Modified coagulation factors with prolonged in vivo half-life
The present invention relates to nucleic acid sequences coding for modified coagulation factors, preferably coagulation factor VIII, and their derivatives; recombinant expression vectors containing such nucleic acid sequences; host cells transformed with such recombinant expression vectors; and recombinant polypeptides and derivatives coded for by said nucleic acid sequences, whereby said recombinant polypeptides and derivatives have biological activities and prolonged in vivo half-lives compared to the unmodified wild-type proteins. The invention also relates to corresponding sequences that result in improved in vitro stability. The present invention further relates to processes for the manufacture of such recombinant proteins and their derivatives. The invention also relates to a transfer vector for use in human gene therapy, which comprises such nucleic acid sequences.
1. A modified factor VIII (FVIII) polypeptide, comprising a FVIII polypeptide having an N-terminal amino acid and a C-terminal amino acid, and a half-life enhancing polypeptide (HLEP) inserted within the B-domain between the N-terminal amino acid and the C-terminal amino acid of the FVIII polypeptide,
wherein the FVIII polypeptide is capable of being cleaved from the HLEP moiety during activation in vivo, wherein the modified FVIII polypeptide exhibits a prolonged half-life prior to activation during a bleeding event and a half-life substantially the same as that of an unmodified FVIII peptide following activation, and
wherein the HLEP comprises albumin or an immunoglobulin constant region polypeptide.
2. The modified FVIII polypeptide according to claim 1 , wherein the modified FVIII polypeptide has a prolonged functional half-life as compared to a FVIII polypeptide lacking an inserted HLEP.
3. The modified FVIII polypeptide according to claim 1 , wherein the modified FVIII polypeptide has a prolonged antigenic half-life as compared to the FVIII polypeptide lacking an inserted HLEP.
4. The modified FVIII polypeptide according to claim 2 , wherein the functional half-life is increased by at least 25% as compared to the functional half-life of the FVIII polypeptide lacking an inserted HLEP.
5. The modified FVIII polypeptide according to claim 3 , wherein the antigenic half-life is increased by at least 25% as compared to the antigenic half-life of the FVIII polypeptide lacking an inserted HLEP.
6. The modified FVIII polypeptide according to claim 1 , wherein the modified FVIII polypeptide has an improved in vivo recovery as compared to the FVIII polypeptide lacking an inserted HLEP.
7. The modified FVIII polypeptide according to claim 6 , wherein the in vivo recovery is increased by at least 10% as compared to the in vivo recovery of the FVIII polypeptide lacking an inserted HLEP.
8. The modified FVIII polypeptide according to claim 1 , wherein the modified FVIII polypeptide has increased stability in serum-free culture media and/or in animal protein-free culture media as compared to the FVIII polypeptide lacking an inserted HLEP.
9. The modified FVIII polypeptide according to claim 1 , wherein the B-domain of FVIII or a part thereof is replaced with the HLEP.
10. The modified FVIII polypeptide according to claim 9 , wherein more than 75% of the B-domain is deleted, or more than 75% of the B-domain is replaced by linker sequences.
11. The modified FVIII polypeptide according to claim 1 , wherein the modified FVIII polypeptide has at least 10% of the biological activity of the FVIII polypeptide lacking an inserted HLEP.
12. The modified FVIII polypeptide according to claim 1 , wherein the half-life enhancing polypeptide is albumin.
13. The modified FVIII polypeptide according to claim 1 , wherein the B-domain of FVIII has been replaced partly or completely with human albumin.
14. A pharmaceutical composition comprising a modified FVIII polypeptide according to claim 1 and a pharmaceutically acceptable carrier or excipient.
15. The modified FVIII polypeptide according to claim 1 , wherein the immunoglobulin constant region polypeptide is an immunoglobulin G Fc domain.