Protease resistant modified interferon alpha polypeptides

• US 7,611,700 B2
• Filed: 09/08/2003
• Issued: 11/03/2009
• Est. Priority Date: 09/09/2002
• Status: Active Grant

• Current Assignee: HanAll Biopharma Co. Ltd.
• Original Assignee: Hanall Pharmaceuticals Company Limited
• Inventors: Guyon, Thierry, Drittanti, Lila, Vega, Manuel, Gantier, Rene
• Primary Examiner(s): Spector; Lorraine
• Assistant Examiner(s): STOICA, ELLY GERALD
• Application Number:
  US10/658,834
• Publication Number:
  US 20040132977A1
• Time in Patent Office: 2,248 Days
• Field of Search: None
• US Class Current: 424/85.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/53 Colony-stimulating factor [CSF]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 14/555 : Interferons [IFN]
• CPC Class Codes: C07K 14/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin

Abstract

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.

Claims

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.

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.

10. The interferon alpha cytokine of claim 1, comprising two amino acid replacements.

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.

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.

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-α

2b 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.

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.

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.

9. An interferon alpha cytokine, comprising the sequence of amino acids set forth in SEQ ID NO:

87.

17. A pharmaceutical composition, comprising a cytokine of claim 9 in a pharmaceutically acceptable carrier.

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.