Modified vitamin K dependent polypeptides
The present invention relates to modified cDNA sequences coding for vitamin K-dependent polypeptides, in particular human Factor VII, human Factor VIIa, human Factor IX and human protein C and their derivatives with improved stability and extended plasma half life, recombinant expression vectors containing such cDNA sequences, host cells transformed with such recombinant expression vectors, recombinant polypeptides and derivatives which do have biological activities of the unmodified wild type protein but having improved stability and processes for the manufacture of such recombinant proteins and their derivatives. The invention also covers a transfer vector for use in human gene therapy, which comprises such modified DNA sequences.
1. A modified human Factor VII, human Factor Vila, or human Factor IX polypeptide having a lengthened activation domain amino acid sequence as compared to a corresponding unmodified, native human Factor VII, human Factor Vila, or human Factor IX, the modified polypeptide comprising
(a) a human Factor VII, human Factor Vila, or human Factor IX polypeptide wherein all or a portion of an activation domain from human Factor IX, human Factor X, or human Prothrombin is inserted into the native activation domain of the human Factor VII, human Factor Vila, or human Factor IX polypeptide, such that the modified polypeptide has a lengthened activation domain amino acid sequence; or
(b) a human Factor VII, human Factor Vila, or human Factor IX polypeptide wherein all or a portion of the native activation domain is removed and all or a portion of an activation domain from human Factor IX, human Factor X, or human Prothrombin is inserted, such that the modified polypeptide has a lengthened activation domain amino acid sequence;
wherein the modified human Factor VII, human Factor Vila, or human Factor IX polypeptide is modified only in the activation domain and has an increased half-life as compared to the unmodified, native human Factor VII, human Factor Vila, or human Factor IX.
2. The modified polypeptide of claim 1 , wherein the modified polypeptide does not contain an N-glycosylation site within its activation peptide.
3. The modified polypeptide of claim 1 , wherein the half-life of the modified polypeptide is increased by at least 50%, as compared to the corresponding unmodified polypeptide.
4. The modified polypeptide of claim 1 , wherein the modified polypeptide has increased stability in an in vitro functional assay, as compared to the corresponding unmodified polypeptide.
5. The modified polypeptide of claim 1 , wherein the modified polypeptide has coagulant activity, as measured in an in vitro coagulation assay.
6. The modified polypeptide of claim 1 , wherein: (i) the activation peptide of the modified polypeptide is capable of being cleaved off during activation, and/or (ii) the zymogen form of the modified polypeptide has an increased half-life as compared to the zymogen form of the corresponding unmodified polypeptide.
7. The modified polypeptide of claim 1 , wherein: (i) the activation peptide of the modified polypeptide is not capable of being cleaved off during activation, and/or (ii) the activated form of the modified polypeptide has an increased half-life as compared to the activated form of the corresponding unmodified polypeptide.
8. The modified polypeptide of claim 1 , comprising an activation domain from human Factor IX, human Factor X, or human Prothrombin in which 1 to 7 amino acids have been added, deleted, and/or substituted.
9. The modified polypeptide of claim 1 , comprising an activation domain from human Factor IX, human Factor X, or human Prothrombin in which 1 to 5 amino acids have been added, deleted, and/or substituted.
10. The modified polypeptide of claim 1 , comprising an activation domain from human Factor IX, human Factor X, or human Prothrombin in which 1 to 3 amino acids have been added, deleted, and/or substituted.
11. The modified polypeptide of claim 1 , wherein the activation domain of the modified polypeptide comprises side chains with glutamic acid residues that can be carboxylated.
12. The modified polypeptide of claim 1 , wherein at least 2 contiguous amino acids have been deleted from the native activation peptide.
13. The modified polypeptide of claim 1 , wherein at least 4 contiguous amino acids have been deleted from the native activation peptide.
14. The modified polypeptide of claim 1 , wherein at least 6 contiguous amino acids have been deleted from the native activation peptide.
15. The modified polypeptide of claim 1 , wherein at least 8 contiguous amino acids have been deleted from the native activation peptide.
16. The modified polypeptide of claim 1 , wherein at least 8 contiguous amino acids from the activation peptide of human Factor IX, human Factor X, or human Prothrombin have been inserted into the native activation peptide.
17. The modified polypeptide of claim 1 , wherein at least 12 contiguous amino acids from the activation peptide of human Factor IX, human Factor X, or human Prothrombin have been inserted into the native activation peptide.
18. The modified polypeptide of claim 1 , wherein at least 15 contiguous amino acids from the activation peptide of human Factor IX, human Factor X, or human Prothrombin have been inserted into the native activation peptide.
19. A pharmaceutical composition comprising the modified polypeptide of claim 1 .