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

• US 20080075672A1
• Filed: 02/06/2007
• Published: 03/27/2008
• Est. Priority Date: 09/09/2002
• Status: Abandoned Application

• Current Assignee: Hanall Pharmaceutical Company Limited
• Original Assignee: Hanall Pharmaceutical Company Limited
• Inventors: Guyon, Thierry, Drittanti, Lila, Vega, Manuel, Gantier, Rene
• Application Number:
  US11/703,610
• Publication Number:
  US 20080075672A1
• Time in Patent Office: Days
• US Class Current: 424/45
• 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 granulocyte colony stimulating factor (G-CSF) cytokine, comprising one or more amino acid replacements in its sequence of amino acid residues, wherein:

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

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

W61S, W61H, P63S, P63A, P68A, L72V, L721, F86I, F86V, E96N, E96H, P100S, P100A, E101N, E101H, P131S, P131A, L133V, L1331, P135S, P135A, F1471, F147V, R169H, R169Q, R172H, R172Q, P177S and P177A; and

the one or more amino acid replacements occur in a mature G-CSF cytokine having the sequence set forth in SEQ ID NO;

210 or in a sequence-related G-CSF cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

210.

2. The modified G-CSF cytokine of claim 1, wherein the unmodified G-CSF cytokine contains the amino acids residues having the sequence set forth in SEQ ID NO:

210.

3. The G-CSF 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 of the amino acid replacements in its sequence of amino acid residues.

4. The G-CSF 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.

5. The G-CSF 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;

W61S, W61H, P63S, P63A, P68S, P68A, L72V, L721, F861, F86V, E96Q, E96N, E96H, P100S, P100A, E101Q, E101N, E101H, P131S, P131A, L133V, L1331, P135S, P135A, F147I, F147V, R169H, R169Q, R172H, R172Q, P177S and P177A; and

the one or more amino acid replacements occur in a mature G-CSF cytokine having the sequence set forth in SEQ ID NO;

210 or in a sequence-related G-CSF cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

210.

6. The G-CSF cytokine of claim 1, wherein only the primary amino acid sequence is modified, and the G-CSF cytokine exhibits increased resistance to proteolysis.

7. A G-CSF cytokine of claim 1, wherein the cytokine comprises the sequence of amino acids set forth in any of SEQ ID NOS:

631, 632, 633, 634, 636, 637, 638, 639, 640, 642, 643, 644, 645, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661 or 662.

8. The G-CSF cytokine of claim 1 that exhibits increased stability compared to the unmodified G-CSF cytokine.

9. The G-CSF cytokine of claim 8, wherein the G-CSF cytokine exhibits increased stability to proteases, human blood lysate or human serum.

10. The G-CSF cytokine of claim 1 that exhibits increased protein half-life in vitro or in vivo compared to the unmodified G-CSF cytokine.

11. The G-CSF cytokine of claim 1 that exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.

12. The G-CSF cytokine of claim 1 that exhibits increased resistance to proteolysis by a protease in the serum.

13. The G-CSF 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 G-CSF cytokine that increase resistance of the G-CSF cytokine to digestion by a protease.

14. The G-CSF cytokine of claim 1 that exhibits increased biological activity compared to the unmodified G-CSF cytokine.

15. The G-CSF cytokine of claim 1 that exhibits decreased biological activity compared to the unmodified G-CSF cytokine.

16. A nucleic acid molecule encoding a modified G-CSF cytokine of claim 1.

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

18. A eukaryotic cell, comprising the nucleic acid molecule of claim 16.

19. A eukaryotic cell, comprising the vector of claim 17.

20. A collection of nucleic acid molecules, comprising a plurality of the molecules of claim 16.

21. A collection of nucleic acid molecules, comprising a plurality of the vectors of claim 17.

22. A method for expression of a modified G-CSF cytokine, comprising:

introducing a nucleic acid of claim 16 into a host; and

culturing the cell under conditions, whereby the encoded modified G-CSF cytokine is expressed.

23. The method of claim 22, further comprising isolating the modified G-CSF cytokine.

24. The method of claim 22, wherein the host cell is a eukaryotic cell or a bacterial cell.

25. A pharmaceutical composition, comprising a G-CSF cytokine of claim 1.

26. The pharmaceutical composition of claim 25, further comprising a pharmaceutically acceptable carrier or excipient.

27. The pharmaceutical composition of claim 26, wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant, and a wetting agent.

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

29. The pharmaceutical composition of claim 28, wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle, and a preservative.

30. The pharmaceutical composition of claim 25, wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule.

31. The pharmaceutical composition of claim 25, formulated for oral, parenteral, intravenous, intradermal, subcutaneous, buccal, inhalation, intramuscular, rectal or topical administration.

32. The pharmaceutical composition of claim 31, formulated for oral administration.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition is formulated for oral administration to the mouth or gastrointestinal tract.

34. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for controlled-release of the G-CSF cytokine.

50. A pharmaceutical composition, comprising a nucleic acid molecule of claim 16.

51. A method, comprising treating a subject by administering the pharmaceutical composition of claim 25, wherein the subject has a disease or condition that is treated by administration of a G-CSF cytokine.

52. The method of claim 51, wherein in the disease or condition is selected from among Crohn'"'"'s disease, cardiac disease, acquired and congenital neutropenias and asthma.

55. A method, comprising treating a subject by administering the pharmaceutical composition of claim 50, wherein the subject has a disease or condition that is treated by administration of a G-CSF cytokine.

56. The method of claim 55, wherein in the disease or condition is selected from among Crohn'"'"'s disease, cardiac disease, acquired and congenital neutropenias and asthma.

35. A pharmaceutical composition formulated for oral administration, comprising a G-CSF cytokine that contains one or more amino acid modification(s), whereby the G-CSF cytokine exhibits increased protease resistance compared to a G-CSF cytokine that does not contain the modification(s).

36. The pharmaceutical composition of claim 35, wherein the modified G-CSF cytokine has been modified by an insertion, a deletion and/or a replacement of one or more amino acid residues, whereby the cytokine is rendered resistant to proteolysis.

37. The pharmaceutical composition of claim 35, wherein:

the modified G-CSF cytokine comprises one or more amino acid replacements at one or more amino acid target positions in the unmodified cytokine.

38. The pharmaceutical composition of claim 37, wherein:

the modified G-CSF cytokine comprises one or more amino acid replacements selected from among replacement of;

W61S, W61H, P63S, P63A, P68S, P68A, L72V, L721, F861, F86V, E96Q, E96N, E96H, P100S, P100A, E101Q, E101N, E101H, P131S, P131A, L133V, L1331, P135S, P135A, F1471, F147V, R169H, R169Q, R172H, R172Q, P177S and P177A; and

the one or more amino acid replacements occur in a mature G-CSF cytokine having the sequence set forth in SEQ ID NO;

210 or in a sequence-related G-CSF cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

210.

39. The pharmaceutical composition of claim 35, further comprising a pharmaceutically acceptable carrier or excipient.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant, and a wetting agent.

41. The pharmaceutical composition of claim 35, further comprising a pharmaceutically acceptable additive.

42. The pharmaceutical composition of claim 41, wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle and a preservative.

43. The pharmaceutical composition of claim 35, wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule.

44. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition is formulated for controlled-release of the G-CSF cytokine.

45. The pharmaceutical composition of claim 35, wherein the G-CSF cytokine has been modified by removing proteolytic digestion sites in the G-CSF cytokine.

46. The pharmaceutical composition of claim 35, wherein the G-CSF cytokine has an increased half-life compared to the unmodified G-CSF cytokine.

47. The pharmaceutical composition of claim 35, wherein the G-CSF cytokine exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.

48. The pharmaceutical composition of claim 35, wherein the G-CSF cytokine exhibits increased biological activity compared to the unmodified G-CSF cytokine.

49. The pharmaceutical composition of claim 35, wherein the G-CSF cytokine exhibits decreased biological activity compared to the unmodified G-CSF cytokine.

53. A method, comprising treating a subject by orally administering the pharmaceutical composition of claim 35, wherein the subject has a disease or condition that is treated by administration of a G-CSF cytokine.

54. The method of claim 53, wherein in the disease or condition is selected from among Crohn'"'"'s disease, cardiac disease, acquired and congenital neutropenias and asthma.