Glycopegylated factor IX
Conjugates between Factor IX and PEG moieties. are disclosed in the present application. The conjugates are linked via a glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. Conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.
1. A PEGylated Factor IX protein comprising SEQ ID NO:1 and a poly(ethylene glycol) moiety, wherein the poly(ethylene glycol) moiety is conjugated to an amino acid selected from Asn 157 and Asn 167 of SEQ ID NO:1 via a glycosyl linking group of a glycosyl residue, and having a composition according to the formula:
wherein
R 2 is COOH;
R 3 is H;
R 4 is OH;
D is OH; and
G is R 1 -L-
wherein
L is C(O)CH 2 NHC(O); and
R 1 is the poly(ethylene glycol) moiety, wherein the poly(ethylene glycol) moiety comprises a branched poly(ethylene glycol) having a core glycerol moiety, and wherein the branched poly(ethylene glycol) has a molecular weight of about 40 kDa.
2. A pharmaceutical formulation comprising the Factor IX conjugate of claim 1 and a pharmaceutically acceptable carrier.
3. A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal the PEGylated Factor IX conjugate of claim 1 .
4. A method of treating hemophilia in a subject, said method comprising administering to said subject said Factor IX conjugate of claim 1 .
5. The method of treating hemophilia of claim 4 , wherein hemophilia is hemophilia B.
6. A Factor IX conjugate comprising a Factor IX protein comprising SEQ ID NO:1 and a poly(ethylene glycol) (PEG) moiety, wherein the PEG moiety is conjugated to an amino acid of SEQ ID NO:1 via a glycosyl linking group of a glycosyl residue, and having a composition according to the formula:
wherein
D is OH; and
G has the formula
wherein
X 2 is CH 2 ,
X 3′ is CH 2 OC(O)NHCH 2 C(O)NH,
X 4 is a bond,
X 5 is H, and
R 16 and R 17 are poly(ethylene) gycol, that compose a branched PEG moiety.
7. The Factor IX conjugate of claim 6 , wherein the PEG moiety is conjugated to an amino acid of SEQ ID NO:1 selected from the group consisting of Asn157 and Asn167 1 via a glycosyl linking group of a glycosyl residue.
8. The Factor IX conjugate of claim 7 , wherein the PEG moiety is conjugated to amino acids Asn 157 and Asn167 of SEQ ID NO:11 via a glycosyl linking group of a glycosyl residue.
9. The Factor IX conjugate of claim 6 , wherein the branched PEG moiety has a molecular weight of about 40 kD.
10. A pharmaceutical formulation comprising the Factor IX conjugate of claim 6 and a pharmaceutically acceptable carrier.
11. A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal the Factor IX conjugate of claim 6 .
12. A method of treating hemophilia in a subject, said method comprising administering to said subject said Factor IX conjugate of claim 6 .
13. The method of treating hemophilia of claim 12 , wherein hemophilia is hemophilia B.
14. A Factor IX conjugate comprising a Factor IX protein comprising SEQ ID NO:1 and a poly(ethylene glycol) (PEG) moiety, wherein the PEG moiety is conjugated to an amino acid selected from Asn 157 and Asn 167 of SEQ ID NO:1 via a glycosyl linking group of a glycosyl residue, and having a composition according to the formula:
wherein
D is OH; and
G is a composition according to the formula
wherein m and n are integers independently selected from 1 to 2500.
15. The Factor IX conjugate of claim 14 , wherein the PEG moiety has a molecular weight of about 40 kD.
16. A pharmaceutical formulation comprising the Factor IX conjugate of claim 14 and a pharmaceutically acceptable carrier.
17. A method of stimulating blood coagulation in a mammal, said method comprising administering to said mammal said Factor IX conjugate of claim 14 .
18. A method of treating hemophilia in a subject, said method comprising administering to said subject said Factor IX conjugate of claim 14 .
19. A Factor IX conjugate comprises a glycosyl residue having the formula:
wherein
a, b, c, d, i, r, s, t, and u are integers independently selected from 0 and 1;
q is 1;
e, f, g, and h are members independently selected from the integers from 0 to 6;
j, k, l, and m are members independently selected from the integers from 0 and 100;
v, w, x, and y are independently selected from 0 and 1, and at least one of v, w, x, and y is 1;
AA is an amino acid residue of said Factor IX peptide selected from the group consisting of Asn 157, Asn 167 and combinations thereof;
Sia-(R) has the formula:
wherein
D is —OH; and
G is R 1 -L-
wherein
L is C(O)CH 2 NHC(O); and
R 1 is the poly(ethylene glycol) moiety, wherein the poly(ethylene glycol) moiety comprises a branched poly(ethylene glycol) having a core glycerol moiety, and wherein the branched poly(ethylene glycol) has a molecular weight of about 40 kDa.
20. A Factor IX conjugate of claim 19 , wherein j, k, l, and m are 1, and e, f, g, and h are 3 or less.