IP Library › Granted Patent US 12,617,839

Granted Patent B2

US 12,617,839 · App. 17/519,719 · Granted May 5, 2026

Factor VIII chimeric proteins and uses thereof

Inventors: Ekta Seth Chhabra (Framingham, MA); Tongyao Liu (Lexington, MA); Robert T. Peters (Needham, MA); John Kulman (Belmont, MA)

Assignee: BIOVERATIV THERAPEUTICS INC.

C07K14/755 C07K16/00 A61K38/00 C07K2319/00 C07K2319/30 C07K2319/31

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Quick Facts

Patent No.: US 12,617,839
App. No.: 17/519,719
Granted: May 5, 2026
Kind: B2

External Links

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Abstract

The present invention provides a chimeric protein comprising a first polypeptide which comprises a FVIII protein and a first Ig constant region or a portion thereof and a second polypeptide which comprises a VWF protein comprising the D′ domain and D3 domain of VWF, a XTEN sequence having less than 288 amino acids in length, and a second Ig constant region or a portion thereof, wherein the first polypeptide and the second polypeptide are associated with each other. The invention also includes nucleotides, vectors, host cells, methods of using the chimeric proteins.

Claims (299)

1 . A method of treating a disorder caused by a deficiency in Factor VIII comprising administering to a subject in need thereof a therapeutically effective amount of a chimeric protein comprising:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a Factor VIII (“FVIII”) protein comprising a N-terminal portion and a C-terminal portion;

wherein the N-terminal portion of the FVIII protein comprises the A1 domain, A2 domain, and a portion of the B domain of full length mature FVIII (SEQ ID NO: 65);

such that the N-terminal portion comprises the amino acid sequence of residues 1 to 745 of SEQ ID NO: 65;

wherein the N-terminal portion is fused to a first XTEN sequence inserted immediately downstream of amino acid 745 of SEQ ID NO: 65; and

wherein the C-terminal portion comprises the A3 domain, the C1 domain, and the C2 domain, such that the C-terminal portion comprises residues 1690-2332 of SEQ ID NO: 65;

(b) a first immunoglobulin (“Ig”) constant region or a portion thereof,

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a von Willebrand Factor (“VWF”) protein comprising a D′ domain and a D3 domain of VWF, wherein the VWF protein contains a residue other than cysteine substituted for residues corresponding to residues 1099 and 1142 of SEQ ID NO: 21;

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues;

(c) a cleavable linker comprising an a2 region of FVIII which comprises the amino acid sequence of Glu720 to Arg740 corresponding to SEQ ID NO: 65, wherein the a2 region is capable of being cleaved by thrombin; and

(d) a second Ig constant region or a portion thereof,

wherein the first polypeptide chain is associated with the second polypeptide chain through the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof.

2 . The method of claim 1 , wherein the disorder is hemophilia A.

3 . The method of claim 2 , wherein wherein the first Ig constant region or portion thereof is associated with the second Ig constant region or portion thereof through two disulfide bonds.

4 . The method of claim 3 , wherein wherein the first Ig constant region or portion thereof is a first Fc region and the second Ig constant region or portion thereof is a second Fc region.

5 . The method of claim 4 , wherein the cleavable linker is 20 to 50 amino acids long.

6 . The method of claim 3 , wherein the cleavable linker is about 30 amino acids long.

7 . The method of claim 2 , wherein the chimeric protein is administered prophylactically.

8 . The method of claim 2 , wherein the chimeric protein is administered for on-demand treatment.

9 . The method of claim 2 , wherein the chimeric protein is administered prior to, during, or after surgery.

10 . The method of claim 2 , wherein the chimeric protein is administered to control an acute bleeding episode.

11 . A method of treating a disorder that comprises a deficiency in Factor VIII comprising intravenously administering to a subject in need thereof a therapeutically effective amount of a chimeric protein comprising:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a Factor VIII (“FVIII”) protein comprising the amino acid sequence of residues 1 to 745 of SEQ ID NO: 65, fused to a first XTEN sequence inserted immediately downstream of residue 745 of SEQ ID NO: 65, fused to residues 1649 to 2332 of SEQ ID NO: 65, and

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a von Willebrand Factor (“VWF”) protein comprising a D′ domain and a D3 domain of VWF, wherein the VWF protein comprises the amino acid sequence of residues 764 to 1240 of SEQ ID NO: 21 with alanine substitutions at residues 1099 and 1142 of SEQ ID NO: 21,

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58,

(d) a second Fc region;

wherein the first Fc region is associated with the second Fc region through a disulfide bond.

12 . A method of treating a disorder that comprises a deficiency in Factor VIII comprising intravenously administering to a subject in need thereof a therapeutically effective amount of a chimeric protein comprising:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a Factor VIII (“FVIII”) protein comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 67 with a first XTEN sequence inserted immediately downstream of the residue corresponding to residue 745 of SEQ ID NO: 67; and

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a von Willebrand Factor (“VWF”) protein comprising a D′ domain and a D3 domain of VWF, wherein the VWF protein contains a residue other than cysteine substituted for residues 1099 and 1142 of SEQ ID NO: 21;

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues;

(d) a second Fc region, wherein the first Fc region is associated with the second Fc region through a disulfide bond.

13 . A method of treating a disorder that comprises a deficiency in Factor VIII comprising intravenously administering to a subject in need thereof a therapeutically effective amount of a chimeric protein comprising:

(i) a first polypeptide chain which comprises a Factor VIII (“FVIII”) protein fused to a first immunoglobulin (“Ig”) constant region or a portion thereof,

wherein the FVIII protein comprises the amino acid sequence of residues 1 to 745 of SEQ ID NO: 202, fused to a first XTEN sequence inserted immediately downstream of residue 745 of SEQ ID NO: 202, fused to residues 746 to 1429 of SEQ ID NO: 202; and

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises a von Willebrand Factor (“VWF”) protein comprising a D′ domain and a D3 domain of VWF fused to a second Ig constant region or a portion thereof by a second XTEN sequence in-between,

wherein the VWF protein comprises the amino acid sequence of SEQ ID NO: 201;

wherein the second XTEN sequence comprises the amino acid sequence of SEQ ID NO: 58; and

wherein the second XTEN sequence is linked to the second Ig constant region or a portion thereof by a linker comprising the amino acid sequence of SEQ ID NO: 88;

wherein the first polypeptide chain is associated with the second polypeptide chain through the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof.

14 . The method of claim 1 , wherein the disorder is Von Willebrand Disease.

15 . The method of claim 14 , wherein the Von Willebrand Disease is Von Willebrand Disease type 3.

16 . The method of claim 2 , wherein the C-terminal portion of the FVIII protein comprises an amino acid sequence at least 95% identical to residues 1641 to 2332of SEQ ID NO: 65.

17 . The method of claim 16 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

18 . The method of claim 17 , wherein the first Fc region and the second Fc region are the same.

19 . The method of claim 18 , wherein the first Fc region and the second Fc region are derived from human IgG1.

20 . The method of claim 19 , wherein the VWF protein consists of the D′ domain and the D3 domain.

21 . The method of claim 11 , wherein the disorder is hemophilia A.

22 . The method of claim 11 , wherein the disorder is Von Willebrand Disease.

23 . The method of claim 22 , wherein the Von Willebrand Disease is Von Willebrand Disease type 3.

24 . The method of claim 21 , wherein the first Fc region is associated with the second Fc region through two disulfide bonds.

25 . The method of claim 21 , wherein the second XTEN sequence links the VWF protein to the cleavable linker, such that the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22.

26 . The method of claim 25 , wherein the first XTEN sequence is inserted into the FVIII protein such that the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2.

27 . The method of claim 26 , wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 173.

28 . The method of claim 27 , wherein the VWF protein consists of the D′ domain and the D3 domain.

29 . The method of claim 12 , wherein the disorder is hemophilia A.

30 . The method of claim 12 , wherein the disorder is Von Willebrand Disease.

31 . The method of claim 30 , wherein the Von Willebrand Disease is Von Willebrand Disease type 3.

32 . The method of claim 29 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

33 . The method of claim 32 , wherein the first Fc region and the second Fc region are the same.

34 . The method of claim 33 , wherein the first Fc region and the second Fc region are derived from human IgG1.

35 . The method of claim 34 , wherein the VWF protein consists of the D′ domain and the D3 domain.

36 . The method of claim 33 , wherein the cleavable linker comprises an a2 region of FVIII comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106.

37 . The method of claim 33 , wherein the cleavable linker is 20 to 50 amino acids long.

38 . The method of claim 37 , wherein the cleavable linker is about 30 amino acids long.

39 . The method of claim 13 , wherein the disorder is hemophilia A.

40 . The method of claim 13 , wherein the disorder is Von Willebrand Disease.

41 . The method of claim 40 , wherein the Von Willebrand Disease is Von Willebrand Disease type 3.

42 . The method of claim 39 , wherein the first Ig constant region or a portion thereof comprises a first Fc region and the second Ig constant region or a portion thereof comprises a second Fc region.

43 . The method of claim 39 , wherein the first Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof by a covalent bond.

44 . The method of claim 39 , wherein the FVIII protein comprises a deletion of residues 746-1648 corresponding to native mature human FVIII protein (SEQ ID NO: 65).

45 . The method of claim 39 , wherein the first Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof by a disulfide bond.

46 . The method of claim 39 , wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 173.

47 . The method of claim 39 , wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173.

48 . The method of claim 39 , wherein the second XTEN sequence is fused to the linker such that the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22.

49 . The method of claim 39 , wherein the second XTEN sequence is linked to the second Ig constant region or a portion thereof by a linker consisting of the amino acid sequence of SEQ ID NO: 88.

50 . The method of claim 48 , wherein the first XTEN sequence is inserted into the FVIII protein such that the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2.

51 . The method of claim 42 , wherein the first Fc region and the second Fc region are identical.

52 . The method of claim 42 , wherein the first Fc region and the second Fc region are derived from human IgG1.

53 . The method of claim 39 , wherein the first Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof by two disulfide bonds.

54 . The method of claim 39 , wherein the VWF protein consists of the D′ domain and the D3 domain.

55 . A method of treating a disorder that comprises a deficiency in Factor VIII comprising intravenously administering to a subject in need thereof a therapeutically effective amount of a chimeric protein comprising:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a von Willebrand Factor (“VWF”) protein comprising a D′ domain and a D3 domain of VWF, wherein the VWF protein comprises the amino acid sequence of residues 764 to 1240of SEQ ID NO: 21 with alanine substitutions at residues 1099 and 1142 of SEQ ID NO: 21,

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues;

(d) a second Fc region;

wherein the first Fc region is associated with the second Fc region through a disulfide bond.

56 . The method of claim 55 , wherein the disorder is hemophilia A.

57 . The method of claim 55 , wherein the disorder is Von Willebrand Disease.

58 . The method of claim 57 , wherein the Von Willebrand Disease is Von Willebrand Disease type 3.

59 . The method of claim 56 , wherein the first Fc region and the second Fc region are the same.

60 . The method of claim 59 , wherein the first Fc region and the second Fc region are derived from human IgG1.

61 . The method of claim 60 , wherein the VWF protein consists of the D′ domain and the D3 domain.

62 . The method of claim 59 , wherein the cleavable linker comprises an a2 region of FVIII comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106.

63 . The method of claim 59 , wherein the cleavable linker is 20 to 50 amino acids long.

64 . The method of claim 63 , wherein the cleavable linker is about 30 amino acids long.

65 . A method of treating hemophilia A comprising intravenously administering a therapeutically effective amount of a chimeric protein to a subject in need thereof, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58;

(d) a second Fc region, wherein the first Fc region is associated with the second Fc region through a disulfide bond, and wherein the chimeric protein is administered prophylactically.

66 . A method of treating hemophilia A comprising intravenously administering a therapeutically effective amount of a chimeric protein to a subject in need thereof, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises from the N-terminus to the C-terminus thereof: a Factor VIII (“FVIII”) protein and a first Fc region;

wherein the FVIII protein comprises residues 1 to 745 and 1649 to 2332 of SEQ ID NO: 65with a first XTEN sequence inserted immediately downstream of residue 745; and

wherein the first XTEN sequence consists of at least about 288 amino acids and comprises an amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or any combination thereof, and

(ii) a second polypeptide chain which comprises from the N-terminus to the C-terminus thereof:

a von Willebrand Factor (“VWF”) protein comprising a D′ domain and a D3 domain of VWF, a second XTEN sequence, a cleavable linker, and a second Fc region,

wherein the D′ domain and D3 domain comprises the amino acid sequence of residues 764 to 1240 of SEQ ID NO: 21 with alanine substitutions at the residues that correspond to positions 1099 and 1142 of SEQ ID NO: 21,

wherein the second XTEN sequence consists of at least about 144 amino acids and less than 288 amino acids and comprises an amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 or any combination thereof,

wherein the cleavable linker comprises an a2 region of FVIII which comprises the amino acid sequence of SEQ ID NO: 194, wherein the a2 region is capable of being cleaved by thrombin,

wherein the first Fc region is associated with the second Fc region through a disulfide bond.

67 . The method of claim 66 , wherein the cleavable linker is 30 to 35 amino acids long.

68 . The method of claim 66 , wherein the cleavable linker is about 30 amino acids long.

69 . The method of claim 66 , herein the first Fc region is associated with the second Fc region through two disulfide bonds.

70 . The method of claim 69 , wherein the first Fc region and the second Fc region are the same, and wherein the first Fc region and the second Fc region are derived from human IgG1.

71 . The method of claim 70 , wherein the VWF protein consists of the D′ domain and the D3 domain.

72 . The method of claim 66 , wherein the chimeric protein is administered prophylactically.

73 . The method of claim 65 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

74 . The method of claim 66 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

75 . The method of claim 1 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

76 . The method of claim 12 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

77 . The method of claim 66 , wherein the first XTEN sequence consists of about 288 amino acids.

78 . The method of claim 77 , wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8.

79 . The method of claim 66 , wherein the second XTEN consists of about 144 amino acids.

80 . The method of claim 79 , wherein the second XTEN comprises the amino acid sequence of SEQ ID NO: 58.

81 . A method of administering a chimeric protein to a subject who has hemophilia A, comprising intravenously administering about 0.1 to 400,000 μg/kg of the chimeric protein to the subject, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(a) a Factor VIII (“FVIII”) protein comprising a N-terminal portion and a C-terminal portion;

wherein the N-terminal portion of the FVIII protein comprises the A1domain, A2 domain, and a portion of the B domain of full length mature FVIII (SEQ ID NO: 65)

such that the N-terminal portion comprises the amino acid sequence of residues 1 to 745 of SEQ ID NO: 65;

wherein the N-terminal portion is fused to a first XTEN sequence inserted immediately downstream of amino acid 745 of SEQ ID NO: 65; and

wherein the C-terminal portion comprises the A3 domain, the C1 domain, and the C2 domain, such that the C-terminal portion comprises residues 1690-2332 of SEQ ID NO: 65;

(b) a first immunoglobulin (“Ig”) constant region or a portion thereof, wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues;

(d) a second Ig constant region or a portion thereof, wherein the first polypeptide chain is associated with the second polypeptide chain through the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof.

82 . The method of claim 81 , wherein wherein the first Ig constant region or portion thereof is associated with the second Ig constant region or portion thereof through two disulfide bonds.

83 . The method of claim 82 , wherein wherein the first Ig constant region or portion thereof is a first Fc region and the second Ig constant region or portion thereof is a second Fc region.

84 . The method of claim 83 , wherein the cleavable linker is 20 to 50 amino acids long.

85 . The method of claim 84 , wherein the cleavable linker is about 30 amino acids long.

86 . The method of claim 81 , wherein the chimeric protein is administered prophylactically.

87 . The method of claim 81 , wherein the chimeric protein is administered for on-demand treatment.

88 . The method of claim 81 , wherein the chimeric protein is administered prior to, during, or after surgery.

89 . The method of claim 81 , wherein the chimeric protein is administered to control an acute bleeding episode.

90 . A method of administering a chimeric protein to a subject who has hemophilia A, comprising intravenously administering about 0.1 to 400,000 μg/kg of the chimeric protein to the subject, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58,

(d) a second Fc region;

wherein the first Fc region is associated with the second Fc region through a disulfide bond.

91 . A method of administering a chimeric protein to a subject who has hemophilia A, comprising intravenously administering about 0.1 to 400,000 μg/kg of the chimeric protein to the subject, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues;

(d) a second Fc region,

wherein the first Fc region is associated with the second Fc region through a disulfide bond.

92 . A method of administering a chimeric protein to a subject who has hemophilia A, comprising intravenously administering about 0.1 to 400,000 μg/kg of the chimeric protein to the subject, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises a Factor VIII (“FVIII”) protein fused to a first immunoglobulin (“Ig”) constant region or a portion thereof,

wherein the VWF protein comprises the amino acid sequence of SEQ ID NO: 201;

wherein the second XTEN sequence comprises the amino acid sequence of SEQ ID NO: 58; and

wherein the second XTEN sequence is linked to the second Ig constant region or a

portion thereof by a linker comprising the amino acid sequence of SEQ ID NO: 88; wherein the first polypeptide chain is associated with the second polypeptide chain through the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof.

93 . The method of claim 81 , wherein the C-terminal portion of the FVIII protein comprises an amino acid sequence at least 95% identical to residues 1641 to 2332 of SEQ ID NO: 65.

94 . The method of claim 93 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

95 . The method of claim 94 , wherein the first Fc region and the second Fc region are the same.

96 . The method of claim 95 , wherein the first Fc region and the second Fc region are derived from human IgG1.

97 . The method of claim 96 , wherein the VWF protein consists of the D′ domain and the D3 domain.

98 . The method of claim 81 , wherein the first Fc region is associated with the second Fc region through two disulfide bonds.

99 . The method of claim 81 , wherein the second XTEN sequence is fused to the linker such that the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22.

100 . The method of claim 99 , wherein the first XTEN sequence is inserted into the FVIII protein such that the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2.

101 . The method of claim 100 , wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 173.

102 . The method of claim 101 , wherein the VWF protein consists of the D′ domain and the D3 domain.

103 . The method of claim 101 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

104 . The method of claim 103 , wherein the first Fc region and the second Fc region are the same.

105 . The method of claim 104 , wherein the first Fc region and the second Fc region are derived from human IgG1.

106 . The method of claim 105 , wherein the VWF protein consists of the D′ domain and the D3 domain.

107 . The method of claim 104 , wherein the cleavable linker comprises an a2 region of FVIII comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106.

108 . The method of claim 104 , wherein the cleavable linker is 20 to 50 amino acids long.

109 . The method of claim 108 , wherein the cleavable linker is about 30 amino acids long.

110 . The method of claim 92 , wherein the first Ig constant region or a portion thereof comprises a first Fc region and the second Ig constant region or a portion thereof comprises a second Fc region.

111 . The method of claim 92 , wherein the first Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof by a covalent bond.

112 . The method of claim 92 , wherein the FVIII protein comprises a deletion of residues 746-1648 corresponding to native mature human FVIII protein (SEQ ID NO: 65).

113 . The method of claim 92 , wherein the first Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof by a disulfide bond.

114 . The method of claim 92 , wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 173.

115 . The method of claim 92 , wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173.

116 . The method of claim 92 , wherein the second XTEN sequence is fused to the linker such that the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22.

117 . The method of claim 92 , wherein the second XTEN sequence is linked to the second Ig constant region or a portion thereof by a linker consisting of the amino acid sequence of SEQ ID NO: 88.

118 . The method of claim 116 , wherein the first XTEN sequence is inserted into the FVIII protein such that the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2.

119 . The method of claim 113 , wherein the first Fc region and the second Fc region are the same.

120 . The method of claim 113 , wherein the first Fc region and the second Fc region are derived from human IgG1.

121 . The method of claim 92 , wherein the first Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof by two disulfide bonds.

122 . The method of claim 92 , wherein the VWF protein consists of the D′ domain and the D3 domain.

123 . A method of administering a chimeric protein to a subject who has hemophilia A, comprising intravenously administering about 0.1 to 400,000 μg/kg of the chimeric protein to the subject, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58, wherein the second XTEN sequence contains less than 288 amino acid residues;

(d) a second Fc region;

wherein the first Fc region is associated with the second Fc region through a disulfide bond.

124 . The method of claim 123 , wherein the first Fc region and the second Fc region are the same.

125 . The method of claim 124 , wherein the first Fc region and the second Fc region are derived from human IgG1.

126 . The method of claim 125 , wherein the VWF protein consists of the D′ domain and the D3 domain.

127 . The method of claim 124 , wherein the cleavable linker comprises an a2 region of FVIII comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106.

128 . The method of claim 124 , wherein the cleavable linker is 20 to 50 amino acids long.

129 . The method of claim 128 , wherein the cleavable linker is about 30 amino acids long.

130 . A method of administering a chimeric protein to a subject who has hemophilia A, comprising intravenously administering about 0.1 to 400,000 μg/kg of the chimeric protein to the subject, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a first Fc region;

wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8; and

(ii) a second polypeptide chain which comprises, from the N-terminus to the C-terminus thereof:

(b) a second XTEN sequence comprising the amino acid sequence of SEQ ID NO: 58;

(d) a second Fc region,

wherein the first Fc region is associated with the second Fc region through a disulfide bond, and

wherein the chimeric protein is administered prophylactically.

131 . A method of administering a chimeric protein to a subject who has hemophilia A, comprising intravenously administering about 0.1 to 400,000 μg/kg of the chimeric protein to the subject, wherein the chimeric protein comprises:

(i) a first polypeptide chain which comprises from the N-terminus to the C-terminus thereof: a Factor VIII (“FVIII”) protein and a first Fc region;

wherein the FVIII protein comprises residues 1 to 745 and 1649 to 2332 of SEQ ID NO: 65 with a first XTEN sequence inserted immediately downstream of residue 745; and

and

(ii) a second polypeptide chain which comprises from the N-terminus to the C-terminus thereof: a von Willebrand Factor (“VWF”) protein comprising a D′ domain and a D3 domain of VWF, a second XTEN sequence, a cleavable linker, and a second Fc region,

wherein the cleavable linker comprises an a2 region of FVIII which comprises the amino acid sequence of SEQ ID NO: 194, wherein the a2 region is capable of being cleaved by thrombin,

wherein the first Fc region is associated with the second Fc region through a disulfide bond.

132 . The method of claim 131 , wherein the cleavable linker is 30 to 35 amino acids long.

133 . The method of claim 131 , wherein the cleavable linker is about 30 amino acids long.

134 . The method of claim 131 , herein the first Fc region is associated with the second Fc region through two disulfide bonds.

135 . The method of claim 134 , wherein the first Fc region and the second Fc region are the same, and wherein the first Fc region and the second Fc region are derived from human IgG1.

136 . The method of claim 135 , wherein the VWF protein consists of the D′ domain and the D3 domain.

137 . The method of claim 131 , wherein the chimeric protein is administered prophylactically.

138 . The method of claim 130 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

139 . The method of claim 131 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

140 . The method of claim 81 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

141 . The method of claim 91 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 88.

142 . The method of claim 131 , wherein the first XTEN sequence consists of about 288 amino acids.

143 . The method of claim 142 , wherein the first XTEN sequence comprises the amino acid sequence of SEQ ID NO: 8.

144 . The method of claim 131 , wherein the second XTEN consists of about 144 amino acids.

145 . The method of claim 144 , wherein the second XTEN comprises the amino acid sequence of SEQ ID NO: 58.

146 . The method of claim 81 , comprising administering about 0.1 to 100,000 μg/kg of the chimeric protein to the subject.

147 . The method of claim 81 , comprising administering about 1,000 to 400,000 μg/kg of the chimeric protein to the subject.

148 . The method of claim 90 , comprising administering about 0.1 to 100,000 μg/kg of the chimeric protein to the subject.

149 . The method of claim 90 , comprising administering about 1,000 to 400,000 μg/kg of the chimeric protein to the subject.

150 . The method of claim 91 , comprising administering about 0.1 to 100,000 μg/kg of the chimeric protein to the subject.

151 . The method of claim 91 , comprising administering about 1,000 to 400,000 μg/kg of the chimeric protein to the subject.

152 . The method of claim 92 , comprising administering about 0.1 to 100,000 μg/kg of the chimeric protein to the subject.

153 . The method of claim 92 , comprising administering about 1,000 to 400,000 μg/kg of the chimeric protein to the subject.

154 . The method of claim 123 , comprising administering about 0.1 to 100,000 μg/kg of the chimeric protein to the subject.

155 . The method of claim 123 , comprising administering about 1,000 to 400,000 μg/kg of the chimeric protein to the subject.

156 . The method of claim 130 , comprising administering about 0.1 to 100,000 μg/kg of the chimeric protein to the subject.

157 . The method of claim 130 , comprising administering about 1,000 to 400,000 μg/kg of the chimeric protein to the subject.

158 . The method of claim 131 , comprising administering about 0.1 to 100,000 μg/kg of the chimeric protein to the subject.

159 . The method of claim 131 , comprising administering about 1,000 to 400,000 μg/kg of the chimeric protein to the subject.

160 . The method of claim 1 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

161 . The method of claim 81 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

Assignments (2)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 5, 2021

From: KULMAN, JOHN

To: BIOGEN MA INC.

Reel/Frame 058028/0486 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 5, 2021

From: BIOGEN MA INC.

To: BIOVERATIV THERAPEUTICS INC.

Reel/Frame 058028/0535 →