Modified factor IX polypeptides and uses thereof
Modified Factor IX (FIX) polypeptides and uses thereof are provided. Such modified FIX polypeptides include FIXa and other forms of FIX. Among the modified FIX polypeptides provided are those that have altered activities, typically altered procoagulant activity, including increased procoagulant activities. Hence, such modified polypeptides are therapeutics.
1. A modified FIX polypeptide, comprising the amino acid replacements R318Y, R338E and T343R in an unmodified FIX polypeptide, wherein:
corresponding amino acid residues are identified by alignment of the unmodified FIX polypeptide with the polypeptide of SEQ ID NO:3;
the unmodified FIX polypeptide comprises the sequence of amino acids set forth in any of SEQ ID NOS: 2, 3, 20 or 325, or a sequence of amino acid residues having at least 95% sequence identity to the FIX polypeptide sequence set forth in any of SEQ ID NOS: 2, 3, 20 or 325; and
the modified FIX polypeptide, when an active form, exhibits one or both of increased catalytic activity and increased procoagulant activity compared with the unmodified FIX polypeptide.
2. The modified FIX polypeptide of claim 1 , comprising an amino acid replacement at residue E410 or at an amino acid residue corresponding to 410 in an unmodified FIX polypeptide, wherein the replacement amino acid is N or S.
3. The modified FIX polypeptide of claim 2 , wherein the amino acid replacement is N.
4. The modified FIX polypeptide of claim 1 , comprising an amino acid replacement at residue 403of a mature FIX polypeptide having a sequence set forth in SEQ ID NO:3 or at amino acid residues corresponding to residue 403 in an unmodified FIX polypeptide, wherein the replacement is E.
5. The modified FIX polypeptide of claim 1 , comprising the amino acid replacements R318Y, R338E, T343R, R403E and E410N in a mature FIX polypeptide having a sequence set forth in SEQ ID NO: 3 or the same replacements at corresponding amino acid residues in an unmodified FIX polypeptide.
6. The modified FIX polypeptide of claim 1 that comprises amino acid replacements selected from among replacements R318Y/R338E/T343R/R403E/E410N, R318Y/R338E/T343R/E410N, Y155F/R318Y/R338E/T343R/R403E, Y155F/K228N/K247N/N249S/R318Y/R338E/T343R/R403E/E410N, Y155F/K247N/N249 5 /R318Y/R338E/T343R/R403E, K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R, Y155F/K247N/N249S/R318Y/R338E/T343R, K228N/R318Y/R338E/T343R/R403E/E410N, K228N/K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R/R403E/E410S, K247N/N249S/R318Y/R338E/T343R, K247N/N249S/R318Y/R338E/T343R/E410N, Y155F/K247N/N249S/R318Y/R338E/T343R/E410N, Y155F/K247N/N249S/R318Y/T343R/R403E/E410N, Y155F/R318Y/R338E/T343R and K228N/K247N/N249S/R318Y/R338E/T343R/E410N, or the same replacements at corresponding amino acid residues in an unmodified FIX polypeptide.
7. The modified FIX polypeptide of claim 1 , comprising an amino acid replacement R318Y/R338E/T343R/R403E/E410N, R318Y/R338E/T343R/E410N, Y155F/R318Y/R338E/T343R/R403E, Y155F/K228N/K247N/N249S/R318Y/R338E/T343R/R403E/E410N, Y155F/K247N/N249S/R318Y/R338E/T343R/R403E, K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R, Y155F/K247N/N249S/R318Y/R338E/T343R, K228N/R318Y/R338E/T343R/R403E/E410N, K228N/K247N/N249S/R318Y/R338E/T343R/R403E, R318Y/R338E/T343R/R403E/E410S or K247N/N249S/R318Y/R338E/T343R.
8. The modified FIX polypeptide of claim 1 , comprising amino acid replacements R318Y/R338E/T343R, R318Y/R338E/T343R/R403E/E410N or R318Y/R338E/T343R/E410N.
9. The modified FIX polypeptide of claim 1 , further comprising the amino acid replacement Y155F or the same replacement at a corresponding amino acid residue in an unmodified FIX polypeptide.
10. A modified FIX polypeptide, comprising the sequence of amino acids set forth in any of SEQ ID NOS: 267, 330, 331, 334, 336, 345-348, 353, 354, 357-360, 366, 368, 369, 378, 379, 393, 394, 397, 398 and 405-407, or a sequence of amino acids that exhibits at least 95% amino acid sequence identity with the sequence of amino acids set forth in any of SEQ ID NOS: 267, 330, 331, 334, 336, 345-348, 353, 354, 357-360, 366, 368, 369, 378, 379, 393, 394, 397, 398 and 405-407 and comprises the amino replacements corresponding to R318Y, R338E and T343R, wherein corresponding replacements are identified by alignment with SEQ ID NO:3, wherein the modified FIX polypeptide, when activated, exhibits one or both of increased catalytic activity and increased procoagulant activity compared with the unmodified FIX polypeptide lacking the R318Y, R338E and T343R replacements.
11. The modified FIX polypeptide of claim 1 that comprises one or more modifications selected from a chemical modification or a post-translational modification, wherein the modified FIX polypeptide is glycosylated, carboxylated, hydroxylated, sulfated, phosphorylated, albuminated, or conjugated to a polyethylene glycol (PEG) moiety.
12. A pharmaceutical composition, comprising the modified FIX polypeptide of claim 1 , in a pharmaceutically acceptable vehicle.
13. The pharmaceutical composition of claim 12 that is formulated for local, systemic, or topical administration.
14. The pharmaceutical composition of claim 12 that is formulated for oral, nasal, pulmonary, buccal, transdermal, subcutaneous, intraduodenal, enteral, parenteral, intravenous, or intramuscular administration.
15. The pharmaceutical composition of claim 12 that is formulated for controlled-release.
16. The pharmaceutical composition of claim 12 that is formulated for single-dosage administration.
17. An article of manufacture, comprising packaging material and the pharmaceutical composition of claim 12 contained within the packaging material.
18. A kit, comprising the pharmaceutical composition of claim 12 , a device for administration of the composition and, optionally, instructions for administration.
19. The pharmaceutical composition of claim 12 , wherein the modified FIX polypeptide comprises the sequence of amino acids set forth in SEQ ID NO:394.
20. A factor IX (FIX) polypeptide consisting of the polypeptide sequence of amino acid residues of SEQ ID NO:394.
21. The FIX polypeptide of claim 20 that is glycosylated.
22. The FIX polypeptide of claim 20 that is hyperglycosylated.
23. The modified FIX polypeptide of claim 1 that is hyperglycosylated.
24. The modified FIX polypeptide of claim 1 that is a mature polypeptide.
25. The modified FIX polypeptide of claim 1 that is a two-chain polypeptide.
26. The modified FIX polypeptide of claim 1 that is a single-chain polypeptide.
27. The modified FIX polypeptide of claim 1 that is activated.
28. A pharmaceutical composition, comprising the FIX polypeptide of claim 20 , in a pharmaceutically acceptable vehicle.
29. A factor IX (FIX) polypeptide zymogen comprising the polypeptide sequence of amino acid residues of SEQ ID NO:394.
30. A method, comprising treating a subject by administering the pharmaceutical composition of claim 12 , wherein the subject has a disease or condition that is treated by administration of FIX or a procoagulant.
31. The method of claim 30 , wherein the disease or condition is treated by administration of active FIX (FIXa).
32. The method of claim 30 , wherein treatment with the pharmaceutical composition ameliorates or alleviates the symptoms associated with the disease or condition.
33. The method of claim 30 , further comprising monitoring the subject for changes in the symptoms associated with disease or condition that is treated by administration of FIX or a procoagulant.
34. The method of claim 30 , wherein the disease or condition to be treated is selected from among blood coagulation disorders, hematologic disorders, hemorrhagic disorders, hemophilias, and bleeding disorders.
35. The method of claim 34 , wherein the hemophilia is hemophilia B.
36. The method of claim 30 , further comprising administering one or more additional coagulation factors.
37. The method of claim 36 , wherein the one or more additional coagulation factors are selected from among plasma purified or recombinant coagulation factors, procoagulants, such as vitamin K, vitamin K derivative and protein C inhibitors, plasma, platelets, red blood cells and corticosteroids.