Method for detecting anti-drug antibodies (ADAs) against anti-TNF-alpha antibodies

Abstract

This technology provides a method for detecting anti-drug antibodies (ADAs) that bind to anti-TNFα antibodies (target antibodies) in a sample, comprising: incubating the sample with a composition containing affinity antibodies and detection antibodies; and isolating and detecting ADAs that form complexes with the affinity antibodies and detection antibodies. The technology also includes a method for determining the concentration of ADAs in a sample and confirming that the sample contains ADAs. The technology also includes a kit for carrying out any of the methods of this technology.

Claims

1. A method for detecting anti-drug antibodies (ADAs) that bind to target antibodies, including anti-TNFα antibodies, in a sample, (i) The sample is (a) A first affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label, and (b) Incubating with a composition comprising a detection antibody containing an anti-TNFα antibody conjugated to a detection label, (ii) Isolating ADA which forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label which binds to the first affinity label, (iii) A method comprising detecting the ADA by measuring the level of the detection indicator that exceeds a predetermined cut point.

2. Isolating the aforementioned ADA (a) To provide a solid phase containing the second affinity label, (b) Contacting the sample and the composition with the solid phase, (c) The method according to claim 1, comprising washing the solid phase.

3. The method according to claim 1 or 2, wherein the target antibody, the affinity antibody, and / or the detection antibody comprises a chimeric human-mouse monoclonal IgG1-kappa antibody.

4. The method according to any one of claims 1 to 3, wherein the target antibody, affinity antibody, and / or detection antibody include SEQ ID NOs: 1 and 4.

5. The method according to any one of claims 2 to 4, wherein the solid phase is a plate.

6. The method according to any one of claims 1 to 5, wherein the first affinity label is biotin.

7. The method according to any one of claims 1 to 6, wherein the second affinity label is streptavidin.

8. The method according to any one of claims 1 to 7, wherein the detection antibody comprises a sulfotag-labeled anti-TNFα antibody.

9. Measuring the level of the aforementioned detection indicator, (1) Contacting the solid phase with a marker, wherein the marker is configured to generate a chemiluminescent signal or an electrochemiluminescent signal when the marker is in close proximity to the detection marker, (2) The method according to any one of claims 2 to 8, comprising detecting the chemiluminescence signal or electrochemiluminescence signal.

10. The method according to claim 9, wherein the marker is tripropylamine (TPA), and measuring the level of the detection label further comprises applying an electric current to the marker to generate the chemiluminescence signal or electrochemiluminescence signal.

11. The method according to any one of claims 2 to 10, wherein the solid phase is blocked by a blocking buffer before the sample and the composition come into contact with the solid phase.

12. The method according to any one of claims 1 to 11, further comprising performing steps (i) to (iii) on a control sample.

13. The method according to claim 12, wherein the control sample is incubated with the composition and brought into contact with the second affinity label.

14. The method according to any one of claims 1 to 13, wherein the predetermined cut point corresponds to a level having a false positive rate of 5%.

15. The method according to claim 14, wherein the 5% false positive rate is determined by measuring the detection label in a plurality of control samples and calculating the 5% false positive rate.

16. The method according to claim 9, wherein the chemiluminescence signal or electrochemiluminescence signal is measured as electrochemiluminescence (ECL).

17. The method according to claim 16, wherein the predetermined cut point is at least one ECL value.

18. The method according to claim 16 or 17, wherein the predetermined cut point is an ECL value of approximately 1.

10.

19. The method according to any one of claims 1 to 18, wherein the method has a sensitivity of less than 6 ng / mL.

20. The method according to claim 19, wherein the sensitivity is less than 4 ng / mL.

21. The method according to any one of claims 1 to 20, wherein the method has a drug resistance of about 100 ng / mL.

22. A method for detecting anti-drug antibodies (ADAs) that bind to target antibodies, including anti-TNFα antibodies, in a sample, (i) Contacting the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugates, (ii) Measuring the level of the anti-TNFα conjugate in the complex, The method described above is a method for detecting ADA with a sensitivity of less than 5 ng / mL.

23. The method according to claim 22, wherein the one or more anti-TNFα conjugates include affinity antibodies containing an anti-TNFα antibody conjugated to an affinity label.

24. The method according to claim 22 or 23, wherein the one or more anti-TNFα conjugates include a detection antibody containing an anti-TNFα antibody conjugated to a detection label.

25. The method according to claim 23, wherein the affinity label comprises biotin.

26. The method according to claim 24, wherein the detection label includes a sulfotag.

27. The method according to any one of claims 22 to 26, wherein the method detects the ADA with a sensitivity of less than 3 ng / mL.

28. The method according to any one of claims 22 to 27, wherein the method detects the ADA with a sensitivity of 2.77 ng / mL or less.

29. A method for estimating the amount of anti-drug antibodies (ADAs) that bind to target antibodies, including anti-TNFα antibodies, in a sample, (i) The sample is (a) an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label, and (b) Incubating with a composition comprising a detection antibody containing an anti-TNFα antibody conjugated to a detection label, (ii) Isolating ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label that binds to the first affinity label, (iii) Detecting the ADA by measuring the level of the detection indicator that exceeds a predetermined cut point, (iv) Perform steps (i) to (iii) on a known sample having a known concentration of ADA, (v) A method comprising comparing the level of the detection label in the sample with the level of the detection label in a known sample.

30. The method according to claim 29, wherein the known sample is successively diluted into a plurality of titer samples, and the level of the detection label in the sample is compared with the level of the detection label in the known sample to generate a curve of the level of the detection label in each of the titer samples, and the detection label in the sample is compared with the curve.

31. A method for confirming that an anti-drug antibody (ADA) binds to a target antibody containing an anti-TNFα antibody in a sample by performing a competitive binding assay, comprising performing the method according to claim 1 on the sample, wherein the composition further comprises an unlabeled anti-TNFα antibody.

32. The method according to claim 31, wherein the ADA is confirmed in the sample by measuring the level of the detection label which is a predetermined coefficient lower than the level of the detection label when the unlabeled anti-TNFα antibody is not present.

33. A kit for detecting anti-drug antibodies (ADAs) that bind to target antibodies, including anti-TNFα antibodies, in a sample, (i) an affinity antibody containing an anti-TNFα antibody conjugated to a first affinity label, (ii) A detection antibody containing an anti-TNFα antibody conjugated to a detection label, and (iii) A kit comprising a solid phase having a second affinity label, wherein the second affinity label is bound to the first affinity label.

34. The kit according to claim 33, wherein the solid phase is a plate.

35. The kit according to claim 33 or 34, wherein the second affinity label is streptavidin.

36. The kit according to any one of claims 33 to 35, wherein the first affinity label is biotin.

37. The kit according to any one of claims 33 to 36, wherein the detection antibody comprises a sulfotag-labeled anti-TNFα antibody.

38. The kit according to any one of claims 33 to 37, further comprising a marker, the marker configured to generate a chemiluminescent signal or an electrochemiluminescent signal when the marker is in proximity to the detection label.

39. The kit according to claim 38, wherein the marker is tripropylamine (TPA).

40. The kit according to any one of claims 33 to 39, further comprising a blocking buffer.

41. The kit according to any one of claims 33 to 40, further comprising a washing buffer.

42. A kit for estimating the amount of anti-drug antibodies (ADAs) that bind to target antibodies, including anti-TNFα antibodies, in a sample. (i) an affinity antibody containing an anti-TNFα antibody conjugated to a first affinity label, (ii) Detection antibody containing an anti-TNFα antibody conjugated to a detection label, (iii) A solid phase comprising a second affinity label, wherein the second affinity label is bound to the first affinity label, and (iv) A kit including a positive control.

43. The kit according to claim 42, wherein the positive control is a plurality of titer samples.

44. A kit for confirming that an anti-drug antibody (ADA) binds to a target antibody containing an anti-TNFα antibody, (i) an affinity antibody containing an anti-TNFα antibody conjugated to a first affinity label, (ii) Detection antibody containing an anti-TNFα antibody conjugated to a detection label, (iii) Unlabeled anti-TNFα antibody, and (iv) A kit comprising a solid phase having a second affinity label, wherein the second affinity label is bound to the first affinity label.

45. The use of a target antibody comprising an anti-TNFα antibody for detecting ADA bound to the target antibody in a sample, (i) The sample is (a) A first affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label, and (b) Incubating with a composition comprising a detection antibody containing an anti-TNFα antibody conjugated to a detection label, (ii) Isolating ADA which forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label which binds to the first affinity label, (iii) Use comprising detecting the ADA by measuring the level of the detection indicator that exceeds a predetermined cut point.

46. Isolating the aforementioned ADA (a) To provide a solid phase containing the second affinity label, (b) Contacting the sample and the composition with the solid phase, (c) The use according to claim 45, comprising washing the solid phase.

47. The use according to claim 45 or 46, wherein the target antibody, affinity antibody, and / or detection antibody comprises a chimeric human-mouse monoclonal IgG1-kappa antibody.

48. The use according to any one of claims 45 to 47, wherein the target antibody, affinity antibody, and / or detection antibody include SEQ ID NOs: 1 and 4.

49. The use according to any one of claims 46 to 48, wherein the solid phase is a plate.

50. The use according to any one of claims 45 to 49, wherein the first affinity label is biotin.

51. The use according to any one of claims 45 to 50, wherein the second affinity label is streptavidin.

52. The use according to any one of claims 45 to 51, wherein the detection antibody comprises a sulfotag-labeled anti-TNFα antibody.

53. Measuring the level of the aforementioned detection indicator, (1) Contacting the solid phase with a marker, wherein the marker is configured to generate a chemiluminescent signal or an electrochemiluminescent signal when the marker is in close proximity to the detection marker, (2) The use according to any one of claims 46 to 52, comprising detecting the chemiluminescence signal or electrochemiluminescence signal.

54. The use according to claim 53, wherein the marker is tripropylamine (TPA), and measuring the level of the detection label further comprises applying an electric current to the marker to generate the chemiluminescent signal or electrochemiluminescent signal.

55. The use according to any one of claims 46 to 54, wherein the solid phase is blocked by a blocking buffer before the sample and the composition come into contact with the solid phase.

56. The use according to any one of claims 45 to 55, further comprising performing steps (i) to (iii) on a control sample.

57. The use according to claim 56, wherein the control sample is incubated with the composition and brought into contact with the second affinity label.

58. The use according to any one of claims 45 to 57, wherein the predetermined cut point corresponds to a level having a false positive rate of 5%.

59. The use according to claim 58, wherein the 5% false positive rate is determined by measuring the detection label in a plurality of control samples and calculating the 5% false positive rate.

60. The use according to claim 53, wherein the chemiluminescence signal or electrochemiluminescence signal is measured as electrochemiluminescence (ECL).

61. The use according to claim 60, wherein the predetermined cut point is at least one ECL value.

62. The use according to claim 60 or 61, wherein the predetermined cut point has an ECL value of approximately 1.

10.

63. The use according to any one of claims 45 to 62, wherein the method has a sensitivity of less than 6 ng / mL.

64. The use according to claim 63, wherein the sensitivity is less than 4 ng / mL.

65. The use according to any one of claims 45 to 64, wherein the method has a drug tolerance of about 100 ng / mL.

66. The use of one or more anti-TNFα conjugates for detecting anti-drug antibodies (ADAs) that bind to a target antibody containing an anti-TNFα antibody in a sample, (i) Contacting the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate, (ii) Measuring the level of the anti-TNFα conjugate in the complex, The method described above detects the ADA with a sensitivity of less than 5 ng / mL.

67. The use according to claim 66, wherein the one or more anti-TNFα conjugates include affinity antibodies containing an anti-TNFα antibody conjugated to an affinity label.

68. The use according to claim 66 or 67, wherein the one or more anti-TNFα conjugates comprise a detection antibody containing an anti-TNFα antibody conjugated to a detection label.

69. The use according to claim 67, wherein the affinity label comprises biotin.

70. The use according to claim 68, wherein the detection label includes a sulfotag.

71. The use according to any one of claims 66 to 70, wherein the method detects the ADA with a sensitivity of less than 3 ng / mL.

72. The use according to any one of claims 66 to 71, wherein the method detects the ADA with a sensitivity of 2.77 ng / mL or less.

73. The use of the target antibody containing the anti-TNFα antibody for estimating the amount of anti-drug antibody (ADA) that binds to the target antibody containing the anti-TNFα antibody in a sample, (i) The sample is (a) an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label, and (b) Incubating with a composition comprising a detection antibody containing an anti-TNFα antibody conjugated to a detection label, (ii) Isolating ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label that binds to the first affinity label, (iii) Detecting the ADA by measuring the level of the detection indicator that exceeds a predetermined cut point, (iv) Perform steps (i) to (iii) on a known sample having a known concentration of ADA, (v) Use comprising comparing the level of the detection label in the sample with the level of the detection label in the known sample.

74. The use according to claim 73, wherein the known sample is successively diluted into a plurality of titer samples, and the level of the detection label in the sample is compared with the level of the detection label in the known sample, thereby generating a curve of the level of the detection label in each of the titer samples, and the detection label in the sample is compared with the curve.

75. A use of the target antibody comprising an anti-TNFα antibody to confirm that the anti-drug antibody (ADA) binds to the target antibody comprising an anti-TNFα antibody in a sample by performing a competitive binding assay, comprising performing the method of claim 1 on the sample, wherein the composition further comprises an unlabeled anti-TNFα antibody.

76. The use according to claim 75, wherein the ADA is confirmed in the sample by measuring the level of the detection label which is a predetermined coefficient lower than the level of the detection label when the unlabeled anti-TNFα antibody is not present.

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