Factor VIII chimeric proteins and uses thereof

1. 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.

2. The chimeric protein of claim 1 , 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.

3. The chimeric protein of claim 1 , 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.

4. The chimeric protein of claim 1 , wherein the FVIII protein comprises a deletion of residues 746-1648 corresponding to native mature human FVIII protein (SEQ ID NO: 65).

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

6. The chimeric protein of claim 1 , 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.

7. The chimeric protein of claim 1 , wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 173.

8. The chimeric protein of claim 1 , wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173.

9. The chimeric protein of claim 1 , 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.

10. The chimeric protein of claim 1 , 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.

11. The chimeric protein of claim 9 , 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.

12. The chimeric protein of claim 2 , wherein the first Fc region and the second Fc region are identical.

13. The chimeric protein of claim 2 , wherein the first Fc region and the second Fc region are derived from human IgG1.

14. The chimeric protein of claim 1 , 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.

15. The chimeric protein of claim 1 , wherein the VWF protein consists of the D′ domain and the D3 domain.

16. The chimeric protein of claim 1 , wherein the VWF protein further comprises the D1 and D2 domains of VWF.

17. The chimeric protein of claim 16 , wherein the VWF protein further comprises a signal peptide of VWF.

18. The chimeric protein of claim 17 , wherein the FVIII protein further comprises a signal peptide of FVIII.

19. The chimeric protein of claim 17 , wherein the second polypeptide chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 197.

20. The chimeric protein of claim 1 , wherein the second polypeptide chain comprises an amino acid sequence at least 99% identical to SEQ ID NO: 197.

21. A chimeric protein comprising:

(i) a first polypeptide chain comprising a Factor VIII (“FVIII”) protein, a first XTEN sequence that is inserted in the FVIII protein, and a first Fc region, wherein the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 173; and

(ii) a second polypeptide chain comprising a von Willebrand Factor (“VWF”) protein, a second XTEN sequence, a linker comprising the amino acid sequence of SEQ ID NO: 88, and a second Fc region, wherein the second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 197;

wherein the first polypeptide chain and the second polypeptide chain are associated through a disulfide bond between the first Fc region and the second Fc region.

22. The chimeric protein of claim 21 , wherein the FVIII protein further comprises a signal peptide of FVIII.

23. The chimeric protein of claim 21 , wherein the first polypeptide chain and the second polypeptide chain are associated through two disulfide bonds between the first Fc region and the second Fc region.

24. 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.

25. The chimeric protein of claim 24 , wherein the first Fc region is associated with the second Fc region through two disulfide bonds.

26. The chimeric protein of claim 24 , 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.

27. The chimeric protein of claim 26 , 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.

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

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

30. 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);

wherein the N-terminal portion comprises the amino acid sequence of residues 1 to 745 of SEQ ID NO: 65 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 a residue 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.

31. The chimeric protein of claim 30 , 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.

32. The chimeric protein of claim 31 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

33. The chimeric protein of claim 32 , 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.

34. The chimeric protein of claim 33 , 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.

35. The chimeric protein of claim 34 , 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.

36. The chimeric protein of claim 35 , wherein the first Fc region and the second Fc region are the same.

37. The chimeric protein of claim 36 , wherein the first Fc region and the second Fc region are derived from human IgG1.

38. The chimeric protein of claim 37 , wherein the VWF protein consists of the D′ domain and the D3 domain.

39. The chimeric protein of claim 37 , wherein the VWF protein further comprises the D1 and D2 domain of VWF.

40. The chimeric protein of claim 39 , wherein the VWF protein further comprises a signal peptide of VWF.

41. The chimeric protein of claim 40 , wherein the FVIII protein further comprises a signal peptide of FVIII.

42. The chimeric protein of claim 30 , wherein the cleavable linker comprises an a2 region of FVIII comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106.

43. A pharmaceutical composition comprising the chimeric protein of claim 30 and a pharmaceutically acceptable carrier.

44. 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 a2region 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.

45. The chimeric protein of claim 44 , wherein the VWF protein contains an alanine substitution at residue 1099 and residue 1142 of SEQ ID NO: 21.

46. The chimeric protein of claim 45 , wherein the first Fc region and the second Fc region are the same.

47. The chimeric protein of claim 45 , wherein the first Fc region and the second Fc region are derived from human IgG1.

48. The chimeric protein of claim 47 , wherein the VWF protein consists of the D′ domain and the D3 domain.

49. The chimeric protein of claim 47 , wherein the VWF protein further comprises the D1 and D2 domain of VWF.

50. The chimeric protein of claim 49 , wherein the VWF protein further comprises a signal peptide of VWF.

51. The chimeric protein of claim 50 , wherein the FVIII protein further comprises a signal peptide of FVIII.

52. The chimeric protein of claim 47 , wherein the first Fc region is associated with the second Fc region through two disulfide bonds.

53. The chimeric protein of claim 44 , wherein the cleavable linker comprises an a2 region of FVIII comprising an amino acid sequence at least 90% identical to SEQ ID NO: 106.

54. A pharmaceutical composition comprising the chimeric protein of claim 44 and a pharmaceutically acceptable carrier.

55. The chimeric protein of claim 30 , wherein the cleavable linker is 20 to 50 amino acids long.

56. The chimeric protein of claim 44 , wherein the cleavable linker is 20 to 50 amino acids long.

57. The chimeric protein of claim 30 , wherein the cleavable linker is about 30 amino acids long.

58. The chimeric protein of claim 44 , wherein the cleavable linker is about 30 amino acids long.