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

• US 20060020116A1
• Filed: 07/06/2005
• Published: 01/26/2006
• 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, Gantier, Rene, Vega, Manuel
• Application Number:
  US11/176,830
• Publication Number:
  US 20060020116A1
• Time in Patent Office: Days
• US Class Current: 530/351
• 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 pharmaceutical composition, comprising a modified cytokine, wherein:

the modified cytokine comprises a sequence of amino acids set forth in any of SEQ ID Nos. 2-181, 233-1303 or a structural homolog thereof;

the modified cytokine is more resistant to proteolysis compared to a wild-type cytokine; and

the composition is formulated for oral delivery.

2. The pharmaceutical composition of claim 1, wherein the cytokine is selected from among interleukin-10 (IL-10), interferon beta (IFN-β

), interferon alpha-2a (IFN α

-2a), interferon alpha-2b (IFN α

-2b), interferon gamma (IFN-γ

), granulocyte colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), human growth hormone (hGH), ciliary neurotrophic factor (CNTF), leptin, oncostatin M, interleukin-6 (IL-6) and interleukin-12 (IL-12), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), Flt3 ligand and stem cell factor (SCF).

3. The pharmaceutical composition of claim 1, wherein the modified cytokine is selected from among modified cytokines comprising amino acids set forth in any of SEQ ID Nos. 2-181, 233-1303 or a structural homolog thereof.

4. The pharmaceutical composition of claim 1, wherein the modified cytokine is more resistant to proteolysis by a protease of the luminal gastrointestinal tract.

5. The pharmaceutical composition of claim 1, wherein the cytokine is selected from among interleukin-10 (IL-10), interferon beta (IFN-β

), interferon alpha-2a (IFN α

-2a), interferon alpha-2b (IFN α

-2b), interferon α

-2c (IFN α

-2c), interferon gamma (IFN-γ

), granulocyte colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), human growth hormone (hGH), ciliary neurotrophic factor (CNTF), leptin, oncostatin M, interleukin-6 (IL-6) and interleukin-12 (IL-12), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), Flt3 ligand and stem cell factor (SCF).

6. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, wherein the excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant and a wetting agent.

8. The pharmaceutical composition of claim 1, further comprising a pharmaceutically-acceptable additive.

9. The pharmaceutical composition of claim 8, wherein the additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle and a preservative.

10. The pharmaceutical composition of claim 6 that is formulated as a tablet.

11. The pharmaceutical composition of claim 6 that is formulated as a capsule.

12. The pharmaceutical composition of claim 6 that is formulated as a liquid.

13. The pharmaceutical composition of claim 6 that is formulated as a lozenge.

14. The pharmaceutical composition of claim 13 that is formulated for controlled-release.

15. The pharmaceutical composition of claim 1, formulated for oral administration to the mouth or gastrointestinal tract.

16. The pharmaceutical composition of claim 1, wherein the cytokine has been modified by removing proteolytic digestion sites in the cytokine thereby increasing the overall stability of the cytokine structure.

17. The pharmaceutical composition of claim 16, wherein stability is assessed by measuring residual biological activity.

18. The pharmaceutical composition of claim 1, wherein the modified cytokine has an increased half-life compared to an unmodified or wild-type cytokine.

19. The pharmaceutical composition of claim 1, wherein the modified cytokine comprises one or more amino acid replacements at one or more target positions.

20. The pharmaceutical composition of claim 19,

wherein the modified cytokine is a human interferon, and

the one or more target positions are selected from among positions 41, 58, 78, 107, 117, 125, 133 and 159 of SEQ ID NO:

182, positions 41, 59, 79, 108, 118, 126, 134 and 160 of SEQ ID NO;

183, positions 27, 33, 41, 58, 78, 89, 107, 109, 110, 111, 113, 117, 121, 125, 133 and 159 of SEQ ID NO;

185, positions 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134 and 160 of SEQ ID NOS;

186-195, positions 39, 42, 45, 47, 52, 67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128, 130, 134, 136, 137, 163 and 165 of SEQ ID NO;

196, and positions 33, 37, 40, 41, 42, 58, 61, 64, 65 and 66 of SEQ ID NO;

199.

21. The pharmaceutical composition of claim 19,

wherein the modified cytokine comprises one or more mutations at one or more target positions in a cytokine,

wherein the one or more mutations comprises an insertion, a deletion and/or a replacement of one or more native amino acid residues, and

wherein the one or more target positions are selected from among positions 41, 58, 78, 107, 117, 125, 133 and 159 of SEQ ID NO:

182, positions 41, 59, 79, 108, 118, 126, 134 and 160 of SEQ ID NO;

183, positions 27, 33, 41, 58, 78, 89, 107, 109, 110, 111, 113, 117, 121, 125, 133 and 159 of SEQ ID NO;

185, positions 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134 and 1600 of SEQ ID NOS;

186-195 positions 39, 42, 45, 47, 52, 67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128, 130, 134, 136, 137, 163 and 165 of SEQ ID NO;

196, and positions 33, 37, 40, 41, 42, 58, 61, 64, 65 and 66 of SEQ ID NO;

199, positions 49, 50, 52, 53, 54, 55, 56, 57, 59, 60, 67, 68, 71, 72, 74, 75, 78, 81, 84, 85, 86, and 88 of SEQ ID NO;

200, positions 43, 45, 48, 49, 52, 53, 55, 72, 75, 76, 123, 129, 130, 131, 162, and 165 of SEQ ID NO;

201, positions 38, 41, 45, 46, 48, 49, 51, 60, 63, 67, 92, 93, 119, 120, 123, and 124 of SEQ ID NO;

202, positions 3, 40, 42, 43, 55, 58, 59, 61, 89, 90, 91, 95, and 96 of SEQ ID NO;

203, positions 43, 45, 48, 49, 52, 53, 60, 61, 65, 67, 68, 72, 100, 103, 104, 106, 107, 109, 110, and 132 of SEQ ID NO;

204, positions 37, 43, 46, 59, 63, 66, 96, 100, 101, and 103 of SEQ ID NO;

205, positions 27, 31, 34, 37, 54, 58, 61, 62, 63, 96, 98, 99, 100, 102, 103, 106, 107, 108, 109, 134, and 137 of SEQ ID NO;

206, positions 26, 37, 53, 60, 61, 64, 66, 100, 102, 103, and 126 of SEQ ID NO;

207, positions 32, 34, 39, 46, 47, 56, 84, 85, 88, 89, 90, 102, 110, and 111 of SEQ ID NO;

208, positions 32, 34, 38, 48, 79, 82, 85, 86, 88, 107, 108, 110, and 111 of SEQ ID NO;

209, positions 62, 64, 66, 67, 86, 89, 92, 100, 102, 104, 131, 132, 133, 135, 136, 138, 140, 143, 148, and 151 of SEQ ID NO;

212, positions 69, 70, 85, 99, 102, 104, 106, 109, 137, 143, 146, 148, 149, 153, 154, and 156 of to SEQ ID NO;

213, positions 59, 60, 63, 65, 84, 87, 89, 91, 94, 97, 99, 100, 103, and 106 of SEQ ID NO;

214, positions 56, 61, 66, 67, 68, 70, 72, 75, 78, 79, 82, 89, 92, 93, 107, 110, 111, 115, 117, 124, 125, 127, 128, 129, and 189 of SEQ ID NO;

215, positions 56, 59, 64, 65, 66, 88, 92, 94, 101, 129, 130, 133, 134, 140, 143, 145, 146, 147, 183, and 186 of SEQ ID NO;

216, and positions 64, 65, 66, 68, 69, 75, 77, 92, 98, 103, 105, 108, 133, 138, 139, 140, 149, 156, 178, and 181 of SEQ ID NO;

217.

22. A method, comprising treating a subject by administering the pharmaceutical composition of claim 1, where in the subject has a disease or condition that is treated by administration of a cytokine.

23. The method of claim 22, wherein the subject has a disease or condition that is selected from among an infectious disease, an allergic disease, asthma, a microbial disease, a pregnancy-related disease, a bacterial disease, a heart disease, a viral disease, a histological disease, a genetic disease, a blood-related disease, a fungal disease, an adrenal disease, a cancer, a liver disease, an autoimmune disease, a growth disorder, diabetes, a neurodegenerative disease, multiple sclerosis, Parkinson'"'"'s disease, Alzheimer'"'"'s disease, a fibrosis, a liver disease or condition, a degenerative disease, obesity, tissue damage, a hematopoietic-associated disease, a sclerosis and an inflammatory disease.

24. The method of claim 22, wherein the modified cytokine is an IFN-β

.

25. The method of claim 24, wherein the subject has multiple sclerosis.

26. The method of claim 22, wherein the modified cytokine is an IFNα

.

27. The method of claim 26, wherein the subject has a disease or condition selected from among type B chronic hepatitis, type C chronic hepatitis, a herpes infection, a melanoma, a Kaposi sarcoma, and a lymphoma.

28. The method of claim 22, wherein the modified cytokine is an IL-10.

29. The method of claim 28, wherein the subject has a disease or condition selected from among the disease or condition is selected from among an inflammatory disease or condition selected from chronic liver injury, chronic liver disease and a myeloma.

30. The method of claim 22, wherein the modified cytokine is IFN-γ

.

31. The method of claim 30, wherein the subject has a disease or condition selected from among interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis and adjunctive immunotherapy for immunosuppressed patients.

32. The method of claim 22, wherein the modified cytokine is a G-CSF.

33. The method of claim 32, wherein the subject has a disease or condition selected from among Crohn'"'"'s disease, a cardiac disease, an acquired congenital neutropenia, a congenital neutropenia and asthma.

34. The method of claim 22, wherein the modified cytokine is an LIF.

35. The method of claim 34, wherein the subject has a disease or condition selected from among myocardial infarction, multiple sclerosis, olfactory epithelium replacement stimulation and axonal atrophy.

36. The method of claim 22, wherein the modified cytokine is an hGH.

37. The method of claim 36, wherein the subject has a disease or condition selected from among growth hormone deficiency and acromegaly.

38. The method of claim 22, wherein the modified cytokine is a CNTF.

39. The method of claim 38, wherein subject has a disease or condition selected from among retinal degeneration, a neurodegenerative disease and an auditory degenerative disease.

40. The method of claim 39, wherein the neurodegenerative disease is Huntingdon'"'"'s disease.

41. The method of claim 22, wherein the modified cytokine is a leptin.

42. The method of claim 41, wherein the subject has a disease or condition selected from among obesity, pancreatitis and endometriosis.

43. The method of claim 22, wherein the modified cytokine is oncostatin M.

44. The method of claim 43, wherein the subject has a disease or condition selected from among a chronic inflammatory disease, rheumatoid arthritis, multiple sclerosis and tissue damage.

45. The method of claim 22, wherein the modified cytokine is an IL-6.

46. The method of claim 45, wherein the subject has a disease or condition selected from among liver injury, Crohn'"'"'s disease and a hematopoietic-associated disease.

47. The method of claim 22, wherein the modified cytokine is an IL-12.

48. The method of claim 47, wherein the subject has a disease or condition selected from among coxsackievirus, a neuroblastoma, a melanoma, renal cell carcinoma, and mucosal immunity induction.

49. The method of claim 22, wherein the modified cytokine is an erythropoietin (EPO).

50. The method of claim 49, wherein the subject has a disease or condition selected from among hypoxia, myocardial ischemia, anemia with renal failure and a cancer.

51. The method of claim 22, wherein the modified cytokine is a GM-CSF.

52. The method of claim 51, wherein the subject has a disease or condition selected from among leukemia, a melanoma, a breast carcinoma, a liver carcinoma, a renal cell carcinoma, an autoimmune disease and adjunctive therapy for immunosuppressed patients.

53. The method of claim 22, wherein the modified cytokine is an IL-2.

54. The method of claim 53, wherein the subject has a disease or condition selected from among a melanoma or a colon carcinoma.

55. The method of claim 54, wherein the immune system is reactivated after chemotherapy.

56. The method of claim 22, wherein the modified cytokine is an IL-3.

57. The method of claim 56, wherein the subject has a disease or condition selected from among a leukemia, a motor neuropathy, amyotrophic lateral sclerosis and asthma.

58. The method of claim 22, wherein the modified cytokine is an IL-4.

59. The method of claim 58, wherein the subject has a disease or condition is selected from among allergic asthma and lupus.

60. The method of claim 22, wherein the modified cytokine is an IL-5.

61. The method of claim 60, wherein the subject has a condition selected from among a parasite, asthma, and an allergic disease accompanied by eosinophilia.

62. The method of claim 22, wherein the modified cytokine is an IL-13.

63. The method of claim 62, wherein the subject has a disease or condition selected from among an intracellular infection, a B-cell cancer and asthma.

64. The method of claim 22, wherein the modified cytokine is a Flt3 ligand.

65. The method of claim 64, wherein the subject has a disease or condition selected from among prostate cancer, myeloid leukemia and engraftment of allogenic hematopoietic stem cells.

66. The method of claim 22, wherein the modified cytokine is a SCF.

67. The method of claim 66, wherein the subject has a disease or condition selected from among a hepatic injury, asthma and hematopoietic engraftment.