Antibody titer test

Abstract

We provide cell-based tests for measuring antibody titers. [Solution] A method for determining the activity of a polypeptide that binds to a target antigen and includes an Fc receptor-binding domain, comprising: a) contacting an immobilized target antigen with a polypeptide preparation to form an antigen-polypeptide complex; and b) contacting the antigen-polypeptide complex with a phagocytic cell, the phagocytic cell comprising an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor; the expression of the reporter indicates the activity of the polypeptide, the method for determining the activity of the polypeptide.

Claims

1. A method for determining the activity of a polypeptide that binds to a target antigen and contains an Fc receptor-binding domain, a) Contacting an immobilized target antigen with a polypeptide preparation to form an antigen-polypeptide complex. b) Contacting an antigen-polypeptide complex with a phagocytic cell, wherein the phagocytic cell contains an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor. Including; Reporter expression indicates polypeptide activity. A method for determining the activity of a polypeptide.

2. A method for quantifying the titer of a polypeptide preparation in which the polypeptide binds to a target antigen, a) Contacting multiple populations of immobilized target antigens with polypeptide preparations of different concentrations to form antigen-polypeptide complexes. b) Contacting these antigen-polypeptide complexes with phagocytic cells, wherein the phagocytic cells contain a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor. c) Measuring reporter expression, and d) EC of polypeptide preparations ５０ Determine the EC of the polypeptide preparation. ５０ And the EC of standard samples of polypeptides with known titers ５０ Including comparison with A method for quantifying the potency of polypeptide preparations.

3. Using multi-parameter logistic fit to standard samples, the EC of polypeptide preparations was determined. ５０ The method according to claim 2, further comprising calculating the titer based on the titer.

4. The method according to claim 3, wherein the multi-parameter logistic fit is a 3-parameter, 4-parameter, or 5-parameter logistic fit.

5. EC of standard samples ５０ However, the EC of polypeptide preparations ５０ The method according to any one of claims 2 to 4, which is determined at the same time.

6. The method according to any one of claims 1 to 5, wherein the reporter is luciferase or a fluorescent protein.

7. The method according to claim 6, wherein the luciferase is firefly luciferase, sea lice luciferase, or nanoluciferase.

8. The method according to any one of claims 1 to 7, wherein the response element that responds to activation by the Fcγ receptor is an NFκB response element, an NFAT response element, an AP-1 response element, or an ERK-responsive transcription factor.

9. The method according to any one of claims 1 to 8, wherein the phagocytic cell is a monocyte.

10. The method according to any one of claims 1 to 9, wherein the phagocytic cells are from a cell line.

11. The method according to claim 10, wherein the cell line is the THP-1 cell line or the U-937 cell line.

12. The method according to any one of claims 1 to 11, wherein the Fcγ receptor is FcγRI (CD64), FcγRIIa (CD32a), or FcγRIIII (CD16).

13. The method according to any one of claims 1 to 12, wherein phagocytes are manipulated to overexpress Fcγ receptors.

14. The method according to claim 13, wherein phagocytic cells are manipulated to overexpress FcγRIIa.

15. The method according to any one of claims 1 to 14, wherein phagocytic cells do not express FcγRIII.

16. The method according to any one of claims 1 to 15, wherein the target antigen is beta-amyloid (Aβ) or CD20.

17. The method according to claim 16, wherein the target antigen is beta-amyloid (Aβ).

18. The method according to claim 17, wherein Aβ is human Aβ.

19. The method according to claim 17 or 18, wherein Aβ comprises a monomer and / or oligomer of Aβ.

20. The method according to claim 17, wherein the human Aβ is Aβ1-40 or Aβ1-42.

21. The method according to any one of claims 1 to 20, wherein the polypeptide comprises a full-length Fc domain or an FcR-binding fragment of an Fc domain.

22. The method according to any one of claims 1 to 21, wherein the polypeptide specifically binds to Aβ.

23. The method according to any one of claims 1 to 22, wherein the polypeptide is an antibody or an immunoadhesin.

24. The method according to claim 22 or 23, wherein the polypeptide is crenezumab.

25. The method according to any one of claims 1 to 24, wherein the target antigen is immobilized on the surface.

26. The method according to claim 25, wherein the surface is a plate.

27. The method according to claim 26, wherein the plate is a multiwell plate.

28. The method according to any one of claims 25 to 27, wherein the antigen is immobilized on the surface at or near the N-terminus, at or near the C-terminus, or at or near the N-terminus and or near the C-terminus.

29. The method according to any one of claims 25 to 28, wherein the target antigen is immobilized on a surface using a biotin-streptavidin system.

30. The method according to claim 29, wherein the target antigen is bound to biotin, and the surface contains bound streptavidin.

31. The method according to claim 29 or 30, wherein the target antigen is bound to biotin at or near its N-terminus, at or near its C-terminus, or at or near its N-terminus and / or near its C-terminus.

32. The method according to any one of claims 1 to 31, wherein the reporter is detected after one or more of the following time periods have elapsed: approximately 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20, 24 hours, or more than 24 hours, following contact of the antigen-polypeptide complex with a phagocyte.

33. A kit comprising an immobilized target antigen and a phagocyte for determining the titer of a polypeptide preparation in which the polypeptide binds to a target antigen and contains an Fc receptor-binding domain, wherein the phagocyte comprises an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor, Reporter expression indicates polypeptide titer. kit.

34. A kit comprising an immobilized target antigen, phagocytic cells, and a standard sample for quantifying the titer of a polypeptide preparation in which the polypeptide binds to a target antigen and contains an Fc receptor-binding domain, Phagocytes contain an Fcγ receptor and a nucleic acid encoding a reporter operably linked to a response element that responds to activation by the Fcγ receptor, and the expression of the reporter indicates the titer of the polypeptide. The standard sample includes a preparation of a polypeptide with a known titer. kit.

35. The kit according to claim 33 or 34, wherein the reporter is luciferase or a fluorescent protein.

36. The kit according to claim 35, wherein the luciferase is firefly luciferase, sea lice luciferase, or nanoluciferase.

37. The kit according to any one of claims 33 to 36, further comprising a reagent for detecting the expression of a reporter.

38. The kit according to any one of claims 33 to 37, wherein the response element that responds to activation by the Fcγ receptor is an NFκB response element, an NFAT response element, an AP-1 response element, or an ERK-responsive transcription factor.

39. The kit according to any one of claims 33 to 38, wherein the phagocytic cells are derived from a cell line.

40. The kit according to claim 39, wherein the cell line is the THP-1 cell line or the U-937 cell line.

41. The kit according to any one of claims 33 to 40, wherein the Fcγ receptor is FcγRI (CD64), FcγRIIa (CD32a), or FcγRIIII (CD16).

42. The kit according to any one of claims 33 to 41, wherein phagocytic cells are engineered to overexpress Fcγ receptors.

43. The kit according to claim 42, wherein phagocytic cells are manipulated to overexpress FcγRIIa.

44. The kit according to any one of claims 33 to 43, wherein phagocytic cells do not express FcγRIII.

45. The kit according to any one of claims 33 to 44, wherein the target antigen is beta-amyloid (Aβ) or CD20.

46. The kit according to any one of claims 33 to 45, wherein the target antigen is beta-amyloid (Aβ).

47. The kit according to claim 46, wherein Aβ is human Aβ.

48. The kit according to claim 46 or 47, wherein Aβ comprises monomer and / or oligomeric Aβ.

49. The kit according to claim 48, wherein the human Aβ is Aβ1-40 or Aβ1-42.

50. The kit according to any one of claims 33 to 49, wherein the polypeptide comprises a full-length Fc domain or an FcR-binding fragment of an Fc domain.

51. The kit according to any one of claims 33 to 50, wherein the polypeptide specifically binds to Aβ.

52. The kit according to any one of claims 33 to 51, wherein the polypeptide is an antibody or an immunoadhesin.

53. The kit according to claim 52, wherein the polypeptide is crenezumab.

54. The kit according to any one of claims 33 to 53, wherein the target antigen is immobilized on the surface.

55. The kit according to claim 54, wherein the surface is a plate.

56. The kit according to claim 55, wherein the plate is a multiwell plate.

57. The kit according to claim 55 or 56, wherein the target antigen is bound to biotin at or near its N-terminus, at or near its C-terminus, or at or near both its N-terminus and C-terminus.

58. The kit according to any one of claims 54 to 57, wherein the target antigen is immobilized on the surface using a biotin-streptavidin system.

59. The kit according to claim 58, wherein the target antigen is bound to biotin, and the surface contains bound streptavidin.

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