Mutated truncated von Willebrand Factor
The present invention provides a modified polypeptide which binds Factor VIII. The polypeptide comprises truncated von Willebrand Factor (VWF) which comprises a sequence as shown in SEQ ID NO:3 or a fragment thereof or a sequence 90% identical thereto, wherein the truncated VWF comprises at least one modification in comparison to SEQ ID NO: 3 in at least one position selected from the group consisting of SI, S3, LI 8, V42, S43, K149, N248, S279, V320, T325, Q395 and K418.
1. A polypeptide comprising a truncated von Willebrand Factor (VWF), wherein the truncated VWF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:5-12 and 17, and wherein the truncated VWF binds Factor VIII (FVIII).
2. The polypeptide of claim 1 , wherein the truncated VWF binds to FVIII with an off rate lower than a reference polypeptide comprising SEQ ID NO:3.
3. The polypeptide of claim 2 , wherein the truncated VWF binds to FVIII with an off rate at least 5 fold lower than the reference polypeptide.
4. The polypeptide of claim 3 , wherein the truncated VWF binds to FVIII with an off rate at least 10 fold lower than the reference polypeptide.
5. The polypeptide of claim 2 , wherein the truncated VWF binds to FVIII with a dissociation constant (KD) at least 5 fold lower than the reference polypeptide.
6. The polypeptide of claim 5 , wherein the truncated VWF binds to FVIII with an off rate at least 10 fold lower than the reference polypeptide.
7. The polypeptide of claim 1 , wherein the truncated VWF comprises amino acids 1243 to 1247 of SEQ ID NO:2 or amino acids 1243 to 1270 of SEQ ID NO:2.
8. The polypeptide of claim 1 , wherein the truncated VWF lacks amino acids 1243 to 1247 of SEQ ID NO:2 or amino acids 1243 to 2813 of SEQ ID NO:2.
9. The polypeptide of claim 1 , further comprising a half-life extending moiety.
10. The polypeptide of claim 9 , wherein the half-life extending moiety is a heterologous amino acid sequence fused to the truncated VWF.
11. The polypeptide of claim 10 , wherein the heterologous amino acid sequence comprises a polypeptide selected from the group consisting of immunoglobulin constant regions and portions thereof, transferrin and fragments thereof, the C-terminal peptide of human chorionic gonadotropin, solvated random chains with large hydrodynamic volume known as XTEN, homo-amino acid repeats (HAP), proline-alanine-serine repeats (PAS), albumin, afamin, alpha-fetoprotein, Vitamin D binding protein, polypeptides capable of binding under physiological conditions to albumin or immunoglobulin constant regions, and combinations thereof.
12. The polypeptide of claim 10 , wherein the heterologous amino acid sequence comprises an Fc fragment.
13. The polypeptide of claim 10 , wherein the heterologous amino acid sequence comprises albumin.
14. The polypeptide of claim 13 , wherein the N-terminus of the albumin is fused to the C-terminus of the truncated VWF, either directly or via a spacer.
15. The polypeptide of claim 14 , wherein 1 to 5 amino acids at the natural C-terminus of the truncated VWF have been deleted.
16. The polypeptide of claim 9 , wherein the half-life extending moiety is conjugated to the polypeptide.
17. The polypeptide of claim 16 , wherein the half-life extending moiety is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acids (PSAs), elastin-like polypeptides, heparosan polymers, hyaluronic acid, albumin binding ligands, and combinations thereof.
18. The polypeptide of claim 16 , wherein the half-life extending moiety is a fatty acid chain.
19. The polypeptide of claim 1 , wherein the polypeptide is a glycoprotein comprising N-glycans, and wherein at least 75% of the N-glycans comprise, on average, at least one sialic acid moiety.
20. The polypeptide of claim 19 , wherein at least 60% of the N-glycans comprise, on average, at least one α-2,6-sialic acid moiety.
21. The polypeptide of claim 1 , wherein the polypeptide is a glycoprotein comprising N-glycans, and wherein at least 85% of the N-glycans comprise, on average, at least one sialic acid moiety.
22. The polypeptide of claim 1 , wherein the polypeptide is a glycoprotein comprising N-glycans, and wherein at least 90% of the N-glycans comprise, on average, at least one sialic acid moiety.
23. The polypeptide of claim 1 , wherein the polypeptide is a glycoprotein comprising N-glycans, and wherein at least 95% of the N-glycans comprise, on average, at least one sialic acid moiety.
24. The polypeptide of claim 1 , wherein the polypeptide is a dimer.
25. A complex comprising a FVIII molecule and the polypeptide of claim 1 .
26. A pharmaceutical composition comprising the polypeptide of claim 1 .
27. A method of treating a blood coagulation disorder, comprising administering to a patient in need thereof a pharmaceutically effective amount of the polypeptide of claim 1 .
28. The method of claim 27 , wherein the blood coagulation disorder is von Willebrand's disease (VWD) or hemophilia A.
29. A method of treating a blood coagulation disorder, comprising administering to a subject in need thereof a pharmaceutically effective amount of the polypeptide of claim 1 and a FVIII,
wherein the subject has endogenous VWF, and
wherein the molar ratio of the polypeptide to the FVIII is greater than 50, and/or the molar ratio of the polypeptide to the endogenous VWF is greater than 0.5.
30. The method of claim 29 , wherein the subject is a human.
31. The method of claim 29 , wherein the polypeptide is administered intravenously.
32. The method of claim 29 , wherein the FVIII and the polypeptide are administered separately.
33. The method of claim 29 , wherein the FVIII and the polypeptide are administered simultaneously or sequentially.
34. The method of claim 29 , wherein the mean residence time (MRT) of the FVIII is increased by the co-administration of the polypeptide, as compared to a reference treatment in which the polypeptide and the FVIII are administered in equimolar amounts.
35. The method of claim 29 , wherein the frequency of administration of the FVIII is reduced as compared to a treatment with the FVIII alone.
36. The method of claim 29 , wherein the plasma half-life of the polypeptide is greater than that of the endogenous VWF.
37. The method of claim 36 , wherein the plasma half-life of the polypeptide is at least 25% greater than that of the endogenous VWF.
38. A pharmaceutical composition comprising (i) a FVIII and (ii) the polypeptide of claim 1 , wherein the molar ratio of the polypeptide to the FVIII is greater than 50.
39. A method for improving the plasma half-life of FVIII, and/or for reducing the frequency of administration of FVIII, comprising administering to a subject in need thereof an effective amount of the polypeptide of claim 1 .
40. A method for increasing the half-life of FVIII, comprising mixing the FVIII with the polypeptide of claim 1 .
41. A method of treating a blood coagulation disorder, comprising administering to a subject in need thereof an effective amount of the polypeptide of claim 1 and an effective amount of a FVIII, wherein the polypeptide is administered intravenously or subcutaneously, and wherein the FVIII is administered intravenously.
42. The method of claim 41 , wherein the MRT of the FVIII is increased by the co-administration of the polypeptide, as compared to a treatment with the FVIII alone; and/or wherein the frequency of administration of the FVIII is reduced, as compared to a treatment with the FVIII alone.