids 1 to 1665 of SEQ ID NO: 2), or has a signal sequence and is identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids -19 to 1665 of SEQ ID NO: 2). method. (Item 28) The method according to any one of items 25 to 27, wherein the second polypeptide consists essentially of a sequence that has no signal sequence and is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids 1 to 227 of SEQ ID NO: 4), or has a signal sequence and is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids -20 to 227 of SEQ ID NO: 4). (Item 29) The method according to item 28, wherein the second polypeptide consists essentially of a sequence that has no signal sequence and is identical to the amino acid sequence shown in Table 2A(ii) (amino acids 1 to 227 of SEQ ID NO: 4), or has a signal sequence and is identical to the amino acid sequence shown in Table 2A(ii) (amino acids -20 to 227 of SEQ ID NO: 4). (Item 30) The method according to any one of items 1 to 29, wherein the chimeric polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 31) A method of administering Factor VIII to a subject in need thereof, the method comprising administering to the subject a therapeutic dose of a chimeric polypeptide comprising a Factor VIII portion and a second portion, thereby obtaining an area under the plasma concentration-time curve (AUC) that is at least about 1.25-fold greater than the AUC obtained with an equivalent amount of polypeptide consisting of the Factor VIII portion. (Item 32) The method of item 31, wherein the chimeric polypeptide is administered at dosing intervals of about 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or more. (Item 33) The method according to any one of items 31-32, wherein the subject is in need of prophylactic treatment. (Item 34) The method of item 31, wherein the subject is in need of on-demand treatment. (Item 35) The method of item 32, wherein the subject is in need of treatment for the appearance of bleeding symptoms. (Item 36) The method of item 33, wherein the subject is in need of treatment for joint hemorrhage, muscle bleeding, oral bleed, bleeding, bleeding into muscle, oral hemorrhage, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the iliopsoas sheath. (Item 37) The method of item 34, wherein the subject is in need of treatment for surgical prophylaxis, intraoperative management, or surgery. (Item 38) The method of item 37, wherein the surgery is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hernia repair, synovectomy, total knee arthroplasty, craniotomy, bone fixation, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement. (Item 39) The method according to any one of items 34 to 38, wherein the dosing interval of the chimeric polypeptide is about once every 24 to 36, 24 to 48, 24 to 72, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours or longer. (Item 40) The method according to any one of items 31 to 39, wherein the therapeutic dose is 10 to 100 IU / kg. (Item 41) The method according to item 40, wherein the therapeutic dose is 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100 IU / kg. (Item 42) The method according to item 41, wherein the therapeutic dose is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 IU / kg. (Item 43) The method according to any one of items 31 to 42, wherein the subject is human. (Item 44) The method according to any one of items 31 to 43, wherein the Factor VIII is human Factor VIII. (Item 45) The method according to any one of items 31 to 44, wherein the Factor VIII has a complete or partial deletion of the B domain. (Item 46) The method according to item 44, wherein the Factor VIII portion of the chimeric polypeptide has no signal sequence and is at least 90% or 95% identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). (Item 47) The method according to item 46, wherein the Factor VIII portion of the chimeric polypeptide has no signal sequence and is identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). (Item 48) The method according to item 44, wherein the Factor VIII portion of the chimeric polypeptide has a signal sequence and is at least 90% or 95% identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 49) The method according to item 48, wherein the Factor VIII portion of the chimeric polypeptide has a signal sequence and is identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 50) The method according to item 43 or 44, wherein the second portion of the chimeric polypeptide is at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12). (Item 51) The second portion of the chimeric polypeptide is as follows: the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12), and the method according to item 50, wherein they are identical. (Item 52) The method according to any one of items 31 to 51, wherein the chimeric polypeptide is in the form of a hybrid comprising a second polypeptide bound to the chimeric polypeptide, and the second polypeptide consists essentially of Fc. (Item 53) The method according to item 52, wherein the chimeric polypeptide has no signal sequence and is at least 90% or 95% identical to the factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids 1 to 1665 of SEQ ID NO: 2), or has a signal sequence and is at least 90% or 95% identical to the factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids -19 to 1665 of SEQ ID NO: 2). (Item 54) The method according to item 53, wherein the chimeric polypeptide has no signal sequence and is identical to the factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids 1 to 1665 of SEQ ID NO: 2), or has a signal sequence and is identical to the factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids -19 to 1665 of SEQ ID NO: 2). (Item 55) The method according to any one of items 52 to 54, wherein the second polypeptide consists essentially of a sequence that has no signal sequence and is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids 1 to 227 of SEQ ID NO: 4), or has a signal sequence and is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids -20 to 227 of SEQ ID NO: 4). (Item 56) The method according to item 55, wherein the second polypeptide consists essentially of a sequence that has no signal sequence and is identical to the amino acid sequence shown in Table 2A(ii) (amino acids 1 to 227 of SEQ ID NO: 4), or has a signal sequence and is identical to the amino acid sequence shown in Table 2A(ii) (amino acids -20 to 227 of SEQ ID NO: 4). (Item 57) The method according to any one of items 31 to 56, wherein the chimeric polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 58) A method of administering Factor VIII to a subject in need thereof, the method comprising administering to the subject a therapeutic dose of a polypeptide comprising Factor VIII and Fc at dosing intervals of about 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or more. (Item 59) The method according to item 58, wherein the subject is in need of prophylactic treatment. (Item 60) The method according to any one of items 58 to 59, wherein the therapeutic dose is 10 to 100 IU / kg. (Item 61) The method according to item 60, wherein the therapeutic dose is 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100 IU / kg. (Item 62) The method according to item 61, wherein the therapeutic dose is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 IU / kg. 1. (Item 63) The method according to any one of items 58 to 62, wherein the subject is human. (Item 64) The method according to any one of items 58 to 63, wherein the Factor VIII is human Factor VIII. (Item 65) The method according to any one of items 58 to 64, wherein the Factor VIII has a complete or partial deletion of the B domain. (Item 66) The method according to item 64, wherein the Factor VIII has no signal sequence and is at least 90% or 95% identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). (Item 67) The method according to item 66, wherein the factor VIII is identical to the factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12) and does not have a signal sequence. (Item 68) The method according to item 64, wherein the factor VIII has a signal sequence and is at least 90% or 95% identical to the factor VIII amino acid sequence shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 69) The method according to item 68, wherein the factor VIII has a signal sequence and is identical to the factor VIII amino acid sequence shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 70) The method according to item 64 or 65, wherein the second part is at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12). (Item 71) The method according to item 70, wherein the second part is identical to the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12). (Item 72) The method according to any one of items 58 to 71, wherein the chimeric polypeptide is in the form of a hybrid comprising a second polypeptide bound to the chimeric polypeptide, and the second polypeptide consists essentially of Fc. (Item 73) The method according to item 72, wherein the chimeric polypeptide has no signal sequence and is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids 1 to 1665 of SEQ ID NO: 2), or has a signal sequence and is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids -19 to 1665 of SEQ ID NO: 2). (Item 74) The method according to item 73, wherein the chimeric polypeptide has no signal sequence and is identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids 1 to 1665 of SEQ ID NO: 2), or has a signal sequence and is identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) (amino acids -19 to 1665 of SEQ ID NO: 2). (Item 75) The method according to any one of items 72 to 74, wherein the second polypeptide consists essentially of a sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids 1 to 227 of SEQ ID NO: 4) without a signal sequence, or is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids -20 to 227 of SEQ ID NO: 4) with a signal sequence. (Item 76) The method according to item 75, wherein the second polypeptide consists essentially of a sequence that is identical to the amino acid sequence shown in Table 2A(ii) (amino acids 1 to 227 of SEQ ID NO: 4) without a signal sequence, or is 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids -20 to 227 of SEQ ID NO: 4) with a signal sequence. (Item 77) The method according to any one of items 58 to 76, wherein the polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 78) A polypeptide comprising Factor VIII and Fc that has no signal sequence and is at least 90% or 95% identical to the Factor VIII amino acid sequences shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). (Item 79) The polypeptide according to item 78, wherein the Factor VIII has no signal sequence and is identical to the Factor VIII amino acid sequences shown in Table 2 (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). (Item 80) A polypeptide comprising Factor VIII and Fc that has a signal sequence and is at least 90% or 95% identical to the Factor VIII amino acid sequences shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 81) The polypeptide according to item 80, wherein the Factor VIII has a signal sequence and is identical to the Factor VIII amino acid sequences shown in Table 2 (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12). (Item 82) The polypeptide according to items 78 to 81, wherein the Fc is at least 90% or 95% identical to the Fc amino acid sequences shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12). (Item 83) wherein the Fc is the Fc amino acid sequence shown in Table 2 (amino acids 1439 to 1665 of SEQ ID NO: 2 ; amino acids 2333 to 2559 of SEQ ID NO: 6; amino acids 741 to 967 of SEQ ID NO: 8; amino acids 746 to 972 of SEQ ID NO: 10; amino acids 685 to 924 of SEQ ID NO: 12) and is identical to the polypeptide according to Item 82. (Item 84) A polypeptide according to Item 78, which has no signal sequence and is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences (amino acids 1 to 1665 of SEQ ID NO: 2) shown in Table 2A(i), or which has a signal sequence and is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences (amino acids -19 to 1665 of SEQ ID NO: 2) shown in Table 2A(i). (Item 85) A polypeptide according to Item 84, which has no signal sequence and is identical to the Factor VIII and Fc amino acid sequences (amino acids 1 to 1665 of SEQ ID NO: 2) shown in Table 2A(i), or which has a signal sequence and is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences (amino acids -19 to 1665 of SEQ ID NO: 2) shown in Table 2A(i). (Item 86) A polypeptide according to any one of Items 78 to 85, which is in the form of a hybrid containing a second polypeptide, and the second polypeptide consists essentially of Fc. (Item 87) A polypeptide according to Item 85, wherein the second polypeptide consists essentially of a sequence that has no signal sequence and is at least 90% or 95% identical to the amino acid sequence (amino acids 1 to 227 of SEQ ID NO: 4) shown in Table 2A(ii), or which has a signal sequence and is at least 90% or 95% identical to the amino acid sequence (amino acids -20 to 227 of SEQ ID NO: 4) shown in Table 2A(ii). (Item 88) The polypeptide according to item 86, wherein the second polypeptide consists essentially of an amino acid sequence identical to the amino acid sequence shown in Table 2A(ii) (amino acids 1 to 227 of SEQ ID NO: 4) without a signal sequence, or consists of an amino acid sequence having a signal sequence and being 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) (amino acids -20 to 227 of SEQ ID NO: 4). (Item 89) The polypeptide according to any one of items 78 to 88, which has a half-life that is at least about 1.5 to 6 times, 1.5 to 5 times, 1.5 to 4 times, 1.5 to 3 times, or 1.5 to 2 times longer than the polypeptide consisting of the Factor VIII. (Item 90) A polynucleotide encoding the polypeptide according to any one of items 78 to 85. (Item 91) The polynucleotide according to item 90, which contains the Factor VIII-Fc nucleotide sequence (SEQ ID NO: 1, 5, 7, 9, or 11) in Table 1. (Item 92) A polynucleotide encoding the Factor VIII-Fc polypeptide and the second polypeptide according to any one of items 86 to 89. (Item 93) The polynucleotide according to any one of items 90 to 92, which is a vector, plasmid, phage, or virus. (Item 94) The polynucleotide according to any one of items 90 to 93, which is DNA or RNA. (Item 95) A cultured human embryonic cell containing the polynucleotide according to any one of items 89 to 94. (Item 96) The cell according to item 95, which is HEK293 cell. (Item 97) A method for producing a Factor VIII-Fc hybrid protein, comprising the following: culturing the cell according to item 95 or 96 under conditions that enable the expression of the encoded Factor VIII-Fc chimeric polypeptide and the encoded polypeptide consisting essentially of Fc; and Recovering the encoded Factor VIII-Fc hybrid protein A method comprising. (Item 98) A protein produced by the method according to item 97. (Item 99) The chimeric polypeptide has the following: The ability to interact with phospholipid vesicles comparable to the ability of the polypeptide consisting of the Factor VIII portion; The ability to form an X-ase complex that activates Factor X comparable to the ability of the polypeptide consisting of the Factor VIII portion; The ability to be activated by α-thrombin within 5 minutes comparable to the ability of the polypeptide consisting of the Factor VIII portion; and The ability to interact with Factor IXa comparable to the ability of the polypeptide consisting of the Factor VIII portion The method according to any one of items 1 to 57 having one or more characteristics selected from the group consisting of. (Item 100) The chimeric polypeptide has the following: Forming an X-ase complex that activates Factor X with a Km within about 1, about 1.5, or about 2 standard deviations of the Km of the polypeptide consisting of the Factor VIII portion (where Km is measured as a function of Factor X concentration); Forming an X-ase complex that activates Factor X with a Vmax within about 1, about 1.5, or about 2 standard deviations of the Vmax of the polypeptide consisting of the Factor VIII portion (where Vmax is measured as a function of Factor X concentration); Forming an X-ase complex that activates Factor X with a Kd within about 1, about 1.5, or about 2 standard deviations of the Kd of the polypeptide consisting of the Factor VIII portion (where Kd is measured as a function of Factor IXa concentration); and Forming an X-ase complex that activates Factor X with a Vmax within about 1, about 1.5, or about 2 standard deviations of the Vmax of the polypeptide consisting of the Factor VIII portion (where Vmax is measured as a function of Factor IXa concentration); The method according to item 99, having one or more traits selected from the group consisting of (Item 101) The polypeptide is as follows: The ability to interact with phospholipid vesicles comparable to the ability of the polypeptide consisting of the Factor VIII moiety; The ability to form an X-ase complex that activates Factor X comparable to the ability of the polypeptide consisting of the Factor VIII moiety; The ability to be activated by α-thrombin within 5 minutes comparable to the ability of the polypeptide consisting of the Factor VIII moiety; and The method according to any one of items 58 to 77, having one or more traits selected from the group consisting of the ability to interact with Factor IXa comparable to the ability of the polypeptide consisting of the Factor VIII moiety. (Item 102) The polypeptide is as follows: Forming an X-ase complex that activates Factor X with a Km within about 1, about 1.5, or about 2 standard deviations of the Km of the polypeptide consisting of the Factor VIII moiety (where Km is measured as a function of Factor X concentration); Forming an X-ase complex that activates Factor X with a Vmax within about 1, about 1.5, or about 2 standard deviations of the Vmax of the polypeptide consisting of the Factor VIII moiety (where Vmax is measured as a function of Factor X concentration); Forming an X-ase complex that activates Factor X with a Kd within about 1, about 1.5, or about 2 standard deviations of the Kd of the polypeptide consisting of the Factor VIII moiety (where Kd is measured as a function of Factor IXa concentration); and Forming an X-ase complex that activates Factor X with a Vmax within about 1, about 1.5, or about 2 standard deviations of the Vmax of the polypeptide consisting of the Factor VIII moiety (where Vmax is measured as a function of Factor IXa concentration); The method according to item 101, having one or more traits selected from the group consisting of The method according to item 101, having one or more traits selected from the group consisting of (Item 103) As follows: The ability to interact with phospholipid vesicles comparable to the ability of the polypeptide consisting of the Factor VIII moiety; The ability to form an Xase complex that activates Factor X comparable to the ability of the polypeptide consisting of the Factor VIII moiety; The ability to be activated by α-thrombin within 5 minutes comparable to the ability of the polypeptide consisting of the Factor VIII moiety; and The ability to interact with Factor IXa comparable to the ability of the polypeptide consisting of the Factor VIII moiety The polypeptide according to any one of items 78 to 89 and 98 having 1, about 1.5 or about 2 or more traits selected from the group consisting of. (Item 104) The polypeptide is as follows: Forming an Xase complex that activates Factor X with a Km within about 1, about 1.5 or about 2 standard deviations of the Km of the polypeptide consisting of the Factor VIII moiety (where Km is measured as a function of Factor X concentration); Forming an Xase complex that activates Factor X with a Vmax within about 1, about 1.5 or about 2 standard deviations of the Vmax of the polypeptide consisting of the Factor VIII moiety (where Vmax is measured as a function of Factor X concentration); Forming an Xase complex that activates Factor X with a Kd within about 1, about 1.5 or about 2 standard deviations of the Kd of the polypeptide consisting of the Factor VIII moiety (where Kd is measured as a function of Factor IXa concentration); and Forming an Xase complex that activates Factor X with a Vmax within about 1, about 1.5 or about 2 standard deviations of the Vmax of the polypeptide consisting of the Factor VIII moiety (where Vmax is measured as a function of Factor IXa concentration); The method according to item 101 having one or more traits selected from the group consisting of. (Item 105) The method according to any one of items 1 to 77 having an average incremental recovery rate (K value) (activity: observed value) greater than 1.38 IU / dL per IU / kg. (Item 106) The method according to any one of items 1 to 77 and 104 having an average incremental recovery rate (K value) (activity: observed value) of at least about 1.5, at least about 1.85 or at least about 2.46 IU / dL per IU / kg. (Item 107) The chimeric polypeptide in the patient population or the subject is as follows: Average clearance (CL) (activity) in the patient population of about 2.33 ± 1.08 mL / h / kg or less; Average clearance (CL) (activity) in the patient population of about 1.8 - 2.69 mL / h / kg; Average clearance (CL) (activity) in the patient population that is about 65% of the clearance of the polypeptide consisting of the Factor VIII moiety; Clearance (CL) (activity) in the subject of about 1.22 - 5.19 mL / h / kg; Average residence time (MRT) (activity) in the patient population of at least about 26.3 ± 8.33 hours; Average MRT (activity) in the patient population of about 25.9 - 26.5 hours; Average MRT (activity) in the patient population that is about 1.5 times longer than the average MRT of the polypeptide consisting of the Factor VIII moiety; Average residence time (MRT) (activity) in the subject of about 14 - 41.3 hours; Average t1 / 2 beta (activity) in the patient population of about 18.3 ± 5.79 hours; Average t1 / 2 beta (activity) in the patient population of about 18 - 18.4 hours; Average t1 / 2 beta (activity) in the patient population that is about 1.5 times longer than the average t1 / 2 beta of the polypeptide consisting of the Factor VIII moiety; t1 / 2 beta (activity) in the subject of about 11 - 26.4 hours; Average incremental recovery rate (K value) (activity; observed value) in the patient population of about 2.01 ± 0.44 IU / dL per IU / kg; The average incremental recovery rate (K value) (activity; observed value) in the patient population of about 1.85 - 2.46 IU / dL per IU / kg; The average incremental recovery rate (K value) (activity; observed value) in the patient population which is about 90% of the average incremental recovery rate of the polypeptide consisting of the Factor VIII moiety; The incremental recovery rate (K value) (activity; observed value) in the subject of about 1.38 - 2.88 IU / dL per IU / kg; The average Vss (activity) in the patient population of about 55.1 ± 12.3 mL / kg; The average Vss (activity) in the patient population of about 45.3 - 56.1 mL / kg; The average Vss (activity) in the subject of about 37.7 - 79.4 mL / kg; About 49.9 ± 18.2 IU * per IU / kg of the average AUC / dose (activity) in the patient population of h / dL; About 44.8 - 57.6 IU * per IU / kg of the average AUC / dose (activity) in the patient population of h / dL; and About 19.2 - 81.7 IU * per IU / kg of the AUC / dose in the subject of h / dL; The method described in any of Items 1 - 75 and 104 - 106 showing one or more pharmacokinetic parameters selected from the group consisting of; (Item 108) The method described in any of Items 1 - 77 and 104 - 106 where the second moiety is XTEN or albumin. (Item 109) The method described in any of Items 1 - 77 and 104 - 106 where the therapeutic dose is about 10 - about 150, 100 - 110, 110 - 120, 120 - 130, 130 - 140, 140 - 150, 110, 115, 120, 125, 130, 135, 140, 145 or 150 IU / kg. (Item 110) The method described in any of Items 1 - 77 and 104 - 106 where the dose administration interval is 1.5 - 5, 1.5, 2, 3, 4 or 5 days or more.

Brief Description of the Drawings

[0028]

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DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides a method for treating hemophilia A with factor VIII using longer dosing intervals and / or larger AUCs than are possible with currently known factor VIII products. The present invention further provides improved factor VIII chimeric polypeptides, factor VIII chimeric nucleotides, and methods of production.

[0030] Treatment of hemophilia A aims at replacement therapy to restore FVIII activity to 1 - 5% of normal levels to prevent spontaneous bleeding (Mannucci, P.M., et al., N. Engl. J. Med. 344:1773 - 9 (2001) (incorporated herein by reference in its entirety)). Plasma-derived and recombinant FVIII products are available for on-demand treatment of bleeding symptoms or for prophylactic treatment to prevent the occurrence of bleeding symptoms. Based on the half-life of these products (10 - 12 hours) (White G.C., et al., Thromb. Haemost. 77:660 - 7 (1997); Morfini, M., Haemophilia 9 (suppl 1):94 - 99; discussion 100 (2003)), treatment regimens usually require frequent intravenous administration 2 - 3 times a week for prophylaxis and 1 - 3 times a day for on-demand treatment (Manco-Johnson, M.J., et al., N. Engl. J. Med. 357:535 - 544 (2007)) (incorporated herein by reference in its entirety). Such frequent administration is painful and inconvenient.

[0031] The present invention provides a method of administering factor VIII to a subject in need thereof, the method comprising administering to the subject a therapeutic amount of a chimeric factor VIII polypeptide, such as a chimeric factor VIII-Fc polypeptide, or a hybrid of such a polypeptide, at an administration interval that is at least about 1.5-fold longer than the administration interval required for an equivalent amount of said factor VIII that does not contain a non-factor VIII moiety, e.g., does not contain an Fc moiety (a polypeptide consisting of said factor VIII moiety).

[0032] The administration interval may be at least about 1.5- to 6-fold longer, 1.5- to 5-fold longer, 1.5- to 4-fold longer, 1.5- to 3-fold longer, or 1.5- to 2-fold longer than the administration interval required for an equivalent amount of said factor VIII that does not contain a non-factor VIII moiety, e.g., does not contain an Fc moiety (a polypeptide consisting of said factor VIII moiety). The administration interval may be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6-fold longer than the administration interval required for an equivalent amount of said factor VIII that does not contain a non-factor VIII moiety, e.g., does not contain an Fc moiety (a polypeptide consisting of said factor VIII moiety). The administration interval may be about every 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more.

[0033] The administration interval may be at least about 1.5 to 5, 1.5, 2, 3, 4, or 5 days or more.

[0034] The present invention further provides a method of administering factor VIII to a subject in need thereof, the method comprising administering to the subject a therapeutic amount of a chimeric factor VIII polypeptide, such as a chimeric factor VIII-Fc polypeptide, or a hybrid of such a polypeptide, to obtain an area under the plasma concentration-time curve (AUC) that is at least about 1.25-fold greater than the AUC obtained with an equivalent amount of said factor VIII that does not contain a non-factor VIII moiety, e.g., does not contain an Fc moiety (a polypeptide consisting of said factor VIII moiety).

[0035] The present invention further provides a method of administering factor VIII to a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a polypeptide comprising factor VIII and Fc, or a hybrid of such a polypeptide, at an administration interval of about 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more.

[0036] The method of the invention can be practiced with respect to subjects in need of prophylactic treatment or on-demand treatment.

[0037] As used herein, "administering" means providing to the subject a pharmaceutically acceptable factor VIII polypeptide of the invention by a pharmaceutically acceptable route. Preferred routes of administration are intravenous, such as intravenous injection and intravenous infusion. Further routes of administration include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration. The chimeric polypeptide and hybrid protein can be administered as part of a pharmaceutical composition comprising at least one excipient.

[0038] "Area under the plasma concentration-time curve (AUC)" as used herein has the same meaning as the term in pharmacology and is based on the rate and extent of absorption of factor VIII after administration. The AUC is determined over a specific time, such as 12, 18, 24, 36, 48, or 72 hours, or to infinity using extrapolation based on the slope of the curve. Unless otherwise specified herein, the AUC is determined to infinity. The determination of the AUC can be performed in a single subject or in a population of subjects for which an average is calculated.

[0039] The "B domain" of Factor VIII, as used herein, 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 full-length human Factor VIII. The other human Factor VIII domains are defined by the following amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is commonly referred to as the Factor VIII light chain activation peptide. For porcine, murine, and canine Factor VIII, the positions of the boundaries for all domains, including the B domain, are also known in the art. Preferably, the B domain of Factor VIII is deleted ("B domain deleted Factor VIII" or "BDDFVIII"). An example of BDDFVIII is REFACTO (recombinant BDDFVIII), which has the same sequence as the Factor VIII portion of the sequence of Table 2A(i) (amino acids -19 to 1438 or 1 to 1438 of SEQ ID NO: 2).

[0040] The "Factor VIII lacking the B domain" may have complete or partial deletions disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563 (each of which is incorporated herein by reference in its entirety). In some embodiments, the Factor VIII lacking the B domain sequence of the present invention includes any one of the deletions disclosed in columns 4, lines 4 to 5, column 28 and Examples 1 to 5 of U.S. Patent No. 6,316,226 (also in US6,346,513). In some embodiments, the Factor VIII lacking the B domain of the present invention has the deletions disclosed in columns 2, lines 26 to 51 and Examples 5 to 8 of U.S. Patent No. 5,789,203 (also in US6,060,447, US5,595,886, and US6,228,620). In some embodiments, 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, line 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, domain-deleted Factor VIII has most of the B domain deleted, but still includes the amino-terminal sequence of the B domain that is essential for in vivo proteolytic processing into the two polypeptide chains of the first translation product, as disclosed in International Publication No. 91 / 09122, which is hereby incorporated by reference in its entirety. In some embodiments, domain-deleted Factor VIII is structured such that amino acids 747 to 1638 are deleted. That is, the B domain is substantially completely deleted. Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), which is hereby incorporated by reference in its entirety. Domain-deleted Factor VIII may further have amino acids 771 to 1666 or amino acids 868 to 1562 of Factor VIII deleted. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988) (which is hereby incorporated by reference in its entirety). Further B domain deletions that are part of the present invention include, for example: deletions of amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), deletion of 797-1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), deletion of 741-1646 (Kaufman (PCT Published Application No. WO 87 / 04187)), deletion of 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), deletion of 741-1648 (Pasek (PCT Application No. 88 / 00831)), deletions of 816-1598 or 741-1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)) (each of which is hereby incorporated by reference in its entirety). Each of the said deletions may be made with any factor VIII sequence.

[0041] As used herein, the term "chimeric polypeptide" means a polypeptide that contains at least two polypeptides (or sub-sequences or peptides) from different origins therein. Chimeric polypeptides can include, for example, 2, 3, 4, 5, 6, 7, or more polypeptides from different origins such as different genes, different cDNAs, or different animals or other species. A chimeric polypeptide can include, for example, one or more linkers that join different sub-sequences. Thus, within one chimeric polypeptide, sub-sequences can be joined directly, or indirectly via a linker, or both. A chimeric polypeptide can include additional peptides such as a signal sequence and sequences such as 6His and FLAG that assist in protein purification or detection. In addition, a chimeric polypeptide can have amino acid or peptide additions at the N-terminus and / or C-terminus.

[0042] In some embodiments, the chimeric polypeptide comprises a Factor VIII portion and a non-Factor VIII portion. Exemplary non-Factor VIII portions include, for example, Fc, XTEN, and albumin. Exemplary chimeric polypeptides of the present invention include, for example, chimeric Factor VIII-Fc polypeptides, chimeric Factor VIII-XTEN poly peptides, and chimeric Factor VIII-albumin polypeptides.

[0043] Exemplary chimeric Factor VIII-Fc polypeptides include, for example, SEQ ID NOs: 2, 6, 8, 10, and 12 (Table 2) (regardless of the presence or absence of their signal sequences and the chimeric Fc polypeptide of SEQ ID NO: 4) (Table 2).

[0044] The chimeric polypeptide may comprise a sequence that is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) without a signal sequence (amino acids 1-1665 of SEQ ID NO: 2) or a sequence that is at least 90% or 95% identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) with a signal sequence (amino acids -19-1665 of SEQ ID NO: 2). The chimeric polypeptide may comprise a sequence identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) without a signal sequence (amino acids 1-1665 of SEQ ID NO: 2) or a sequence identical to the Factor VIII and Fc amino acid sequences shown in Table 2A(i) with a signal sequence (amino acids -19-1665 of SEQ ID NO: 2).

[0045] As described above, exemplary chimeric polypeptides include Factor VIII fused with one or more XTEN polypeptides. Schellenburger et al., Nat. Biotech. 27:1186-90 (2009) (which is hereby incorporated by reference in its entirety). Factor VIII may be fused to the N-terminus of the XTEN polypeptide or the C-terminus of the XTEN polypeptide. However, it is assumed that the Factor VIII component of the Factor VIII-XTEN fusion protein can be treated with a protease to obtain a processed Factor VIII-containing polypeptide. A protease site may be included between the XTEN portion and the Factor VIII portion to enable such treatment. Examples of XTEN polypeptides include those disclosed in International Publication No. WO 2009 / 023270, International Publication No. WO 2010 / 091122, International Publication No. WO 2007 / 103515, US 2010 / 0189682, and US 2009 / 0092582 (each of which is hereby incorporated by reference in its entirety).

[0046] As described above, exemplary chimeric polypeptides also include Factor VIII fused with one or more albumin polypeptides. Preferably, the albumin is human albumin. Factor VIII can be fused to either the N-terminus or the C-terminus of the albumin. However, it is assumed that the Factor VIII component of the Factor VIII-albumin fusion protein can be treated with an enzymatically active proprotein convertase to obtain a processed Factor VIII-containing polypeptide. Examples of albumin that can be used in the present invention, such as fragments thereof, are known. For example, U.S. Patent No. 7,592,010; U.S. Patent No. 6,686,179; and Schulte, Thrombosis Res. 124 Suppl. 2:S6-S8 (2009) (each of which is hereby incorporated by reference in its entirety).

[0047] In some embodiments, a chimeric polypeptide comprising a Factor VIII moiety has an increased half-life (t1 / 2) compared to a polypeptide consisting of the same Factor VIII moiety and lacking a non-Factor VIII moiety. A chimeric Factor VIII polypeptide with an increased t1 / 2 may be referred to herein as a long-acting Factor VIII. Examples of long-acting chimeric Factor VIII polypeptides include, for example, Factor VIII fused to Fc (including chimeric Factor VIII polypeptides in hybrid forms such as FVIII-Fc monomer-dimer hybrids; see Example 1, Figure 1, and Table 2A; and U.S. Patent Nos. 7,404,956 and 7,348,004), Factor VIII fused to XTEN, and Factor VIII fused to albumin.

[0048] As used herein, "culturing," "cultivate," and "cultivating" mean incubating cells under in vitro conditions that permit cell growth or division, or maintaining cells in a viable state. As used herein, "cultured cells" means cells that propagate in vitro.

[0049] As used herein, the term "Factor VIII" means the functional Factor VIII polypeptide in its normal role in coagulation, unless otherwise specified. Thus, the term Factor VIII encompasses variant polypeptides that are functional. Preferred Factor VIII proteins are human, porcine, canine, and murine Factor VIII proteins. As described in the Background section, full-length polypeptides and polynucleotide sequences are known, as well as many functional fragments, mutants, and modified versions. Examples of human Factor VIII sequences are shown as sub-sequences in SEQ ID NOs: 2, 6, 8, 10, and 12 (Table 2). Factor VIII polypeptides include, for example, full-length Factor VIII, full-length Factor VIII without an N-terminal Met, mature Factor VIII (without a signal sequence), mature Factor VIII with an additional N-terminal Met, and / or Factor VIII with a complete or partial deletion of the B domain. Preferred Factor VIII variants include a B domain deletion, whether partial or complete.

[0050] As noted above and below, a very large number of functional Factor VIII variants are known. In addition, hundreds of non-functional mutations in Factor VIII have been identified in hemophilia patients, and it has been found that the effect of these mutations on Factor VIII function depends more on where they are within the three-dimensional structure of Factor VIII than on the nature of the substitution (Cutler et al., Hum. Mutat. 19:274-8 (2002)) (which is incorporated herein by reference in its entirety). In addition, comparison of Factor VIII from humans and other species has identified conserved residues that may be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632) (which are incorporated herein by reference in their entirety).

[0051] The human Factor VIII gene has been isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337 (1984); Vehar, G. A., et al., Nature 312:337-342 (1984); International Publication No. WO 87 / 04187; International Publication No. WO 88 / 08035; International Publication No. WO 88 / 03558; U.S. Patent No. 4,757,006) (each of which is incorporated herein by reference in its entirety), and the amino acid sequence was deduced from the cDNA. Capon et al., U.S. Patent No. 4,965,199 (incorporated herein by reference in its entirety) disclose recombinant DNA methods for the production of Factor VIII in mammalian host cells and the purification of human Factor VIII. Expression of human Factor VIII in CHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells) has been reported. Human Factor VIII has been modified to delete some or all of the B domain (U.S. Patent Nos. 4,994,371 and 4,868,112 (each of which is incorporated herein by reference in its entirety)), and substitution of the human Factor VIII B domain with the human Factor V B domain has been carried out (U.S. Patent No. 5,004,803 (incorporated herein by reference in its entirety)). The cDNA sequence encoding human Factor VIII and the predicted amino acid sequence are shown by SEQ ID NOs: 1 and 2, respectively, of U.S. Patent Application Publication No. 2005 / 0100990 (incorporated herein by reference in its entirety).

[0052] U.S. Patent No. 5,859,204, Lollar, J. S. (incorporated herein by reference in its entirety) reports a functional mutant of factor VIII with reduced antigenicity and reduced immunoreactivity. U.S. Patent No. 6,376,463, Lollar, J. S. (incorporated herein by reference in its entirety) also reports a mutant of factor VIII with reduced immunoreactivity. U.S. Patent Application Publication No. 2005 / 0100990 (Saenko et al., incorporated herein by reference in its entirety) reports a functional mutation in the A2 domain of factor VIII.

[0053] Many functional factor VIII molecules containing a B domain deletion are disclosed in the following patents: US6,316,226 and US6,346,513 (both assigned to Baxter); US7,041,635 (assigned to In2Gen); US5,789,203, US6,060,447, US5,595,886, and US6,228,620 (assigned to Chiron); US5,972,885 and US6,048,720 (assigned to Biovitrum), US5,543,502 and US5,610,278 (assigned to Novo Nordisk); US5,171,844 (assigned to Immuno Ag); US5,112,950 (assigned to Transgene S.A.); US4,868,112 (assigned to Genetics Institute) (each of which is incorporated herein by reference in its entirety).

[0054] The porcine Factor VIII sequence has been published (Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)) (which is hereby incorporated by reference in its entirety), and a full-length porcine cDNA sequence obtained from PCR amplification of the Factor VIII sequence from a porcine spleen cDNA library has been reported (Healey, J. F., et al., Blood 88:4209-4214 (1996) (which is hereby incorporated by reference in its entirety). Hybrid human / porcine Factor VIII with substitutions of entire domains, entire subunits, and specific amino acid sequences has been disclosed in U.S. Patent No. 5,364,771 to Lollar and Runge, and International Publication No. 93 / 20093 (which are hereby incorporated by reference in their entireties). More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine Factor VIII and chimeric Factor VIII in which the porcine A1 and / or A2 domains are replaced with the corresponding human domains have been reported in International Publication No. 94 / 11503 (which is hereby incorporated by reference in its entirety). U.S. Patent No. 5,859,204, to Lollar, J.S., also discloses porcine cDNA and the deduced amino acid sequence. U.S. Patent No. 6,458,563, assigned to Emory (which is hereby incorporated by reference in its entirety), discloses B-domain-deleted porcine Factor VIII.

[0055] Factor VIII (or the Factor VIII portion of a chimeric polypeptide) can be at least 90% or 95% identical to the Factor VIII amino acid sequences shown in Table 2 that do not include a signal sequence (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12). Factor VIII (or the Factor VIII portion of a chimeric polypeptide) can be identical to the Factor VIII amino acid sequences shown in Table 2 that do not include a signal sequence (amino acids 1 to 1438 of SEQ ID NO: 2; amino acids 1 to 2332 of SEQ ID NO: 6; amino acids 1 to 740 of SEQ ID NO: 8; amino acids 1 to 745 of SEQ ID NO: 10; or amino acids 1 to 684 of SEQ ID NO: 12).

[0056] Factor VIII (or the Factor VIII portion of a chimeric polypeptide) is the Factor VIII amino acid sequence shown in Table 2 that includes a signal sequence (amino acids -19 to 14 38 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12) and can be at least 90% or 95% identical. Factor VIII (or the Factor VIII portion of a chimeric polypeptide) can be identical to the Factor VIII amino acid sequence shown in Table 2 that includes a signal sequence (amino acids -19 to 1438 of SEQ ID NO: 2; amino acids -19 to 2332 of SEQ ID NO: 6; amino acids -19 to 740 of SEQ ID NO: 8; amino acids -19 to 745 of SEQ ID NO: 10; or amino acids -20 to 684 of SEQ ID NO: 12).

[0057] "Equivalent", as used herein, means the same amount of Factor VIII activity expressed in international units, which is independent of the molecular weight of the polypeptide in question. 1 international unit (IU) of Factor VIII activity approximately corresponds to the amount of Factor VIII in 1 milliliter of normal human plasma. Several assays, including the European Pharmacopoeia chromogenic substrate assay and the one-stage clotting assay, are available for measuring Factor VIII activity.

[0058] As used herein, "Fc", unless otherwise specified, means a functional neonatal Fc receptor (FcRn) binding partner. An FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor, resulting in active transport of the FcRn binding partner by the FcRn receptor. Thus, the term "Fc" includes any variant of an IgG Fc that is functional. 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 (incorporated herein by reference in its entirety)). The major contact region of Fc with FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within one Ig heavy chain. Examples of FcRn binding partners include whole IgG, Fc fragments of IgG, and other fragments of IgG that contain the complete binding region for FcRn. Examples of major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 in the CH2 domain and amino acid residues 385-387, 428, and 433-436 in the CH3 domain. All references to immunoglobulin or immunoglobulin fragment, or amino acid numbering of regions, are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda; MD (incorporated herein by reference in its entirety). (FcRn receptors have been isolated from several mammalian species including humans. The sequences of human FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180: 2377) (which is hereby incorporated 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 No. WO 2004 / 101740 and International Publication No. WO 2006 / 074199 (which are hereby incorporated by reference in their entirety).

[0059] Fc (or the Fc portion of a chimeric polypeptide) can include one or more mutations, and combinations of mutations.

[0060] Fc (or the Fc portion of a chimeric polypeptide) can include mutations that confer an increased half-life, such as M252Y, S254T, T256E, and combinations thereof, as disclosed in Oganesyan et al., Mol. Immunol. 46:1750 (2009) (which is hereby incorporated by reference in its entirety); H433K , N434F, and combinations thereof; mutants disclosed on pages 1-2, paragraph

[0012] , and in Examples 9 and 10 of US2009 / 0264627A1 (which is hereby incorporated by reference in its entirety); and mutants disclosed on page 2, paragraphs

[0014] -

[0021] of US20090163699A1 (which is hereby incorporated by reference in its entirety).

[0061] Examples of the Fc (or the Fc portion of a chimeric polypeptide) further include, for example, the following mutations: The Fc region of IgG can be modified according to well-known procedures such as site-directed mutagenesis to obtain a modified IgG or Fc fragment or a portion thereof, which are bound by FcRn. Such modifications include, for example, modifications far from the FcRn contact site and modifications within the contact site that preserve or even enhance the binding to FcRn. For example, one of the following amino acid residues (Fcy1) in human IgG1 Fc can be substituted without significantly losing the Fc binding affinity for FcRn: 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, A330S, P331A, P331S, 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 (where, for example, P238A represents the wild-type proline substituted by alanine at position number 238. In addition to alanine, other amino acids can be substituted for the wild-type amino acid at the aforementioned positions.Introducing a mutation alone into the Fc can result in more than 100 FcRn binding partners that are different from the native Fc. Furthermore, introducing combinations of two, three, or more of these individual mutations together can result in more than a hundred FcRn binding partners. Some of these mutations may confer new functions on the FcRn binding partner. For example, one embodiment incorporates N297A to remove the highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing the circulating half-life of the FcRn binding partner, and to prevent the FcRn binding partner from being able to bind to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA without impairing the affinity for FcRn. (Routledge et al. 1995, Transplantation 60:847 (incorporated herein by reference in its entirety); Friend et al. 1999, Transplantation 68:1632 (incorporated herein by reference in its entirety); Shields et al. 1995, J. Biol. Chem. 276:6591 (incorporated herein by reference in its entirety)). Furthermore, at least three human Fc gamma receptors appear to recognize binding sites on IgG within the lower hinge region (generally amino acids 234-237). Thus, another example of a new function and reduced immunogenicity results from mutations in this region, such as, for example, by substituting amino acids 233-236 of human IgG1 "ELLG" with the corresponding sequence from IgG2 "PVA" (having one amino acid deletion). There is a possibility. FcyRI, FcyRII, and FcyRIII, which mediate various effector functions, have been proven not to bind IgG1 when such mutations are not introduced (Ward and Ghetie 1995, Therapeutic Immunology 2:77 (incorporated herein by reference in its entirety); and Armour et al. 1999, Eur. J. Immunol. 29:2613 (incorporated herein by reference in its entirety)). As a further example of the novel functions resulting from the said mutations, the affinity for FcRn may increase in some cases compared to the wild-type affinity. This increased affinity may reflect an increased "on" rate, a decreased "off" rate, or both an increased "on" rate and a decreased "off" rate. Examples of mutations thought to confer increased affinity for FcRn include, for example, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591, incorporated herein by reference in its entirety).

[0062] Fc (or the Fc portion of the chimeric polypeptide) can be at least 90% or 95% identical to the Fc amino acid sequences shown in Table 2 (amino acids 1439 - 1665 of SEQ ID NO:2; amino acids 2333 - 2559 of SEQ ID NO:6; amino acids 741 - 967 of SEQ ID NO:8; amino acids 746 - 972 of SEQ ID NO:10; amino acids 685 - 924 of SEQ ID NO:12). Fc (or the Fc portion of the chimeric polypeptide) can be identical to the Fc amino acid sequences shown in Table 2 (amino acids 1439 - 1665 of SEQ ID NO:2; amino acids 2333 - 2559 of SEQ ID NO:6; amino acids 741 - 967 of SEQ ID NO:8; amino acids 746 - 972 of SEQ ID NO:10; amino acids 685 - 924 of SEQ ID NO:12).

[0063] "Hybrid" polypeptides and proteins, as used herein, mean a combination with a second polypeptide of a chimeric polypeptide. The chimeric polypeptide and the second polypeptide in the hybrid can bind to each other by protein-protein interactions, such as charge-charge interactions or hydrophobic interactions. The chimeric polypeptide and the second polypeptide in the hybrid can bind to each other by disulfide bonds or other covalent bonds. Hybrids are described in International Publication Nos. 2004 / 101740 and 2006 / 074199 (each of which is incorporated herein by reference in its entirety). See also U.S. Pat. Nos. 7,404,956 and 7,348,004 (each of which is incorporated herein by reference in its entirety). The second polypeptide can be a second copy of the same chimeric polypeptide or a non-identical chimeric polypeptide. See, e.g., FIG. 1, Example 1, and Table 2. In a preferred embodiment, the second polypeptide is a polypeptide comprising an Fc. In a preferred embodiment, the chimeric polypeptide is a chimeric Factor VIII-Fc polypeptide and the second polypeptide consists essentially of Fc. For example, the hybrid polypeptide of Example 1, which is an rFVIIIFc recombinant fusion protein consisting of one molecule of recombinant B domain-deleted human FVIII (BDD-rFVIII) fused to the dimer Fc domain of human IgG1 without an intervening linker sequence. This hybrid polypeptide is referred to herein as the FVIIIFc monomer Fc fusion protein, the FVIIIFc monomer hybrid, the monomer FVIIIIFc hybrid, and the FVIIIFc monomer-dimer. See Example 1, FIG. 1, and Table 2A. The examples provide preclinical and clinical data for this hybrid polypeptide.

[0064] The second polypeptide in the hybrid may include a sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without a signal sequence (amino acids 1 to 227 of SEQ ID NO: 4) or a sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) including a signal sequence (amino acids -20 to 227 of SEQ ID NO: 4), or may be composed essentially of this sequence. The second poly peptide may include a sequence that is identical to the amino acid sequence shown in Table 2A(ii) without a signal sequence (amino acids 1 to 227 of SEQ ID NO: 4) or a sequence that is identical to the amino acid sequence shown in Table 2A(ii) including a signal sequence (amino acids -20 to 227 of SEQ ID NO: 4), or may be composed essentially of this sequence.

[0065] Figure 1 is a schematic diagram showing the structure of the B-domain-deleted Factor VIII-Fc chimeric polypeptide and its relationship with the second polypeptide which is an Fc polypeptide. To obtain this hybrid, the coding sequence of human recombinant B-domain-deleted FVIII was obtained from human liver polyA RNA (Clontech) using FVIII-specific primers by reverse transcription-polymerase chain reaction (RT-PCR). The FVIII sequence includes the natural signal sequence of FVIII. For the total deletion of 2682 bp, the B-domain deletion was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638; 4969 bp). Next, the coding sequence of human recombinant Fc was obtained from a human leukocyte cDNA library (Clontech) using Fc-specific primers by RT-PCR. The primers were designed to directly fuse the Fc sequence to the N-terminus without a linker intervening the B-domain-deleted FVIII sequence. The FVIII cDNA sequence was cloned under the control of the CMV promoter into the mammalian dual-expression vector pBUDCE4.1 (Invitrogen). A second identical Fc sequence containing the mouse Igk signal sequence was obtained by RT-PCR and cloned downstream of the EF1α, the second promoter, in the expression vector pBUDCE4.1.

[0066] The rFVIIIFc expression vector was transfected into human embryonic kidney 293 cells (HEK293H; Invitrogen) using Lipofectamine 2000 transfection reagent (Invitrogen). A stable clone cell line was generated by selection with Zeocin (Invitrogen). FVIIIFc was generated for in vivo characterization using one clone cell line, 3C4-22. Recombinant FVIIIFc was produced and purified at Biogen Idec (Cambridge, Massachusetts) (McCue et al. 2009). The transfection method was expected to generate three products, namely monomeric rFVIIIFc hybrid, dimeric rFVIIIFc hybrid, and dimeric Fc. However, dimeric rFVIIIFc was essentially not detected in the conditioned medium from these cells. Rather, the conditioned medium contained Fc and monomeric rFVIIIFc. The size of dimeric rFVIIIFc might have been too large to allow sufficient secretion from the cells. This result was beneficial because it made the purification of the monomer easier than when all three proteins were present. The substances used in this study had a specific activity of approximately 9000 IU / mg.

[0067] "Dosing interval", as used herein, means the amount of time that elapses between multiple administrations to a subject. Comparisons of dosing intervals can be made in a single subject or in a population of subjects, and then the average obtained in the population can be calculated.

[0068] When administering a chimeric Factor VIII polypeptide of the present invention, such as a chimeric Factor VIII-Fc factor polypeptide (a polypeptide or hybrid containing Factor VIII), the dosing interval may be at least about 1.5 times longer than the dosing interval required for an equivalent Factor VIII that does not contain a non-Factor VIII portion, for example, does not contain an Fc portion (a polypeptide consisting of the Factor VIII). The dosing interval may be at least about 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than the dosing interval required for an equivalent Factor VIII that does not contain a non-Factor VIII portion, for example, does not contain an Fc portion (a polypeptide consisting of the Factor VIII). The dosing interval may be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times longer than the dosing interval required for an equivalent Factor VIII that does not contain a non-Factor VIII portion, for example, does not contain an Fc portion (a polypeptide consisting of the Factor VIII). The dosing interval is about every 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. The dosing interval may be at least about 1.5 to 5, 1.5, 2, 3, 4, or 5 days or more. For on-demand therapy, the dosing interval of the chimeric polypeptide or hybrid is approximately once every 24 to 36, 24 to 48, 24 to 72, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or more. It may also be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times longer than the dosing interval required for an equivalent Factor VIII that does not contain a non-Factor VIII portion, for example, does not contain an Fc portion (a polypeptide consisting of the Factor VIII). The dosing interval is about every 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. The dosing interval may be at least about 1.5 to 5, 1.5, 2, 3, 4, or 5 days or more. For on-demand therapy, the dosing interval of the chimeric polypeptide or hybrid is approximately once every 24 to 36, 24 to 48, 24 to 72, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or more.

[0069] Preferably, 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, or three times per week, or three times per week or less. Preferably, the effective amount is 65 IU / kg and the dosing interval is once a week or once every 6 to 7 days.

[0070] "Long-acting Factor VIII" is a Factor VIII having an increased half-life (also referred to herein as t1 / 2, t1 / 2 beta, elimination half-life, and HL) compared to the reference Factor VIII. The increased half-life of long-acting Factor VIII can be due to, for example, fusion to one or more non-Factor VIII polypeptides such as Fc, XTEN, or albumin. The increased half-life can be due to one or more modifications such as, for example, pegylation. Exemplary long-acting Factor VIII polypeptides include, for example, chimeric Factor VIII polypeptides containing Fc, chimeric Factor VIII polypeptides containing XTEN, and chimeric Factor VIII polypeptides containing albumin. Further exemplary long-acting Factor VIII polypeptides include, for example, pegylated Factor VIII.

[0071] The "reference" polypeptide in the case of a long-acting chimeric Factor VIII polypeptide is a polypeptide consisting essentially of the Factor VIII portion of the chimeric polypeptide, such as the same Factor VIII portion without an Fc portion, XTEN portion, or albumin portion. Similarly, the reference polypeptide in the case of a modified Factor VIII is the same unmodified Factor VIII, for example, non-pegylated Factor VIII.

[0072] In some embodiments, when the long-acting Factor VIII is administered to a subject, it has one or more of the following characteristics: An average residence time (MRT) (activity) in the subject of about 14 to 41.3 hours; A clearance (CL) (activity) in the subject of about 1.22 to 5.19 mL / h / kg or less; A t1 / 2 beta (activity) in the subject of about 11 to 26.4 hours; An incremental recovery (K value) (activity; observed value) in the subject of about 1.38 to 2.88 IU / dL per 1 IU / kg; A Vss (activity) in the subject of about 37.7 to 79.4 mL / kg; and An AUC / dose in the subject of about 19.2 to 81.7 IU*h / dL per 1 IU / kg.

[0073] In some embodiments, the long-acting Factor VIII has one or more of the following characteristics when administered to a patient population: An average incremental recovery rate (K value) (activity; observed value) greater than 1.38 IU / dL per 1 IU / kg; An average incremental recovery rate (K value) (activity; observed value) of at least about 1.5, at least about 1.85, or at least about 2.46 IU / dL per 1 IU / kg. An average clearance (CL) (activity) in the patient population of about 2.33 ± 1.08 mL / h / kg or less; An average clearance (CL) (activity) in the patient population of about 1.8 to 2.69 mL / h / kg; An average clearance (CL) (activity) in the patient population that is about 65% of the clearance of the polypeptide containing Factor VIII without modification; An average residence time (MRT) (activity) in the patient population of at least about 26.3 ± 8.33 hours; An average MRT (activity) in the patient population of about 25.9 to 26.5 hours; An average MRT (activity) in the patient population that is about 1.5 times longer than the average MRT of the polypeptide containing Factor VIII without modification; Mean t1 / 2 beta (activity) in the patient population approximately 18.3 ± 5.79 hours before; Mean t1 / 2 beta (activity) in the patient population approximately 18 to 18.4 hours; Mean t1 / 2 beta (activity) in the patient population approximately 1.5 times longer than the mean t1 / 2 beta of the polypeptide containing Factor VIII without modification; Mean incremental recovery rate (K value) (activity; observed value) in the patient population approximately 2.01 ± 0.44 IU / dL per 1 IU / kg; Mean incremental recovery rate (K value) (activity; observed value) in the patient population approximately 1.85 to 2.46 IU / dL per 1 IU / kg; Mean incremental recovery rate (K value) (activity; observed value) in the patient population that is approximately 90% of the mean incremental recovery rate of the polypeptide containing Factor VIII without modification; Mean Vss (activity) in the patient population approximately 55.1 ± 12.3 mL / kg; Mean Vss (activity) in the patient population approximately 45.3 to 56.1 mL / kg; Mean AUC / dose (activity) in the patient population approximately 49.9 ± 18.2 IU*h / dL per 1 IU / kg; Mean AUC / dose (activity) in the patient population approximately 44.8 to 57.6 IU*h / dL per 1 IU / kg.

[0074] "On-demand treatment", as used herein, is intended to be performed over a short period of time and means treatment in response to a current condition such as a bleeding symptom or in response to a recognized need such as a planned surgery. Conditions that may require on-demand treatment include, for example, bleeding symptoms, joint hemarthrosis, muscle bleeding, oral bleeding, massive bleeding, massive bleeding into muscle, massive oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial massive bleeding, intra-abdominal massive bleeding, intrathoracic massive bleeding, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the iliopsoas sheath. Subjects may require treatment for surgical prophylaxis, intraoperative management, or surgery. Such surgeries include, for example, minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal hernia incision, synovectomy, total knee arthroplasty, craniotomy, bone fixation, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

[0075] Preferably, on-demand treatment resolves more than 80% (more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or 100%) or 80 - 100%, 80 - 90%, 85 - 90%, 90 - 100%, 90 - 95%, or 95 - 100% of bleeding (e.g., spontaneous bleeding) with a single dose. Preferably, more than 80% (more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more 2%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or 100%) or 80 - 100%, 80 - 90%, 85 - 90%, 90 - 100%, 90 - 95%, or 95 - 100% of bleeding symptoms are evaluated as excellent or good by a physician after on-demand treatment. Preferably, more than 5% (more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%), or 5 - 20%, 5 - 15%, 5 - 10%, 10 - 20%, or 10 - 15% of bleeding symptoms are evaluated as fair by a physician after on-demand treatment.

[0076] "Polypeptide", "peptide" and "protein" are used interchangeably and refer to a polymeric compound composed of covalently linked amino acid residues.

[0077] "Polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymeric compound consisting of covalently linked nucleotide residues. The polynucleotide can be DNA, cDNA, RNA, single-stranded or double-stranded, a vector, plasmid, phage, or virus. Examples of polynucleotides include those in Table 1 encoding the polypeptides in Table 2 (see Table 1). Further examples of polynucleotides include fragments of the polynucleotides in Table 1, for example, fragments of the polypeptides in Table 2, for example, those encoding Factor VIII, Fc, signal sequences, 6His, and other fragments of the polypeptides in Table 2.

[0078] "Prophylactic treatment", as used herein, means administering the Factor VIII polypeptide to a subject in multiple doses over a long period of time to increase the level of Factor VIII activity in the subject's plasma. Preferably, the increased level is sufficient to reduce the incidence of spontaneous bleeding or to prevent bleeding in the event of, for example, an accidental injury. Preferably, during prophylactic treatment, the plasma protein level in the subject does not drop below the subject's baseline level or below the level of Factor VIII characteristic of severe hemophilia (<1 IU / dl [1%]).

[0079] Preferably, the prophylaxis is "adjusted" for each patient by determining the PK data for each patient and administering the factor VIII of the present invention at dosing intervals that maintain trough levels of 1-3% FVIII activity. Adjustment can be made if the subject experiences unacceptable bleeding symptoms defined as two or more breakthrough bleeding episodes over a two-month period. In this case, the adjustment targets trough levels of 3-5%. Preferably, as a result of prophylactic treatment, prevention and control of bleeding, continuous control of bleeding, continuous protection from bleeding, and / or continuous benefit are obtained. Prevention, such as continuous protection, can be demonstrated by increased AUC (AUC-LAST) and decreased clearance up to the final measurement time point, resulting in an increased terminal phase t1 / 2 compared to short-acting FVIII. Preferably, prophylaxis is demonstrated by better Cmax, better Tmax, and / or greater mean residence time compared to short-acting FVIII. Preferably, as a result of prophylaxis, breakthrough bleeding episodes do not occur within about 24, 36, 48, 72, or 96 hours (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours, preferably within 72 hours) after injection (e.g., the last injection). Preferably, as a result of prophylaxis, with once-weekly dosing (e.g., 65 IU / kg), more than 30% (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, or more than 90%, preferably more than 50%) of the average annual reduction in bleeding symptoms.

[0080] As used herein, "subject" means a human or non-human mammal. Non-human mammals include, for example, mice, dogs, primates, monkeys, cats, horses, cows, pigs, and other domestic and small animals.

[0081] As used herein, "therapeutic amount" means the dosage that achieves the therapeutic goal as described herein. The calculation of the required dosage of factor VIII is based on the empirical finding that, on average, 1 IU of factor VIII per kg of body weight raises the plasma factor VIII activity by about 2 IU / dL. The required dosage is given by the formula: Required units = body weight (kg) × desired factor VIII increase (IU / dL or % of normal value) × 0.5 (IU / kg per IU / dL) and is determined using.

[0082] The therapeutic amount that can be used in the method of the present invention is about 10 - 100 IU / kg, and more specifically, 10 - 20, 20 - 30, 30 - 40, 40 - 50, 50 - 60, 60 - 70, 70 - 80, 80 - 90, or 90 - 100 IU / kg, and more specifically, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / kg.

[0083] A further therapeutic amount that can be used in the method of the present invention is about 10 - about 150 IU / kg, and more specifically, about 100 - 110, 110 - 120, 120 - 130, 130 - 140, 140 - 150 IU / kg, and more specifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150 IU / kg.

[0084] As used herein, "variant" refers to a polynucleotide or polypeptide that differs from the original polynucleotide or polypeptide but retains its basic properties, such as factor VIII coagulant activity or Fc (FcRn binding) activity. Generally, variants are overall very similar and are identical to the original polynucleotide or polypeptide in many regions. Variants include, for example, polypeptide and polynucleotide fragments, deletion, insertion, and modified versions of the original polypeptide.

[0085] Variant polynucleotides can include, for example, nucleotide coding sequences (factor VIII portion, Fc portion (individually or together)) in SEQ ID NO: 1, 3, 5, 7, 9, or 11 or their complementary strands, known mutants and recombinant factor VIII or Fc nucleotide coding sequences or their complementary strands as disclosed in the publications and patents referred to herein, nucleotide sequences (factor VIII portion, Fc portion (individually or together)) encoding the polypeptides of SEQ ID NO: 2, 4, 6, 8, 10, or 12, and / or nucleotide sequences that are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to or composed of any polynucleotide fragment of these nucleic acid molecules (e.g., the fragments described herein). Polynucleotides that hybridize to these nucleic acid molecules under stringent hybridization conditions or low stringency conditions are also included as variants, as long as these polynucleotides are functional, in the same way as the polypeptides encoded by these polynucleotides.

[0086] Variant polypeptides are represented, for example, by SEQ ID NO: 2, 4, 6, 8, 10, or 12 a polypeptide sequence (Factor VIII moiety, Fc moiety (individually or together)), and / or a polypeptide fragment of any of these polypeptides (e.g., the fragments described herein), and may comprise or consist of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to such polypeptide sequence or polypeptide fragment.

[0087] A nucleic acid having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may contain mutations up to 5 points per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted, or replaced with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted with a plurality of nucleotides in the reference sequence. The query sequence may be, for example, the entire sequence shown in SEQ ID NO: 1 or 3, an ORF (open reading frame), or any fragment identified herein.

[0088] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequences or polypeptides of the present invention can be determined in the usual way using known computer programs. A preferred method for determining the best overall match between a query sequence (reference or original sequence) and a target sequence (also called global sequence alignment) can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245), which is hereby incorporated by reference in its entirety. In sequence alignment, both the query sequence and the target sequence are DNA sequences. RNA sequences can be compared by converting U to T. The results of the global sequence alignment are expressed as % identity. The preferred parameters used in the FASTDB alignment of DNA sequences for calculating % identity are as follows: Matrix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty 0.05, Window Size = 500 or the length of the target nucleotide sequence (whichever is shorter).

[0089] If the target array is shorter than the query array due to 5' or 3' deletions rather than internal truncations, manual correction must be applied to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the target array when calculating % identity. For a target array truncated at the 5' or 3' end compared to the query array, the % identity is corrected by calculating the number of bases in the query array that are at the 5' and 3' of the target array that do not match / align as a percentage of all bases in the query array. Whether a nucleotide matches / aligns is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the % identity calculated by the FASTDB program using specific parameters to obtain the final % identity score. This corrected score is the one used for the purposes of the present invention. As shown by the FASTDB alignment, only the bases outside the 5' and 3' bases of the target array that do not match / align with the query array are calculated for the purpose of manually adjusting the % identity score.

[0090] For example, a 90-base target array is aligned with a 100-base query array to determine % identity. A deletion occurs at the 5' end of the target array, and thus the FASTDB alignment does not show a match / align for the first 10 bases at the 5' end. The 10 unpaired bases are 10% of the sequence (number of bases at the non-matching 5' and 3' ends / total number of bases in the query array), and thus 10% is subtracted from the % identity score calculated by the FASTDB program. If the remaining 90 bases match perfectly, the final % identity is 90%. In another example, a 90-base target array is compared to a 100-base query array. This time, the deletion is an internal deletion, so the bases that do not match / align with the query are not at the 5' or 3' of the target array. In this case, the % identity calculated by FASTDB is not manually corrected. Again, only the 5' and 3' bases of the target array that do not match / align with the query array are manually corrected. No other manual corrections are made for the purposes of the present invention.

[0091] A polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to the query amino acid sequence of the present invention means that the amino acid sequence of the target polypeptide is identical to the query sequence, except that the target polypeptide sequence may contain up to 5 amino acid modifications per 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the target sequence can be inserted, deleted, (indels) or substituted with another amino acid. These modifications of the reference sequence may occur anywhere between the amino or carboxy terminal positions of the reference amino acid sequence or between those terminal positions, singly among the residues in the reference sequence, or interspersed in any of one or more contiguous groups within the reference sequence.

[0092] As a practical matter, whether any particular polypeptide is, for example, at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2 (Factor VIII moiety, Fc moiety (either or together)), or 4, or the amino acid sequence of a known Factor VIII or Fc polypeptide sequence can be determined in the usual way using known computer programs. A preferred method for determining the best overall match between a query sequence (reference or original sequence) and a target sequence (also called global sequence alignment) can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990) (which is hereby incorporated by reference in its entirety)). In sequence alignment, the query and target sequences can be either both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is expressed as % identity. Preferred parameters used in the FASTDB amino acid alignment are as follows: matrix = PAM0, k-tuple = 2, mismatch penalty = 1, joining penalty = 20, randomization group length = 0, cutoff score = 1, window size = sequence length, gap penalty = 5, gap size penalty = 0.05, window size = 500 or the length of the target amino acid sequence (whichever is shorter).

[0093] If the target array is shorter than the query array due to N- or C-terminal deletion rather than internal deletion, manual correction must be made to the results. This is because the FASTDB program does not account for N-terminal and C-terminal truncations of the target array when calculating global % identity. For a target array truncated at the N- and C-termini compared to the query array, the % identity is corrected by calculating the number of residues in the query array that do not match / align with the corresponding target residues at the N- and C-termini of the target array as a percentage of all bases in the query array. Whether a residue matches / aligns is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the % identity calculated by the FASTDB program using specific parameters to obtain the final % identity score. This final % identity score is the one used for the purposes of the present invention. Only the residues at the N- and C-termini of the target array that do not match / align with the query array are considered for the purpose of manually adjusting the % identity score. That is, only the query residues are located outside the outermost N- and C-terminal residues of the target array.

[0094] For example, a 90 - amino - acid residue target sequence is aligned with a 100 - residue query sequence to determine % identity. The deletion occurs at the N - terminus of the target sequence, and thus, the FASTDB alignment does not show a match / alignment for the first 10 residues at the N - terminus. The 10 unpaired residues are 10% of the sequence (the number of non - matching N - and C - terminal residues / total number of residues in the query sequence), and thus, 10% is subtracted from the % identity score calculated by the FASTDB program. If the remaining 90 residues match perfectly, the final % identity is 90%. In another example, a 90 - residue target sequence is compared with a 100 - residue query sequence. This time, since the deletion is an internal deletion, there are no N - or C - terminal residues of the target sequence that do not match / align with the query. In this case, the % identity calculated by FASTDB is not corrected manually. Again, only the residue positions outside the N - and C - termini of the target sequence that do not match / align with the query sequence, as shown by the FASTDB alignment, are corrected manually. No other manual corrections are made for the purposes of the present invention.

[0095] Polynucleotide variants can include modifications in the coding region, non - coding region, or both. Particularly preferred are polynucleotide variants that result in silent substitutions, additions, or deletions, but do not modify the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Additionally, variants in which 5 - 10, 1 - 5, or 1 - 2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for various reasons, such as to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by a bacterial host such as E. coli).

[0096] 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, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can be diverse at either the polynucleotide and / or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants can be produced by mutagenesis techniques or by direct synthesis.

[0097] Variants can be generated using known methods of protein engineering and recombinant DNA technology to improve or modify the properties of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without substantially losing biological function. Ron et al., J. Biol. Chem. 268: 2984-2988 (1993) (incorporated herein by reference in its entirety) reported a mutant KGF protein that still had heparin-binding activity even after deletion of 3, 8, or 27 amino-terminal amino acid residues. Similarly, interferon gamma showed up to 10-fold higher activity after deletion of 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988) (incorporated herein by reference in its entirety).

[0098] Furthermore, there is sufficient evidence to show 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 hereby incorporated by reference in its entirety) performed a detailed mutagenesis analysis of human cytokine IL-1α. They used random mutagenesis to generate over 3,500 individual IL-1α mutants with an average of 2.5 amino acid changes per mutant over the full length of the molecule. Multiple mutations were investigated at every possible amino acid position. The investigators found that "most of the molecule can be modified with little or no effect on either [binding or biological activity]." (See summary). In fact, out of the over 3,500 nucleotide sequences investigated, only 23 unique amino acid sequences produced proteins with significantly different activity from the wild type.

[0099] As described above, polypeptide mutants include, for example, modified polypeptides. 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 hereby incorporated by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. In some embodiments, Factor VIII is modified, such as pegylated, at any convenient position. In some embodiments, Factor VIII is pegylated with a cysteine that is surface-exposed on Factor VIII, preferably a surface-exposed cysteine that has been engineered. Mei et al. (2010). In some embodiments, the modified Factor VIII, such as pegylated Factor VIII, is a long-acting Factor VIII.

[0100] The "volume of distribution at steady state (Vss)" has the same meaning as this term is used in pharmacology, which is the apparent space (volume) into which a drug is distributed, as used herein. Vss = the amount of drug in the body divided by the plasma concentration at steady state.

[0101] "About," as used herein with respect to a range, modifies both ends of the range. Thus, "about 10-20" means "about 10-about 20."

[0102] Although the invention has been described in detail, it is not intended to be limited, and will be more clearly understood by reference to the following examples, which are included for illustrative purposes only. All patents and publications mentioned herein are expressly incorporated herein by reference.

Examples

[0103] Example 1 Summary A recombinant factor VIII-Fc (rFVIIIFc) fusion protein lacking the B domain was generated to extend the half-life of FVIII. rFVIIIFc was studied in mouse and dog models of severe hemophilia A and compared with rFVIII (ReFacto®). The whole blood clotting time (WBCT) in hemophilia A mice was corrected to be 2- to 3-fold longer, and the elimination half-life in plasma was approximately 2-fold longer for rFVIIIFc compared to ReFacto®. In hemophilia A dogs, intravenous administration of rFVIIIFc (125I U / kg) corrected the WBCT to normal values. The WBCT remained less than 20 minutes (this time corresponds to FVIII:C > 1%) for approximately 96 hours, compared to 48 hours for dogs treated with ReFacto®. The elimination half-life of rFVIIIFc in dog plasma was 15.7 ± 1.7 hours and 15.4 ± 0.3 hours, respectively, when measured using ELISA or chromogenic assay. ReFacto® corrected the WBCT to approximately half the length of rFVIIIFc, and the plasma half-life was 7.0 hours. Thus, by fusing FVIII to Fc, a molecule was produced that has an increased plasma half-life and the ability to protect against bleeding for an extended period.

[0104] Introduction The reduction in mortality, prevention of joint damage, and improvement in quality of life are significant achievements due to the development of plasma-derived recombinant FVIII. Long-term protection from bleeding is another important advancement in the treatment of patients with hemophilia A. The inventors generated recombinant factor VIII-Fc (rFVIIIFc) chimeric proteins and hybrids as a method to extend the half-life of FVIII.

[0105] rFVIIIFc is a heterodimeric hybrid protein composed of B domain-deleted FVIII recombinantly fused to the Fc domain of human immunoglobulin G1 (IgG1) (Figure 1, SEQ ID NO:2; Table 2A) (this protein is also referred to herein as the FVIIIFc monomer Fc fusion protein, FVIIIFc monomer hybrid, monomeric FVIIIIFc hybrid, and FVIIIFc monomer-dimer). Fc enables binding to the neonatal Fc receptor (FcRn), which protects IgG from degradation and confers the 3-week half-life observed for IgG in humans (Ghetie V, and Ward ES., Annu. Rev. Immunol. 2000;18:739-766; Roopenian DC, and Akilesh S., Nature Rev. Immunol. 2007;7:715-725 (each of which is incorporated herein by reference in its entirety)).

[0106] The Fc domain of IgG1 has been fused to the ligand-binding regions of growth factors, cytokines, enzymes and receptors (Ashkanazi A, et al., Int. Rev. Immunol. 1993:10:219-27; Chamow SM, and Ashkanazi A, Trends Biotechnol. 1996:14:52-60; Fisher et al., N. Engl. J. Med. 1996:334(26):1697-702 (each of which is incorporated herein by reference in its entirety). Some of these have become important therapeutic molecules (e.g., etanercept, alefacept, abatacept). In these fusion proteins, two effector molecules bind to two Fc molecules. In this example, rFVIIIFc was constructed as a monomeric Fc fusion protein (one copy of the polypeptide consisting of the sequence in Table 2A(i) (SEQ ID NO: 2), with or without a signal sequence, and one copy of the polypeptide consisting of the sequence in Table 2A(ii) (SEQ ID NO: 4), with or without a signal sequence), i.e., using only one copy of the effector molecule (see Figure 1), and the studies presented herein compare the pharmacodynamics and pharmacokinetics of this novel protein to rFVIII in mouse and dog models of hemophilia A. The signal sequence is cleaved during secretion. This protein construct is referred to herein as the FVIIIFc monomeric Fc fusion protein, the FVIIIFc monomeric hybrid, the monomeric FVIIIIFc hybrid, and the FVIIIFc monomer-dimer. For the structure and production of this protein, see Example 1, Figure 1, Table 2A; and U.S. Patent Nos. 7,404,956 and 7,348,004 (each of which is incorporated herein by reference in its entirety).

[0107] Methods and Materials FVIII Preparations Recombinant FVIIIFc Using FVIII-specific primers, the coding sequence of human recombinant B domain-deleted FVIII was obtained by reverse transcription polymerase chain reaction (RT-PCR) from human liver polyA RNA (Clonetech). The FVIII sequence contains the native signal sequence for FVIII. The B domain deletion was from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638; 4969 bp) for an overall deletion of 2682 bp. For the structure and production of this protein, see Example 1, Figure 1, Table 2A; and U.S. Patent Nos. 7,404,956 and 7,348,004, the disclosures of which are incorporated herein by reference, respectively.

[0108] Using Fc-specific primers, the coding sequence for human recombinant Fc was obtained by RT-PCR from a human leukocyte cDNA library (Clonetech). Primers were designed such that the B domain-deleted FVIII sequence was directly fused to the N-terminus of the Fc sequence without an intervening linker. The FVIIIFc DNA sequence was cloned into the mammalian dual expression vector pBUDCE4.1 (Invitrogen) under the control of the CMV promoter. A second identical Fc sequence containing the mouse Igk signal sequence was obtained by RT-PCR and cloned downstream of the second promoter EF1α in the expression vector pBUDCE4.1.

[0109] Using Lipofectamine 2000 transfection reagent (Invitrogen), the rFVIIIFc expression vector was transfected into human embryonic kidney 293 cells (HEK293H; Invitrogen). Stable clone cell lines were generated by selection with zeocin (Invitrogen, Carlsbad, CA). Using one clone cell line 3C4-22, FVIIIFc was generated for in vivo characterization. Recombinant FVIIIFc was produced and purified (McCue JT, et al., J. Chromatogr. A 2009; 7824-7830 (this description is incorporated herein by reference), Biogen Idec (Cambridge, MA)). The above transfection strategy was expected to yield three products, namely monomeric rFVIIIFc hybrid, dimeric rFVIIIFc hybrid and dimeric Fc. However, essentially no dimeric rFVIIIFc was detected in the conditioned medium from these cells. Rather, the conditioned medium contained Fc and monomeric rFVIIIFc. It is thought that the dimeric rFVIIIFc was too large to allow efficient secretion from the cells. This result was beneficial as monomer purification was not complicated by the presence of all three proteins. The substances used in these tests had a specific activity of approximately 9000 IU / mg. Furthermore, these human cells yielded higher protein levels than the other cells tested in this experiment.

[0110] Recombinant FVIII Recombinant B domain-deleted FVIII (ReFacto®) was purchased from Novis Pharmaceuticals and prepared according to the manufacturer's instructions. ReFacto® (recombinant B domain-deleted FVIII) has the same amino acid sequence as amino acids 1-1438 of SEQ ID NO: 2.

[0111] Hemophilia A animals The hemophilia A mice were obtained from Dr. Kazazian at the University of Pennsylvania (Bi L, et al., Nat. Genet. 1995; 10(1): 119-121; the contents of this description are incorporated herein by reference) and are FVIII exon 16 knockouts on a 129×B6 background bred at Syntonix. These mice show prolonged whole blood clotting time (>60 minutes) and thus are a good model of severe hemophilia A.

[0112] The hemophilia A dogs were from an inbred colony maintained at the Francis Owen Blood Research Laboratory at the University of North Carolina (Chapel Hill) (Graham, JB, et al., J. Exp. Med. 1949; 90: 97-111; the contents of this description are incorporated herein by reference). These dogs have a severe hemophilia phenotype comparable to the severe form of the human disease (Graham, JB, et al., J. Exp. Med. 1949; 90: 97-111; Lozier, JN, et al., Proc. Natl. Acad. Sci. 2002; 99: 12991-12996; the contents of these descriptions are each incorporated herein by reference).

[0113] Study Design Hemophilia A mouse test The effects of rFVIIIFc and ReFacto® on whole blood clotting time (WBCT) were tested in FVIII-deficient mice. Each protein was administered intravenously at 50 IU / kg, and blood was collected from the tail vein of each mouse before dosing and at various time points after dosing. Blood samples were incubated in capillary tubes at 37°C and visually inspected for the presence of a blood clot once per minute. The time to blood clot formation was recorded. If a blood clot did not occur by 60 minutes, the clotting time was recorded as >60 minutes. Blood from normal mice clots in approximately 4 minutes (range 2-7 minutes; n = 10 mice) in the WBCT assay.

[0114] In the second set of tests, hemophilia A mice were administered a single intravenous dose of 50 IU / kg rFVIIIFc, ReFacto® or Advate® (4 mice / time point). At 0.25, 8, 24, 48 and 72 hours after dosing, blood was collected by cardiac puncture into one-tenth volume of 3.2% sodium citrate. Plasma was prepared and stored at -80°C until analysis for FVIII activity using an FVIII-specific chromogenic assay.

[0115] Hemophilia A dog study In a single-dose PK / PD study of rFVIIIFc, two hemophilia A dogs from the Chapel Hill colony were administered a single intravenous dose of 125 IU / kg and blood samples were collected before dosing and at selected time points after dosing for WBCT, activated partial thromboplastin time (aPTT), FVIIIFc plasma concentration, hematology and serum chemistry. Time points for WBCT included before dosing, 5 and 30 minutes after dosing, and 1, 2, 4, 8, 24, 32, 48, 72, 96, 144 and 168 hours after dosing. Blood collections for coagulation activity (aPTT) and FVIIIFc plasma concentration included the above time points for WBCT, as well as 15 minutes and 3, 6, 12 hours after dosing.

[0116] A second study was performed in which ReFacto® (114 IU / kg for dog M12 and 120 IU / kg for dog M38) was administered intravenously. WBCT was measured until the clotting time was ≥20 minutes (corresponding to FVIII:C > 1%), then 125 IU / kg of rFVIIIFc was administered intravenously to the same dogs and blood samples were collected for WBCT, aPTT, FVIIIFc, plasma concentration, hematology and serum chemistry. Time points for WBCT included before dosing, 5 and 30 minutes after dosing, and 1, 2, 4, 8, 24, 32, 48, 72 hours after dosing. Blood was also collected for FVIIIFc at 96, 120, 144 and 168 hours after dosing. Blood collections for coagulation activity (aPTT) and FVIIIFc plasma concentration included the above time points for WBCT, as well as 15 minutes after dosing and It included 3, 6, and 12 hours.

[0117] The WBCT procedure in hemophilia A dogs was slightly different from that in hemophilia A mice. After dosing with rFVIIIFc or ReFacto®, 1 mL of blood was collected at various time points, 0.5 mL was dispensed into two siliconized glass tubes, and then this was placed in a 28 °C water bath. Starting at 1 minute, one test tube was shaken up and down every 30 seconds, and the second test tube remained static. Once a blood clot formed in the shaken test tube, the second test tube was then shaken up and down every 30 seconds until a blood clot formed. The time to a fully gelled blood clot in the second test tube was recorded as WBCT.

[0118] FVIII activity in plasma Measurement of FVIII activity in plasma by FVIII-specific chromogenic assay Plasma samples were tested for FVIII activity by an automated chromogenic method using the Sysmex CA1500 instrument and reagents from Siemens Healthcare Diagnostics (Dallas, TX; kit number B4238 - 40). The activity of rFVIIIFc was determined using a standard curve prepared with the 7th International Standard Factor FVIII concentrate (NIBSC code 99 / 678) added to human FVIII - deficient plasma (Stago USA) at concentrations in the range of 1.5 - 0.016 IU / mL.

[0119] Measurement of rFVIIIFc or FVIII by ELISA FVIIIFc in canine plasma by ELISA An FVIII antibody specific for the A1 domain (Green Mountain Antibodies: GMA - 8002) was coated onto a 96 - well plate and incubated at 37 °C for 1 hour. The coated plate was blocked with Tris - buffered saline containing Tween 20, CaCl 2 and bovine serum albumin at room temperature for 1 hour, and then standards, controls, and samples prepared in normal dog plasma were diluted 1:10 and then added to the plate and incubated at 37 °C for 1 hour. The plate was washed, and then donkey (F(ab)’ 2) Anti-human Fc-HRP (Jackson: 709-036-098) was added and incubated at 37 °C for 1 hour. After washing, TMB (BioFx Ultra-Sensitive Substrate: TMBS-0100-01) was added to the plate and the substrate reaction was quenched with acid, and absorbance was measured at 450 nm using a SpectraMax Plus plate reader (Molecular Devices).

[0120] ReFacto® in canine plasma by ELISA An anti-FVIII antibody specific for the A1 domain on the heavy chain (Green Mountain Antibodies: GMA-8002) was coated onto a 96-well plate and incubated at room temperature for 2 hours. The coated plate was blocked at 37 °C for 1 hour. After washing, standards, controls and samples were prepared in normal canine plasma and then diluted 1:10 and added to the plate and incubated at room temperature for 2 hours. The plate was washed and then treated with a detection antibody, a pre-diluted anti-FVIII horseradish peroxidase conjugate (Affinity Biologicals: F8C-EIA-D) and incubated at room temperature for 1 hour. After washing, TMB (BioFx Ultra-Sensitive Substrate: TMBS-0100-01) was added to the plate for 10 minutes. The substrate reaction was quenched with acid and the signal was measured at a wavelength of 450 nm using a SpectraMax Plus plate reader (Molecular Devices).

[0121] Measurement of fibrinogen According to the manufacturer's instructions, the concentration of fibrinogen in plasma was measured at Esoterix (Research Triangle Park, NC) using a kit containing HemosIL® PT-Fibrinogen-HS reagent (ILL, MA, catalog number 0008468210) and an ACL 7000 coagulation analyzer (Beckman Coulter).

[0122] Measurement of platelets ​Platelets were counted in EDTA-anticoagulated whole blood by an automated method using a Vet-ABC-Diff hematology analyzer (SCIL Animal Care Co., Gurnee, IL) programmed with species-specific smart cards.

[0123] Pharmacokinetic analysis Pharmacokinetic parameters were calculated by non-compartmental analysis using WinNonlin software, version 5.2 (Pharsight, Mountain View, Ca). PK parameters included the maximum concentration in plasma (C max ), the area under the plasma concentration-time curve (AUC), the elimination half-life (t 1 / 2 ), the volume of distribution (Vss), and the clearance (Cl).

[0124] Results Recombinant FVIII-Fc rFVIIIFc is a recombinant fusion of human B domain-deleted FVIII without an intervening linker sequence and Fc from human IgG1 (rFVIIIFc: Figure 1).

[0125] Purified rFVIIIFc has a specific activity of approximately 9000 IU / mg as measured using a chromogenic activity assay. Recombinant B domain-deleted FVIII (ReFacto®) has reported specific activities of 9110 - 13700 IU / mg. Conversion of specific activity to IU / nmol to account for the size difference between FVIIIFc and ReFacto® (216 kDa and 170 kDa, respectively) indicates that the two proteins have approximately equivalent specific activities (1970 IU / nmol for rFVIIIFc and 1521 - 2287 IU / nmol for ReFacto®). Thus, the FVIII activity of rFVIIIFc is not affected by the fusion of the C-terminus of human FVIII and the N-terminus of human Fc.

[0126] Administration to hemophilia A mice A single dose of 50 IU / kg of rFVIIIFc or ReFacto® was administered intravenously to FVIII-deficient mice (n = 6 / group). Blood samples were collected before dosing and up to 120 hours after dosing, and WBCT was determined as described in Materials and Methods. Baseline WBCT was greater than 60 minutes. Data from a representative experiment are shown in Figure 2 and Table 3. Immediately after dosing with rFVIIIFc or ReFacto®, WBCT was corrected to 2 - 17 minutes. Blood from mice treated with ReFacto® lost the ability to clot by 42 hours, while blood from all mice treated with rFVIIIFc was still clotting at 96 hours, and blood from 1 of 6 mice was clotting at 113 hours but had lost the ability to clot by 120 hours. These data suggest that the duration of action for rFVIIIFc is approximately 2 - 3 hours longer than that of ReFacto®.

[0127] The chromogenic activity of rFVIIIFc, ReFacto® or Advate® (full-length recombinant FVIII) was tested in FVIII-deficient mice after a single intravenous dose of 50 IU / kg. Blood was collected before dosing and at 8, 24, 48 and 72 hours after dosing. Activity was measured using an FVIII-specific chromogenic assay. This is shown in Figure 3. Pharmacokinetic parameters are reported in Table 4. The circulating half-life for rFVIIIFc was approximately 1.6 - 2-fold longer (11.1 hours) compared to Advate® (7 hours) and ReFacto® (5 hours). Cmax was 0.47 ± 0.30 IU / mL for Advate® and ReFacto® Compared to 0.67 ± 0.44 IU / mL for the (trademark), it was 1.6 ± 0.36 IU / mL for rFVIIIFc. The systemic exposure of rFVIIIFc was significantly greater for rFVIIIFc (22.6 hour·IU / mL) compared to ReFacto® (6.94 hour·IU / mL) and Advate® (3.90 hour·IU / mL), and in hemophilia A mice, the clearance for rFVIIIFc was significantly lower (2.09 mL / hour / kg) compared to both ReFacto® (7.2 mL / hour / kg) and Advate® (12.8 hour / mL / kg).

[0128] Administration to Hemophilia A Dogs The pharmacodynamics (PD) and pharmacokinetics (PK) of rFVIIIFc were tested in the Chapel Hill colony of hemophilia A dogs. A single intravenous dose of 125 IU / kg of rFVIIIFc was administered to each of 4 hemophilia A dogs, and WBCT was immediately corrected to normal (Figure 4). The range of WBCT in normal dogs is 8 - 12 minutes. WBCT remained below 20 minutes for approximately 96 hours, which coincides with FVIII:C > 1%. However, 1 dog showed WBCT < 20 minutes for 72 hours. Additionally, aPTT was also immediately corrected to normal (Table 6). Plasma concentrations of rFVIIIFc were measured using a specific ELISA intended to detect both the FVIII and Fc portions of the molecule. The plasma concentration vs. time curve is shown in Figure 5. PK analysis of the data showed that t 1 / 2 was 15.7 ± 1.7 hours (Table 5). Similar results were obtained when rFVIIIFc was measured using an FVIII-specific chromogenic activity assay (t 1 / 2 = 15.4 ± 0.3 hours, Table 5). The plasma concentration vs. time curves were similar using both methods (Figures 5 and 6). When activity data were converted from IU / mL to ng / mL using the specific activity for rFVIIIFc, a good correlation with ELISA data was observed, demonstrating that the protein measured by ELISA was sufficiently active.

[0129] Of the dogs treated with rFVIIIFc, two also received a single dose of ReFacto® 72 hours before rFVIIIFc administration (dog M12 received 114 IU / kg and dog M38 received 120 IU / kg). WBCT and aPTT were corrected normally immediately after ReFacto® administration. However, normalization of WBCT after a single dose of rFVIIIFc continued for approximately twice as long as compared to ReFacto® (Figure 4). Furthermore, when protein concentrations in plasma were measured by ELISA, the plasma half-life of rFVIIIFc (15.7 ± 1.7 hours) was approximately twice as long for rFVIIIFc compared to ReFacto® (7.0 and 6.7 hours) (Table 5). Similar results were obtained when the two molecules were measured by FVIII-specific chromogenic activity.

[0130] To assess the potential risk of thrombogenicity, platelets and fibrinogen were measured. After dosing with rFVIIIFc or ReFacto®, the platelet count and plasma fibrinogen concentration did not change from pre-dose values (data not shown).

[0131] Discussion Recombinant FVIIIFc was produced in human embryonic kidney 293 (HEK293) cells from a stably transfected cell line and purified from cell culture medium. Production in a human cell line represents a significant change in manufacturing compared to commonly available rFVIII products produced in Chinese hamster ovary cells or hamster neonatal kidney cells. The rationale for this change was that human cells were predicted to be best equipped to perform the post-translational modifications required for the FVIII portion of this molecule.

[0132] To account for the difference in molecular weight between rFVIIIFc and recombinant B domain-deleted FVIII (ReFacto®), the conversion of specific activity to IU / nmol showed that the specific activity was similar for both proteins (1970 IU / nmol for rFVIIIFc and 1521 - 2287 IU / nmol for ReFacto®). Since the C1 and C2 domains of FVIII are involved in phospholipid binding that is essential for all FVIII activity, it is somewhat surprising that the specific activity for rFVIIIFc is not affected by the fusion of the C-terminus of FVIII and the N-terminus of Fc (Fay, PJ, J. Hematology 83: 103 - 8 (2006) and Raut, S, et al., Br. J. Haematol. 107: 323 (1999); the disclosures of which are hereby incorporated by reference in their entireties).

[0133] The treatment of hemophilia A is demand - responsive at the time of bleeding symptom onset or by prophylaxis for bleeding prevention. Although on - demand treatment is still frequently used, there is a trend towards prophylaxis and prevention of joint damage (Blanchette P, et al., Haematophilia 2004: 10; 679 - 683; Manco - Johnson, MJ. Et al., N. Engl. J. Med. 2007; 357: 535 - 544; the contents of these descriptions are each incorporated herein by reference). To maintain FVIII:C above 1% in patients, due to the relatively short half - life of 10 - 12 hours, for prophylaxis, general FVIII products are administered every 2 - 3 days (Morfini, M, Haemophilia 2003; 9 (suppl 1): 94 - 99; discussion 100; White GC, et al., Thromb. Haemost. 1997: 77: 660 - 7; Blanchette, P, et al., J. Thromb. Haemost. 2008 Aug; 6(8): 1319 - 26; the contents of these descriptions are each incorporated herein by reference). Long - acting FVIII therapies that provide long - term protection from bleeding show a significant improvement in the quality of life of patients with hemophilia A. Strategies to extend the half - life of clotting factors have been successful for other molecules, for example, pegylation (Rostin J. et al., Bioconj. Chem. 2000; 11: 387 - 96; the content of this description is incorporated herein by reference), glycopegylation (Stennicke HR, et al., Thromb. Haemost. 2008; 100: 920 - 8; the content of this description is incorporated herein by reference), formulations using pegylated liposomes (Spira J, et al., Blood ;2006; 108: 3668 - 3673; Pan J, et al., Blood 2009; 114: 2802-2811; each of these disclosures is incorporated herein by reference) and conjugation with albumin (Schulte S., Thromb. Res. 2008; 122 Suppl 4: S14-9; this disclosure is incorporated herein by reference). Pegylation represents an approach to reduce clearance, however, the effect of in vivo modification is currently unknown. The result of direct pegylation of FVIII in vivo is currently unknown, while FVIII formulated with pegylated liposomes has been clinically studied and shown to have a moderate or no effect on bleeding duration (Spira J, et al., Blood ;2006; 108: 3668-3673; Spira J, et al., Thromb. Haemost. 2008 Sep; 100(3): 429-34; each of these disclosures is incorporated herein by reference).

[0134] The approach of the present invention for extending the half-life of FVIII was to recombinantly fuse FVIII with the Fc domain of IgG1. Fc binds to the natural receptor FcRn, and its normal function is the protection of IgG from degradation. The results described herein are for hemophilia A mice and hemophilia A dogs, representing the initial pharmacokinetic and efficacy characterization of rFVIIIFc compared to rFVIII products. In both species, the half-life of rFVIIIFc was approximately 2-fold that of rFVIII when measured by FVIII activity or ELISA (dogs only). These data correlated well with the WBCT results from both animal models, i.e., the duration of the effect of rFVIIIFc on WBCT was approximately 2-fold longer compared to ReFacto®. In dogs, C maxAnd the clearance was similar for rFVIIIFc and ReFacto®, however, the AUC and volume of distribution at steady state were approximately 1.5-fold and 2-fold, respectively, for rFVIIIFc compared to ReFacto®. The PK parameters for ReFacto® in this animal model are consistent with the values reported in the literature (Brinkhous K, et al., Sem. Thromb. Haemost. 2002; 28: 269-272; the contents of this description are incorporated herein by reference).

[0135] If these findings translate to a similar half-life in humans, this represents a significant advance in the treatment of patients with hemophilia A.

[0136] Additional references (the contents of each of these descriptions are incorporated herein by reference)

[0137]

Number

[0138] Materials and Methods rFVIIIFc (Biogen Idec): Supplied as a frozen solution at a concentration of 1.2 mg / mL and 9882 IU / mL. The specific activity was 8235 IU / mg. Stored at -70°C. Diluted before injection.

[0139] Name: Xyntha® (Novis Pharmaceuticals); Supplied as a lyophilized powder. This was reconstituted according to the manufacturer's instructions to yield a solution with a nominal concentration of 525 IU / mL. Stored according to the manufacturer's recommendations.

[0140] Animals Using cynomolgus monkeys from the New Iberia Research Center (NIRC) colony, the study was conducted at NIRC (New Iberia, LA) under the approved NIRC IACUC protocol (APS 2008 - 8733 - 058) (NIRC study number 8733 - 0903).

[0141] Six naïve cynomolgus monkeys (3 males and 3 females) determined to be in good health were used in the study.

[0142] The study was conducted according to the protocol and UL Lafayette - NIRC standard operating procedures.

[0143] Study Design Each of the six monkeys (3 males and 3 females) was administered rFVIIIFc intravenously at 125 IU / kg. In a crossover study, the same animals were administered Xyntha® (BDD - rFVIII) intravenously at 125 IU / kg. Group 1 animals (n = 3) received Xyntha on day 0 and rFVIIIFc on day 3, while Group 2 animals (n = 3) received rFVIIIFc on day 0 and then Xyntha on day 4. For Group 2, the additional day between doses was to ensure that rFVIIIFc had sufficient time to decline below the planned baseline level. Blood was collected from each animal at pre - dose, 0.25, 4, 12, 24, 36, 48, and 72 hours post - dose into plasma in one - tenth volume of 3.2% sodium citrate for measurement of rFVIIIFc or Xyntha by ELISA as well as FVIII - specific chromogenic activity assay.

[0144] ELISA for Measuring rFVIIIFc and FVIII in Plasma Method for measuring rFVIIIFc in monkey plasma To quantify rFVIIIFc in monkey plasma, this enzyme-linked immunosorbent assay (ELISA) was designed. In this ELISA method, a goat anti-human IgG-(H+L) antibody (monkey, adsorbed) (catalog number A80-319A) from Bethyl Laboratories was diluted in coating buffer and immobilized on a 96-well microtiter sample plate. The plate was aspirated, and all non-adsorbed sites were blocked by adding blocking buffer (3% BSA / 1× Tris) at 37 °C for 2 hours. Plasma samples were diluted 1:20 with high calcium sample dilution buffer (3% non-fat dry milk / TBST, containing 30 mM CaCl 2 and dispensed onto the sample plate. The plate was incubated at 37 °C for approximately 2 hours. Then, the plate was washed, and a mouse anti-B domain deleted (α.BDDA1) factor VIII (A1 domain) antibody (catalog number GMA-8002) from Green Mountain Antibodies was added to the plate and incubated at 37 °C for approximately 1 hour. After washing the plate, an HRP-conjugated goat anti-mouse IgG2a antibody (catalog number 1080-05) from Southern Biotech was added to the plate and incubated at room temperature for approximately 30 minutes. The plate was washed again, a tetramethylbenzidine (TMB) peroxidase substrate solution was added, and the plate was incubated at room temperature for approximately 30 minutes. The reaction was stopped by adding a non-acidic stop solution. Color development was proportional to the amount of rFVIIIFc in the sample. The plate was read using an absorbance plate reader at a single detection wavelength of 650 nm. The rFVIIIFc concentration was determined using a standard curve obtained by plotting the optical density (OD) against the concentration using a 4-parameter logistic curve fitting program. The calibration curve range of this method was 0.400 ng / mL to 51.2 ng / mL in 5% monkey plasma (8.00 ng / mL to 1024 ng / mL in 100% monkey plasma). A single calibrator outside the assay qualification range at 0.200 ng / mL in 5% monkey plasma could be included and serve as an anchor point to facilitate curve fitting. Based on the optimal fit of the curve, the anchor point was removed or retained (i.e., the highest number of standards were read within the limited precision, %RE ).

[0145] Method for measuring FVIII in monkey plasma To quantify FVIII in monkey plasma, this enzyme-linked immunosorbent assay (ELISA) was designed. In this ELISA method, a mouse αBDDA1 FVIII antibody (catalog number GMA-8002) from Green Mountain Antibodies was diluted in coating buffer and immobilized on a 96-well microtiter sample plate. The plate was aspirated and all non-adsorbing sites were blocked by adding blocking buffer (3% BST / 1× Tris) at 37 °C for 1 hour. Plasma samples were diluted 1:20 with high calcium sample dilution buffer (blocking buffer containing 100 mM CaCl 2 and distributed onto the sample plate. The plate was incubated at 37 °C for approximately 2 hours. After washing the plate, the detection antibody from the Affinity Biologicals Kit, an HRP-labeled polyclonal antibody (catalog number F8C-EIA-D), was further diluted in TBS / 0.05% Tween 20, added to the plate, and incubated at room temperature for approximately 1 hour. The plate was washed again, tetramethylbenzidine (TMB) peroxidase substrate solution was added, and the plate was incubated at room temperature for approximately 30 minutes. The reaction was stopped by adding acidic stop solution. Color development is proportional to the amount of FVIIIFc in the sample. The plate was read with an absorbance plate reader using a single detection wavelength of 450 nm. The FVIII concentration was determined using a standard curve obtained by plotting optical density (OD) vs. concentration with a 4-parameter logistic curve fitting program. The calibration curve range of this method is 0.625 ng / mL to 20 ng / mL in 5% monkey plasma (12.5 ng / mL to 400 ng / mL in 100% monkey plasma). Two calibration materials outside the assay qualification range at 0.313 and 0.156 ng / mL in 5% monkey plasma are included and can serve as anchor points to facilitate curve fitting. Based on the optimal fit of the curve, the anchor points are removed or retained (i.e., the highest number of standards are read within the limit of precision, %RE).

[0146] FVIII-specific chromogenic assay The FVIII activity in cynomolgus monkey plasma samples was estimated based on the administered dose and then diluted to approximately 0.25 - 1 IU / ml in human FVIII - deficient plasma (Diagnostica Stago). Samples were analyzed on a Sysmex CA1500 (Siemens Diagnostic Healthcare) using an FVIII chromogenic kit (Siemens). In this chromogenic assay, rFVIIIFc in the plasma sample is activated by thrombin. Next, activated factor VIII (FVIIIa) promotes the conversion of factor X (FX) to factor Xa (FXa) in the presence of activated factor IX (FIXa), phospholipid (PL), and calcium ions. The FXa activity is assayed by hydrolysis of a p - nitroanilide substrate specific for FXa. The initial rate of release of p - nitroaniline (pNA) measured at 405 nm is proportional to the FXa activity and thus proportional to the FVIII activity in the sample. The limit of quantification of FVIII activity by rFVIIIFc in this assay is ~0.3 IU / ml. The assay can measure total FVIII activity lower than the lower limit of approximately 0.06 IU / ml with an accuracy of ±20%. The calculated activity of pre - dose samples for each individual animal was subtracted from the values at each time point to generate a PD curve (FVIII activity vs. time).

[0147] A standard curve was generated from NIBSC 7th International Standard Factor FVIII concentrate diluted to 1 IU / ml in human FVIII - deficient plasma. The 6A19 standard curve was serially diluted on the Sysmex instrument to obtain concentrations of 0.15, 0.1, 0.05, 0.025, 0.0053, and 0.0026 IU / ml. Since the instrument dilutes all samples internally by 1:10, the FVIII standard concentrations correspond to plasma concentrations of 1.5 - 0.026 IU / ml, which is the range of FVIII activity that can be measured.

[0148] PK Analysis Using the WinNonlin software program (version 5.2; Pharsight Corporation, Mountain View, CA), the concentration - time profiles were evaluated using the non - compartmental analysis module.

[0149] Results The concentration of rFVIIIFc in monkey plasma was measured using a sandwich ELISA format that measures both the FVIII and Fc portions of the molecule. The data are reported in Table 7. All pre-dose samples were below the limit of quantification. Figure 7 shows the group mean rFVIIIFc and Xyntha plasma concentrations over time. Individual plasma concentration versus time curves are shown in Figure 8. Summaries of the PK parameters for rFVIIIFc and Xyntha are shown in Tables 9 and 10, respectively. The mean t1 / 2 for rFVIIIFc was 11.9 ± 1.7 hours (range 9.3 - 14.1 hours), and for Xyntha, the mean elimination t1 / 2 was 12.7 ± 4.4 hours (range 9.2 - 19.9 hours).

[0150] FVIII activity was measured using an FVIII-specific chromogenic assay. The data are reported in Table 8. The pre-dose activity due to endogenous FVIII was subtracted from all samples. A graph of the mean group data is shown in Figure 9. Individual plasma concentration versus time curves are shown in Figure 10. Summaries of the PK parameters for rFVIIIFc and Xyntha are shown in Tables 9 and 10, respectively. The mean t1 / 2 for rFVIIIFc was 16.1 ± 6.9 hours (range 11.6 - 29.4 hours), and for Xyntha it was 12.5 ± 1.7 hours (range 10.4 - 14.3 hours).

[0151] Discussion and Conclusions The elimination half-life was approximately the same for rFVIIIFc and Xyntha following a single intravenous dose of 125 IU / kg.

[0152] Example 3 This is a Phase I / IIa, non-blind, crossover, dose-escalating, multi-center, first-in-human study intended to evaluate the safety, tolerability, and pharmacokinetics of a single-dose administration of rFVIIIFc in subjects with severe (<1 IU / dL [1%] factor VIII [FVIII] endogenous) hemophilia A. A total of approximately 12 treatment-experienced patients will be enrolled and dosed with rFVIIIFc at 25 or 65 IU / kg. After screening (planned within 28 days prior to the first dose of the reference comparator, Advate® [rFVIII]) and a minimum of 4 days (96 hours) of FVIII-free treatment elapsed prior to the first injection, approximately 6 subjects will receive a single 25 IU / kg dose of Advate® followed by a 3-day (72-hour) pharmacokinetic (PK) profile, then a crossover study and a single, non-blind dose of 25 IU / kg of rFVIIIFc for 7-day (168-hour) PK profiling. The first 3 subjects will be dosed sequentially. For the first 3 subjects dosed with 25 IU / kg of rFVIIIFc, each subject will receive an inhibitor assay on day 14 (336 hours) after injection of rFVIIIFc. Dosing of the next subjects (for the first 3 subjects only) will occur once the inhibitor test is complete. After the 3rd subject completes the 14-day inhibitor assay, the remaining 3 subjects (25 IU / kg) and 6 subjects (65 IU / kg) will begin to enroll sequentially, at least 1 day apart, within each dose group.

[0153] One week after the last subject has received the 25 IU / kg dose of rFVIIIFc, approximately 6 additional subjects will be recruited for the 65 IU / kg cohort. Each subject in the 65 IU / kg cohort will then receive a 4-day (96-hour) PK profile followed by a crossover study and a single, non-blind dose of 65 IU / kg of rFVIIIFc for 10-day (240-hour) profiling Administer the dose. If bleeding symptoms occur prior to the first injection of rFVIIIFc in any cohort, the subject's pre - trial FVIII product shall be used for treatment and the first injection of rFVIIIFc for the PK profile shall not be administered until at least 4 days have elapsed.

[0154] All subjects are followed for safety for 14 days (336 hours) and during the 28 - day safety evaluation period after administration of rFVIIIFc at 25 IU / kg or 65 IU / kg. All subjects undergo pharmacokinetic sampling before and after dose administration, along with blood samples for analysis of FVIII activity at the intended time points.

[0155] Example 4 Activity within the Xase complex To examine the binding of FVIII proteins (rBDD FVIII and rFVIIIFc) to FIXa and to measure the ability of these proteins to activate FX, kinetic assays were performed to examine these interactions in the context of the Xase complex. This assay involves activated FIX and activated rBDD in the presence of calcium on a phospholipid surface Monitoring the formation of the Xase complex with FVIII or rFVIIIFc proteins and the conversion of FX to FXa as measured by cleavage of a chromogenic or fluorogenic substrate.

[0156] Briefly, FVIII is first activated with α-thrombin for 5 minutes and then mixed with FIXa in the presence of Ca2+ and synthetic phospholipid vesicles (25% phosphatidylserine (PS) / 75% phosphatidylcholine (PC)) or platelets. Under the following conditions, FVIIIa and FIXa interact in the presence of a phospholipid surface and calcium ions to form an active Xase complex, which mediates the conversion of FX to FXa by proteolytic processing. FXa cleaves an FXa-specific chromogenic or fluorogenic substrate. The cleaved substrate is chromogenic, and thus the amount of cleaved substrate in the solution indicates the amount of FXa produced. This is quantified by measuring the absorbance of the solution at 405 nm.

[0157] A. Activation of Factor X The ability of rBDD FVIII and rFVIIIFc to activate FX was tested in the context of the Xase complex described above. Thrombin-activated FVIII protein was incubated with FIXa and phospholipids in the presence of calcium and then added to different concentrations of FX in the presence of an FX-specific substrate to determine the rate of FXa generation (Figure 11).

[0158] Based on these data, the Km and Vmax for different FVIII proteins in the context of the Xase complex were calculated (Chang 1997) (Table 11). The data are presented as the mean ± corresponding standard deviation for six analytes (three experiments containing duplicate runs). Based on these data, these proteins (rBDD FVIII and rFVIIIFc) were found to have comparable Km and Vmax values within the variation of the assay. Thus, with respect to the ability to interact with phospholipids and activate FX, the Xase complex formed with rFVIIIFc behaves similarly to the Xase complex formed with the approved product rBDD FVIII (ReFacto). It should be noted that these comparable data also demonstrate that rFVIIIFc is activated to a comparable extent to rBDD FVIII after a short incubation with thrombin.

[0159] B. Interaction with FIXa The interaction of rBDD FVIII and rFVIIIFc with FIXa was also observed in the Xase complex. The rFVIIIFc protein was examined in a combined context. Xase complexes were assembled as above with a constant amount of FX and various FIXa levels, and the FXa production rate was determined (Figure 12). From these data, Kd values ​​for the Xase complexes formed with both FVIII proteins to FIXa are established (Chang 1997). Data are presented as the mean of six assays (three experiments containing duplicate runs) ± corresponding standard deviation (Table 12). Both proteins were found to have similar Kd and Vmax values, indicating that rFVIIIFc has a comparable interaction with FIXa similar to the licensed rBDD FVIII product.

[0160] Example 5 Interim pharmacokinetic data from the Phase I / IIa clinical trial discussed in Example 3 demonstrated the following results for FVIIIFc: FVIIIFc significantly reduced systemic exposure (AUC INF ) by approximately 50%, a reduction in clearance (Cl) by approximately 50%, and an increase in elimination half-life and MRT by approximately 50-70%. Furthermore, FVIIIFc showed increased C168, TBLP1, TBLP3, and TBLP5 values ​​compared to ADVATE.

[0161] AUC INF : Area under the concentration-time curve from zero to infinity Beta HL: elimination half-life; t 1 / 2β Also referred to as C168: Estimated FVIIIFc activity above baseline at approximately 168 hours after dose administration Cl: Clearance MRT: Average residence time TBLP1: Model-predicted time after dose administration when FVIIIFc activity declines to approximately 1 IU / dL above baseline TBLP3: Model predicted time after dosing when FVIIIFc activity decreased to approximately 3 IU / dL above baseline TBLP5: Model predicted time after dosing when FVIIIFc activity decreased to approximately 5 IU / dL above baseline Example 6 Recombinant B domain-deleted factor VIII-Fc (rFVIIIFc) fusion protein has been developed as an approach to extend the half-life of FVIII. The pharmacokinetics (PK) of rFVIIIFc were compared with rFVIII in hemophilia A mice. It was found that the terminal half-life was twice as long for rFVIIIFc compared to rFVIII. To confirm that the fundamental mechanism for the extended half-life is due to the protection of rFVIIIFc by FcRn, PK was evaluated in FcRn knockout and human FcRn transgenic mice. A single intravenous dose (125 IU / kg) was administered and plasma concentrations were measured using a chromogenic activity assay. Cmax was similar between rFVIIIFc and rFVIII (XYNTHA®) in both mouse strains. However, the half-life for rFVIIIFc was comparable to that of rFVIII in FcRn knockout mice, while the half-life for rFVIIIFc was extended approximately 2-fold longer than rFVIII in hFcRn transgenic mice. These results confirm that FcRn mediates or is involved in the extended half-life of rFVIIIFc compared to rFVIII. Since whole blood flow occlusion measured by rotational thromboelastometry (ROTEM) has been shown to correlate with the efficacy of coagulation factors in a bleeding model of hemophilic mice as well as in clinical applications, an attempt was made to evaluate the ex vivo efficacy of rFVIIIFc in hemophilia A mice using ROTEM. Hemophilia A mice were administered a single intravenous dose of 50 IU / kg rFVIIIFc, XYNTHA® (FVIII) or ADVATE® (FVIII). Five minutes after dosing, clot formation was evaluated in terms of clotting time (CT), clot formation time (CF The same was true for T) and the α angle. However, rFVIIIFc showed a significant improvement in CT at 72 and 96 hours after dosing, and both CFT and the α angle were also improved at 96 hours compared to both XYNTHA® (FVIII) and ADVATE® (FVIII), in line with the PK prolongation of rFVIIIFc. Thus, the construction of the FVIII Fc-fusion results in the production of a molecule with an elucidated mechanism of action that has the ability to provide increased half-life and extended protection from bleeding.

[0162] Example 7 This example shows the final analysis results regarding FVIII activity from 16 patients treated with FVIII products at 25 and 65 IU / kg. See Examples 3 and 5.

[0163] In this example, rFVIIIFc is a recombinant fusion protein consisting of a single molecule of recombinant B domain-deleted human FVIII (BDD-rFVIII) fused to the dimeric Fc domain of human IgG1 and having no intervening linker sequence. This protein construct is also referred to herein as the heterodimeric hybrid protein, FVIIIFc monomeric Fc-fusion protein, FVIIIFc monomeric hybrid, monomeric FVIIIFc hybrid, and FVIIIFc monomer-dimer. See Figure 1 and Table 2A.

[0164] Preclinical studies using rFVIIIFc have shown an approximately two-fold prolongation of the half-life of rFVIII activity compared to commercially available rFVIII products. The rationale for this study was to evaluate the safety and tolerability of a single-dose administration of rFVIIIFc in a frozen liquid formulation and to provide data on PK in subjects with severe hemophilia A. For this study, 16 evaluable subjects were available for PK evaluation. A single administration of two doses of both rFVIIIFc and Advate at nominal doses of 25 (n = 6) and 65 IU / kg body weight (n = 10) was infused intravenously over approximately 10 minutes. Blood samples for plasma PK assays were obtained prior to infusion and up to 10 days after dose administration. PK of FVIII activity for both Advate and rFVIIIFc was characterized in this study using a model-dependent method.

[0165] Objective The first objective of this study was to assess the safety and tolerability of a single administration of two doses of rFVIIIFc (25 and 65 IU / kg) in previously treated patients (PTPs) 12 years of age and older with severe hemophilia A.

[0166] The second objective was to determine the pharmacokinetic (PK) parameters as determined by the pharmacodynamic (PD) activity of FVIII over time after a single administration of 25 or 65 IU / kg of rFVIIIFc compared to Advate® in one-stage clotting and chromogenic assays.

[0167] Study Plan (see Example 3) Blood samples were collected for FVIII activity PK evaluation at screening visit (within 28 days before Advate dose administration); at pre-injection (day 0 before Advate injection) and at 10 and 30 minutes and 1, 3, 6, and 9 hours after injection; at 24 hours after Advate injection on day 1; at 48 hours after Advate injection on day 2; at 72 hours after Advate injection on day 3; at 96 hours after high-dose Advate administration on day 4 (cohort B only).

[0168] Immediately before administration of rFVIIIFc, at 10 and 30 minutes and at 1, 3, 6, and 9 hours after injection of rFVIIIFc on the day of rFVIIIFc injection; at 2 4 hours on day 1; at 48, 72, 96, and 120 hours after injection of rFVIIIFc on days 2 to 5; at 168 hours after injection of rFVIIIFc on day 7; at 192, 216, and 240 hours after administration of high-dose rFVIIIFc on days 8, 9, and 10 (cohort B only), blood samples were collected for FVIII activity PK evaluation. FVIII activity was also measured at the final study visit at 672 hours after injection of rFVIIIFc (day 28 after injection of rFVIIIFc).

[0169] Pharmacokinetic modeling and calculations Abbreviations TBLP1 = Model-predicted time after dose administration when FVIII activity decreased to approximately 1 IU / dL above baseline.

[0170] TBLP3 = Model-predicted time after dose administration when FVIII activity decreased to approximately 3 IU / dL above baseline.

[0171] KV_M = Cmax_M / actual dose (IU / kg) KV_OB = Cmax_OB / actual dose (IU / kg) IVR_M = 100 × Cmax_M × plasma volume (dL) / total dose (IU); where plasma volume (mL) = (23.7 × body length (cm)) + (9.0 × body weight (kg)) - 1709.

[0172] IVR_OB = 100 × Cmax_OB × plasma volume (dL) / total dose (IU); where plasma volume (mL) = (23.7 × body length (cm)) + (9.0 × body weight (kg)) - 1709.

[0173] Results Figure 13. Observed group mean (+SE) FVIII activity vs. time profile. Categorized by dose level. Grouped by compound (one-stage assay; 25 IU / kg (A) and 65 IU / kg (B) and chromogenic assay; 25 IU / kg (C) and 65 IU / kg (D)).

[0174] Figure 14. Observed group mean (+SE) FVIII activity vs. time profile. Grouped by dose level and compound (one-stage assay; A) (chromogenic assay; B).

[0175] Single bolus pharmacokinetics (one-stage assay) Observed FVIII activity increased rapidly after a short IV infusion of Advate or rFVIIIFc, and the mean (±SD) model-predicted Cmax values were 56.6 ± 4.74 and 121 ± 28.2 IU / dL for Advate and 55.6 ± 8.18 and 108 ± 16.9 IU / dL for rFVIIIFc, respectively, for the 25 and 65 IU / kg dose groups. All Advate- and rFVIIIFc-treated patients showed a dose-related increase in FVIII activity. The increases observed in both Cmax and AUCINF were slightly lower than proportional to doses above the dose range evaluated.

[0176] After the end of the infusion, the decrease in observed FVIII activity showed a monoexponential decay characteristic until reaching the baseline level. The rate of decrease in FVIII activity was lower for rFVIIIFc than for Advate, and the mean (±SD) model-predicted elimination half-life values were 11.9 ± 2.98 and 10.4 ± 3.03 hours for Advate and 18.0 ± 3.88 and 18.4 ± 6.99 hours for rFVIIIFc, respectively, for the 25 and 65 IU / kg dose groups. The elimination half-life values appeared to be dose-independent across the dose range evaluated for both FVIII products.

[0177] Total body FVIII exposure (assessed by AUCINF) was ~48% and 61% greater after administration of rFVIIIFc than Advate at 25 and 65 IU / kg dose levels, respectively. Mean (±SD) model-predicted AUCINF values were 974 ± 259 and 1810 ± 606 hours * IU / dL for Advate and 1440 ± 316 and 2910 ± 1320 hours * IU / dL for rFVIIIFc, respectively.

[0178] Similar to the elimination half-life, MRT was prolonged for rFVIIIFc compared to Advate. Mean (±SD) model-predicted MRT values were 17.1 ± 4.29 and 14.9 ± 4.38 hours for Advate and 25.9 ± 5.60 and 26.5 ± 10.1 hours for rFVIIIFc at 25 and 65 IU / kg dose groups, respectively. MRT values appeared to be dose-independent across the dose ranges evaluated for both FVIII products.

[0179] Furthermore, the first PK parameter values for CL and V were determined. The CL value for rFVIIIFc simply accounted for ~66% of the value observed for Advate at equivalent doses. The mean (±SD) model-predicted CL values were 2.70 ± 0.729 and 4.08 ± 1.69 mL / hour / kg for Advate and 1.80 ± 0.409 and 2.69 ± 1.25 mL / hour / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. The V values were similar between Advate and rFVIIIFc, and the mean (±SD) model-predicted V values were 43.9 ± 4.27 and 56.1 ± 13.4 mL / kg for Advate and 45.3 ± 7.23 and 61.6 ± 10.6 mL / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. With increasing doses of Advate and rFVIIIFc, a slight increase in the mean CL and V values was observed; however, the increase in the standard deviation at the 65 IU / kg dose, which was associated with dose-level limitations, confounded the assessment of the dose-dependence of these parameters. For example, the CV% geometric mean CL value for the rFVIIIFc treatment group increased from 23.0% (25 IU / kg) to 48.6% (65 IU / kg).

[0180] In addition to the first PK parameters, second PK parameters (e.g., K value, IVR, etc.) were determined to evaluate the duration of action of FVIII. Evidence of PK differences was also observed with rFVIIIFc, which demonstrated an increase in the TBLP1 and TBLP3 values when compared to Advate at equivalent doses. The IVR and K values for Advate and rFVIIIFc were considered to be similar. A slight increase in TBLP1 and TBLP3 was observed with increasing doses of Advate and rFVIIIFc. In contrast, a slight decrease in the mean IVR and K values was observed with increasing doses of Advate and rFVIIIFc. As noted above, the assessment of the dose-dependence of these parameters was confounded by the limited dose levels.

[0181] The mean (±SD) observed TBLP1 was 2.88 ± 0.733 and 2.93 ± 0.848 IU / dL (per IU / kg) for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 4.28 ± 0.873 and 5.16 ± 2.02 IU / dL (per IU / kg) for rFVIIIFc. The mean (±SD) observed TBLP3 was 2.06 ± 0.527 and 2.26 ± 0.666 IU / dL (per IU / kg) for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 3.09 ± 0.623 and 3.93 ± 1.59 IU / dL (per IU / kg) for rFVIIIFc.

[0182] The mean IVR and K values (subtracting baseline and residual drug in the model) calculated using the observed Cmax values were generally greater than those determined using the model-predicted Cmax values; they were in agreement with approximate values slightly lower than the peak activity observed using a one-compartment model. The mean (±SD) observed K values were 2.57 ± 0.198 and 2.13 ± 0.598 IU / dL (per IU / kg) for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 2.46 ± 0.330 and 1.85 ± 0.332 IU / dL (per IU / kg) for rFVIIIFc. The mean (±SD) observed IVR values were 94.1 ± 15.6 and 85.8 ± 16.5% for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 89.5 ± 11.9 and 74.8 ± 6.72% for rFVIIIFc.

[0183] Single-dose pharmacokinetics (chromogenic assay) The observed FVIII activity increased rapidly after a short IV infusion of Advate or rFVIIIFc, and the mean (±SD) model-predicted Cmax values were 70.2 ± 9.60 and 157 ± 38.6 IU / dL for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 70.3 ± 10.0 and 158 ± 34.7 IU / dL for rFVIIIFc.

[0184] All patients treated with Advate and rFVIIIFc showed a dose-related increase in FVIII activity. The increases observed in both Cmax and AUCINF were slightly lower than proportional to the doses above the dose range evaluated.

[0185] After the end of infusion, the decline in observed FVIII activity showed mono-exponential decay characteristics until reaching the baseline level. The rate of decline in FVIII was lower for rFVIIIFc than for Advate, and the mean (±SD) model-predicted elimination half-life values were 10.7 ± 1.98 and 10.3 ± 3.27 hours for Advate and 16.2 ± 2.92 and 19.0 ± 7.94 hours for the 25 and 65 IU / kg dose groups, respectively, for rFVIIIFc. The elimination half-life values appeared to be dose-independent across the dose range evaluated for both FVIII products.

[0186] Total systemic FVIII exposure (assessed by AUCINF) was ~53% and 84% greater after administration of rFVIIIFc than Advate at the 25 and 65 IU / kg dose levels, respectively. The mean (±SD) model-predicted AUCINF values were 1080 ± 236 and 2320 ± 784 hours * IU / dL for Advate and 1650 ± 408 and 4280 ± 1860 hours * IU / dL for rFVIIIFc.

[0187] Similar to the elimination half-life, the MRT was prolonged for rFVIIIFc compared to Advate. The mean (±SD) model-predicted MRT values were 15.3 ± 2.86 and 14.8 ± 4.72 hours for Advate and 23.4 ± 4.22 and 27.3 ± 11.4 hours for the 25 and 65 IU / kg dose groups, respectively, for rFVIIIFc. The MRT values appeared to be dose-independent across the dose range evaluated for both FVIII products.

[0188] Furthermore, the first PK parameter values for CL and V were determined. The CL value for rFVIIIFc simply accounted for ~58 - 66% of the values observed for Advate at equivalent doses. The mean (±SD) model-predicted CL values were 2.39 ± 0.527 and 3.21 ± 1.40 mL / hour / kg for Advate and 1.57 ± 0.349 and 1.86 ± 0.970 mL / hour / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. The V values were similar between Advat e and rFVIIIFc, and the mean (±SD) model-predicted V values were 35.8 ± 5.52 and 43.6 ± 11.2 mL / kg for Advate and 35.9 ± 6.65 and 42.7 ± 8.91 mL / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. Increases in mean CL and V values were observed with increasing doses of Advate and rFVIIIFc; however, the increase in standard deviation at the 65 IU / kg dose, which may be associated with dose-level limitations, confounded the assessment of the dose-dependence of these parameters.

[0189] In addition to the first PK parameters, second PK parameters (e.g., K value, IVR, etc.) were determined to evaluate the duration of action of FVIII. Evidence of PK differences was also observed with rFVIIIFc, which demonstrated increased TBLP1 and TBLP3 values when compared to Advate at equivalent doses. The IVR and K values for Advate and rFVIIIFc were considered to be similar.

[0190] Slight increases in TBLP1 and TBLP3 were observed with increasing doses of Advate and rFVIIIFc. In contrast, slight decreases in mean IVR and K values were observed with increasing doses of Advate and rFVIIIFc. As noted above, the assessment of the dose-dependence of these parameters was confounded by the limited dose levels.

[0191] The mean (±SD) observed TBLP1 was 2.70 ± 0.511 and 3.09 ± 0.978 IU / dL (per IU / kg) for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 4.06 ± 0.798 and 5.66 ± 2.38 IU / dL (per IU / kg) for rFVIIIFc. The mean (±SD) observed TBLP3 was 1.98 ± 0.377 and 2.39 ± 0.718 IU / dL (per IU / kg) for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 3.04 ± 0.598 and 4.44 ± 1.84 IU / dL (per IU / kg) for rFVIIIFc.

[0192] The mean IVR and K values calculated using the observed Cmax values (subtracting the baseline and residual drug in the model) were generally greater than the values determined using the model-predicted Cmax values; they were consistent with approximate values slightly lower than the peak activity observed using a one-compartment model. The mean (±SD) observed K values were 3.08 ± 0.429 and 2.85 ± 0.721 IU / dL (per IU / kg) for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 3.12 ± 0.451 and 2.92 ± 0.985 IU / dL (per IU / kg) for rFVIIIFc. The mean (±SD) observed IVR values were 112 ± 14.5 and 116 ± 26.9% for the 25 and 65 IU / kg dose groups, respectively, for Advate, and 113 ± 16.3 and 117 ± 33.6% for rFVIIIFc.

[0193] Conclusion All Advate- and rFVIIIFc-treated patients showed comparable dose-related increases in Cmax and AUCINF across the evaluated dose ranges. Peak plasma levels of Advate and rFVIIIFc activity were generally observed within 1 hour after the end of infusion and remained detectable for several days after dosing. After the end of infusion, the decrease in baseline-corrected FVIII activity showed monoexponential decay characteristics for both products until reaching baseline levels. Parameter values for elimination half-life and MRT appeared to be dose-independent across the dose ranges evaluated for both FVIII products. With increasing doses of Advate and rFVIIIFc, a slight increase in mean CL and V values was noted; however, the increase in inter-subject variability at the 65 IU / kg dose, which may be associated with dose-level limitations, confounded the assessment of dose-dependence of these parameters.

[0194] Comparison of rFVIIIFc and Advate active PK demonstrated an approximately 48 - 61% (one-stage assay) or 53 - 84% (chromogenic assay) increase in systemic exposure, an approximately 30 - 40% reduction in clearance, and an approximately 50 - 80% increase in both elimination half-life and MRT for rFVIIIFc compared to Advate at comparable doses. Evidence of PK differences was also observed for rFVIIIFc, which demonstrated increased TBLP1 and TBLP3 values when compared to Advate at equivalent doses. IVR and K values for Advate and rFVIIIFc were considered similar.

[0195] PK parameters derived from chromogenic assay results generally agreed with those from the one-stage assay, although the chromogenic assay yielded higher estimated values for exposure parameters (e.g., Cmax, AUCINF, etc.).

[0196] With the PK improvements observed, rFVIIIFc can provide extended protection from bleeding and lower the injection frequency for individuals with hemophilia A.

[0197] Example 8 Based on the preliminary PK analysis from the first human trial of rFVIII:Fc (Example 3), the A-LONG trial was planned. A-LONG is an open-label, multi-center evaluation of the safety, pharmacokinetics, and efficacy of recombinant factor VIII Fc fusion (FVIII-Fc) in the prevention and treatment of bleeding in treated subjects with severe hemophilia A (defined as <1 IU / dL [<1%] endogenous FVIII).

[0198] Approximately 106 subjects will be enrolled in one of three regimens: a prophylaxis regimen tailored to the purpose (arm 1), a weekly regimen (arm 2), and a regimen on demand (arm 3).

[0199] Arm 1: Regimen tailored to the purpose Arm 1 includes the entire group and the PK subgroup. Approximately 66 subjects will be enrolled. The initial regimen is 25 IU / kg on the first day, twice a week, and then 50 IU / kg on the 4th day of that week. Subjects will receive rFVIIIFc in this weekly prophylaxis regimen until PK results for rFVIIIFc are obtained. Based on this result, a prophylaxis regimen tailored to each individual will be established, where the dose and interval are determined to maintain a trough level of 1 - 3% FVIII activity. Then each subject will receive their individual prophylaxis regimen tailored to the purpose throughout the study.

[0200] Subjects will be monitored throughout the study, and ongoing dose and interval adjustments will be made. Adjustments will only be made if the subject experiences unacceptable bleeding episodes defined as the occurrence of ≥2 spontaneous bleeding episodes over a period of around 2 months. In this case, the adjustment targets a trough level of 3 - 5%.

[0201] Arm 2: Weekly regimen Approximately 20 subjects are enrolled / randomized to receive the following simplified rFVIIIFc PK profiling: at least 96-hour washout; single-dose administration of rFVIIIFc at 65 IU / kg; starting with rFVIIIFc on Day 0, with simple sample collection before injection and at 10 (±2) minutes, 3 hours (±15 minutes), 72 (±2) hours [Day 3], and 96 (±2) hours [Day 4] after the start of injection. After the simplified PK profiling, the subjects then receive a fixed dose of 65 IU / kg every 7 days.

[0202] Arm 3: Regimen on demand At least 10 major surgeries in at least 5 subjects are evaluated in this trial. Major surgery is defined as any surgical procedure (elective or emergency) involving general anesthesia and / or respiratory support, in which a large body cavity is penetrated, exposed, or there is a substantial impairment of physical or physiological function (e.g., laparotomy, thoracotomy, craniotomy, joint replacement, and amputation).

[0203] For prophylaxis during surgery, subjects are treated with rFVIIIFc at 35 - 50 IU / kg every 12 - 24 hours. Before surgery, the physician reviews the subject's rFVIIIFc PK profile and determines the dosage regimen of factor VIII replacement generally required for the type of planned surgery and the subject's clinical condition. Recommendations for appropriate dosing of rFVIIIFc during the surgical treatment period, including any rehabilitation time, take these factors into account.

[0204] The primary objectives of this trial are to (a) evaluate the safety and tolerability of rFVIIIFc administered as prophylaxis, on demand, and as a surgical treatment regimen; and (b) evaluate the efficacy of rFVIIIFc administered as prophylaxis, on demand, and as a surgical treatment regimen. The secondary objectives of this trial are to (a) characterize the PK profile of rFVIIIFc and compare the PK of FVIIIFc with that of the current commercial product, Advate; (b) evaluate the individual response to FVIIIFc; and (c) evaluate FVIIIFc consumption.

[0205] The first objective · To evaluate the safety and tolerability of rFVIIIFc administered as prophylaxis, on demand, and as a surgical treatment regimen · To evaluate the efficacy of rFVIIIFc administered as prophylaxis, on demand, and as a surgical treatment regimen The second objective · To characterize the PK profile of rFVIIIFc and compare the PK of FVIIIFc with that of the current commercial product, Advate® · To evaluate the individual response to rFVIIIFc · To appropriately prevent bleeding in the prophylactic regimen; to maintain constancy in the surgical setting; or to characterize the range of doses and schedules required to treat the onset of bleeding symptoms in the weekly treatment or prophylactic setting on demand · To evaluate rFVIIIFc consumption (e.g., total annual rFVIIIFc consumption / subject).

[0206] Example 9 Clinical ROTEM assay In the study of Example 8, in addition to measuring plasma FVIII activity by the one-stage activated partial thromboplastin time (aPTT) assay, whole blood rotational thromboelastometry (ROTEM) was also examined to assess the improvement of overall blood flow occlusion by rFVIIIFc and Advate in two subjects, specifically, one subject in the low-dose cohort and one subject in the high-dose cohort.

[0207] rFVIIIFc and Advate appear to be similarly active in clot formation when added to the subject's blood prior to rFVIIIFc treatment. The clotting time (CT) was linear with respect to the doses of rFVIIIFc and Advate in the range of approximately 1% to 100% of the market, and the dose response was similar between rFVIIIFc and Advate in the same subject.

[0208] After administration of doses of Advate and then rFVIIIFc, citrated blood samples were taken at various time points Whole blood was sampled, and clot formation after calcium readdition was monitored by ROTEM. Despite variable baseline CTs according to residual FVIII levels before Advate or rFVIIIFc dosing, both products effectively corrected CT to comparable levels at 30 minutes after injection. Furthermore, the improvement in CT was more sustained in subjects dosed at this low dose of 25 IU / kg rFVIIIFc at the time of and 3 hours after injection, compared to Advate. However, the differential improvement of rFVIIIFc vs Advate was not as readily perceptible at the 65 IU / kg dose.

[0209] Table Table 1: Polynucleotide Sequences A. B Domain-Deleted FVIIIFc (i) B Domain-Deleted FVIIIFc Chain DNA Sequence (Underline: FVIII Signal Peptide; Bold: Fc Region) (SEQ ID NO: 1: This Encodes SEQ ID NO: 2)

[0210]

Chem.

[0211]

Chem.

[0212]

Chem.

[0213]

Chem.

[0214]

Chem.

[0215]

Chem.

[0216]

Chem.

[0217]

Chem.

[0218]

Chem.

[0219]

Chem.

[0220]

Chem.

[0221]

Chem.

[0222]

Chem.

[0223]

Chem.

[0224] i) B domain-deleted FVIII-Fc chain (underlined: 19-amino acid signal sequence) (SEQ ID NO: 2)

[0225]

Chem.

[0226]

Chem.

[0227] i) Full-length FVIII Fc chain (underlined: FVIII signal peptide) (SEQ ID NO: 6)

[0228]

Chemical formula

[0229]

Chemical formula

[0230]

Chemical formula

[0231] (i) HC-Fc (bold: Fc sequence; underlined: signal peptide) (SEQ ID NO: 8)

[0232]

Chemical formula

[0233]

Chemical Structure

[0234]

Chemical Structure

[0235]

Table 3 - A

[0236]

Table 3 - B

[0237]

Table 4

[0238]

Table 5

[0239]

Table 6

[0240]

Table 7

[0241]

Table 8

[0242]

Table 9

[0243]

Table 10

[0244]

Table 11

[0245]

Table 12

Claims

1. 1. A pharmaceutical composition for reducing the incidence of bleeding episodes in a human subject with hemophilia A in need of treatment for spontaneous bleeding, comprising a therapeutic dose of a long-acting factor VIII polypeptide comprising functional factor VIII (FVIII) having clotting activity and Fc, The pharmaceutical composition is administered in multiple doses, the therapeutic dose being about 25 IU / kg to about 65 IU / kg with a dosing interval of 3 to 5 days, and is administered intravenously.

2. 2. The composition of claim 1, wherein each of the multiple doses is 25 IU / kg.

3. 2. The composition of claim 1, wherein each of the multiple doses is 50 IU / kg.

4. 2. The composition of claim 1, wherein each of the multiple doses is 65 IU / kg.

5. The composition according to any one of claims 1 to 4, wherein the administration interval is 3 days.

6. The composition according to any one of claims 1 to 4, wherein the administration interval is 4 days.

7. The composition according to any one of claims 1 to 4, wherein the administration interval is 5 days.

8. 2. The composition of claim 1, wherein the therapeutic dose is about 25 IU / kg, about 30 IU / kg, about 35 IU / kg, about 40 IU / kg, about 45 IU / kg, about 50 IU / kg, about 55 IU / kg, about 60 IU / kg, or about 65 IU / kg.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein said long-acting Factor VIII polypeptide is PEGylated.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein said long-acting factor VIII polypeptide is a FVIIIFc monomer-dimer hybrid.

11. The FVIIIFc monomer-dimer hybrid comprises two Fc molecules, The composition of claim 10, wherein one of the following is fused to the C-terminus of the light chain of factor VIII.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the functional factor VIII comprises factor VIII in which the B domain is fully or partially deleted.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the functional factor VIII comprises an amino acid sequence that is at least 90% identical to amino acids 1 to 1438 of SEQ ID NO:2 or amino acids 1 to 2332 of SEQ ID NO:

6.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the functional factor VIII comprises amino acids 1 to 1438 of SEQ ID NO:2 or amino acids 1 to 2332 of SEQ ID NO:

6.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the Fc is at least 90% or 95% identical to amino acids 1439 to 1665 of SEQ ID NO:2 or amino acids 2333 to 2559 of SEQ ID NO:

6.

16. The pharmaceutical composition according to any one of claims 1 to 15, further comprising at least one excipient.

17. The composition of any one of claims 1 to 16, wherein the plasma Factor VIII:C trough level in the subject is maintained above 1 IU / dl.

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