Protease resistant modified interferon alpha polypeptides
View Patent ↗Processes and systems for the high throughput directed evolution of peptides and proteins, particularly cytokines that act in complex biological settings, are provided. Also provided is a rational method for generating protein variants and the resulting variants.
1. An isolated interferon (IFN) alpha cytokine, comprising an amino acid replacement in its sequence of amino acids, wherein:
an amino acid replacement is E41 Q, whereby the interferon alpha cytokine exhibits increased resistance to proteolysis so that it can be administered orally compared to the unmodified interferon alpha cytokine that does not comprise the amino acid replacement; and
in the interferon-alpha cytokine,
the interferon is not glycosylated at E41Q; and
the unmodified interferon alpha cytokine is selected from among an interferon α-2b (IFNα-2b), an interferon α-2a (IFNα-2a), an interferon α-2c (IFNα-2c) and consensus interferon whose sequences are set forth in SEQ ID Nos. 1, 182, 185 and 232, respectively.
2. An interferon alpha cytokine selected from among IFNα-2b, IFNα-2a, IFNα-2c, consensus interferon, IFNα-c, IFNα-d, IFNα-5, IFNα-6, IFNα-4, IFNα-4b, IFNα-I, IFNα-J, IFNα-H, IFNα-F and IFNα-8, each comprising the modification E41Q, wherein:
the sequence of a human wild-type interferon alpha for each of IFNα-2b, IFNα-2a, IFNα-2c, consensus interferon, IFNα-c, IFNα-d, IFNα-5, IFNα-6, IFNα-4, IFNα-4b, IFNα-I, IFNα-J, IFNα-H, IFNα-F and IFNα-8; is set forth in SEQ ID Nos. 1, 182, 185, 232, 183 and 186-195, respectively; the modification at E41Q increases protease resistance of the interferon alpha cytokine so that it can be administered orally.
the interferon alpha cytokine is not glycosylated at E41Q; and
residue 1 in each SEQ ID corresponds to residue 1 of the mature interferon-alpha.
3. An interferon alpha cytokine of claim 2 , comprising two, three, four or five amino acid replacements in its sequence of amino acids.
4. The interferon alpha cytokine of claim 3 that is a modified IFNα-2b cytokine.
5. A pharmaceutical composition, comprising a cytokine of claim 1 in a pharmaceutically acceptable carrier.
6. An interferon alpha cytokine of claim 1 that is an IFNα-2b.
7. The interferon alpha cytokine of claim 1 that contains only a single amino acid replacement compared to the interferon cytokine that does not include the E41Q replacement.
8. The pharmaceutical composition of claim 5 , formulated for oral administration.
9. An interferon alpha cytokine, comprising the sequence of amino acids set forth in SEQ ID NO:87.
10. The interferon alpha cytokine of claim 1 , comprising two amino acid replacements.
11. A modified interferon (IFN) alpha cytokine of claim 2 , comprising one or two amino acid replacements.
12. The modified IFN alpha cytokine of claim 11 that is an IFN-α2b cytokine.
13. A pharmaceutical composition, comprising a cytokine of claim 11 in a pharmaceutically acceptable carrier.
14. A pharmaceutical composition, comprising a cytokine of claim 12 in a pharmaceutically acceptable carrier.
15. A pharmaceutical composition, comprising a cytokine of claim 6 in a pharmaceutically acceptable carrier.
16. The pharmaceutical composition of claim 15 that is formulated as a tablet or capsule for oral administration.
17. A pharmaceutical composition, comprising a cytokine of claim 9 in a pharmaceutically acceptable carrier.
18. The modified interferon alpha cytokine of claim 4 , comprising amino acid replacements corresponding to E41Q and D94G.
19. A pharmaceutical composition, comprising a modified interferon alpha cytokine of claim 2 .
20. The pharmaceutical composition of claim 17 that is formulated for oral administration.
21. The pharmaceutical composition of claim 20 that is formulated as tablet or capsule.
22. The interferon alpha cytokine of claim 2 that contains only a single amino acid replacement compared to the interferon cytokine that does not include the E41Q replacement.
23. The pharmaceutical composition of claim 19 that is formulated for oral administration.
24. The pharmaceutical composition of claim 23 that is formulated as a tablet or capsule.