Factor VII or VIIa - Like Molecules
Conjugates of Factor VII (FVII) and Factor VIIa (FVIIA) are provided, as are methods for preparing them. Methods for producing novel polypeptides contributing to the production of such conjugates are provided. Methods of treatment by administering a FVII or FVIIa conjugate are provided.
1 . A conjugate comprising at least one non-polypeptide moiety covalently attached to a polypeptide, wherein the amino acid sequence of the polypeptide differs from that of wild-type FVII or FVIIa shown in SEQ ID NO:1 in that at least one amino acid residue comprising an attachment group for said non-polypeptide moiety has been introduced or removed,
wherein if said non-polypeptide moiety is a sugar moiety and if said attachment group is an attachment group for an in vivo N-glycosylation site, then said at least one amino acid residue does not comprise the substitution T106N or V253N.
2 . The conjugate according to claim 1 , wherein the attachment group is selected from the group consisting of lysine, cysteine, aspartic acid and glutamic acid.
3 . The conjugate according to claim 2 , wherein the attachment group is lysine.
4 . The conjugate according to claim 3 , wherein the amino acid sequence of the polypeptide differs from SEQ ID NO:1 in that at least one lysine residue has been removed.
5 . The conjugate according to claim 4 , wherein the lysine residue has been removed by substitution.
6 . The conjugate according to claim 5 , wherein the removed lysine residue is selected from the group consisting of K18, K32, K38, K62, K85, K109, K137, K143, K148, K157, K161, K197, K199, K316, K337, K341, K389 and combinations thereof.
7 . The conjugate according to claim 6 , wherein the lysine residue is selected from the group consisting of K18, K62, K85, K197, K341 and combinations thereof.
8 . The conjugate according to claim 7 , wherein the lysine residue is substituted with an amino acid residue selected from the group consisting of R, Q, N and H.
9 . The conjugate according to claim 8 , wherein the lysine residue is substituted with R.
10 . The conjugate according to claim 3 , wherein the amino acid sequence of the polypeptide differs from SEQ ID NO:1 in that at least one lysine residue has been introduced.
11 . The conjugate according to claim 10 , wherein the lysine residue has been introduced by substitution.
12 . The conjugate according to claim 11 , wherein the substitution is selected from the group consisting of 142K, Y44K, L288K, D289K, R290K, G291K, A292K, T293K, Q313K, S314K, R315K, V317K. L390K, M391K, R392K, S393K, E394K, P395K, R396K, P397K, G398K, V399K, L400K, L401K, R402K, A403K, P404K, F405K and combinations thereof.
13 . The conjugate according to claim 12 , wherein the substitution is selected from the group consisting of R290K, R315K, R392K, R396K, R402K and combinations thereof.
14 . The conjugate according to claim 3 , wherein at least one lysine residue has been removed and at least one lysine residue has been introduced.
15 . The conjugate according to claim 2 , wherein the attachment group is cysteine.
16 . The conjugate according to claim 15 , wherein the cysteine residue has been introduced by substitution.
17 . The conjugate according to claim 16 , wherein the substitution is selected from the group consisting of 130C, K32C, D33C, A34C, T37C, K38C, W41C, Y44C, S45C, D46C, L141C, E142C. K143C, R144C, L288C, D289C, R290C, G291C, A292C, S314C, R315C, K316C. V317C, L390C, M391C, R392C, S393C, E394C, P395C, R396C, P397C, G398C, V399C, L401C, R402C, A403C, P404C and combinations thereof.
18 . The conjugate according to claim 17 , wherein the substitution is selected from the group consisting of K32C, Y44C, K143C, R290C, R315C, K341C, R392C, R396C, R402C and combinations thereof.
19 . The conjugate according to claim 2 , wherein the attachment group is aspaitic acid and/or glutamic acid.
20 . The conjugate according to claim 19 , wherein the amino acid sequence of the polypeptide differs from SEQ ID NO:1 in that at least one aspartic acid residue and/or glutamic acid residue has been introduced.
21 . The conjugate according to claim 20 , wherein the aspartic acid residue and/or the glutamic acid residue has been introduced by substitution.
22 . The conjugate according to claim 21 , wherein the substitution is selected from the group consisting of I130D/E, K32D/E, A34D/E, T37D/E, K38D/E, W41D/E, Y44D/E, S45D/E, D46C, L141D/E, E142D/E, K143D/E, R144D/E, L288D/E, R290D/E, G291D/E, A292D/E, Q313D/E, S314D/E, R315D/E, K316D/E, V317D/E, L390D/E, M391D/E, R392D/E, S393D/E, P395D/E, R396D/E, P397D/E, G398D/E, V399D/E, L401D/E, R402D/E, A403D/E, P404D/E, and combinations thereof.
23 . The conjugate according to claim 22 , wherein the substitution is selected from the group consisting of K32D/E, Y44D/E, K143D/E, R290D/E, R315D/E, K341D/E, R392D/E, R396D/E, R402D/E and combinations thereof.
24 . The conjugate according to claim 19 , wherein the amino acid sequence of the polypeptide differs from SEQ ID NO:1 in that at least one aspartic acid residue and/or glutamic acid residue has been removed.
25 . The conjugate according to claim 24 , wherein the aspartic acid residue and/or glutamic acid residue has been removed by substitution.
26 . The conjugate according to claim 25 , wherein the substitution is selected from the group consisting of D33, D46, D48, E77, E82, D86, D87, E94, E99, D104, E116, D123, E132, E142, E163, D196, E210, D212, E215, D217, D219, E220, D256, E265, E270, D289, E296, D309, D319, E325, D334, D338, D343, E385, E394 and combinations thereof.
27 . The conjugate according to claim 19 , wherein at least one aspartic acid and/or glutamic acid residue has been introduced and at least one aspartic acid and/or glutamic acid residue has been removed.
28 . The conjugate according to claim 1 , wherein the non-polypeptide moiety is a polymer molecule.
29 . The conjugate according to claim 28 , wherein the polymer molecule is selected from the group consisting of natural or synthetic homopolymers and heteropolymers.
30 . The conjugate according to claim 29 , wherein the polymer molecule is a synthetic homo-polymer or hetero-polymer selected from the group consisting of linear and branched polyethylene glycol, polyvinyl alcohol (PVA), polycarboxylic acids and poly-(vinylpyrrolidone).
31 . The conjugate according to claim 30 , wherein the polymer is a linear or branched polyethylene glycol.
32 . The conjugate according to claim 31 , wherein the polyethylene glycol has a molecular weight of about 300 to 100,000 Da.
33 . The conjugate according to claim 1 , wherein the polypeptide differs in 1-15 amino acid residues from the amino acid sequence shown in SEQ ID NO:1.
34 . The conjugate according to claim 1 , wherein said non-polypeptide moiety is a sugar moiety and wherein said attachment group is an attachment group for a sugar moiety.
35 . The conjugate according to claim 34 , wherein said attachment group is a glycosylation site.
36 . The conjugate according to claim 35 , wherein at least one non-naturally occurring glycosylation site has been introduced.
37 . The conjugate according to claim 35 , wherein at least one naturally occurring glycosylation site has been removed.
38 . The conjugate according to claim 35 , wherein at least one non-naturally occurring glycosylation site has been introduced and at least one naturally occurring glycosylation site has been removed.
39 . The conjugate according to claim 36 , wherein the introduced glycosylation site is an iii vivo glycosylation site.
40 . The conjugate according to claim 39 , wherein the glycosylation site is an ill vivo O-glycosylation site.
41 . The conjugate according to claim 39 , wherein the glycosylation site is an in vivo N-glycosylation site.
42 . The conjugate according to claim 41 , wherein the amino acid sequence of the polypeptide differs from SEQ ID NO:1 in that at least one naturally occurring N-X′-X sequence is substituted with a N-X′-S or N-X′-T sequence, wherein X′ is any amino acid except P, and X is any amino acid except for S and T.
43 . The conjugate according to claim 41 , wherein the amino acid sequence of the polypeptide differs from SEQ ID NO:1 in that at least one naturally occurring X-X′-S or X-X′-T sequence naturally present in SEQ ID NO:1 is substituted with a N-X′-S or a N-X′-T sequence, wherein X′ is any amino acid except P, and X is any amino acid except for N.
44 . The conjugate according to claims 42 or 43 , wherein the substitution is selected from the group consisting of F4S/T, P10N, Q21N, W41N, S43N, A51N, G58N, L65N, G59S/T, E82S/T, N95S/T, G97S/T, Y101N, D104N, K109N, G117N, G124N, S126N, T128N, A175S/T, G179N, I186S/T, V188N, R202S/T, I205S/T, D212N, E220N, I230N, P231N, P236N, G237N, E265N, T267N, E270N, R277N, L280N, G291N, P303S/T, L305N, Q312N, G318N, G331N, D334N, K337N, G342N, H348N, R353N, Y357N, I361N, V376N, R379N, M39 IN, and combinations thereof.
45 . The conjugate according to claim 44 , wherein the substitution is selected from the group consisting of F4S/T, P10N, Q21N, W41N, A51N, G58N, G59S/T, N95S/T, G97S/T, Y101N, D104, K109N, G117N, G124N, S126N, T128N, A175S/T, I186S/T, V188N, R202S/T, I205S/T, D212N, E220N, E265N, T267N, E270N, L280N, G291N, P303S/T, G318N. G331N, D334N, K337N, R353N, Y357N, M391N, and combinations thereof.
46 . The conjugate according to claim 41 , wherein the amino acid sequence of the polypeptide differs from SEQ ID NO:1 in that at least one K-X′-X sequence or R-X′-X sequence naturally present in SEQ ID NO:1 is substituted with a N-X′-S or a N-X′-T sequence, wherein X′ is any amino acid except P, and X is any amino acid except for S and T.
47 . The conjugate according to claim 46 , wherein the substitution is selected from the group consisting of K32N+A34S/T, K38N+F40S/T, K62N+Q64S/T, K85N+D87S/T, K137N+P139S/T, K143N+N145S/T, K148N+Q149S/T, K161N+E163S/T, K197N+K199S/T, K199N+W201S/T, K316N+G318S/T, K337N, K341N+D343S/T, K389N+M391S/T and combinations thereof.
48 . The conjugate according to claim 41 , wherein a non-naturally occurring in vivo N-glycosylation site is introduced by substitution, the substitution being selected from the group consisting of K32N+A34S/T, F31N+D33S/T, I30N+K32S/T, A34N+R36S/T, K38N+F40S/T, T37N+L39S/T, R36N+K38S/T, L39N+W41S/T, F40N+142S/T, W41N, I42N+Y44S/T, S43N, Y44N+D46S/T, S45N+G47S/T, D46N+D48S/T, G47N+Q49S/T, K1443N+N145S/T, E142N+R144S/T, L141N+K143S/T, I140N+E142S/T, R144N+A146S/T, A146N+K148S/T, S147N+P149S/T, R290N+A292S/T, D289N+G291S/T, L288N+R290S/T, L287N+D289S/T, G291N, A292N+A294S/T, T293N+L295S/T, R315N+V317S/T, S314N+K316S/T, Q313N+R315S/T, Q312N, K316N+G318S/T, V317N+D319S/T, G318N. K341N+D343S/T, S339N+K341S/T, G342N, D343N+G345S/T, R392N+E394S/T, M391N, L390N+R392S/T, K389N+M391S/T, S393N+P395S/T, E394N+R396S/T, P395N+P397S/T, R396N+G398S/T, P397N+V399S/T, G398N+L400S/T, V399N+L401S/T, L400N+R402S/T, L401N+A403S/T, R402N+P404S/T, A403N+F405S/T, P404N+P406S/T, K143N+N145S/T+R315N+V317S/T and combinations thereof.
49 . The conjugate according to claim 48 , wherein the substitution is selected from the group consisting of K32N+A34S/T, K38N+F40S/T, Y44N+D46S/T, K143N+N145S/T, R290N+A292S/T, K341N+D343S/T, R392N+E394S/T, R396N+G398S/T, R402N+P404S/T, K143N+N145S/T+R315N+V317S/T and combinations thereof.
50 . The conjugate according to claim 49 , wherein the substitution is selected from the group consisting of K32N+A34T, K38N+F40T, Y44N+D46T, K143N+N145T, R290N+A292T, K341N+D343T, R392N+E394T, R396N+G398T, R402N+P404T, K143N+N145T+R315N+V317T and combinations thereof.
51 . The conjugate according to claim 34 , wherein the polypeptide differs in 1-15 amino acid residues from the amino acid sequence shown in SEQ ID NO:1.
52 . The conjugate according to claim 39 , wherein two or more in vivo glycosylation sites have been introduced.
53 . The conjugate according to claim 34 , which further comprises at least one non-polypeptide moiety covalently attached to an amino acid residue of the polypeptide, wherein the non-polypeptide moiety is different from a sugar moiety.
54 . The conjugate according to claim 53 , wherein the non-polypeptide moiety is a polymer molecule as defined in claim 29 .
55 . The conjugate according to claim 1 , wherein the conjugate has a molecular weight of at least 67 kDa.
56 . The conjugate according to claim 1 , wherein the conjugate has been inactivated.
57 - 62 . (canceled)
63 . A composition comprising a conjugate as defined in claim 1 and a pharmaceutical acceptable carrier or excipient.
64 - 67 . (canceled)