Recombinant factor VIII having reduced inactivation by activated protein C
The present invention relates to a recombinant factor VIII that is characterized by one or more mutations within a region surrounding an activated protein C cleavage site, which one or more mutations result in a reduced rate of inactivation by activated protein C. Isolated nucleic acid molecules, recombinant expression vectors, and host cells suitable for expression of the recombinant factor VIII are also disclosed. The recombinant factor VIII can be used for the treatment of clotting disorders, such as hemophilia A.
1. A recombinant factor VIII comprising an A1 domain comprising one or more substitutions within the amino acid sequence EEPQLRMKNNE (residues 331-341 of SEQ ID NO: 2), wherein the one or more substitutions exclude the arginine residue and said recombinant factor VIII has a reduced rate of inactivation by activated protein C compared to wildtype factor VIII.
2. The recombinant factor VIII according to claim 1 , wherein the one or more substitutions comprises substitution of proline at the third residue of said amino acid sequence.
3. The recombinant factor VIII according to claim 1 , wherein the one or more substitutions comprises substitution of glutamine at the fourth residue of said amino acid sequence.
4. The recombinant factor VIII according to claim 1 , wherein the one or more substitutions comprises substitution of leucine at the fifth residue of said amino acid sequence.
5. The recombinant factor VIII according to claim 1 , wherein the one or more substitutions comprises substitution of methionine at the seventh residue of said amino acid sequence.
6. The recombinant factor VIII according to claim 1 , wherein the one or more substitutions comprises substitution of lysine at the eighth residue of said amino acid sequence.
7. The recombinant factor VIII according to claim 1 , wherein the one or more substitutions comprises substitution of asparagine at the ninth residue of said amino acid sequence.
8. The recombinant factor VIII according to claim 1 , wherein the one or more substitutions comprise substitution of -PQL- with -VDQ-, substitution of -MKN-with -GNQ- , or both, within said amino acid sequence.
9. The recombinant factor VIII according to claim 1 , wherein the recombinant factor VIII consists of domains A1, A2, A3, C1, and C2, or portions thereof.
10. The recombinant factor VIII according to claim 1 , wherein the recombinant factor VIII is substantially pure.
11. The recombinant factor VIII according to claim 1 further comprising a point mutation of a glutamic acid residue corresponding to position 113 of SEQ ID NO: 2 (Glu113).
12. A pharmaceutical composition comprising the recombinant factor VIII according to claim 1 .
13. The pharmaceutical composition according to claim 12 further comprising a stabilizer, a delivery vehicle, or a pharmaceutically acceptable carrier.
14. A method of treating an animal for hemophilia A, the method comprising:
administering to an animal exhibiting hemophilia A an effective amount of the recombinant factor VIII according to claim 1 , whereby the animal exhibits effective blood clotting following vascular injury.
15. The method according to claim 14 , wherein the effective amount comprises between about 10 to about 50 units/kg body weight of the animal.
16. The method according to claim 14 , wherein the animal is a human.
17. The method according to claim 14 , further comprising periodically repeating said administering.
18. The recombinant factor VIII according to claim 1 wherein the recombinant factor VIII further comprises a substitution of a glutamic acid residue corresponding to position 287 of SEQ ID NO: 2(G 1 u287), a substitution of an aspartic acid residue corresponding to position 302 of SEQ ID NO: 2 (Asp302), a substitution of an aspartic acid residue corresponding to position 519 of SEQ ID NO: 2 (Asp519), a substitution of a glutamic acid residue corresponding to position 665 of SEQ ID NO: 2 (G 1 u665), a substitution of a glutamic acid residue corresponding to position 1984 of SEQ ID NO: 2 (G 1 u1984), or combinations thereof.
19. The recombinant factor VIII according to claim 18 , wherein the substitution of the Asp302 residue is Asp302Ala.
20. The recombinant factor VIII according to claim 18 , wherein the substitution of the G 1 u287 residue is Glu287Ala.
21. The recombinant factor VIII according to claim 18 , wherein the substitution of the G 1 u665 residue is Glu665Ala or Glu665Val.
22. The recombinant factor VIII according to claim 18 , wherein the substitution of the Asp519 residue is Asp519Ala or Asp519Val.
23. The recombinant factor VIII according to claim 18 , wherein the substitution of the G 1 u 1 984 residue is Glu1984Ala or Glu1984Val.