Glycopegylated interferon α
View Patent ↗The present invention provides IFN-α conjugates including IFN-α peptides and modifying groups such as PEG moieties. The IFN-α peptide and modifying group are linked via an intact glycosyl linking group interposed between and covalently attached to the IFN-α peptide and the modifying group. The IFN-α conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar onto an amino acid or a glycosyl residue on the IFN-α peptide. Also provided are methods for preparing the IFN-α conjugates, methods for treating various disease conditions with the IFN-α conjugates, and pharmaceutical formulations including the IFN-α conjugates.
1. An IFN-α conjugate comprising:
an IFN-α peptide; and
a glycosyl group attached to an asparagine, lysine, serine, or threonine residue of said IFN-α peptide, said glycosyl group comprising a modified sialyl residue having the formula:
wherein
R 2 is H, CH 2 OR 7 , COOR 7 or OR 7
wherein
R 7 represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl;
R 3 and R 4 are members independently selected from H, substituted or unsubstituted alkyl, OR 8 , and NHC(O)R 9
wherein
R 8 and R 9 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and sialic acid;
L a is a linker selected from NHC(O), C(O)NH, and
(CH 2 ) j C(O)NH(CH 2 ) h ,(CONH), wherein h and j are integers independently selected from 0 to 10;
R 16 and R 17 are independently selected from a poly(ethylene glycol) moiety and a poly(amino acid) moiety;
X 2 and X 4 are linkage fragments independently selected from S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH, NHC(O)O, OC(O)NH, CH 2 S, CH 2 O, CH 2 CH 2 O, CH 2 CH 2 S, (CH 2 ) O O, (CH 2 ) O S, and (CH 2 ) O Y′-PEG
wherein
o is an integer from 1 to 50;
Y′ is a member selected from S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH, and O; and
X 5 is a member selected from H, CH 3 , and a poly(ethylene glycol) moiety.
2. The IFN-α conjugate according to claim 1 , wherein
has a formula that is a member selected from:
wherein
Q is selected from H and substituted or unsubstituted C 1 -C 6 alkyl;
e and f are integers independently selected from 1 to 2500; and
q is an integer from 0 to 20.
3. The IFN-α conjugate according to claim 2 , wherein
has a formula that is a member selected from:
wherein
Q is selected from H and substituted or unsubstituted C 1 -C 6 alkyl;
e, f and f′ are integers independently selected from 1 to 2500; and
q and q′ are integers independently selected from 1 to 20.
4. The IFN-α conjugate according to claim 1 , wherein said glycosyl group having a formula that is a member selected from:
5. The IFN-α conjugate according to claim 1 , wherein said glycosyl group having the formula:
6. The IFN-α conjugate according to claim 5 , wherein said glycosyl group attached to said amino acid residue has the formula:
wherein
AA is said amino acid residue of said IFN-α peptide.
7. The IFN-α conjugate according to claim 1 wherein said glycosyl group has the formula which is a member selected from:
8. The IFN-α conjugate according to claim 1 wherein said glycosyl group has the formula which is a member selected from:
9. The IFN-α conjugate according to claim 8 wherein said glycosyl group has the formula which is a member selected from:
10. The IFN-α conjugate according to claim 1 , wherein said amino acid residue is a member selected from asparagine and lysine.
11. The IFN-α conjugate according to claim 1 , wherein said amino acid residue is a member selected from serine and threonine.
12. The IFN-α conjugate according to claim 1 , wherein said peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
13. An IFN-α conjugate having a formula which is a member selected from the group consisting of:
wherein AA is a threonine residue of an IFN-α peptide at a position corresponding to amino acid 106 of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and
m and n are integers independently selected from 1 to 2500.
14. The IFN-α conjugate according to claim 1 , wherein said IFN-α peptide comprises at least one glycosyl group comprising a structure having the formula:
wherein
R 15 is said modified sialyl residue; and
p is an integer from 1 to 10.
15. The IFN-α conjugate according to claim 14 , wherein said at least one glycosyl linking group attached to an amino acid of said IFN-α peptide has a formula selected from:
and combinations thereof
wherein
AA is said amino acid residue of said peptide;
t is an integer equal to 0 or 1;
p is an integer from 1 to 10; and
R 15 ′ is a member selected from H, OH, sialic acid, said modified sialyl residue and Sia-Sia p
wherein
Sia p is said modified sialyl residue,
wherein at least one R 15 ′ is selected from said modified sialyl residue and Sia-Sia p .
16. The IFN-α conjugate according to claim 15 , wherein said amino acid residue is an asparagine residue.
17. A method of preparing an IFN-α conjugate according to claim 1 , said method comprising:
(a) contacting a substrate IFN-α peptide comprising a glycosyl moiety selected from:
with a PEG-sialic acid donor having the formula:
wherein
c is 0 or 1; and
(b) contacting said IFN-α peptide and said PEG-sialic acid donor with an enzyme that transfers PEG-sialic acid from said donor onto said glycosyl moiety, under conditions appropriate for said transfer.
18. The method of claim 17 , further comprising, prior to step (a), expressing a nucleic acid encoding said substrate IFN-α peptide in a suitable host.
19. The method of claim 18 , wherein said host is selected from a bacterial cell, an insect cell and a mammalian cell.
20. The method of claim 19 , wherein said host is an E. coli cell.
21. The method of claim 19 , wherein said host is a Spodoptera frugiperda cell.
22. A method of inhibiting viral replication, increasing the lytic potential of NK cells, and modulating MHC molecule expression, said method comprising administering to a mammal an effective amount of the peptide conjugate according to claim 1 .
23. A method of treating a disease in a mammal, wherein said disease is a member selected from hairy cell leukemia, malignant melanoma, follicular lymphoma, condylomata acuminata, AIDS-related Kaposi's sarcoma, Hepatitis C, Hepatitis B, human papilloma virus infection, Chronic Myeloid Leukemia (CML), chronic phase Philadelphia chromosome (Ph) positive Chronic Myelogenous Leukemia, non-Hodgkin's lymphoma (NHL), lymphoma, bladder cancer, and a renal cancer, said method comprising administering to said mammal an effective amount of a peptide conjugate according to claim 1 .
24. A method of treating a viral infection in a subject in need thereof, said method comprising the step of administering to the subject an amount of a peptide conjugate according to claim 1 effective to treat said viral infection in said subject.
25. A pharmaceutical formulation comprising the IFN-α conjugate according to claim 1 , and a pharmaceutically acceptable carrier.
26. A method of preparing an IFN-α conjugate according to claim 1 , said method comprising:
(a) contacting a substrate IFN-α peptide comprising a glycosyl moiety selected from:
with a PEG-sialic acid donor having the formula:
wherein
c is 0 or 1; and
(b) contacting the IFN-α peptide and the PEG-sialic acid donor with an enzyme that transfers PEG-sialic acid from the donor onto the glycosyl moiety, under conditions appropriate for the transfer.
27. The method of claim 26 , further comprising, prior to step (a), expressing a nucleic acid encoding the substrate IFN-α peptide in a suitable host.
28. The method of claim 27 , wherein the host is selected from a bacterial cell, an insect cell and a mammalian cell.
29. The method of claim 28 , wherein the host is an E. coli cell.
30. The method of claim 28 , wherein the host is a Spodoptera frugiperda cell.
31. The method of claim 28 , wherein the host is a Chinese hamster ovary (CHO) cell.