Anti-ceruloplasmin antibodies and their use

Abstract

Provided herein are antibodies that bind to ceruloplasmin and are useful for a variety of applications, including the detection and immunocapture of ceruloplasmin in biological samples. The antibodies are useful in methods for measuring non-ceruloplasmin-bound copper and labile-bound copper concentrations in biological samples.

Claims

1. It binds to human ceruloplasmin (SEQ ID NO: 1), (a) Sequence IDs 25 and 26, respectively (b) Sequence IDs 49 and 50, respectively, and (c) Sequence IDs 73 and 74, respectively An isolated antibody comprising heavy chain variable region CDR1, CDR2, and CDR3 sequences and light chain variable region CDR1, CDR2, and CDR3 sequences of a heavy chain and light chain variable region pair selected from the group consisting of the above.

2. An isolated antibody that binds to human ceruloplasmin (SEQ ID NO: 1), (a) Heavy chain variable region CDR1, CDR2, and CDR3 sequences containing the amino acid sequences shown in SEQ ID NOs. 5, 6, and 7, respectively, and light chain variable region CDR1, CDR2, and CDR3 sequences containing the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively; (b) Heavy chain variable region CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences shown in SEQ ID NOs. 29, 30, and 31, respectively, and light chain variable region CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences shown in SEQ ID NOs. 32, 33, and 34, respectively; or (c) Heavy chain variable regions CDR1, CDR2, and CDR3 sequences containing the amino acid sequences shown in SEQ ID NOs. 53, 54, and 55, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences containing the amino acid sequences shown in SEQ ID NOs. 56, 57, and 58, respectively. An isolated antibody that contains and binds to human ceruloplasmin (SEQ ID NO: 1).

3. An isolated antibody that binds to human ceruloplasmin (SEQ ID NO: 1) and includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 49, and 73.

4. An isolated antibody that binds to human ceruloplasmin (SEQ ID NO: 1) and includes a heavy chain variable region and a light chain variable region, wherein the light chain variable region contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 50, and 74.

5. It binds to human ceruloplasmin (SEQ ID NO: 1), (a) Sequence IDs 25 and 26, respectively (b) Sequence IDs 49 and 50, respectively, and (c) Sequence IDs 73 and 74, respectively An isolated antibody comprising a heavy chain variable region and a light chain variable region containing an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of the above.

6. It binds to human ceruloplasmin (SEQ ID NO: 1), (a) Sequence IDs 25 and 26, respectively (b) Sequence IDs 49 and 50, respectively, and (c) Sequence IDs 73 and 74, respectively An isolated antibody comprising a heavy chain variable region and a light chain variable region containing an amino acid sequence selected from the group consisting of the following.

7. It binds to human ceruloplasmin (SEQ ID NO: 1), (a) Sequence IDs 27 and 28, respectively (b) Sequence IDs 51 and 52, respectively, and (c) Sequence IDs 75 and 76, respectively An isolated antibody comprising a heavy chain and a light chain containing an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of the following.

8. It binds to human ceruloplasmin (SEQ ID NO: 1), (a) Sequence IDs 27 and 28, respectively (b) Sequence IDs 51 and 52, respectively, and (c) Sequence IDs 75 and 76, respectively An isolated antibody comprising a heavy chain and a light chain containing an amino acid sequence selected from the group consisting of the following.

9. An isolated antibody that binds to human ceruloplasmin (SEQ ID NO: 1) and competes with the antibody described in any one of claims 1 to 8 for binding to ceruloplasmin.

10. An isolated antibody that binds to human ceruloplasmin (SEQ ID NO: 1) and to the same epitope on human ceruloplasmin as the antibody described in any one of claims 1 to 9.

11. The antibody according to any one of claims 1 to 10, which is a monoclonal antibody.

12. An antibody according to any one of claims 1 to 6 and 9 to 11, selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or variants thereof.

13. The antibody according to any one of claims 1 to 6 and 9 to 11, which is a rabbit antibody.

14. The antibody according to claim 13, which is an IgG antibody.

15. An antibody according to any one of claims 1 to 14, which binds to ceruloplasmin in a human biological sample.

16. The antibody according to any one of claims 1 to 15, wherein the human biological sample is human plasma or human serum.

17. The antibody according to any one of claims 1 to 16, wherein the plasma is human plasma containing lithium heparin.

18. An antibody according to any one of claims 1 to 17, which binds to purified human ceruloplasmin.

19. An immunoconjugate comprising an antibody according to any one of claims 1 to 18, linked to a drug.

20. The immunoconjugate according to claim 19, wherein the aforementioned drug is a detectable label.

21. A nucleic acid encoding a heavy chain variable region and / or a light chain variable region of an antibody according to any one of claims 1 to 18.

22. A nucleic acid containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 197 to 228.

23. An expression vector comprising the nucleic acid described in claim 21 or one or more nucleic acids described in claim 22.

24. Cells transformed with the expression vector described in claim 23.

25. An antibody mixture comprising two or three antibodies that bind to human ceruloplasmin (SEQ ID NO: 1), wherein the two or three antibodies are (a) an isolated antibody comprising heavy chain variable region CDR1, CDR2, and CDR3 sequences containing the amino acid sequences shown in SEQ ID NOs. 5, 6, and 7, respectively, and light chain variable region CDR1, CDR2, and CDR3 sequences containing the amino acid sequences shown in SEQ ID NOs. 8, 9, and 10, respectively, (b) an isolated antibody comprising heavy chain variable region CDR1, CDR2, and CDR3 sequences containing amino acid sequences shown in SEQ ID NOs. 29, 30, and 31, respectively, and light chain variable region CDR1, CDR2, and CDR3 sequences containing amino acid sequences shown in SEQ ID NOs. 32, 33, and 34, respectively, (c) an isolated antibody comprising heavy chain variable region CDR1, CDR2, and CDR3 sequences containing amino acid sequences shown in SEQ ID NOs. 53, 54, and 55, respectively, and light chain variable region CDR1, CDR2, and CDR3 sequences containing amino acid sequences shown in SEQ ID NOs. 56, 57, and 58, respectively, A mixture of antibodies selected from the group consisting of the following.

26. The two or three antibodies mentioned above, (a) Sequence IDs 25 and 26, respectively (b) Sequence IDs 49 and 50, respectively, and (c) Sequence IDs 73 and 74, respectively The antibody mixture according to claim 25, comprising a heavy chain variable region and a light chain variable region, each containing an amino acid sequence selected from the group consisting of the following.

27. The two or three antibodies mentioned above, (a) Sequence IDs 27 and 28, respectively (b) Sequence IDs 51 and 52, respectively, and (c) Sequence IDs 75 and 76, respectively The antibody mixture according to claim 25 or 26, comprising a heavy chain and a light chain having an amino acid sequence selected from the group consisting of the following.

28. An antibody mixture according to any one of claims 25 to 27, comprising two antibodies selected from subparts (a) to (c).

29. The antibody mixture according to claim 28, comprising two antibodies selected from the group consisting of subparts (a) and (b), (a) and (c), and (b) and (c).

30. The antibody mixture according to claim 28 or 29, wherein the two antibodies are present in a ratio of 2:1 in the order (a):(b), (a):(c), (b):(a), (b):(c), (c):(a), or (c):(b).

31. An antibody mixture according to any one of claims 25 to 30, comprising the antibodies of subparts (a), (b), and (c).

32. The antibody mixture according to claim 31, wherein the antibodies of subparts (a), (b), and (c) are present in a ratio of 2:1:1 in the order of (a):(b):(c), (a):(c):(b), (b):(a):(c), (b):(c):(a), (c):(a):(b), or (c):(b):(a).

33. The antibody or antibody mixture according to any one of claims 1 to 18 and 25 to 32, wherein one or more antibodies in the antibody mixture are immobilized on a solid support.

34. The antibody or antibody mixture according to any one of claims 1 to 18 and 25 to 32, wherein one or more antibodies in the antibody mixture are configured to be immobilized on a solid support after complex formation with ceruloplasmin.

35. The antibody or antibody mixture according to any one of claims 1 to 18 and 25 to 34, wherein one or more antibodies in the antibody mixture are immobilized on immunocapture beads.

36. The antibody or antibody mixture according to claim 35, wherein the immunocapture beads are streptavidin-coated beads, protein G beads, protein A beads, protein A / G beads, and tosyl-activated beads.

37. The antibody or antibody mixture according to claim 35 or claim 36, wherein the beads are magnetic immunocapture beads.

38. The antibody or antibody mixture according to any one of claims 25 to 37, wherein one or more antibodies in the antibody mixture are irreversibly linked to the immunocapture beads.

39. The antibody or antibody mixture according to claim 33 or 34, wherein the solid support comprises at least one of agarose resin, a chromatography plate, a streptavidin plate, and a titer plate.

40. A kit for measuring copper concentration in a biological sample, comprising an antibody or antibody mixture according to any one of claims 1 to 18 and 25 to 39, and instructions for use.

41. The kit according to claim 40, further comprising a chelating agent.

42. A method for measuring the concentration of non-ceruloplasmin-bound copper in a biological sample, (a) Contacting the biological sample with an immunocapsulation reagent containing an antibody or antibody mixture according to any one of claims 1 to 18 and 25 to 39 to form immunocapsulation ceruloplasmin, (b) Obtaining a non-ceruloplasmin sample by removing the immunocaptured ceruloplasmin, (c) Measuring the copper concentration in the non-ceruloplasmin sample, A method for measuring the concentration of non-ceruloplasmin-bound copper in a biological sample, including [specific component].

43. The method according to claim 42, wherein the copper concentration in the non-ceruloplasmin sample is measured using inductively coupled plasma mass spectrometry (ICP-MS).

44. A method for measuring the concentration of unstable copper in a biological sample, (a) Contacting the biological sample with an immunocapsulation reagent containing an antibody or antibody mixture according to any one of claims 1 to 18 and 25 to 39 to form immunocapsulation ceruloplasmin, (b) Removing the immunocaptured ceruloplasmin to obtain a non-ceruloplasmin sample, (c) Contacting the non-ceruloplasmin sample with a chelating agent that binds to unstable copper bonds, (d) Remove the non-unstable bonded copper to obtain an unstable bonded copper sample, (e) Measuring the copper concentration in the unstable copper sample, A method for measuring the concentration of unstable bound copper in a biological sample, including [specific component].

45. The method according to claim 44, wherein the copper concentration in the unstable-coupled copper sample is measured using inductively coupled plasma mass spectrometry (ICP-MS).

46. The method according to claim 45, further comprising introducing an internal standard into the unstable bonded copper sample before measuring the copper concentration.

47. The method according to claim 46, wherein the internal standard includes at least one of copper and rhodium.

48. The method according to any one of claims 44 to 47, wherein the chelating agent is selected from the group consisting of penicillamine, trientine hydrochloride, trientine tetrahydrochloride, and EDTA.

49. The method according to claim 48, wherein the chelating agent comprises EDTA.

50. The method according to any one of claims 44 to 49, further comprising the removal of the non-unstable bonded copper to obtain a non-unstable bonded copper sample.

51. The method according to claim 50, further comprising measuring the copper concentration in the non-unstable bonded copper sample.

52. The method according to any one of claims 50 or 51, wherein the non-unstable bonded copper sample contains molybdenum.

53. The method according to claim 52, further comprising measuring the molybdenum concentration in the non-unstable bonded copper sample.

54. The method according to any one of claims 42 to 53, further comprising removing the immunocaptured ceruloplasmin to obtain an immunocaptured ceruloplasmin sample.

55. The method according to claim 54, further comprising measuring the ceruloplasmin concentration in the immunocapsulated ceruloplasmin sample.

56. The method according to claim 55, wherein the ceruloplasmin concentration is measured using mass spectrometry.

57. The method according to claim 56, wherein the mass spectrometry has an analyte detection limit of at least 5 μg / mL.

58. The method according to claim 56 or 57, wherein the mass spectrometry includes liquid chromatography-mass spectrometry (LC-MS).

59. The method according to any one of claims 55 to 58, wherein the ceruloplasmin concentration in the biological sample is less than 200 μg / mL.

60. The method according to any one of claims 55 to 59, further comprising measuring the copper concentration in the immunocaptured ceruloplasmin sample.

61. The method according to claim 60, wherein the copper concentration in the immunocapsulated ceruloplasmin sample is measured using inductively coupled plasma mass spectrometry.

62. The method according to any one of claims 42 to 61, wherein the biological sample is human plasma or human serum.

63. The method according to any one of claims 42 to 62, wherein the biological sample is derived from a patient who has or is suspected to have a copper metabolism-related disease or disorder.

64. The method according to claim 63, wherein the copper metabolism-related disease or disorder is selected from the group consisting of Wilson's disease, copper toxicity, copper deficiency, Menkes disease, and aceruloplasminemia.

65. A composition for use in a method for treating a patient diagnosed with a copper metabolism-related disease or disorder according to the method of claim 64, wherein the composition comprises a therapeutic agent, and the method comprises administering to the patient an effective amount of the therapeutic agent to treat the copper metabolism-related disease or disorder.

66. The composition according to claim 65, wherein the therapeutic agent is selected from the group consisting of bis-cholinetetrathiomolybdate, zinc, trientine hydrochloride, trientine tetrahydrochloride, and penicillamine.

67. The composition according to claim 65, wherein the therapeutic agent is bis-cholinetetrathiomolybdate.

68. The composition according to claim 67, wherein the effective amount of bis-cholinetetrathiomolybdate is in the range of 15 mg to 60 mg per day.

69. The composition according to claim 67 or 68, wherein the effective amount of bis-cholinetetrathiomolybdate is 15 mg per day.

70. The composition according to claim 67, wherein the effective dose of bis-cholinetetrathiomolybdate is 15 mg every other day.

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