Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules

• US 20090131318A1
• Filed: 06/06/2008
• Published: 05/21/2009
• Est. Priority Date: 09/09/2002
• Status: Active Grant

• Current Assignee: HanAll Biopharma Co. Ltd.
• Original Assignee: HanAll Biopharma Co. Ltd.
• Inventors: Guyon, Thierry, Drittanti, Lila, Vega, Manuel, Gantier, Rene
• Granted Patent: US 8,114,839 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• 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

Compositions of modified cytokines and uses thereof generated using 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 are modified cytokines formulated for oral delivery and uses thereof to treat diseases and conditions mediated by cytokines.

Claims

1. A modified erythropoietin cytokine, comprising one or more amino acid replacements in its sequence of amino acid residues, wherein:

the modified erythropoietin cytokine exhibits increased resistance to proteolysis compared to the unmodified erythropoietin cytokine that does not comprise the one or more amino acid replacements;

one amino acid replacement is R by Q at position 131; and

the one or more amino acid replacements correspond to amino acid replacements in a mature erythropoietin cytokine having the sequence set forth in SEQ ID NO;

201 or in a sequence-related erythropoietin at a corresponding amino acid position(s) relative to SEQ ID NO;

201.

2. The modified erythropoietin cytokine of claim 1, wherein the unmodified erythropoietin cytokine comprises a sequence of amino acids set forth in SEQ ID NO:

201.

3. The modified erythropoietin cytokine of claim 1 that comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid replacements in its sequence of amino acid residues.

4. The modified erythropoietin cytokine of claim 1, further comprising one or more additional amino acid replacements in its sequence of amino acids, wherein:

the one or more amino acid replacements and positions thereof are selected from among replacement of;

D by Q at position 43;

D by N at position 43;

K by Q at position 45;

K by N at position 45;

F by I at position 48;

F by V at position 48;

Y by H at position 49;

Y by I at position 49;

K by Q at position 52;

K by N at position 52;

R by H at position 53;

R by Q at position 53;

E by Q at position 55;

E by N at position 55;

E by H at position 55;

E by Q at position 72;

E by N at position 72;

E by H at position 72;

L by V at position 75;

L by I at position 75;

R by H at position 76;

R by Q at position 76;

P by S at position 121;

P by A at position 121;

P by S at position 122;

P by A at position 122;

D by Q at position 123;

D by N at position 123;

P by S at position 129;

P by A at position 129;

L by V at position 130;

L by I at position 130;

R by H at position 162;

R by Q at position 162;

D by Q at position 165; and

D by N at position 165; and

the one or more amino acid replacements correspond to amino acid replacements in a mature erythropoietin cytokine having the sequence set forth in SEQ ID NO;

201 or in a sequence-related erythropoietin at a corresponding amino acid position(s) relative to SEQ ID NO;

201.

5. The modified erythropoietin cytokine of claim 1, wherein only the primary amino acid sequence is modified, and the erythropoietin cytokine exhibits increased resistance to proteolysis.

6. The modified erythropoietin cytokine of claim 1 that exhibits increased stability compared to the unmodified erythropoietin cytokine.

7. The modified erythropoietin cytokine of claim 6, wherein the erythropoietin cytokine exhibits increased stability to proteases, human blood lysate or human serum.

8. The modified erythropoietin cytokine of claim 1 that exhibits increased protein half-life in vitro or in vivo compared to the unmodified erythropoietin cytokine.

9. The modified erythropoietin cytokine of claim 1 that exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.

10. The modified erythropoietin cytokine of claim 1 that exhibits increased resistance to proteolysis by a protease in the serum.

11. The modified erythropoietin cytokine of claim 1, wherein increased resistance to proteolysis is due to replacement of one or more amino acids at target positions in an unmodified erythropoietin cytokine that increases resistance of the modified erythropoietin cytokine to digestion by a protease.

12. The modified erythropoietin cytokine of claim 1 that exhibits increased biological activity compared to the unmodified erythropoietin cytokine.

13. The modified erythropoietin cytokine of claim 1 that exhibits decreased biological activity compared to the unmodified erythropoietin cytokine.

14. The modified erythropoietin cytokine of claim 1, comprising a sequence of amino acids set forth in SEQ ID NO:

969.

15. A nucleic acid molecule encoding a modified erythropoietin cytokine of claim 1.

16. A vector, comprising a nucleic acid molecule of claim 15.

17. A eukaryotic cell, comprising the nucleic acid molecule of claim 15.

18. A eukaryotic cell, comprising the vector of claim 16.

19. A method for expression of a modified erythropoietin cytokine, comprising:

introducing a nucleic acid of claim 15 into a host; and

culturing the cell under conditions, whereby the encoded modified erythropoietin cytokine is expressed.

20. The method of claim 19, further comprising isolating the modified erythropoietin cytokine.

21. The method of claim 19, wherein the host cell is a eukaryotic host cell or a bacterial cell.

22. A pharmaceutical composition, comprising a modified erythropoietin cytokine of claim 1.

23. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable carrier or excipient.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant, and a wetting agent.

25. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable additive.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle, and a preservative.

27. The pharmaceutical composition of claim 22, wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule.

28. The pharmaceutical composition of claim 27, wherein the composition is in the form of a tablet or a capsule.

29. The pharmaceutical composition of claim 22, formulated for oral, parenteral, intravenous, intradermal, subcutaneous, buccal, inhalation, intramuscular, rectal or topical administration.

30. The pharmaceutical composition of claim 29, formulated for oral administration.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition is formulated for oral administration to the mouth or gastrointestinal tract.

32. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulated for controlled-release of the modified erythropoietin cytokine.

33. A pharmaceutical composition, comprising a nucleic acid molecule of claim 15.

34. A method, comprising treating a subject by administering the pharmaceutical composition of claim 22, wherein the subject has a disease or condition that is treated by administration of an erythropoietin cytokine.

35. The method of claim 34, wherein the disease or condition is selected from among hypoxia, myocardial ischemia, or anemia associated with renal failure, or cancer.

36. A method, comprising treating a subject by orally administering the pharmaceutical composition of claim 30, wherein the subject has a disease or condition that is treated by administration of an erythropoietin cytokine.

37. The method of claim 36, wherein the disease or condition is selected from among hypoxia, myocardial ischemia, or anemia associated with renal failure, or cancer.

38. A method, comprising treating a subject by administering the pharmaceutical composition of claim 33, wherein the subject has a disease or condition that is treated by administration of an erythropoietin cytokine.

39. The method of claim 38, wherein the disease or condition is selected from among hypoxia, myocardial ischemia, or anemia associated with renal failure, or cancer.