Factor VII or VIIa Polypeptide Variants
The present invention relates to novel polypeptide variants of factor VII (FVII) or factor VIIa (FVIIa) polypeptides, where said variants comprise an amino acid substitution in position 10 and 32 and where said variants further comprise a sugar moiety covalently attached to an introduced in vivo N-glycosylation site located outside of the Gla domain. Such polypeptide variants are useful in therapy, in particular for the treatment of a variety of coagulation-related disorders, such as trauma.
1 . A Factor VII (FVII) or Factor VIIa (FVIIa) polypeptide variant having an amino acid sequence comprising 3-15 amino acid modifications relative to human Factor VII (hFVII) or human Factor VIIa (hFVIIa) having the amino acid sequence shown in SEQ ID NO:2, wherein said amino acid sequence of the variant comprises an amino acid substitution in position 10 and 32 and wherein a sugar moiety is covalently attached to an introduced in vivo N-glycosylation site located outside the Gla domain.
2 . The variant according to claim 1 , wherein said substitution in position 10 is P10Q.
3 . The variant according to claim 1 , wherein said substitution in position 32 is K32E.
4 . The variant according to claim 1 , wherein said substitution in position 10 is P10Q and said substitution in position 32 is K32E.
5 . The variant according to claim 1 , wherein said variant comprises at least one further amino acid modification in the Gla domain.
6 .- 15 . (canceled)
16 . The variant according to claim 5 , wherein said further modification in the Gla domain comprises an amino acid substitution in position 34.
17 . The variant according to claim 16 , wherein a negatively charged amino acid residue is introduced by substitution in position 34.
18 . The variant according to claim 17 , wherein said substitution is A34E.
19 . The variant according to claim 18 , wherein said variant comprises the substitutions P10Q+K32E+A34E.
20 .- 25 . (canceled)
26 . The variant according to claim 1 , wherein said in vivo N-glycosylation site is introduced by substitution.
27 .- 28 . (canceled)
29 . The variant according to claim 26 , wherein said in vivo N-glycosylation site is introduced by a substitution selected from the group consisting of A51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T, I205S, I205T, V253N, T267N, T267N+S269T, S314N+K316S, S314N+K316T, R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318N, D334N and combinations thereof.
30 . The variant according to claim 29 , wherein said in vivo N-glycosylation site is introduced by a substitution selected from the group consisting of A51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T, J205T, V253N, T267N+S269T, S314N+K316T, R315N+V317T, K316N+G318T, G318N, D334N and combinations thereof.
31 . The variant according to claim 30 , wherein said in vivo N-glycosylation site is introduced by a substitution selected from the group consisting of T106N, A175T, I205T, V253N, T267N+S269T and combinations thereof.
32 . The variant according to claim 16 , wherein one in vivo N-glycosylation site has been introduced by substitution.
33 . The variant according to claim 16 , wherein two or more in vivo N-glycosylation sites have been introduced by substitution.
34 .- 40 . (canceled)
41 . The variant according to claim 1 , wherein said variant is in its activated form.
42 . The variant according to claim 1 , wherein said variant, in its activated form, has at least 10% of the amidolytic activity of rhFVIla when assayed in the “Amidolytic Assay” described herein.
43 . The variant according to claim 1 , wherein said variant, in its activated form, has at least 10% of the clotting activity of rhFVIIa when assayed in the “Clotting Assay” described herein.
44 . A nucleotide sequence encoding the variant of claim 1 .
45 . An expression vector comprising the nucleotide sequences of claim 44 .
46 . A host cell comprising the nucleotide sequence of claim 44 .
47 . The host cell according to claim 46 , wherein said host cell is a gammacarboxylating cell capable of in vivo glycosylation.
48 . A pharmaceutical composition comprising the variant of claim 1 , and a pharmaceutical acceptable carrier or excipient.
49 .- 54 . (canceled)
55 . A method for treating a mammal having a disease or a disorder wherein clot formation is desirable, comprising administering to a mammal in need thereof an effective amount of the pharmaceutical composition of claim 48 .
56 . The method according to claim 55 , wherein said disease or disorder is selected from the group consisting of hemorrhages, including brain hemorrhages, severe uncontrolled bleedings, such as trauma, bleedings in patients undergoing living transplantations, bleeding in patients undergoing resection and variceal bleedings.
57 .- 59 . (canceled)