Factor VIII-XTEN fusions and uses thereof

• US 10,548,953 B2
• Filed: 08/14/2014
• Issued: 02/04/2020
• Est. Priority Date: 08/14/2013
• Status: Active Grant

• Current Assignee: Bioverativ Therapeutics Inc. (Sanofi )
• Original Assignee: Bioverativ Therapeutics Inc. (Sanofi )
• Inventors: Liu, Tongyao, Chang, Pei-Yun, Kulman, John, Schellenberger, Volker, Jiang, Haiyan, Peters, Robert T., Chhabra, Ekta Seth
• Primary Examiner(s): Wehbe, Ann Marie S
• Application Number:
  US14/912,047
• Publication Number:
  US 20160199454A1
• Time in Patent Office: 2,000 Days
• Field of Search: 514 141, 514 44 R, 530383, 4353201, 435325
• 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 38/37 Factors VIII
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C12N 15/85 for animal cells

Abstract

The present invention provides chimeric proteins comprising a Factor VIII (FVIII) polypeptide and XTEN polypeptides. The FVIII polypeptide can have a reduced affinity for von Willebrand Factor (VWF). XTEN polypeptides of appropriate sizes are inserted into the FVIII polypeptide at particular locations in order to extend the half-life of the FVIII polypeptide. The invention also includes nucleotides, vectors, host cells, and methods of using the chimeric proteins, e.g., to treat bleeding diseases and disorders.

Claims

1. A chimeric protein comprising (i) a Factor VIII (FVIII) polypeptide and (ii) four XTENs wherein the FVIII polypeptide has reduced affinity for von Willebrand Factor (VWF), and wherein the FVIII polypeptide lacks amino acids 746-1685 corresponding to the mature human FVIII polypeptide (SEQ ID NO:

4).

2. The chimeric protein of claim 1, wherein at least three of the four XTENs are inserted immediately downstream of residues selected from the group consisting of:

(a) residues 18, 745, and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO;

     4);

(b) residues 745, 1720 and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO;

     4); and

(c) residues 745, 1900, and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO;

     4).

3. The chimeric protein of claim 1, wherein at least two of the four XTENs are inserted immediately downstream of residues 745 and 2332 corresponding to the mature FVIII polypeptide (SEQ ID NO:

4).

4. The chimeric protein of claim 1, wherein at least one of the four XTENs is 36 to 72 amino acids in length or 42 to 78 amino acids in length.

5. The chimeric protein of claim 1, wherein at least one of the four XTENs is selected from the group consisting of SEQ ID NO:

22 (36AE), SEQ ID NO;

36 (42AE), SEQ ID NO;

23 (72AE), and SEQ ID NO;

24 (78AE).

6. The chimeric protein of claim 1, wherein at least one of the four XTENs is selected from the group consisting of SEQ ID NO:

37 (144AE), SEQ ID NO;

38 (144AG), SEQ ID NO;

98 (150AE), SEQ ID NO;

99 (150 AG), and any combination thereof.

7. The chimeric protein of claim 1, wherein:

(a) an XTEN of SEQ ID NO;

22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO;

37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO;

39 (288 AE) is inserted immediately downstream of residue 2332;

(b) an XTEN of SEQ ID NO;

36 (42AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO;

98 (150AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

98 (150 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO;

100 (294 AE) is inserted immediately downstream of residue 2332;

(c) an XTEN of SEQ ID NO;

37 (144AE) or SEQ ID NO;

39 (288 AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

22 (36AE) is inserted immediately downstream of residue 1720, an XTEN of SEQ ID NO;

37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO;

39 (288 AE) is inserted immediately downstream of residue 2332;

(d) an XTEN of SEQ ID NO;

98 (150AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

36 (42AE) is inserted immediately downstream of residue 1720, an XTEN of SEQ ID NO;

98 (150 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO;

100 (294AE) is inserted immediately downstream of residue 2332;

(e) an XTEN of SEQ ID NO;

22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO;

37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

22 (36 AE) is inserted immediately downstream of residue 1720, and an XTEN of SEQ ID NO;

39 (288 AE) is inserted immediately downstream of residue 2332;

or

(f) an XTEN of SEQ ID NO;

36 (42AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO;

98 (150AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

36 (42 AE) is inserted immediately downstream of residue 1720, and an XTEN of SEQ ID NO;

100 (294 AE) is inserted immediately downstream of residue 2332.

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

9. A vector or a set of vectors comprising the polynucleotide or the set of polynucleotides of claim 8 and one or more promoter operably linked to the polynucleotide or the set of polynucleotides.

10. A host cell comprising the polynucleotide of claim 8.

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

12. 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.

13. A method of making a chimeric protein, comprising transfecting one or more host cells with the polynucleotide of claim 8 and expressing the chimeric protein in the host cell.

14. A method of extending or increasing the half-life of a FVIII protein, wherein the method comprises administering an effective amount of the chimeric protein of claim 1 to a subject in need thereof, wherein the XTEN sequence or sequences extend or increase the half-life of the FVIII protein.

15. The chimeric protein claim 1, wherein the four XTENs comprise an XTEN of SEQ ID NO:

22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO;

37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO;

39 (288 AE) is inserted immediately downstream of residue 2332.

16. The chimeric protein claim 1, wherein the four XTENs comprise an XTEN of SEQ ID NO:

37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

22 (36AE) is inserted immediately downstream of residue 1720, an XTEN of SEQ ID NO;

37 (144 AE) is inserted immediately downstream of residue 1900, and an XTEN of SEQ ID NO;

39 (288 AE) is inserted immediately downstream of residue 2332.

17. The chimeric protein of claim 1, wherein the four XTENs comprise an XTEN of SEQ ID NO:

22 (36AE) is inserted immediately downstream of residue 18, an XTEN of SEQ ID NO;

37 (144AE) is inserted immediately downstream of residue 745, an XTEN of SEQ ID NO;

22 (36 AE) is inserted immediately downstream of residue 1720, and an XTEN of SEQ ID NO;

39 (288 AE) is inserted immediately downstream of residue 2332.