FACTOR VIII COMPLEX WITH XTEN AND VON WILLEBRAND FACTOR PROTEIN, AND USES THEREOF

• US 20150266943A1
• Filed: 07/10/2013
• Published: 09/24/2015
• Est. Priority Date: 07/11/2012
• Status: Active Grant

• Current Assignee: Bioverativ Therapeutics Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc., Biogen MA Inc. (Biogen, Inc.)
• Inventors: Chhabra, Ekta Seth, Liu, Tongyao, Peters, Robert T., Kulman, John, Chang, Pei-yun
• Granted Patent: US 10,138,291 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/36 Blood coagulation or fibrinolysis factors
• CPC Class Codes: A61K 38/37 Factors VIII
• CPC Class Codes: A61K 47/55 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity

Abstract

The present invention includes a chimeric protein comprising a VWF protein with D′ domain and D3 domain of VWF, one or more XTEN sequence, and a FVIII protein, wherein the VWF fragment, the XTEN sequence, or the FVIII protein are linked to or associated with each other. The chimeric protein can further comprise one or more Ig constant region or a portion thereof (e.g., an Fc region). A polypeptide chain of a VWF fragment is associated with a FVIII polypeptide chain linked to an XTEN sequence. The VWF fragment polypeptide chain can prevent or inhibit binding of endogenous VWF to FVIII protein linked to the XTEN sequence. By preventing or inhibiting binding of endogenous VWF to FVIII protein, VWF fragment can extend half-life of chimeric protein comprising FVIII protein. The invention includes nucleotides, vectors, host cells, use of VWF fragment, or chimeric proteins.

Claims

1. A chimeric protein comprising (i) a von Willebrand Factor (VWF) fragment comprising a D′

domain and a D3 domain of VWF, (ii) an extended length polypeptide (XTEN) sequence, and (iii) a factor VIII (FVIII) protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker, and wherein, the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein.

5. The chimeric protein of claim 1, which comprises

(a) a single polypeptide chain comprising the VWF fragment, the XTEN sequence and the FVIII protein;

or

(b) two polypeptides comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the FVIII protein and the second polypeptide comprises the VWF fragment and the XTEN sequence.

7. The chimeric protein of claim 1, further comprising (iv) an immunoglobulin (Ig) constant region or a portion thereof linked to either the VWF fragment, the XTEN sequence, or the FVIII protein, or any combination thereof.

8. The chimeric protein of claim 1, comprising a formula, which comprises:

(a) V-X-FVIII;

(b) FVIII-X-V;

(c) V-X;

FVIII;

(d) X-V;

FVIII;

(e) FVIII;

V-X;

(f) FVIII;

X-V;

(g) V-L2-X-L1-F1;

FVIII-L3-F2;

(h) V-L2-X-L1-F1;

F2-L3-FVIII;

(i) F1-L1-X-L2-V;

FVIII-L3-F2;

(j) F1-L1-X-L2-V;

F2-L3-FVII;

(k) V-L2-X-L1-F1-L4-FVIII-L3-F2;

(l) F2-L3-FVIII-L4-F1-L1-X-L2-V;

(m) FVIII-L3-F2-L4-V-L2-X-L1-F1; and

(n) F1-L1-X-L2-V-L4-F2-L3-FVIII,

wherein V comprises the VWF fragment,

each of L1, L2, and L3 comprises an optional linker,

L4 is an optional linker,

FVIII comprises the FVIII protein,

X comprises one or more XTEN sequences,

F1 comprises an optional Ig constant region or a portion thereof,

F2 comprises an optional additional Ig constant region or a portion thereof;

(-) is a peptide bond or one or more amino acids; and

(;

) is a covalent bond or a non-covalent bond.

10. The chimeric protein of claim 7, wherein the Ig constant region or a portion thereof comprises a first Fe region, which is linked to the XTEN sequence or the VWF fragment.

12. The chimeric protein of claim 7, wherein the Ig constant region or a portion thereof is linked to the XTEN sequence by a cleavable linker.

13. The chimeric protein of claim 7, further comprising an additional Ig constant region or a portion thereof linked to the FVIII protein.

14. The chimeric protein of claim 13, wherein the additional Ig constant region or a portion thereof comprises a second Fe region.

17. The chimeric protein of claim 14, wherein the second Fc region is further linked to the VWF fragment by a processable linker.

20. The chimeric protein of claim 13, wherein the additional Ig constant region or a portion thereof is associated with the Ig constant region or a portion thereof.

22. The chimeric protein of claim 20, wherein the additional Ig constant region or a portion thereof is associated with the Ig constant region or a portion thereof by a disulfide bond.

24. The chimeric protein of claim 1, wherein the half-life of the FVIII protein is extended compared to a FVIII protein without the VWF fragment or compared to wild type FVIII.

48. The chimeric protein of claim 1, wherein:

(a) the VWF fragment does not bind to a VWF clearance receptor;

or

(b) the VWF fragment is capable of (i) protecting the FVIII protein from cleavage by one or more proteases, (ii) protecting the FVIII protein from activation, (iii) stabilizing the heavy chain and/or the light chain of the FVIII protein, or (iv) preventing clearance of the FVIII protein by one or more scavenger receptors.

66. The chimeric protein of claim 13, which comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises a heavy chain of the FVIII protein and a XTEN sequence, the second polypeptide comprises a light chain of the FVIII protein and the Ig constant region or a portion thereof, and the third polypeptide comprises the VWF fragment, the XTEN sequence, and the additional Ig constant region or a portion thereof;

wherein the heavy chain of the FVIII protein and the light chain of the FVIII protein are associated with each other by a metal bond; and

wherein the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are associated by a disulfide bond.

67. The chimeric protein of claim 1, wherein the chimeric protein comprises a first polypeptide and a second polypeptide,

wherein the first polypeptide comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:

101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, and 117; and

wherein the second polypeptide comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from SEQ ID NOs;

118, 119, and 120.

69. The chimeric protein of claim 1, wherein the FVIII protein is linked to at least two XTEN sequences, at least three XTEN sequences, at least four XTEN sequences, at least five X XTEN sequences, or at least six XTEN sequences.

72. The chimeric protein of claim 1, wherein an additional XTEN is inserted within one or more insertion sites in the FVIII protein selected from the group consisting of the amino acid residues in Table 7, Table 8, Table 9 and Table 10.

75. The chimeric protein of claim 1, wherein the FVIII protein comprises a B domain or a portion thereof.

76. The chimeric protein of claim 75, wherein the FVIII protein is SQ B domain deleted FVIII.

77. The chimeric protein of claim 75, wherein an additional XTEN sequence is inserted within the B domain or a portion thereof.

93. The chimeric protein of claim 1, wherein the VWF fragment comprises at least two VWF fragments, at least three VWF fragments, at least our VWF fragments, at least five VWF fragments, or at least six VWF fragments.

94. The chimeric protein of claim 1, wherein the VWF fragment comprises an amino acid at least 90% identical to amino acids 764 to 1240 of SEQ ID NO:

2.

95. The chimeric protein of claim 94, wherein the VWF fragment consists essentially of or consists of amino acids 764 to 1240 of SEQ ID NO:

2.

108. The chimeric protein of claim 1, wherein the XTEN sequence is selected from the group consisting of AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144.

109. The chimeric protein of claim 108, wherein the XTEN sequence is selected from the group consisting of SEQ ID NO:

36;

SEQ ID NO;

37;

SEQ ID NO;

38;

SEQ ID NO;

39;

SEQ ID NO;

40;

SEQ ID NO;

41;

SEQ ID NO;

42;

SEQ ID NO;

43, SEQ ID NO;

44, and SEQ ID NO;

127.

110. The chimeric protein of claim 108, wherein the XTEN sequence is AE288.

120. A polynucleotide or a set of polynucleotides encoding the chimeric protein of claim 1.

122. A vector comprising the polynucleotide or the set of polynucleotides of claim 120 and one or more promoter operably linked to the polynucleotide or the set of polynucleotides.

124. A host cell comprising the polynucleotide or the set polynucleotides of claim 120.

127. A pharmaceutical composition comprising the chimeric protein of claim 1, and a pharmaceutically acceptable carrier.

128. A pharmaceutical composition comprising the polynucleotide or the set of polynucleotides of claim 120 and a pharmaceutically acceptable carrier.

137. A method of preventing or inhibiting binding of a FVIII protein with endogenous VWF, extending or increasing half-life of a FVIII protein, or preventing or inhibiting clearance of a FVIII protein, from a cell, comprising adding an effective amount of the chimeric protein of claim 1 to a subject in need thereof.

138. A method of preventing or inhibiting binding of a FVIII protein with endogenous VWF, extending or increasing half-life of a FVIII protein, or preventing or inhibiting clearance of a FVIII protein from a cell, comprising adding an effective amount of the polynucleotide or the set of polynucleotides of claim 120 to a subject in need thereof.

143. A method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein of claim 1.

146. A method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the polynucleotide or the set of polynucleotides of claim 120.

148. A method of making a chimeric protein, comprising transfecting one or more host cell with the polynucleotide or the set of polynucleotides of claim 120 and expressing the chimeric protein in the host cell.

2-4. -4. (canceled)

6. (canceled)

9. (canceled)

11. (canceled)

15-16. -16. (canceled)

18-19. -19. (canceled)

21. (canceled)

23. (canceled)

25-47. -47. (canceled)

49-65. -65. (canceled)

68. (canceled)

70-71. -71. (canceled)

73-74. -74. (canceled)

78-92. -92. (canceled)

96-107. -107. (canceled)

111-119. -119. (canceled)

121. (canceled)

123. (canceled)

125-126. -126. (canceled)

129-136. -136. (canceled)

139-142. -142. (canceled)

144-145. -145. (canceled)

147. (canceled)

149-165. -165. (canceled)