COMPOSITIONS AND METHODS FOR THE THERAPY AND DIAGNOSIS OF INFLAMMATORY BOWEL DISEASE

• US 20090142778A1
• Filed: 02/25/2008
• Published: 06/04/2009
• Est. Priority Date: 12/17/2001
• Status: Active Grant

• Current Assignee: Corixa Corporation (GSK plc)
• Original Assignee: Corixa Corporation (GSK plc)
• Inventors: Hershberg, Robert M., Mohamath, Raodoh, Hosken, Nancy Ann, Lodes, Michael J., Targan, Stephan R.
• Granted Patent: US 9,290,543 B2
• Time in Patent Office: Days
• US Class Current: 435/7.210
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/523 expressing foreign proteins
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• 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: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/40 bacterial
• CPC Class Codes: A61K 39/46 Cellular immunotherapy
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4648 Bacterial antigens
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/205 from Campylobacter (G)
• CPC Class Codes: C07K 16/1232 from Escherichia (G)
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: G01N 2800/065 : Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
• CPC Class Codes: G01N 33/505 : involving T-cells
• CPC Class Codes: G01N 33/6893 : related to diseases not provided for elsewhere

Abstract

Compositions and methods for the therapy and diagnosis of Inflammatory Bowel Disease (IBD), including Crohn'"'"'s Disease and Ulcerative Colitis, are disclosed. Illustrative compositions comprise one or more bacterial polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of IBD.

Claims

1. An isolated polynucleotide comprising a sequence selected from the group consisting of:

(a) sequences provided in SEQ ID NOs;

75, 83, 85, 1-37, 51-74, 76-78 and 86;

(b) complements of the sequences provided in SEQ ID NOs;

75, 83, 85, 1-37, 51-74, and 76-78;

(c) sequences consisting of at least 20 contiguous residues of a sequence provided in SEQ ID NOs;

75, 83, 85, 1-37, 51-74, 76-78 and 87;

(d) sequences that hybridize to a sequence provided in SEQ ID NOs;

75, 83, 85, 1-37, 51-74, 76-78 and 86, under highly stringent conditions;

(e) sequences having at least 75% identity to a sequence of SEQ ID NOs;

75, 83, 85, 1-37, 51-74, 76-78 and 87;

(f) sequences having at least 90% identity to a sequence of SEQ ID NOs;

75, 83, 85, 1-37, 51-74, 76-78 and 87; and

(g) degenerate variants of a sequence provided in SEQ ID NOs;

75, 83, 85, 1-37, 51-74, 76-78 and 87.

2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) sequences encoded by a polynucleotide of claim 1;

(b) sequences having at least 70% identity to a sequence encoded by a polynucleotide of claim 1;

(c) sequences having at least 90% identity to a sequence encoded by a polynucleotide of claim 1;

(d) sequences set forth in SEQ ID NOs;

79, 84, 86, 80-82, 38-50 and 88-89;

(e) sequences having at least 70% identity to a sequence set forth in SEQ ID NOs;

79, 84, 86, 80-82, 38-50 and 88-89; and

(f) sequences having at least 90% identity to a sequence set forth in SEQ ID NOs;

79, 84, 86, 80-82, 38-50 and 88-89.

3. An expression vector comprising a polynucleotide of claim 1 operably linked to an expression control sequence.

4. A host cell transformed or transfected with an expression vector according to claim 3.

5. A method of making a polypeptide comprising:

culturing a host cell transformed or transfected with an expression vector according to claim 3 under conditions whereby the polynucleotide is expressed and the polypeptide is produced;

and recovering the polypeptide.

6. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to a polypeptide of claim 2.

7. The antibody according to claim 6 wherein said antibody is a monoclonal antibody.

8. The antibody according to claim 6 wherein said antibody is a human antibody.

9. The antibody according to claim 6 wherein said antibody has been humanized.

10. The antibody according to claim 6 wherein said antibody binds to a flagellin protein.

11. The antibody according to claim 10 wherein said antibody is a neutralizing antibody against said flagellin protein.

12. The antibody according to claim 6 wherein said antibody blocks the interaction between a flagellin protein and a Toll-like receptor.

13. The antibody according to claim 12 wherein said Toll-like receptor is TLR5.

14. A fusion protein comprising at least one polypeptide according to claim 2.

15. An oligonucleotide that hybridizes to a polynucleotide according to claim 1 under highly stringent conditions.

16. The oligonucleotide of claim 15 wherein said polynucleotide according to claim 1 comprises a polynucleotide that encodes a flagellin protein.

17. A method of stimulating and/or, expanding T cells specific for an enteric bacterial protein, comprising contacting T cells with at least one component selected from the group consisting of:

(a) polypeptides according to claim 2;

(b) polynucleotides according to claim 1; and

(c) antigen-presenting cells that express a polynucleotide according to claim 1, under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.

18. The method according to claim 17 wherein said T cells are CD4+ T cells.

19. The method according to claim 18 wherein said CD4+ T cells mediate a decrease in inflammation in the colon.

20. The method according to claim 17 wherein said bacterial antigen comprises a flagellin polypeptide.

21. An isolated T cell population, comprising T cells prepared according to the method of claim 17.

22. The population of T cells according to claim 19 wherein said T cells produce a cytokine selected from the group consisting of interleukin 10 (IL-10), interleukin 4 (IL-4), interleukin 5 (IL-5) and transforming growth factor beta (TGFβ

).

23. The population of T cells according to claim 19 wherein said T cells produce IL-10.

24. The population of T cells according to claim 19 wherein said T cells produce transforming growth factor beta (TGFβ

).

25. The population of T cells according to claim 19 wherein said T cells produce IL-10 and TGFβ

.

26. A composition comprising a first component selected from the group consisting of physiologically acceptable carriers and a second component selected from the group consisting of:

(a) polypeptides according to claim 2;

(b) polynucleotides according to claim 1;

(c) antibodies according to claim 5;

(d) fusion proteins according to claim 14;

(e) T cell populations according to claim 21; and

(f) antigen presenting cells that express a polypeptide according to claim 2.

27. The composition of claim 26 further comprising an immunostimulant.

28. The composition of claim 25 wherein said immunostimulant is an adjuvant selected from the group consisting of Freund'"'"'s Incomplete Adjuvant;

Freund'"'"'s Complete Adjuvant;

Merck Adjuvant 65;

AS-1, AS-2;

aluminum hydroxide gel;

aluminum phosphate;

a salt of calcium, iron or zinc;

an insoluble suspension of acylated tyrosine acylated sugars;

cationically or anionically derivatized polysaccharides;

polyphosphazenes;

biodegradable microspheres;

monophosphoryl lipid A, QS21, aminoalkyl glucosaminide 4-phosphates, and quil A.

29. A method of detecting the presence of inflammatory bowel disease in a mammal, comprising the steps of:

(a) obtaining a biological sample from the mammal, wherein said biological sample comprises antibodies;

(b) contacting the biological sample with a polypeptide of claim 2;

(c) detecting in the sample an amount of antibody that binds to the polypeptide; and

(d) comparing the amount of bound antibody to a predetermined cut-off value and therefrom determining the presence of inflammatory bowel disease in the mammal.

30. The method of claim 29 wherein said biological sample is selected from the group consisting of sera, stool, tissue or other material obtained by colonoscopy, tissue or other material obtained by ileoscopy, tissue or other material obtained by esophagogastroduodenoscopy (EGP), and tissue or other material obtained by surgery.

31. The method of claim 29 wherein said polypeptide of claim 2 comprises a flagellin polypeptide.

32. A method of detecting the presence of inflammatory bowel disease in a mammal, comprising the steps of:

(a) obtaining a biological sample from said mammal, wherein said biological sample comprises polynucleotides;

(b) contacting said sample with at least one oligonucleotide that is at least in part complementary to a polynucleotide of claim 1;

(c) detecting in the sample an amount of a polynucleotide that hybridizes to said oligonucleotide; and

(d) comparing the amount of said polynucleotide that hybridizes to said oligonucleotide to a predetermined cut-off value, and therefrom determining the presence of inflammatory bowel disease in a mammal.

33. The method of claim 32 wherein said oligonucleotide hybridizes to a polynucleotide of claim 1 under moderately stringent conditions.

34. The method according to claim 32, wherein the amount of polynucleotide that hybridizes to said oligonucleotide is determined using a polymerase chain reaction.

35. The method according to claim 32, wherein the amount of polynucleotide that hybridizes to said oligonucleotide is determined using a hybridization assay.

36. The method according to claim 32 wherein said biological sample is selected from the group consisting of sera, stool, tissue or other material obtained by colonoscopy, tissue or other material obtained by ileoscopy, tissue or other material obtained by esophagogastroduodenoscopy (EGP), and tissue or other material obtained by surgery.

37. The method according to claim 32 wherein said polynucleotide of claim 1 comprises a polynucleotide that encodes a flagellin protein.

38. A method of stimulating and/or expanding B cells that produce antibodies specific for an enteric bacterial protein, comprising contacting B cells with at least one component selected from the group consisting of:

(a) polypeptides according to claim 2; and

(b) polynucleotides according to claim 1;

under conditions and for a time sufficient to permit the stimulation and/or expansion of B cells.

39. The method according to claim 38 wherein said B cells produce antibodies that bind a flagellin protein.

40. The method according to claim 39 wherein said antibodies are neutralizing antibodies against said flagellin protein.

41. The method according to claim 39 wherein said antibodies block the interaction between a flagellin protein and a Toll-like receptor.

42. The method according to claim 41 wherein said Toll-like receptor comprises TLR5.

43. An isolated B cell population, comprising B cells prepared according to any one of the methods of claims 39-42.

44. A method of stimulating an immune response in a mammal, comprising administering to the mammal the composition of any one of claims 26-28.

45. The method according to claim 44 wherein said immune response comprises T cells that produce a cytokine selected from the group consisting of interleukin 10 (IL-10), interleukin 4 (IL-4), interleukin 5 (IL-5) and transforming growth factor beta (TGFβ

).

46. The method according to claim 45 wherein said cytokine comprises IL-10 and/or TGFβ

.

47. A method of decreasing gastrointestinal inflammation associated with inflammatory bowel disease in a mammal, comprising administering to said mammal the composition of any one of claims 26-28.

48. A method of identifying bacterial antigens associated with inflammatory bowel disease in a mammal, comprising the steps of:

(a) obtaining a biological sample from said mammal wherein said biological sample comprises T cells;

(b) contacting said biological sample with at least one component selected from the group consisting of;

(i) polypeptides according to claim 2;

(ii) polynucleotides according to claim 1; and

(iii) antigen-presenting cells that express a polynucleotide according to claim 1;

under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells;

(c) detecting in the sample the magnitude of said stimulation and/or expansion of T cells; and

(d) comparing the magnitude of said stimulation and/or expansion to a predetermined cut-off value, and therefrom identifying bacterial antigens associated with inflammatory bowel disease in the mammal.

49. The method of claim 48 wherein said mammal is a mouse.

50. The method of claim 49 wherein said mouse is of a strain selected from the group consisting of C3H/HeJ Bir, BALB/c IL-10−

/−

, B6 IL-10−

/−

, B10 IL-10−

/−

, MDR1a−

/−

, TCRα

−

/−

, IL-2−

/−

, IL-2R−

/−

, mice with DSS (Dextransodiumsulfate) induced colitis, G α

_ai−

/−

, and CD45 RB transgenic mice.

51. The method of claim 49 wherein said mouse is of the C3H/HeJ Bir strain.

52. A method of monitoring the progression of inflammatory bowel disease in a mammal, comprising the steps of:

(a) obtaining a biological sample from the mammal, wherein said biological sample comprises antibodies;

(b) contacting the biological sample with a polypeptide of claim 2;

(c) detecting in the sample an amount of antibody that binds to the polypeptide; and

(d) repeating steps (a), (b), and (c) using a biological sample obtained from the mammal at a subsequent point in time; and

(e) comparing the amount of bound antibody in step (c) to the amount of bound antibody in step (d) and therefrom monitoring the progression of inflammatory bowel disease in the mammal.

57. A method of monitoring the progression of inflammatory bowel disease in a mammal, comprising the steps of:

(a) obtaining a biological sample from said mammal, wherein said biological sample comprises polynucleotides;

(b) contacting said sample with at least one oligonucleotide that is at least in part complementary to a polynucleotide of claim 1;

(c) detecting in the sample an amount of a polynucleotide that hybridizes to said oligonucleotide;

(d) repeating steps (a), (b), and (c) using a biological sample obtained from said mammal at a subsequent point in time; and

(e) comparing the amount of said polynucleotide that hybridizes to said oligonucleotide in step (c) to the amount of said polynucleotide that hybridizes to said oligonucleotide in step (d);

and therefrom monitoring the progression of inflammatory bowel disease in a mammal.

58. The method of claim 57 wherein said oligonucleotide hybridizes to a polynucleotide of claim 1 under moderately stringent conditions.

59. The method according to claim 57, wherein the amount of polynucleotide that hybridizes to said oligonucleotide is determined using a polymerase chain reaction.

60. The method according to claim 57, wherein the amount of polynucleotide that hybridizes to said oligonucleotide is determined using a hybridization assay.

61. The method according to claim 57 wherein said biological sample is selected from the group consisting of sera, stool, tissue or other material obtained by colonoscopy, tissue or other material obtained by ileoscopy, tissue or other material obtained by esophagogastroduodenoscopy (EGP), and tissue or other material obtained by surgery.

62. The method according to claim 57 wherein said polynucleotide of claim 1 comprises a polynucleotide that encodes a flagellin protein.

63. A diagnostic kit comprising at least one oligonucleotide according to claim 15.

64. A diagnostic kit comprising at least one antibody according to claim 5 and a detection reagent, wherein the detection reagent comprises a reporter group.

65. A diagnostic kit, comprising:

(a) a portion of at least one or more polypeptides according to claim 2 wherein said portion can be bound by an antibody; and

(b) a detection reagent comprising a reporter group.

66. A kit according to claim 65, wherein said portion of at least one or more polypeptides is immobilized on a solid support.

67. A kit according to claim 65, wherein the detection reagent comprises an anti-immunoglobulin, protein G, protein A, or a lectin.

68. A kit according to claim 65, wherein the reporter group is selected from the group consisting of radioactive groups, fluorescent groups, luminescent groups, enzymes, biotin, and dyes.

69. An immunogenic composition comprising a compound of claim 2, an antigen and a suitable carrier.

70. A method for identifying an inflammatory bowel disease type in a patient, comprising:

(a) obtaining an antibody comprising biological sample from a patient;

(b) contacting the biological sample with a polypeptide of claim 2;

(c) detecting in the sample an amount of antibody that binds to the polypeptide; and

(d) comparing the amount of bound antibody to a predetermined value associated with and therefrom subdividing the inflammatory bowel disease type.

71. A method of claim 70, wherein the disease type is selected from ulcerative colitis and Crohn'"'"'s Disease.

72. The method of claim 70 wherein said polypeptide of claim 2 comprises a flagellin polypeptide.

53. A method of identifying bacterial antigens associated with inflammatory bowel disease in a first mammal, comprising the steps of:

(a) obtaining a biological sample from said first mammal wherein said biological sample comprises DNA from cecal bacteria;

(b) constructing an expression library with said DNA;

(c) screening said expression library with sera from either said first mammal or a second mammal with inflammatory bowel disease;

thereby identifying bacterial antigens associated with inflammatory bowel disease.

54. The method of 53 wherein said first and said second mammal are both mice.

55. The method of 53 wherein said first mammal is a C3H/HeJ Bir mouse and said second mammal is a mouse of a strain selected from the group consisting of BALB/c IL-10−

/−

, B6 IL-10−

/−

, B10 IL-10−

/−

, MDR1a−

/−

, TCRα

−

/−

, IL-2−

/−

, IL-2R−

/−

, mice with DSS (Dextransodiumsulfate) induced colitis, G α

_ai−

/−

, and CD45 RB transgenic mice.

56. The method of 53 wherein said first mammal is a mouse and said second mammal is a human.