High molecular weight derivatives of vitamin K-dependent polypeptides
Modifications of vitamin K-dependent polypeptides that lead to enhanced protein function on a weight or molar basis and/or increase of protein lifetime in the circulation are described. Both objectives are important for using vitamin K-dependent polypeptides for pro- and anti-coagulation therapies, as well as for other uses in the circulation.
1. An isolated or recombinant mutant Factor VII or Factor VIIa polypeptide linked to a polyethylene glycol (PEG) polymer, wherein the polypeptide comprises a modified gamma-carboxyglutamic acid (GLA) domain having an amino acid sequence with two, three, four, or five substitutions relative to the amino acid sequence of the wild-type human Factor VII or Factor VIIa GLA domain, wherein the substitutions comprise P10Q and K32E, and wherein the circulation lifetime of the polypeptide linked to PEG is increased as compared to a corresponding polypeptide not linked to PEG, and offsets any reduction in coagulation function as compared to a corresponding polypeptide not linked to PEG.
2. The polypeptide of claim 1 , wherein the polypeptide is a mutant Factor VIIa polypeptide.
3. The polypeptide of claim 1 , wherein the PEG polymer is linked to the polypeptide via an amino group or a carbohydrate group on the polypeptide.
4. The polypeptide of claim 1 , wherein the PEG polymer is linked to the polypeptide via a lysine residue of the polypeptide.
5. The polypeptide of claim 1 , wherein the polypeptide is linked to at least two PEG polymers.
6. The polypeptide of claim 5 , wherein the polypeptide is linked to three PEG polymers.
7. The polypeptide of claim 1 , wherein the PEG polymer has a molecular weight of 10,000 to 40,000 Daltons.
8. The polypeptide of claim 7 , wherein the PEG polymer has a molecular weight of 10,000 Daltons.
9. The polypeptide of claim 7 , wherein the PEG polymer has a molecular weight of 20,000 Daltons.
10. The polypeptide of claim 7 , wherein the PEG polymer has a molecular weight of 40,000 Daltons.
11. The polypeptide of claim 1 , wherein the PEG polymer is linked to the polypeptide via an amino group or a carbohydrate group on the polypeptide.
12. The polypeptide of claim 1 , wherein the PEG polymer is linked to the polypeptide via a lysine residue of the polypeptide.
13. The polypeptide of claim 1 , wherein the mutant Factor VIIa polypeptide has an increased membrane binding affinity relative to wild-type human Factor VIIa.
14. The polypeptide of claim 1 , wherein the PEG polymer is directly linked to the polypeptide.
15. The polypeptide of claim 1 , wherein the PEG polymer is indirectly linked to the polypeptide.
16. A composition comprising a carrier and a recombinant mutant Factor VII or Factor VIIa polypeptide linked to a PEG polymer, wherein the polypeptide comprises a modified GLA domain having an amino acid sequence with two, three, four, or five substitutions relative to the amino acid sequence of the wild-type human Factor VII or Factor VIIa GLA domain, wherein the substitutions comprise P10Q and K32E, and wherein the circulation lifetime of the polypeptide linked to PEG is increased as compared to a corresponding polypeptide not linked to PEG, and offsets any reduction in coagulation function as compared to a corresponding polypeptide not linked to PEG.
17. The composition of claim 16 , wherein the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.
18. The composition of claim 17 , wherein the polypeptide is a recombinant mutant Factor VII polypeptide.
19. The composition of claim 17 , wherein the polypeptide is a recombinant mutant Factor VIIa polypeptide.
20. The composition of claim 16 , wherein the polypeptide is a mutant Factor VIIa polypeptide.
21. The composition of claim 16 , wherein the PEG polymer is linked to the polypeptide via an amino group or a carbohydrate group on the polypeptide.
22. The composition of claim 16 , wherein the PEG polymer is linked to the polypeptide via a lysine residue of the polypeptide.
23. The composition of claim 16 , wherein the polypeptide is linked to at least two PEG polymers.
24. A method for increasing blood clot formation in a subject having a condition in which increased clot formation is desirable, comprising administering to the subject an amount of the mutant Factor VII or Factor VIIa polypeptide of claim 1 effective to increase blood clot formation.
25. The method of claim 24 , wherein the condition is hemophilia.
26. A method for preparing a mutant Factor VII or Factor VIIa polypeptide linked to a PEG polymer, the polypeptide comprising an amino acid sequence with two, three, four, or five substitutions relative to the amino acid sequence of wild-type human Factor VII or Factor VIIa GLA domain, wherein the substitutions comprise P10Q and K32E, wherein the method comprises incubating a reactive PEG polymer with the mutant Factor VII or Factor VIIa polypeptide for a time sufficient to link the PEG polymer to the polypeptide, wherein the PEG polymer is reactive with amino groups or carbohydrate groups of the polypeptide, and wherein the circulation lifetime of the polypeptide linked to PEG is increased as compared to a corresponding polypeptide not linked to PEG, and offsets any reduction in coagulation function as compared to a corresponding polypeptide not linked to PEG.
27. The method of claim 26 , wherein the polypeptide is a mutant Factor VIIa polypeptide.
28. The method of claim 26 , wherein the PEG polymer is linked to the polypeptide via an amino group or a carbohydrate group on the polypeptide.
29. The method of claim 26 , wherein the PEG polymer is linked to the polypeptide via a lysine residue of the polypeptide.
30. The method of claim 26 , wherein the polypeptide is linked to at least two PEG polymers.