Method for Identifying Agents Capable of Inducing Respiratory Sensitization and Array and Analytical Kits for Use in the Method

• US 20150111771A1
• Filed: 04/26/2013
• Published: 04/23/2015
• Est. Priority Date: 04/26/2012
• Status: Abandoned Application

• Current Assignee: SenzaGen AB
• Original Assignee: SenzaGen AB
• Inventors: Lindstedt, Malin, Borrebaeck, Carl, Johansson, Henrik, Albrekt, Ann-Sofie
• Application Number:
  US14/396,422
• Publication Number:
  US 20150111771A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2800/24 Immunology or allergic disorders SLE G01N2800/104
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The present invention relates to an in vitro method for identifying agents capable of inducing respiratory sensitization in a mammal and arrays and diagnostic kits for use in such methods. In particular, the methods include measurement of the expression of the biomarkers listed in Table 1A, Table 1B and/or Table 1C in MUTZ-3 cells exposed to a test agent.

Claims

1. A method for identifying agents capable of inducing respiratory sensitization in a mammal comprising or consisting of the steps of:

a) exposing a population of dendritic cells or a population of dendritic-like cells to a test agent; and

b) measuring in the cells the expression of one or more biomarker(s) selected from the group defined in Table 1A and/or Table 1B;

wherein the expression in the cells of the one or more biomarkers measured in step (b) is indicative of the sensitizing effect of the sample to be tested.

2. The method according to claim 1 further comprising:

c) exposing a separate population of the dendritic cells or dendritic-like cells to one or more negative control agent that is not a respiratory sensitizer in a mammal; and

d) measuring in the cells the expression of the one or more biomarker(s) measured in step (b)

wherein the test agent is identified as a respiratory sensitizer in the event that the presence and/or amount in the test sample of the one or more biomarker measured in step (d) differs from the presence and/or amount in the control sample of the one or more biomarker measured in step (b).

3. The method according to claim 1 or 2 further comprising:

e) exposing a separate population of the dendritic cells or dendritic-like cells to one or more positive control agent that is a respiratory sensitizer in a mammal; and

f) measuring in the cells the expression of the one or more biomarker(s) measured in step (b)

wherein the test agent is identified as a respiratory sensitizer in the event that the presence and/or amount in the test sample of the one or more biomarker measured in step (f) corresponds to the presence and/or amount in the positive control sample of the one or more biomarker measured in step (b).

4. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of one or more biomarkers defined in Table 1A, for example, at least 2 of the biomarkers defined in Table 1A.

5. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of OR5B21.

6. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of SLC7A7.

7. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of OR5B21 and SLC7A7.

8. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of one or more of the biomarkers defined in Table 1B, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the biomarkers defined in Table 1B.

9. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of all of the biomarkers defined in Table 1B.

10. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of one or more of the biomarkers defined in Table 1C, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286 or 287 of the biomarkers defined in Table 1C.

11. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of all of the biomarkers defined in Table 1C.

12. The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of all of the biomarkers defined in Table 1.

13. The method according to any one of the preceding claims wherein step (b) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarker(s).

14. The method according to claim 13 wherein the nucleic acid molecule is a cDNA molecule or an mRNA molecule.

15. The method according to claim 13 wherein the nucleic acid molecule is an mRNA molecule.

16. The method according to claim 13 wherein the nucleic acid molecule is an cDNA molecule.

17. The method according to any one of claims 13 to 16 wherein measuring the expression of the one or more biomarker(s) in step (b) is performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (q RT-PCR), nanoarray, microarray, macroarray, autoradiography and in situ hybridisation.

18. The method according to any one of claims 13 to 17 wherein measuring the expression of the one or more biomarker(s) in step (b) is determined using a DNA microarray.

19. The method according to any one of the preceding claims wherein measuring the expression of the one or more biomarker(s) in step (b) is performed using one or more binding moieties, each capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table 1.

20. The method according to claim 19 wherein the one or more binding moieties each comprise or consist of a nucleic acid molecule.

21. The method according to claim 20 wherein the one or more binding moieties each comprise or consist of DNA, RNA, PNA, LNA, GNA, TNA or PMO.

22. The method according to claim 19 or 20 wherein the one or more binding moieties each comprise or consist of DNA.

23. The method according to any one of claims 20 to 22 wherein the one or more binding moieties are 5 to 100 nucleotides in length.

24. The method according to any one of claims 20 to 23 wherein the one or more nucleic acid molecules are 15 to 35 nucleotides in length.

25. The method according to any one of claims 20 to 24 wherein the binding moiety comprises a detectable moiety.

26. The method according to claim 25 wherein the detectable moiety is selected from the group consisting of:

a fluorescent moiety;

a luminescent moiety;

a chemiluminescent moiety;

a radioactive moiety (for example, a radioactive atom);

or an enzymatic moiety.

27. The method according to claim 26 wherein the detectable moiety comprises or consists of a radioactive atom.

28. The method according to claim 27 wherein the radioactive atom is selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.

29. The method according to claim 26 wherein the detectable moiety of the binding moiety is a fluorescent moiety.

30. The method according to any one of claims 1 to 21 wherein step (b) comprises or consists of measuring the expression of the protein of the one or more biomarker defined in step (b).

31. The method according to claim 30 wherein measuring the expression of the one or more biomarker(s) in step (b) is performed using one or more binding moieties each capable of binding selectively to one of the biomarkers identified in Table 1.

32. The method according to claim 31 wherein the one or more binding moieties comprise or consist of an antibody or an antigen-binding fragment thereof.

33. The method according to claim 32 wherein the antibody or fragment thereof is a monoclonal antibody or fragment thereof.

34. The method according to claim 32 or 33 wherein the antibody or antigen-binding fragment is selected from the group consisting of intact antibodies, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′

fragments and F(ab)₂ fragments), single variable domains (e.g. V_H and V_L domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).

35. The method according to claim 34 wherein the antibody or antigen-binding fragment is a single chain Fv (scFv).

36. The method according to claim 31 wherein the one or more binding moieties comprise or consist of an antibody-like binding agent, for example an affibody or aptamer.

37. The method according to any one of claims 31 to 36 wherein the one or more binding moieties comprise a detectable moiety.

38. The method according to claim 37 wherein the detectable moiety is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety and an enzymatic moiety.

39. The method according to any one of the preceding claims wherein step (b) is performed using an array.

40. The method according to claim 39 wherein the array is a bead-based array.

41. The method according to claim 40 wherein the array is a surface-based array.

42. The method according to any one of claims 39 to 41 wherein the array is selected from the group consisting of:

macroarray;

microarray;

nanoarray.

43. An array for use in a method according any one of the preceding claims, the array comprising one or more first binding agents as defined in any one of claims 19 to 29 and 31 to 38.

44. An array according to claim 43 comprising binding agents which are collectively capable of binding to all of the biomarkers defined in Table 1.

45. An array according to claim 43 or 44 wherein the first binding agents are immobilised.

46. The method according to any one of the preceding claims for identifying agents capable of inducing a respiratory hypersensitivity response.

47. The method according to any one of the preceding claims wherein the hypersensitivity response is a humoral hypersensitivity response.

48. The method according to claim 46 or 47 wherein the hypersensitivity response is a type I hypersensitivity response.

49. The method according to any one of the preceding claims for identifying agents capable of inducing respiratory allergy.

50. The method according to any one of the preceding claims wherein the population of dendritic cells or population of dendritic-like cells is a population of dendritic-like cells.

51. The method according to claim 50 wherein the dendritic-like cells are myeloid dendritic-like cells.

52. The method according to claim 51 wherein the myeloid dendritic-like cells are derived from myeloid dendritic cells.

53. The method according to claim 52 wherein the cells derived from myeloid dendritic cells are myeloid leukaemia-derived cells.

54. The method according to claim 53 wherein the myeloid leukaemia-derived cells are selected from the group consisting of KG-1, THP-1, U-937, HL-60, Monomac-6, AML-193 and MUTZ-3.

55. The method according to any one of the preceding claims wherein the dendritic-like cells are MUTZ-3 cells.

56. The method according to any one of the preceding claims wherein the one or more negative control agent provided in step (c) is selected from the group consisting of 1-butanol, 4-aminobenzoic acid, chlorobenzene, dimethyl formamide, ethyl vanillin, isopropanol, methyl salicylate, propylene glycol, potassium permanganate, Tween 80™

(polyoxyethylene (20) sorbitan monooleate) and zinc sulphate.

57. The method according to claim 56 wherein at least 2 control non-sensitizing agents are provided, for example, at least 3, 4, 5, 6, 7, 8, 9, 10 or at least 11 control non-sensitizing agents.

58. The method according to any one of the preceding claims wherein the one or more positive control agent provided in step (e) comprises or consists of one or more agent selected from the group consisting of ammonium hexachloroplatinate, ammonium persulfate, glutaraldehyde, hexamethylen diisocyanate, maleic anhydride, methylene diphenol diisocyanate, phtalic anhydride, toluendiisocyanate and trimellitic anhydride.

59. The method according to claim 58 wherein at least 2 control sensitizing agents are provided, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 control sensitizing agents.

60. The method according to any one of the preceding claims wherein the method is indicative of the sensitizing potency of the sample to be tested.

61. An array for use in a method according any one of the preceding claims, the array comprising one or more binding moieties as defined in any one of claims 19 to 29 and 31-38.

62. An array according to claim 61 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table 1A.

63. An array according to claim 61 or 62 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table 1B.

64. An array according to claim 61, 62 or 63 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table 1C.

65. An array according to any one of claims 61 to 64 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table 1.

66. An array according to any on of claims 61 to 64 wherein the binding moieties are immobilised.

69. An analytical kit for use in a method according any one of claims 1 to 60 comprising:

A) an array according to any one of claims 61 to 66; and

B) instructions for performing the method as defined in any one of claims 1 to 60 (optional).

70. An analytical kit according to claim 69 further comprising one or more control samples.

71. An analytical kit according to claim 69 comprising one or more non-sensitizing agent(s).

72. An analytical kit according to claim 69, 70 or 71 comprising one or more sensitizing agent(s).

67. Use of two or more biomarkers selected from the group defined in Table 1 in combination for identifying respiratory hypersensitivity response sensitising agents.

68. The use according to claim 67 wherein all of the biomarkers defined in Table 1 are used collectively for identifying hypersensitivity response sensitising agents.

73. A method or use substantially as described herein.

74. An array or kit substantially as described herein.