Interferon-β variants and conjugates
View Patent ↗The present invention provides new interferon β conjugates, methods of preparing such conjugates and the use of such conjugates in therapy, in particular for the treatment of multiple sclerosis.
1. An isolated nucleic acid comprising a nucleotide sequence encoding an interferon β polypeptide variant exhibiting an interferon β activity, the variant comprising a variant sequence which differs from the wild-type human interferon β sequence SEQ ID NO:2 in no more than 8 amino acid residues and which comprises at least one introduced N-glycosylation site selected from the group consisting of S2N+N4T/S, L9N+R11T/S, R11N, S12N+N14T/S, F15N+C16S/T, Q16N+Q18T/S, K19N+L21T/S, Q23N+H25T/S, G26N+L28T/S, R27N+E29T/S, L28N+Y30T/S, D39T/S, K45N+L47T/S, Q46N+Q48T/S, Q48N+F50T/S, Q49N+Q51T/S, Q51N+E53T/S, R71N+D73T/S, Q72N, D73N, S75N, S76N+G78T/S, L88T/S, Y92T/S, H93N+I95T/S, L98T/S, E103N+K105T/S, E104N+L106T/S, E107N+E109T/S, K108N+D110T/S, D110N, F111N+R113T/S, and L116N relative to SEQ ID NO:2.
2. The nucleic acid of claim 1 , wherein the variant sequence comprises two introduced N-glycosylation sites comprising the substitutions Q49N+Q51T/S and F111N+R113T/S.
3. The nucleic acid of claim 2 , wherein the variant sequence further comprises a substitution of the aspartic acid residue at position 110 relative to SEQ ID NO:2.
4. The nucleic acid of claim 3 , wherein the variant sequence further comprises the substitution C17S.
5. The nucleic acid of claim 4 , wherein the variant sequence further comprises at least one substitution selected from the group consisting of K19R, K33R, and K45R.
6. The nucleic acid of claim 5 , wherein the variant sequence comprises the substitutions K19R, K33R, and K45R.
7. An expression vector comprising the nucleic acid of claim 6 .
8. An isolated host cell comprising the nucleic acid of claim 6 .
9. A method of producing an interferon β polypeptide variant, the method comprising:
providing a host cell of claim 8 and a culture medium;
culturing the host cell under conditions in which the variant is expressed; and recovering the variant.
10. An expression vector comprising the nucleic acid of claim 4 .
11. An isolated host cell comprising the nucleic acid of claim 4 .
12. A method of producing an interferon β polypeptide variant, the method comprising:
providing a host cell of claim 11 and a culture medium;
culturing the host cell under conditions in which the variant is expressed;
and recovering the variant.
13. The method of claim 12 , wherein the host cell is a glycosylating host cell.
14. The nucleic acid of claim 1 , wherein the variant sequence comprises at least one introduced N-glycosylation site selected from the group consisting of Q49N+Q51T/S, D110N, and F111N+R113T/S.
15. An expression vector comprising the nucleic acid of claim 1 .
16. An isolated host cell comprising the nucleic acid of claim 1 .
17. The isolated host cell of claim 16 , which host cell is a glycosylating host cell.
18. The isolated host cell of claim 17 , which glycosylating host cell is a CHO cell, a BHK cell, a HEK293 cell, or an SF9 cell.
19. A cell culture comprising the host cell of claim 17 and a culture medium.
20. The cell culture of claim 19 , comprising the interferon β polypeptide variant produced by expression of the nucleotide sequence.
21. A method of making an interferon β polypeptide variant, the method comprising:
providing a glycosylating host cell and a culture medium, the glycosylating host cell comprising a nucleic acid of claim 1 , culturing the host cell under conditions which permit expression and glycosylation of the interferon β polypeptide variant, and recovering the resulting interferon β polypeptide variant.
22. The method of claim 21 , further comprising attaching a polymer molecule to at least one attachment group of the variant.
23. The method of claim 22 , wherein the polymer molecule comprises a linear polyethylene glycol or a branched polyethylene glycol.
24. The method of claim 21 , wherein the glycosylating host cell is selected from an S. cerevisiae cell, a Pichia pastoris cell, a CHO cell, a BHK cell, an HEK cell, and an SF9 cell.