Search

US-12617839-B2 - Factor VIII chimeric proteins and uses thereof

US12617839B2US 12617839 B2US12617839 B2US 12617839B2US-12617839-B2

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.

Inventors

  • Ekta Seth Chhabra
  • Tongyao Liu
  • Robert T. Peters
  • John Kulman

Assignees

  • BIOVERATIV THERAPEUTICS INC.

Dates

Publication Date
20260505
Application Date
20211105

Claims (20)

  1. 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. 2 . The method of claim 1 , wherein the disorder is hemophilia A.
  3. 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. 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. 5 . The method of claim 4 , wherein the cleavable linker is 20 to 50 amino acids long.
  6. 6 . The method of claim 3 , wherein the cleavable linker is about 30 amino acids long.
  7. 7 . The method of claim 2 , wherein the chimeric protein is administered prophylactically.
  8. 8 . The method of claim 2 , wherein the chimeric protein is administered for on-demand treatment.
  9. 9 . The method of claim 2 , wherein the chimeric protein is administered prior to, during, or after surgery.
  10. 10 . The method of claim 2 , wherein the chimeric protein is administered to control an acute bleeding episode.
  11. 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, (c) a cleavable linker comprising the amino acid sequence of SEQ ID NO: 88, and (d) a second Fc region; wherein the first Fc region is associated with the second Fc region through a disulfide bond.
  12. 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; (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 Fc region, wherein the first Fc region is associated with the second Fc region through a disulfide bond.
  13. 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. 14 . The method of claim 1 , wherein the disorder is Von Willebrand Disease.
  15. 15 . The method of claim 14 , wherein the Von Willebrand Disease is Von Willebrand Disease type 3.
  16. 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. 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. 18 . The method of claim 17 , wherein the first Fc region and the second Fc region are the same.
  19. 19 . The method of claim 18 , wherein the first Fc region and the second Fc region are derived from human IgG1.
  20. 20 . The method of claim 19 , wherein the VWF protein consists of the D′ domain and the D3 domain.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a division of U.S. patent application Ser. No. 15/110,673, filed Jul. 8, 2016, which is a 35 U.S.C. § 371 filing of International Patent Application No. PCT/US2015/010738, filed Jan. 9, 2015, which claims priority to U.S. Provisional Patent Application Ser. Nos. 61/988,104, filed May 2, 2014, and 61/926,226, filed Jan. 10, 2014, the entire disclosures of which are hereby incorporated herein by reference. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The content of the electronically submitted sequence listing in ASCII text file (Name: 723903_SA9-448USDIV_ST25.txt; Size: 820,859 bytes; and Date of Creation: Nov. 3, 2021) is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION Haemophilia A is a bleeding disorder caused by defects in the gene encoding coagulation factor VIII (FVIII) and affects 1-2 in 10,000 male births. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients affected with hemophilia A can be treated with infusion of purified or recombinantly produced FVIII. All commercially available FVIII products, however, are known to have a half-life of about 8-12 hours, requiring frequent intravenous administration to the patients. See Weiner M. A. and Cairo, M. S., Pediatric Hematology Secrets, Lee, M. T., 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number of approaches have been tried in order to extend the FVIII half-life. For example, the approaches in development to extend the half-life of clotting factors include pegylation, glycopegylation, and conjugation with albumin. See Dumont et al., Blood. 119(13): 3024-3030 (Published online Jan. 13, 2012). Regardless of the protein engineering used, however, the long acting FVIII products currently under development are reported to have limited half-lives—only to about 1.5 to 2 hours in preclinical animal models. See id. Consistent results have been demonstrated in humans, for example, rFVIIIFc was reported to improve half-life up to ˜1.7 fold compared with ADVATE® in hemophilia A patients. See Id. Therefore, the half-life increases, despite minor improvements, may indicate the presence of other T½ limiting factors. See Liu, T. et al., 2007 ISTH meeting, abstract #P-M-035; Henrik, A. et al., 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011 ISTH meeting abstract #P-WE-131. Plasma von Willebrand Factor (VWF) has a half-life of approximately 16 hours (ranging from 13 to 18 hours). Goudemand J, et al. J Thromb Haemost 2005; 3:2219-27. The VWF half-life may be affected by a number of factors: glycosylation pattern, ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin motif-13), and various mutations in VWF. In plasma, 95-98% of FVIII circulates in a tight non-covalent complex with full-length VWF. The formation of this complex is important for the maintenance of appropriate plasma levels of FVIII in vivo. Lenting et al., Blood. 92(11): 3983-96 (1998); Lenting et al., J. Thromb. Haemost. 5(7): 1353-60 (2007). The full-length wild-type FVIII is mostly present as a heterodimer having a heavy chain (MW 200 kD) and a light chain (MW 73 kD). When FVIII is activated due to proteolysis at positions 372 and 740 in the heavy chain and at position 1689 in the light chain, the VWF bound to FVIII is removed from the activated FVIII. The activated FVIII, together with activated factor IX, calcium, and phospholipid (“tenase complex”), induces the activation of factor X, generating large amounts of thrombin. Thrombin, in turn, then cleaves fibrinogen to form soluble fibrin monomers, which then spontaneously polymerize to form the soluble fibrin polymer. Thrombin also activates factor XIII, which, together with calcium, serves to crosslink and stabilize the soluble fibrin polymer, forming crosslinked (insoluble) fibrin. The activated FVIII is cleared fast from the circulation by proteolysis. Due to the frequent dosing and inconvenience caused by the dosing schedule, there is still a need to develop FVIII products requiring less frequent administration, i.e., a FVIII product that has a half-life longer than the 1.5 to 2 fold half-life limitation. BRIEF SUMMARY OF THE INVENTION The present invention provides a chimeric protein comprising (i) a first polypeptide which comprises a Factor VIII (“FVIII”) protein fused to a first immunoglobulin (“Ig”) constant region or a portion thereof and (ii) a second polypeptide 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 an XTEN sequence in-between, wherein the XTEN sequence contains less than 288 amino acid residues and wherein the first polypeptide is linked to or associated with the second polypeptide. Certain embodiments include the chimeric protein as described herein, wherein the XTEN sequence in the second polypeptide