Anti-drug antibody assay

Abstract

The present invention relates to methods for detecting anti-drug antibodies and kits for using such assays.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 373,426, filed August 24, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith is incorporated by reference in its entirety and identified as follows: 280,159 byte XML file entitled "10200-US01-PRI_Sequence_listing", created August 24, 2022.

[0003] The present invention relates to methods for detecting anti-drug antibodies and kits for using such assays. [Background technology]

[0004] Assays for detecting anti-drug antibodies (ADAs) facilitate understanding of potential immune responses to biological drug candidates. Determining the presence of ADAs and assessing their clinical significance is a necessary part of any macromolecule development program.

[0005] All biopharmaceuticals are potentially immunogenic and can induce ADA. The clinical effects of ADA formation are highly variable and can lead to serious adverse events that put patients at risk. Therefore, developing and validating ADA assays with appropriate sensitivity, specificity, and selectivity for detection is critical, both to guide drug development decisions and as a regulatory requirement. Because ADA responses can affect the pharmacokinetics (PK), pharmacodynamics (PD), safety, and efficacy of therapeutic candidates, ADA assays should be designed to detect antibodies that may mediate hypersensitivity responses or have the ability to interfere with the interaction between the therapeutic and its target for a neutralizing effect.

[0006] In 2019, the FDA issued a guidance for industry entitled "Immunogenicity Testing of Therapeutic Protein Products - Developing and Validating Assays for Anti-Drug Antibody Detection," which provides recommendations to facilitate the development and validation of assays for immunogenicity assessment of therapeutic protein products during clinical trials in the industry. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Immunogenicity Testing of Therapeutic Protein Products - Developing and Validating Assays for Anti-Drug Antibody Detection Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need to develop ADA assays that address accuracy, target interference, and meet the 2019 FDA guidance. [Means for solving the problem]

[0009] Given the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, which equivalents are intended to be encompassed by embodiment (E) below.

[0010] E1. A method for detecting anti-drug antibodies (ADA) in a sample, wherein the ADA binds to denosumab, the method comprising: (a) a first antibody or antibody conjugate that binds to RANKL, the first antibody or antibody conjugate comprising a heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208, which can be bound to a solid substrate; (b) (i) a second antibody that binds to RANKL, the second antibody comprising an amino acid sequence that differs from the first antibody by at least one amino acid in one of six CDR sequences, or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; and (c) a third antibody that binds to the ADA and that can be linked to a detectable marker. The method of claim 1, further comprising mixing the

[0011] E2. The method of E1, wherein said sample contains soluble RANKL that interferes with the detection of said ADA.

[0012] E3. A method for reducing false signals in an anti-drug antibody (ADA) assay, wherein the ADA assay is for detecting the presence of anti-denosumab antibodies in a sample, the method comprising: (a) a first antibody or antibody conjugate that binds to RANKL, the first antibody or antibody conjugate comprising a heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208, which can be bound to a solid substrate; (b) (i) a second antibody that binds to RANKL, the second antibody comprising an amino acid sequence that differs from the first antibody by at least one amino acid in one of six CDR sequences, or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; and (c) a third antibody that binds to the ADA and that can be linked to a detectable marker. The method of claim 1, further comprising mixing the

[0013] E4. The method of E3, wherein the false signal is caused by the presence of soluble RANKL, which interferes with the detection of the ADA.

[0014] E5. The method of any one of E1-E4, wherein said first antibody conjugate comprises an antibody molecule covalently linked to an immobilization moiety.

[0015] E6. The method of any one of E1-E4, wherein said first antibody conjugate comprises an antibody molecule non-covalently linked to an immobilization moiety.

[0016] E7. The method of E5 or E6, wherein the solid substrate comprises a binding partner, and the immobilized moiety can be attached to the solid substrate by binding to the binding partner.

[0017] E8. The method of E7, wherein the immobilization moiety is biotin and the binding partner is streptavidin or avidin, or the immobilization moiety is avidin or streptavidin and the binding partner is biotin.

[0018] E9. The method of E7, wherein the immobilization moiety and the binding partner are one of the following binding pairs: an antigen and an antibody that binds to the antigen, a lectin and a polysaccharide, a steroid and a steroid-binding protein, a hormone and a hormone receptor, an enzyme and a substrate for the enzyme, IgG and Protein A, IgG and Protein G, or a ligand and a receptor for the ligand.

[0019] E10. The method of any one of E1-E4, wherein the first antibody is immobilized on a solid substrate comprising Protein A or Protein G.

[0020] E11. The method of any one of E1 to E10, wherein the first antibody or antibody conjugate comprises CDR-H1 comprising SEQ ID NO: 200, CDR-H2 comprising SEQ ID NO: 201, and CDR-H3 comprising SEQ ID NO: 202, and CDR-L1 comprising SEQ ID NO: 205, CDR-L2 comprising SEQ ID NO: 206, and CDR-L3 comprising SEQ ID NO: 207.

[0021] E12. The method of any one of E1-E11, wherein the first antibody or antibody conjugate comprises a heavy chain variable region (VH) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203.

[0022] E13. The method of any one of E1-E12, wherein the first antibody or antibody conjugate comprises a light chain variable region (VL) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 208.

[0023] E14. The method of any one of E1-E13, wherein the first antibody or antibody conjugate comprises a human CH1 domain, such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1.

[0024] E15. The method of E14, wherein said human IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 182.

[0025] E16. The human IgG2 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 194; or the human IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 190; or The method of E14, wherein the human IgG4 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 186.

[0026] E17. The method of any one of E1 to E16, wherein the first antibody or antibody conjugate comprises a human Fc domain, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0027] E18. The method of E17, wherein said human IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 184.

[0028] E19. The human IgG2 Fc comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, or the human IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 192; or The method of E17, wherein the human IgG4 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 188.

[0029] E20. The method of any one of E1 to E13, wherein said first antibody or antibody conjugate comprises a mouse CH1 domain, such as mouse IgG1 CH1, mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1.

[0030] E21. The method of E20, wherein said murine IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 172.

[0031] E22. The method of E20, wherein said murine IgG2a CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 175.

[0032] E23. The method of E20, wherein said murine IgG2b CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 176.

[0033] E24. The method of E20, wherein said murine IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 177.

[0034] E25. The method according to any one of E1 to E13 and E20 to E24, wherein the first antibody or antibody conjugate comprises a mouse Fc domain, such as mouse IgG1 Fc, mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc.

[0035] E26. The method of E25, wherein said murine IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 174.

[0036] E27. The method of E25, wherein said murine IgG2a Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 175.

[0037] E28. The method of E25, wherein said murine IgG2b Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 176.

[0038] E29. The method of E25, wherein said murine IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 177.

[0039] E30. The method of any one of E1-E29, wherein said first antibody or antibody conjugate comprises a human CL domain, such as human kappa CL or human lambda CL.

[0040] E31. The method of E30, wherein said human kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 178 or 179.

[0041] E32. The method of E30, wherein said human lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 180 or 181.

[0042] E33. The method of any one of E1 to E29, wherein said first antibody or antibody conjugate comprises a mouse CL domain, such as mouse kappa CL or mouse lambda CL.

[0043] E34. The method of E33, wherein said mouse kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 171.

[0044] E35. The method of E33, wherein said mouse lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 169 or 170.

[0045] E36. The method of any one of E1-E35, wherein said third antibody is covalently linked to a detectable marker.

[0046] E37. The method of any one of E1-E35, wherein said third antibody is non-covalently linked to a detectable marker.

[0047] E38. The method of any one of E1-E37, wherein the detectable marker is an isotope, an enzyme, a fluorescent moiety, a luminescent moiety, a chromogenic moiety, a metal, or an electrical charge.

[0048] E39. The method of any one of E1-E38, wherein the third antibody comprises six CDR sequences identical to those of the first antibody.

[0049] E40. The third antibody (i) a heavy chain (HC) CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 208, or (ii) CDR-H1 comprising SEQ ID NO: 200, CDR-H2 comprising SEQ ID NO: 201, and CDR-H3 comprising SEQ ID NO: 202, and CDR-L1 comprising SEQ ID NO: 205, CDR-L2 comprising SEQ ID NO: 206, and CDR-L3 comprising SEQ ID NO: 207 The method according to any one of E1 to E39, comprising:

[0050] E41. The method of any one of E1 to E40, wherein the third antibody comprises a heavy chain variable region (VH) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203.

[0051] E42. The method of any one of E1-E41, wherein the third antibody comprises a light chain variable region (VL) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 208.

[0052] E43. The method of any one of E1 to E42, wherein the third antibody comprises a human CH1 domain, such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1.

[0053] E44. The method of E43, wherein said human IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 182.

[0054] E45. The human IgG2 CH1 comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 194, or the human IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 190; or The method of E43, wherein the human IgG4 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 186.

[0055] E46. The method of any one of E1 to E45, wherein the third antibody comprises a human Fc domain, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0056] E47. The method of E46, wherein said human IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 184.

[0057] E48. The human IgG2 Fc comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, or the human IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 192; or The method of E46, wherein the human IgG4 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 188.

[0058] E49. The method of any one of E1 to E42, wherein the third antibody comprises a mouse CH1 domain, such as mouse IgG1 CH1, mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1.

[0059] E50. The method of E49, wherein said murine IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 172.

[0060] E51. The method of E49, wherein said murine IgG2a CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 175.

[0061] E52. The method of E49, wherein said murine IgG2b CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 176.

[0062] E53. The method of E49, wherein said murine IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 177.

[0063] E54. The method of any one of E1 to E42 and E49 to E53, wherein the third antibody comprises a mouse Fc domain, such as mouse IgG1 Fc, mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc.

[0064] E55. The method of E54, wherein said murine IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 174.

[0065] E56. The method of E54, wherein said murine IgG2a Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 175.

[0066] E57. The method of E54, wherein said murine IgG2b Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 176.

[0067] E58. The method of E54, wherein said murine IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 177.

[0068] E59. The method of any one of E1-E58, wherein the third antibody comprises a human CL domain, such as human kappa CL or human lambda CL.

[0069] E60. The method of E59, wherein said human kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 178 or 179.

[0070] E61. The method of E59, wherein said human lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 180 or 181.

[0071] E62. The method of any one of E1 to E58, wherein the third antibody comprises a mouse CL domain, such as mouse kappa CL or mouse lambda CL.

[0072] E63. The method of E62, wherein said mouse kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 171.

[0073] E64. The method of E62, wherein said mouse lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 169 or 170.

[0074] E65. The method of any one of E1 to E64, wherein the third antibody comprises a VH domain comprising the same sequence as the VH of the first antibody and a VL domain comprising the same sequence as the VL domain of the first antibody.

[0075] E66. The method of any one of E1 to E65, wherein the third antibody comprises a heavy chain (HC) comprising the same sequence as the HC of the first antibody and a light chain (LC) comprising the same sequence as the LC of the first antibody.

[0076] E67. The method of any one of E1-E66, wherein the detectable marker comprises a secondary antibody that binds to the constant domain of the third antibody.

[0077] E68. The method of any one of E1-E67, wherein the detectable marker comprises a secondary antibody that binds to the Fc domain of the third antibody.

[0078] E69. The method of any one of E1-E66, wherein said third antibody is covalently linked to a fluorescent, luminescent, or chromogenic moiety.

[0079] E70. The method of any one of E1-E69, wherein said second antibody binds to the same epitope as denosumab.

[0080] E71. The method of any one of E1-E70, wherein said second antibody competes with denosumab for RANKL binding.

[0081] E72. The method of any one of E1-E71, wherein the second antibody comprises an amino acid sequence that differs from the first antibody by at least one amino acid in each of six CDR sequences.

[0082] E73. The method of any one of E1 to E72, wherein the second antibody comprises an amino acid sequence that differs from the first antibody by at least 2, at least 3, at least 4, at least 5, or at least 6 amino acids in the CDR sequences.

[0083] E74. The method of any one of E1 to E73, wherein the ADA does not bind to the second antibody.

[0084] E75. The method of any one of E1 to E74, wherein the second antibody comprises (i) a heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167, and (ii) a light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

[0085] E76. The second antibody (i) a CDR-H1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115 or 121; (ii) a CDR-H2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116 or 122; (iii) a CDR-H3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, or 123; (iv) a CDR-L1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118 or 124; (v) a CDR-L2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119 or 125; (vi) a CDR-L3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, or 126 The method of any one of E1 to E75, comprising:

[0086] E77. The second antibody (1) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 1 to 6; (2) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 6 to 12; (3) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 13 to 18; (4) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 19 to 24; (5) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 25 to 30; (6) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 31 to 36; (7) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 37 to 42; (8) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 43 to 48; (9) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 49 to 54; (10) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 55 to 60; (11) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 61 to 66; (12) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 67 to 72; (13) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 73 to 78; (14) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 79 to 84; (15) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 85 to 90; (16) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 91 to 96; (17) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 97 to 102; (18) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 103 to 108; (19) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 109 to 114; (20) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 115 to 120; (21) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each containing SEQ ID NOs: 121 to 126 The method of any one of E1 to E76, comprising:

[0087] E78. The method according to E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 1 to 6.

[0088] E79. The method according to E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 6 to 12.

[0089] E80. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 13 to 18.

[0090] E81. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 19 to 24.

[0091] E82. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 25 to 30.

[0092] E83. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 31 to 36.

[0093] E84. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 37 to 42.

[0094] E85. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 43 to 48.

[0095] E86. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 49 to 54.

[0096] E87. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 55 to 60.

[0097] E88. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 61 to 66.

[0098] E89. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 67 to 72.

[0099] E90. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 73 to 78.

[0100] E91. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 79 to 84.

[0101] E92. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 85 to 90.

[0102] E93. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 91 to 96.

[0103] E94. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 97 to 102.

[0104] E95. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 103 to 108.

[0105] E96. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 109 to 114.

[0106] E97. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 115 to 120.

[0107] E98. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 121 to 126.

[0108] E99. The method of any one of E1 to E77, wherein the second antibody comprises a heavy chain variable region (VH) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167.

[0109] E100. The method of any one of E1 to E78, wherein the second antibody comprises a light chain variable region (VL) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

[0110] E101. The second antibody (1) A VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 127, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 128; (2) a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 129, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 130; (3) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 131, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 132; (4) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 133, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 134; (5) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 135, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 136; (6) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 137, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 138; (7) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 139, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 140; (8) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 141, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 142; (9) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 143, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 144; (10) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 145, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 146; (11) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 147, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 148; (12) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 149, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 150; (13) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 151, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 152; (14) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 153, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154; (15) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 155, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 156; (16) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 157, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 158; (17) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 159, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 160; (18) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 161, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 162; (19) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 163, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 164; (20) A VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 165, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 166; or (21) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 167, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 168. The method according to any one of E1 to E100, comprising:

[0111] E102. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 127, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 128.

[0112] E103. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 129, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 130.

[0113] E104. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 131, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 132.

[0114] E105. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 133, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 134.

[0115] E106. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 135, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 136.

[0116] E107. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 137, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 138.

[0117] E108. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 139, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 140.

[0118] E109. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 141, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 142.

[0119] E110. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 143, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 144.

[0120] E111. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 145, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 146.

[0121] E112. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 147, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 148.

[0122] E113. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 149, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 150.

[0123] E114. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 151, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 152.

[0124] E115. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 153, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154.

[0125] E116. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 155, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 156.

[0126] E117. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 157, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 158.

[0127] E118. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 159, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 160.

[0128] E119. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 161, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 162.

[0129] E120. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 163, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 164.

[0130] E121. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 165, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 166.

[0131] E122. The method of E101, wherein said second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 167, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 168.

[0132] E123. The method of any one of E1 to E122, wherein the second antibody comprises a human CH1 domain, such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1.

[0133] E124. The method of E123, wherein said human IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 182.

[0134] E125. The human IgG2 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 194, or the human IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 190; or The method of E123, wherein the human IgG4 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 186.

[0135] E126. The method of any one of E1 to E125, wherein the second antibody comprises a human Fc domain, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0136] E127. The method of E126, wherein said human IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 184.

[0137] E128. The human IgG2 Fc comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, or the human IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 192; or The method of E126, wherein the human IgG4 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 188.

[0138] E129. The method of any one of E1 to E122, wherein the second antibody comprises a mouse CH1 domain, such as mouse IgG1 CH1, mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1.

[0139] E130. The method of E129, wherein said murine IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 172.

[0140] E131. The method of E129, wherein said murine IgG2a CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CH1 domain of SEQ ID NO: 175.

[0141] E132. The method of E129, wherein said murine IgG2b CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CH1 domain of SEQ ID NO: 176.

[0142] E133. The method of E129, wherein said murine IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 177.

[0143] E134. The method of any one of E1 to E122 and E129 to E133, wherein the second antibody comprises a mouse Fc domain, such as mouse IgG1 Fc, mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc.

[0144] E135. The method of E134, wherein said murine IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 174.

[0145] E136. The method of E134, wherein said murine IgG2a Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 175.

[0146] E137. The method of E134, wherein said murine IgG2b Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 176.

[0147] E138. The method of E134, wherein said murine IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 177.

[0148] E139. The method of any one of E1 to E138, wherein the second antibody comprises a human CL domain, such as human kappa CL or human lambda CL.

[0149] E140. The method of E139, wherein said human kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 178 or 179.

[0150] E141. The method of E139, wherein said human lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 180 or 181.

[0151] E142. The method of any one of E1 to E138, wherein said second antibody comprises a mouse CL domain, such as mouse kappa CL or mouse lambda CL.

[0152] E143. The method of E142, wherein said mouse kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 171.

[0153] E144. The method of E142, wherein said mouse lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 169 or 170.

[0154] E145. The second antibody is 10 -4 M or less, 10 -5 M or less, 10-6 M or less, 10 -7 M or less, 10 -8 M or less or 10 -9 The method according to any one of E1 to E144, wherein the compound binds to RANKL with a KD value of M or less.

[0155] E146. The method of any one of E1 to E145, wherein the sample is pretreated with the second antibody when mixed with the first antibody or antibody conjugate and the third antibody.

[0156] E147. The method of any one of E1 to E145, wherein the sample, when mixed with the first antibody or antibody conjugate and the third antibody, is pretreated with the OPG.

[0157] E148. The method according to any one of E1 to E145, wherein the sample is simultaneously mixed with the first antibody or antibody conjugate, the second antibody, and the third antibody.

[0158] E149. The method of any one of E1 to E145, wherein the sample is simultaneously mixed with the first antibody or antibody conjugate, the OPG, and the third antibody complex.

[0159] E150. The method of any one of E1 to E149, wherein said RANKL is human RANKL.

[0160] E151. The method of any one of E1 to E150, wherein both the first antibody and the second antibody bind to a human RANKL epitope comprising SEQ ID NO: 214 (DLATE).

[0161] E152. The method of any one of E1-E150, wherein both the first antibody and the second antibody bind to a human RANKL epitope comprising one or more residues from SEQ ID NO:215.

[0162] E153. A diagnostic kit for detecting anti-drug antibodies (ADA) in a sample, wherein the ADA binds to denosumab, the diagnostic kit comprising: (a) a first antibody or antibody conjugate that binds to RANKL, the first antibody or antibody conjugate comprising a heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208, which can be bound to a solid substrate; (b) (i) a second antibody that binds to RANKL, wherein the second antibody comprises an amino acid sequence that differs from the first antibody by at least one amino acid in one of six CDR sequences; or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; (c) a third antibody that binds to the ADA and that can be linked to a detectable marker; and (d) Instructions for use A diagnostic kit comprising:

[0163] E154. The diagnostic kit of E153, wherein said first antibody conjugate comprises an antibody molecule covalently linked to an immobilization moiety.

[0164] E155. The diagnostic kit of E153 or E154, wherein said first antibody conjugate comprises an antibody molecule non-covalently linked to an immobilization moiety.

[0165] E156. The diagnostic kit of E154 or E155, wherein said solid substrate comprises a binding partner, and said immobilized moiety can be attached to the solid substrate by binding to said binding partner.

[0166] E157. The diagnostic kit according to E156, wherein the immobilization moiety is biotin and the binding partner is streptavidin or avidin, or the immobilization moiety is avidin or streptavidin and the binding partner is biotin.

[0167] E158. The diagnostic kit according to E156, wherein the immobilization moiety and the binding partner are one of the following binding pairs: an antigen and an antibody that binds to the antigen, a lectin and a polysaccharide, a steroid and a steroid-binding protein, a hormone and a hormone receptor, an enzyme and a substrate for the enzyme, IgG and Protein A, IgG and Protein G, or a ligand and a receptor for the ligand.

[0168] E159. The diagnostic kit of E153, wherein said first antibody is immobilized on a solid substrate comprising Protein A or Protein G.

[0169] E160. The diagnostic kit of any one of E153 to E159, wherein the first antibody or antibody conjugate comprises CDR-H1 comprising SEQ ID NO: 200, CDR-H2 comprising SEQ ID NO: 201, and CDR-H3 comprising SEQ ID NO: 202, and CDR-L1 comprising SEQ ID NO: 205, CDR-L2 comprising SEQ ID NO: 206, and CDR-L3 comprising SEQ ID NO: 207.

[0170] E161. The diagnostic kit of any one of E153 to E160, wherein the first antibody or antibody conjugate comprises a heavy chain variable region (VH) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 203.

[0171] E162. The diagnostic kit of any one of E153 to E161, wherein the first antibody or antibody conjugate comprises a light chain variable region (VL) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 208.

[0172] E163. The diagnostic kit of any one of E153 to E162, wherein said first antibody or antibody conjugate comprises a human CH1 domain, such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1.

[0173] E164. The diagnostic kit of E163, wherein said human IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 182.

[0174] E165. The human IgG2 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 194, or the human IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 190; or The diagnostic kit of E163, wherein the human IgG4 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 186.

[0175] E166. The diagnostic kit of any one of E153 to E165, wherein the first antibody or antibody conjugate comprises a human Fc domain, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0176] E167. The diagnostic kit of E166, wherein said human IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 184.

[0177] E168. The human IgG2 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, or the human IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 192; or The diagnostic kit of E166, wherein the human IgG4 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 188.

[0178] E169. The diagnostic kit of any one of E153 to E162, wherein said first antibody or antibody conjugate comprises a mouse CH1 domain, such as mouse IgG1 CH1, mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1.

[0179] E170. The diagnostic kit of E169, wherein said murine IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 172.

[0180] E171. The diagnostic kit of E169, wherein said murine IgG2a CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CH1 domain of SEQ ID NO: 175.

[0181] E172. The diagnostic kit of E169, wherein the murine IgG2b CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CH1 domain of SEQ ID NO: 176.

[0182] E173. The diagnostic kit of E169, wherein said murine IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CH1 domain of SEQ ID NO: 177.

[0183] E174. The diagnostic kit of any one of E153 to E162 and E169 to E173, wherein the first antibody or antibody conjugate comprises a mouse Fc domain, such as mouse IgG1 Fc, mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc.

[0184] E175. The diagnostic kit of E174, wherein the murine IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 174.

[0185] E176. The diagnostic kit of E174, wherein the murine IgG2a Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the Fc domain of SEQ ID NO: 175.

[0186] E177. The diagnostic kit of E174, wherein the murine IgG2b Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the Fc domain of SEQ ID NO: 176.

[0187] E178. The diagnostic kit of E174, wherein the murine IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the Fc domain of SEQ ID NO: 177.

[0188] E179. The diagnostic kit of any one of E153 to E178, wherein said first antibody or antibody conjugate comprises a human CL domain, such as human kappa CL or human lambda CL.

[0189] E180. The diagnostic kit of E179, wherein said human kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 178 or 179.

[0190] E181. The diagnostic kit of E179, wherein the human lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 180 or 181.

[0191] E182. The diagnostic kit of any one of E153 to E178, wherein said first antibody or antibody conjugate comprises a mouse CL domain, such as mouse kappa CL or mouse lambda CL.

[0192] E183. The diagnostic kit of E182, wherein said mouse kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 171.

[0193] E184. The diagnostic kit of E182, wherein the mouse lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 169 or 170.

[0194] E185. The diagnostic kit of any one of E153 to E184, wherein said third antibody is covalently linked to a detectable marker.

[0195] E186. The diagnostic kit of any one of E153 to E184, wherein said third antibody is non-covalently linked to a detectable marker.

[0196] E187. The diagnostic kit of any one of E153 to E186, wherein said detectable marker is an isotope, an enzyme, a fluorescent moiety, a luminescent moiety, a chromogenic moiety, a metal, or an electrical charge.

[0197] E188. The diagnostic kit according to any one of E153 to E187, wherein the third antibody comprises six CDR sequences identical to those of the first antibody.

[0198] E189. The third antibody is (i) a heavy chain (HC) CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 208, or (ii) CDR-H1 comprising SEQ ID NO: 200, CDR-H2 comprising SEQ ID NO: 201, and CDR-H3 comprising SEQ ID NO: 202, and CDR-L1 comprising SEQ ID NO: 205, CDR-L2 comprising SEQ ID NO: 206, and CDR-L3 comprising SEQ ID NO: 207 The diagnostic kit according to any one of E153 to E188, comprising:

[0199] E190. The diagnostic kit of any one of E153 to E189, wherein the third antibody comprises a heavy chain variable region (VH) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 203.

[0200] E191. The diagnostic kit of any one of E153 to E190, wherein the third antibody comprises a light chain variable region (VL) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 208.

[0201] E192. The diagnostic kit according to any one of E153 to E191, wherein the third antibody comprises a human CH1 domain, such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1.

[0202] E193. The diagnostic kit of E192, wherein said human IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 182.

[0203] E194. The human IgG2 CH1 comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 194, or the human IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 190; or The diagnostic kit of E192, wherein the human IgG4 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 186.

[0204] E195. The diagnostic kit according to any one of E192 to E194, wherein the third antibody comprises a human Fc domain, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0205] E196. The diagnostic kit of E195, wherein said human IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 184.

[0206] E197. The human IgG2 Fc comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, or the human IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 192; or The diagnostic kit of E195, wherein the human IgG4 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 188.

[0207] E198. The diagnostic kit according to any one of E153 to E191, wherein the third antibody comprises a mouse CH1 domain, such as mouse IgG1 CH1, mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1.

[0208] E199. The diagnostic kit of E198, wherein said murine IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 172.

[0209] E200. The diagnostic kit of E198, wherein said murine IgG2a CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 175.

[0210] E201. The diagnostic kit of E198, wherein said murine IgG2b CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CH1 domain of SEQ ID NO: 176.

[0211] E202. The diagnostic kit of E198, wherein said murine IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the CH1 domain of SEQ ID NO: 177.

[0212] E203. The diagnostic kit according to any one of E153 to E191 and E198 to E202, wherein the third antibody comprises a mouse Fc domain, such as mouse IgG1 Fc, mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc.

[0213] E204. The diagnostic kit of E203, wherein said murine IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 174.

[0214] E205. The diagnostic kit of E203, wherein the murine IgG2a Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 175.

[0215] E206. The diagnostic kit of E203, wherein the murine IgG2b Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 176.

[0216] E207. The diagnostic kit of E203, wherein the murine IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 177.

[0217] E208. The diagnostic kit of any one of E153 to E207, wherein said third antibody comprises a human CL domain, such as human kappa CL or human lambda CL.

[0218] E209. The diagnostic kit of E208, wherein said human kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 178 or 179.

[0219] E210. The diagnostic kit of E208, wherein said human lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 180 or 181.

[0220] E211. The diagnostic kit of any one of E153 to E207, wherein the third antibody comprises a mouse CL domain, such as mouse kappa CL or mouse lambda CL.

[0221] E212. The diagnostic kit of E211, wherein said mouse kappa CL comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 171.

[0222] E213. The diagnostic kit of E211, wherein said mouse lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 169 or 170.

[0223] E214. The diagnostic kit according to any one of E153 to E213, wherein the third antibody comprises a VH domain comprising the same sequence as the VH of the first antibody and a VL domain comprising the same sequence as the VL domain of the first antibody.

[0224] E215. The diagnostic kit according to any one of E153 to E214, wherein the third antibody comprises a heavy chain (HC) comprising the same sequence as the HC of the first antibody and a light chain (LC) comprising the same sequence as the LC of the first antibody.

[0225] E216. The diagnostic kit of any one of E153 to E215, wherein the detectable marker comprises a secondary antibody that binds to the constant domain of the third antibody.

[0226] E217. The diagnostic kit of any one of E153 to E216, wherein the detectable marker comprises a secondary antibody that binds to the Fc domain of the third antibody.

[0227] E218. The diagnostic kit of any one of E153 to E215, wherein said third antibody is covalently linked to a fluorescent, luminescent, or chromogenic moiety.

[0228] E219. The diagnostic kit according to any one of E153 to E218, wherein the second antibody binds to the same epitope as denosumab.

[0229] E220. The diagnostic kit of any one of E153 to E219, wherein said second antibody competes with denosumab for RANKL binding.

[0230] E221. The diagnostic kit according to any one of E153 to E220, wherein the second antibody comprises an amino acid sequence that differs from that of the first antibody by at least one amino acid in each of six CDR sequences.

[0231] E222. The diagnostic kit according to any one of E153 to E221, wherein the second antibody comprises an amino acid sequence that differs from that of the first antibody by at least 2, at least 3, at least 4, at least 5, or at least 6 amino acids in the CDR sequence.

[0232] E223. The diagnostic kit according to any one of E153 to E222, wherein the ADA does not bind to the second antibody.

[0233] E224. The diagnostic kit of any one of E153 to E223, wherein the second antibody comprises (i) heavy chain CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167, and (ii) light chain CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

[0234] E225. The second antibody (i) a CDR-H1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109, 115 or 121; (ii) a CDR-H2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116 or 122; (iii) a CDR-H3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117, or 123; (iv) a CDR-L1 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118 or 124; (v) a CDR-L2 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119 or 125; (vi) a CDR-L3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, or 126 The diagnostic kit according to any one of E153 to E224, comprising:

[0235] E226. The second antibody (1) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 1 to 6; (2) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 6 to 12; (3) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 13 to 18; (4) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 19 to 24; (5) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 25 to 30; (6) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 31 to 36; (7) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 37 to 42; (8) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 43 to 48; (9) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 49 to 54; (10) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 55 to 60; (11) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 61 to 66; (12) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 67 to 72; (13) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 73 to 78; (14) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 79 to 84; (15) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 85 to 90; (16) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 91 to 96; (17) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 97 to 102; (18) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 103 to 108; (19) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 109 to 114; (20) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each containing SEQ ID NOs: 115 to 120; (21) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each containing SEQ ID NOs: 121 to 126. The diagnostic kit according to any one of E153 to E225, comprising:

[0236] E227. The diagnostic kit according to E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 1 to 6.

[0237] E228. The diagnostic kit according to E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 6 to 12.

[0238] E229. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 13 to 18.

[0239] E230. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 19 to 24.

[0240] E231. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each comprising SEQ ID NOs: 25 to 30.

[0241] E232. The diagnostic kit according to E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 31 to 36.

[0242] E233. The diagnostic kit according to E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 37 to 42.

[0243] E234. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 43 to 48.

[0244] E235. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 49 to 54.

[0245] E236. The diagnostic kit according to E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 55 to 60.

[0246] E237. The diagnostic kit according to E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 61 to 66.

[0247] E238. The diagnostic kit according to E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 67 to 72.

[0248] E239. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 73 to 78.

[0249] E240. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 79 to 84.

[0250] E241. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 85 to 90.

[0251] E242. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 91 to 96.

[0252] E243. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 97 to 102.

[0253] E244. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 103 to 108.

[0254] E245. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 109 to 114.

[0255] E246. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 115 to 120.

[0256] E247. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, each comprising SEQ ID NOs: 121 to 126.

[0257] E248. The diagnostic kit of any one of E153 to E247, wherein the second antibody comprises a heavy chain variable region (VH) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167.

[0258] E249. The diagnostic kit of any one of E153 to E248, wherein the second antibody comprises a light chain variable region (VL) that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

[0259] E250. The second antibody (1) A VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 127, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 128; (2) a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 129, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 130; (3) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 131, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 132; (4) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 133, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 134; (5) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 135, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 136; (6) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 137, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 138; (7) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 139, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 140; (8) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 141, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 142; (9) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 143, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 144; (10) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 145, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 146; (11) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 147, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 148; (12) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 149, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 150; (13) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 151, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 152; (14) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 153, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154; (15) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 155, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 156; (16) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 157, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 158; (17) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 159, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 160; (18) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 161, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 162; (19) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 163, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 164; (20) A VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 165, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 166; or (21) VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 167, and VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 168. The diagnostic kit according to any one of E153 to E249, comprising:

[0260] E251. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 127, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 128.

[0261] E252. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 129 and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 130.

[0262] E253. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 131 and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 132.

[0263] E254. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 133, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 134.

[0264] E255. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 135, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 136.

[0265] E256. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 137, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 138.

[0266] E257. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 139, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 140.

[0267] E258. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 141, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 142.

[0268] E259. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 143, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 144.

[0269] E260. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 145, and a VL comprising a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 146.

[0270] E261. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 147, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 148.

[0271] E262. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 149, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 150.

[0272] E263. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 151 and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 152.

[0273] E264. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 153, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 154.

[0274] E265. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 155, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 156.

[0275] E266. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 157, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 158.

[0276] E267. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 159, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 160.

[0277] E268. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 161, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 162.

[0278] E269. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 163, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 164.

[0279] E270. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 165, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 166.

[0280] E271. The diagnostic kit of E250, wherein the second antibody comprises a VH comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 167, and a VL comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 168.

[0281] E272. The diagnostic kit according to any one of E153 to E271, wherein the second antibody comprises a human CH1 domain, such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1.

[0282] E273. The diagnostic kit of E272, wherein said human IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 182.

[0283] E274. The human IgG2 CH1 comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 194, or the human IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 190; or The diagnostic kit of E272, wherein the human IgG4 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 186.

[0284] E275. The diagnostic kit according to any one of E153 to E274, wherein the second antibody comprises a human Fc domain, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc.

[0285] E276. The diagnostic kit of E275, wherein said human IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 184.

[0286] E277. The human IgG2 Fc comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 196, or the human IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 192; or The diagnostic kit of E275, wherein the human IgG4 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 188.

[0287] E278. The diagnostic kit according to any one of E153 to E271, wherein the second antibody comprises a mouse CH1 domain, such as mouse IgG1 CH1, mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1.

[0288] E279. The diagnostic kit of E278, wherein said murine IgG1 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 172.

[0289] E280. The diagnostic kit of E278, wherein said murine IgG2a CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 175.

[0290] E281. The diagnostic kit of E278, wherein said murine IgG2b CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 176.

[0291] E282. The diagnostic kit of E278, wherein said murine IgG3 CH1 comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the CH1 domain of SEQ ID NO: 177.

[0292] E283. The diagnostic kit according to any one of E153 to E271 and E278 to E282, wherein the second antibody comprises a mouse Fc domain such as mouse IgG1 Fc, mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc.

[0293] E284. The diagnostic kit of E283, wherein said murine IgG1 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 174.

[0294] E285. The diagnostic kit of E283, wherein the murine IgG2a Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 175.

[0295] E286. The diagnostic kit of E283, wherein the murine IgG2b Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 176.

[0296] E287. The diagnostic kit of E283, wherein the murine IgG3 Fc comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the Fc domain of SEQ ID NO: 177.

[0297] E288. The diagnostic kit of any one of E153 to E287, wherein the second antibody comprises a human CL domain, such as human kappa CL or human lambda CL.

[0298] E289. The diagnostic kit of E288, wherein said human kappa CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 178 or 179.

[0299] E290. The diagnostic kit of E288, wherein said human lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 180 or 181.

[0300] E291. The diagnostic kit of any one of E153 to E287, wherein the second antibody comprises a mouse CL domain, such as mouse kappa CL or mouse lambda CL.

[0301] E292. The diagnostic kit of E291, wherein said mouse kappa CL comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 171.

[0302] E293. The diagnostic kit of E291, wherein said mouse lambda CL comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 169 or 170.

[0303] E294. The second antibody is 10 -4 M or less, 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less or 10 -9 The diagnostic kit according to any one of E153 to E293, which binds to RANKL with a KD value of M or less.

[0304] E295. The diagnostic kit according to any one of E153 to E294, wherein the RANKL is human RANKL.

[0305] E296. The diagnostic kit of any one of E153 to E295, wherein both the first antibody and the second antibody bind to a human RANKL epitope comprising SEQ ID NO: 214 (DLATE).

[0306] E297. The diagnostic kit of any one of E153 to E295, wherein both the first antibody and the second antibody bind to a human RANKL epitope comprising one or more residues from SEQ ID NO:215.

[0307] A diagnostic kit for use in the method according to any one of E298.E1 to E152. [Brief explanation of the drawings]

[0308] [Figure 1A] 1 shows an exemplary ADA assay. [Figure 1B]Figure 1 shows that soluble RANKL interferes with the ADA assay. [Figure 2A] We show that a second anti-RANKL antibody or OPG can reduce signal interference by sequestering soluble RANKL. [Figure 2B] We show that a second anti-RANKL antibody or OPG can reduce signal interference by sequestering soluble RANKL. DETAILED DESCRIPTION OF THE INVENTION

[0309] 1. Overview of Anti-Drug Antibody (ADA) Assay In one aspect, the present invention provides a method for detecting anti-drug antibodies (ADA) to denosumab in a sample. Generally, the sample is obtained from a patient treated with denosumab.

[0310] Denosumab (trade names Prolia® and Xgeva®) is a human monoclonal antibody that targets RANKL. Denosumab comprises a heavy chain comprising SEQ ID NO: 198 and a light chain comprising SEQ ID NO: 199. Prolia is indicated for the treatment of postmenopausal women with osteoporosis at high risk of fracture, for the treatment of bone mass increase in men with osteoporosis at high risk of fracture, for the treatment of glucocorticoid-induced osteoporosis in men and women at high risk of fracture, for the treatment of bone mass increase in men at high risk of fracture receiving androgen deprivation therapy for non-metastatic prostate cancer, and for the treatment of bone mass increase in women at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer. Xgeva is a prescription drug used to prevent fractures, spinal cord compression, or the need for bone radiation or surgery in patients with multiple myeloma and bone metastases from solid tumors.

[0311] As taught and illustrated in the Examples, one study observed an unusually high incidence of immunoreactivity in an anti-drug antibody (ADA) assay against denosumab. Based on past experience with denosumab clinical trials, this was thought to be a false positive, possibly due to nuclear factor-κB ligand (RANKL) interfering with the ADA assay (Figures 1A and 1B).

[0312] To address false-positive signals caused by soluble RANKL and develop a reliable ADA assay, we incorporated an exemplary anti-RANKL monoclonal antibody that competes with denosumab for binding to RANKL into an improved ADA assay. Because the complementarity-determining regions (CDRs) of antibodies are the result of random mutations from the germline and subsequently undergo a selection process, the CDRs are the domains most likely to be recognized as foreign by the immune system. Consequently, most anti-drug antibodies against monoclonal antibody therapeutics are CDR-directed. Based on these data, we believe that two strategies can be used to reduce the interference of RANKL ligands in ADA assays: (i) sequester soluble RANKL using OPG (a decoy receptor for RANKL) or (ii) sequester RANKL using an antibody that binds to RANKL. This antibody should have a different CDR sequence from denosumab so that the antibody itself does not bind to ADA (Figures 2A and 2B).

[0313] 2. Immunoassays for detecting ADA The most commonly used ADA assays are based on solid-phase immunoassays. A standard solid-phase immunoassay using monoclonal antibodies involves the formation of a complex between an antibody adsorbed to a solid substrate (a "capture" molecule), a molecule of interest (in this case, ADA for denosumab), and an antibody linked to a detectable marker (a "tracer" molecule). In this way, a sandwich is formed: solid substrate-capture molecule-molecule of interest-tracer molecule. In a sandwich, the amount of detectable marker is typically proportional to the concentration of ADA in the sample. One sandwich assay is the double-antigen bridge immunoassay, in which the capture antibody and tracer antibody bind to different epitopes of the antigen. Hoesel, W., et al., J. Immunol. Methods 294 (2004) 101-110, reported an anti-EPO double-antigen bridge assay using a mixture of immobilized rhEPO bound to amino and carbohydrate groups. Immunoassays such as double-antigen bridge ELISA are common assay types for investigating patients' immunogenic responses to antibody drugs. Mire-Sluis, AR, et al., J. Immunol. Methods 289 (2004) 1-16 summarizes recommendations for the design and optimization of immunoassays used to detect host antibodies against biotechnology products.

[0314] capture molecule The "capture" molecule is typically an antibody covalently or non-covalently linked to an immobilization moiety, and the solid substrate comprises a binding partner that binds to the immobilization moiety. The interaction between the immobilization moiety and its binding partner allows the capture molecule to be immobilized to the solid substrate. The capture molecule is also referred to herein as the "first antibody or antibody conjugate."

[0315] Exemplary binding pairs that can be used as the immobilization moiety and its binding partner include, for example, the following (first component-second component): streptavidin-biotin, avidin-biotin, antibody-antigen (see, e.g., Hermanson, G.T., et al., Bioconjugate Techniques, Academic Press, 1996), lectin-polysaccharide, steroid-steroid binding protein, hormone-hormone receptor, enzyme-substrate, IgG-Protein A, IgG-Protein G, and ligand-receptor. In an exemplary embodiment, the capture molecule is an antibody conjugated to biotin, and immobilization is performed via avidin or streptavidin bound to the surface of the solid substrate.

[0316] In certain embodiments, the conjugation of the antibody to the immobilization moiety is by covalent conjugation via the N-terminus and / or ε-amino group (lysine), the ε-amino groups of different lysines, carboxy, sulfhydryl, hydroxyl and / or phenolic functional groups of the amino acid backbone of the antibody and / or sugar alcohol groups of the carbohydrate structure of the antibody.

[0317] Because ADA is often thought to be caused by the CDR sequence, in order to detect ADA against denosumab, the capture molecule should contain an antibody having the same CDR sequence as denosumab. The amino acid sequences of the heavy chain (HC), light chain (LC), heavy chain variable domain (VH), light chain variable domain (VL), heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3), and light chain complementarity determining regions (CDR-L1, CDR-L2, CDR-L3) of denosumab are shown in Table F of the Sequence Listing.

[0318] As used herein, "complementarity determining regions" (CDRs) may be identified according to the Kabat, Chothia, both Kabat and Chothia accumulation, AbM, contact, North, and / or conformational definitions, or any method of determining CDRs well known in the art. See, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structures). The identity of the amino acid residues of a particular antibody that make up a CDR can be determined using methods well known in the art. The AbM definition of a CDR is a compromise between Kabat and Chothia and uses Oxford Molecular's AbM antibody modeling software (Accelrys®). The definition of CDR "contact" is based on the observed antigen contacts shown in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. The definition of CDR "conformation" is based on the residues that generate enthalpic contributions to antigen binding (see, for example, Makabe et al., 2008, J. Biol. Chem., 283:1156-1166). North used a different set of preferred CDR definitions to identify canonical CDR conformations (North et al., 2011, J. Mol. Biol. 406:228-256). In another approach, referred to herein as CDR "conformation definition," the position of the CDR can be identified as the residue that generates enthalpic contributions to antigen binding (Makabe et al., 2008, J. Biol. Chem. 283:1156-1166). Still other definitions of CDR boundaries may not strictly follow one of the above approaches, but still overlap with at least a portion of the Kabat CDRs, but they may be shortened or lengthened in light of predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding.As used herein, CDRs may refer to CDRs defined by any approach known in the art, including combinations of these approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing multiple CDRs, the CDRs (or other residues of the antibody) may be defined according to any of the Kabat, Chothia, North, extended, AbM, contact, and / or conformational definitions.

[0319] For example, the following table provides some commonly used definitions of CDRs:

[0320] [Table 1]

[0321] The CDR sequences shown in the sequence listing are based on the Kabat definition. However, other definitions of CDRs may also be used. Thus, in some embodiments, a first antibody comprises the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 203 and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 208. In some embodiments, a first antibody comprises the CDR-H1 of SEQ ID NO: 200, the CDR-H2 of SEQ ID NO: 201, the CDR-H3 of SEQ ID NO: 202, the CDR-L1 of SEQ ID NO: 205, the CDR-L2 of SEQ ID NO: 206, and the CDR-L3 of SEQ ID NO: 207.

[0322] Outside of the CDR regions, the sequence of the first antibody may differ from that of denosumab, nevertheless, for convenience, the denosumab VH and / or VL sequences may be used. Thus, in some embodiments, the first antibody or antibody conjugate is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:203. The antibody or antibody fragments comprise a heavy chain variable region (VH) that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 208.

[0323] The constant region of the first antibody (or capture antibody) can be human or mouse. Examples of human and mouse constant region sequences are provided in Sequence Table E. For performing assays with human samples, certain mouse constant region sequences may be preferred in some cases. This may reduce cross-reactivity or false positives. Endogenous human antibodies in the sample will not interfere with proper detection of ADA molecules. Thus, in some embodiments, the first antibody or antibody conjugate comprises a human CH1 domain (such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1), a human Fc domain (such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc), a mouse IgG1 CH1 (such as mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1), a mouse IgG1 Fc (such as mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc), a human CL domain (such as human kappa CL or human lambda CL), or a mouse CL domain (such as mouse kappa CL or mouse lambda CL).

[0324] In an exemplary embodiment, the first antibody or antibody conjugate comprises denosumab conjugated to biotin.

[0325] The heavy and light chain sequences of denosumab are set forth in SEQ ID NOs: 198 and 199, respectively. It should be noted that, in many cases, the terminal lysine and glycine residues of the heavy chain can be clipped during the recombinant production process. C-terminal lysine clipping is a common phenomenon that occurs during the bioproduction of monoclonal antibodies. Lysine residues are often removed via carboxypeptidase D (CpD), resulting in the generation of a mixture of antibody isoforms with zero or one C-terminal lysine residue on each heavy chain. Furthermore, after C-terminal lysine cleavage, peptidylglycine α-amidating monooxygenase (PAM) catalyzes the hydroxylation of glycine and the removal of glyoxylate from the glycine residue, leaving an amidated C-terminal proline. Therefore, during recombinant production of monoclonal antibodies, the product is often a mixture of C-terminal processing variants in which the heavy chain C-terminus is at (amidated) proline, glycine, or lysine. Thus, in some embodiments the terminal lysine of SEQ ID NO: 198 may be absent, in some embodiments the terminal lysine of SEQ ID NO: 198 may be present, in some embodiments the terminal glycine-lysine of SEQ ID NO: 198 may be absent, and in some embodiments the terminal glycine-lysine of SEQ ID NO: 198 may be present.

[0326] "Solid substrate" refers to a non-fluidic substance, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic and ferromagnetic particles), glass, and ceramics; gel materials such as silica, alumina, and polymer gels; capillaries, zeolites, and other porous materials that may be made from polymers, metals, glass, and / or ceramics; electrodes, microtiter plates, solid strips, cuvettes, tubes, or other spectrometer sample containers. In ADA assays, solid substrate generally refers to an inert solid surface containing a "binding partner" (e.g., avidin or streptavidin) on its surface that is intended to interact with a "capture" antibody (in this case, a first antibody or antibody conjugate containing an immobilized moiety (e.g., biotin) recognized by the "binding partner"). The solid phase can be an immobilized component, such as a tube, strip, cuvette, or microtiter plate, or a non-immobilized component, such as beads and microparticles. Microparticles can also be used as the solid phase in homogeneous assay formats. A variety of microparticles can be used that allow for either non-covalent or covalent attachment of proteins and other substances. Such particles include polymer particles such as polystyrene and poly(methyl methacrylate), gold particles such as gold nanoparticles and gold colloids, and ceramic particles such as silica, glass, and metal oxide particles. See, e.g., Martin, CR, et al., Analytical Chemistry-News & Features 70 (1998) 322A-327A.

[0327] Generally, the first antibody or antibody conjugate is immobilized on the solid substrate by passive adsorption, thus binding to the solid phase at a site that does not interfere with its binding to ADA. Passive adsorption is described, for example, in Butler, JE, Solid Phases in Immunoassay, In: Immunoassays, Diamandis, EP and Christopoulos, TK (eds.) Academic Press San Diego (1996), pp. 205-225.

[0328] tracer molecule In a typical ADA assay, a "tracer" molecule (also referred to herein as a "third antibody") must be able to bind to the ADA molecule to form a sandwich (see, e.g., FIG. 1A). Because ADAs are typically triggered by CDR sequences, the most convenient method is for the tracer molecule to have the same CDR sequences as the first antibody. The tracer molecule is also linked to a detectable marker to facilitate detection of ADA.

[0329] Thus, in certain embodiments, the third antibody comprises the same six CDRs as denosumab. As described in detail above, the CDRs may be identified according to the Kabat, Chothia, both Kabat and Chothia accumulation, AbM, contact, North, and / or conformational definitions, or any method of determining CDRs well known in the art. The CDR sequences shown in the Sequence Listing are based on the Kabat definition. However, other definitions of CDRs may also be used. Thus, in some embodiments, the third antibody comprises the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 203 and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 208. In some embodiments, the third antibody comprises an antibody comprising CDR-H1 comprising SEQ ID NO: 200, CDR-H2 comprising SEQ ID NO: 201, CDR-H3 comprising SEQ ID NO: 202, CDR-L1 comprising SEQ ID NO: 205, CDR-L2 comprising SEQ ID NO: 206, and CDR-L3 comprising SEQ ID NO: 207.

[0330] Outside of the CDR regions, the sequence of the third antibody may differ from that of denosumab, nevertheless, for convenience, the denosumab VH and / or VL sequences may be used. Thus, in some embodiments, the third antibody has a heavy chain sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:203. and / or a light chain variable region (VL) that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:208.

[0331] The constant region of the third antibody (or tracer antibody) can be human or murine. Examples of human and murine constant region sequences are provided in Sequence Table E. For performing assays with human samples, certain murine constant region sequences may be preferred in some cases. This may reduce cross-reactivity or false positives. Endogenous human antibodies in the sample will not interfere with proper detection of ADA molecules. Thus, in some embodiments, the third antibody comprises a human CH1 domain (such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1), a human Fc domain (such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc), a mouse IgG1 CH1 (such as mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1), a mouse IgG1 Fc (such as mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc), a human CL domain (such as human kappa CL or human lambda CL), or a mouse CL domain (such as mouse kappa CL or mouse lambda CL).

[0332] In some embodiments, the third antibody comprises a VH domain comprising the same sequence as the VH of the first antibody and a VL domain comprising the same sequence as the VL domain of the first antibody, hi some embodiments, the third antibody comprises a heavy chain (HC) comprising the same sequence as the HC of the first antibody and a light chain (LC) comprising the same sequence as the LC of the first antibody.

[0333] Typically, the "tracer" molecule (or "third antibody" herein) is covalently or non-covalently linked to a detectable marker. Detectable markers can be isotopes, enzymes, fluorescent moieties, luminescent moieties, chromogenic moieties, metals, or charges. For example, detectable markers can be electrochemiluminescent labels such as ruthenium bispyridyl complexes. Chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles, and haptens, such as digoxigenin, are also examples of detectable markers. Detectable markers can also be photoactivatable crosslinking groups, such as azide or azirine groups. Metal chelates that can be detected by electrochemiluminescence are also preferred signal-emitting groups, with ruthenium chelates, such as ruthenium(bispyridyl)3 2+ chelate, being particularly preferred. Suitable ruthenium labeling groups are described, for example, in EP 0 580 979, WO 90 / 05301, WO 90 / 11511 and WO 92 / 14138. Furthermore, if desired, nearly all antibodies can be labeled with biotin, HRP enzyme or one of several fluorophores.

[0334] Indirect detection methods are sometimes used. In this case, a "tracer" molecule is considered a "primary antibody" that binds to ADA, and the primary antibody is not labeled for direct detection. Instead, a "secondary antibody" labeled with a detectable tag is applied in a second step to probe the primary antibody-ADA complex. ADA is thus indirectly detected. Another form of indirect detection involves the use of a primary or secondary antibody labeled with an affinity tag, such as biotin. A secondary (or tertiary) probe, such as streptavidin labeled with a detectable enzyme or fluorophore tag, can then be used to probe the biotin tag to obtain a detectable signal. Several variations of these probing and detection strategies exist. Generally, this strategy relies on a specific probe (e.g., a primary antibody) whose presence is directly or indirectly linked to some kind of detectable marker (e.g., an enzyme whose activity can generate a colored product upon reaction with its substrate).

[0335] Most primary antibodies can be generated or engineered to have one of several non-human constant domains: mouse, rabbit, or several others. Many of these are of the IgG class, particularly the IgG1 subclass. Therefore, it is relatively easy and economical for manufacturers to produce and supply ready-to-use labeled secondary antibodies for most applications and detection systems. The choice of secondary antibody depends on the species of animal (host species) in which the primary antibody was raised. For example, if the primary antibody has a mouse constant domain, the secondary antibody should be an anti-mouse antibody obtained from a host other than a mouse. Ready-made secondary antibodies that bind to the constant domain or Fc domain of the primary antibody are generally readily available.

[0336] Biotin-based systems are often used as detectable markers. When biotin-binding proteins are used as probes, the highly specific affinity interaction between biotin and avidin or streptavidin proteins forms the basis of many types of detection and affinity purification methods. Because biotin is very small (244 daltons), its covalent attachment to antibodies or other probes does little to interfere with their function. However, its presence as a label on the probe allows for efficient and specific secondary detection with either avidin or streptavidin. Both types of biotin-binding proteins are available in purified forms labeled with enzyme or fluorescent tags, which allow for detection in many types of assay systems.

[0337] Enzyme labels are commonly used as secondary antibody (or streptavidin) tags for detection in blotting and immunoassays. Enzymes provide a detectable signal through their activity, resulting in colored, luminescent, or fluorescent products upon reaction with specific substrate chemicals. While reporter enzymes such as beta-galactosidase and luciferase have been successfully used to generate probes, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes most widely used as labels for protein detection. A range of chromogenic, fluorogenic, and chemiluminescent substrates are available for use with either enzyme.

[0338] Alkaline phosphatase (AP), typically isolated from calf intestine, is a large (140 kDa) protein that catalyzes the hydrolysis of phosphate groups from substrate molecules to produce colored or fluorescent products or the emission of light as a by-product of the reaction. AP has optimal enzymatic activity at basic pH (pH 8-10) and can be inhibited by cyanide, arsenate, inorganic phosphate, and divalent cation chelators such as EDTA. AP offers certain advantages as a detectable marker. Because its reaction kinetics is linear, detection sensitivity can be improved by simply allowing the reaction to proceed for a longer period of time.

[0339] Horseradish peroxidase (HRP) is a 40 kDa protein that catalyzes the oxidation of substrates by hydrogen peroxide, resulting in colored or fluorescent products or the release of light as a by-product of the reaction. HRP functions optimally at near-neutral pH and can be inhibited by cyanide, sulfide, and azide. Antibody-HRP conjugates have been shown to be superior to antibody-AP conjugates in terms of the specific activity of both the enzyme and the antibody. Furthermore, its high turnover rate, good stability, low cost, and wide availability of substrates make HRP the enzyme of choice for many applications. Due to the small size of the HRP enzyme, further improvements in sensitivity can be achieved by using poly-HRP-conjugated secondary antibodies.

[0340] Two common fluorophores for labeling probes are fluorescein (fluorescein isothiocyanate, FITC) and rhodamine (tetramethylrhodamine isothiocyanate, TRITC).

[0341] Other detectable markers include fluorescent proteins, such as various forms of green fluorescent protein (GFP) and phycobiliproteins (allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin).

[0342] Reduced signal interference As disclosed herein, in an exemplary ADA assay for detecting denosumab ADA, a large number of samples showed false positive signals, believed to be caused by RANKL interference with the ADA assay (Figures 1A and 1B).

[0343] The sequence of human RANKL, including membrane-bound and soluble forms of RANKL, is provided in Sequence Table F. The soluble form of human RANKL contains residues 69 to 317 (as numbered in WO 98 / 46757) of the membrane-bound form, shown as SEQ ID NOs: 212 and 213. Proteolytic cleavage between residues 139 and 140 of membrane-bound RANKL also yields the soluble form, shown as SEQ ID NO: 214.

[0344] To address false-positive signals caused by soluble RANKL, it is important to include a molecule capable of sequestering soluble RANKL in the ADA assay. Two strategies are provided herein: one uses a second anti-RANKL antibody, and the other uses the decoy receptor OPG.

[0345] To effectively isolate RANKL without interfering with the detection of ADA, the second antibody should have the following characteristics: (1) the second antibody should have a different CDR sequence from denosumab (since ADA is often CDR-directed, having different CDRs generally ensures that there is no cross-reactivity between the second antibody and ADA), and (2) the second antibody should bind to RANKL. Preferably, the second antibody binds to the same epitope of RANKL as denosumab, or the second antibody competes (or at least partially competes) with denosumab for RANKL binding. Signal interference caused by RANKL can be most effectively reduced if the second antibody and denosumab cannot simultaneously bind to RANKL. Nevertheless, competing (or at least partially competing) with denosumab for RANKL binding is not an absolute requirement. For example, in Examples 3-6, an exemplary anti-RANKL mAb was found not to compete with denosumab for RANKL binding. Epitope binning demonstrated that this anti-RANKL mAb binds to a different epitope bin and does not block denosumab or OPG for RANKL binding. Those skilled in the art will be able to easily evaluate the binding behavior of a second antibody and design an ADA assay accordingly based on general knowledge. Example 2B also provides an example of how to evaluate and select a suitable second antibody.

[0346] Furthermore, although it is preferred that the second antibody compete (or at least partially compete) with denosumab for RANKL binding, it is not necessary that the second antibody bind to RANKL with the same affinity (KD value) as denosumab. Even a poor competitor with a lower binding affinity for RANKL may be suitable to reduce / eliminate interference. In that case, the competing antibody can be added to the serum sample in large amounts and only need to sequester a small amount of RANKL.

[0347] KD is the equilibrium dissociation constant, k off / k onKD is the ratio of K to K. KD and K are inversely proportional. The KD value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), and thus the lower the KD value (the lower the concentration required), the higher the affinity of the antibody. In an exemplary embodiment, the KD value of the second antibody against RANKL is about 10 -1 M or less, about 10 -2 M or less, about 10 -3 M or less, about 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10-13 M or about 10 -10 M~about 10 -13 It's M.

[0348] In exemplary embodiments, the KD of the second anti-RANKL antibody is micromolar, nanomolar, picomolar, or femtomolar. -4 ~10 -6 M or 10 -7 ~10 -9 M or 10 -10 ~10 -12 M or 10 -13 ~10 -15 In exemplary embodiments, the second antibody has a mAb concentration within the range of about 1 uM or less, about 900 nM or less, about 800 nM or less, about 700 nM or less, about 600 nM or less, about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less, It binds to its target with a KD value of about 1 nM or less, about 900 pM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 250 pM or less, about 200 pM or less, about 150 pM or less, about 100 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 25 pM or less, about 20 pM or less, about 15 pM or less, about 10 pM or less, about 5 pM or less, or about 1 pM or less.

[0349] KD values ​​can be determined using methods well established in the art. DOne representative method for measuring KD is surface plasmon resonance (SPR), a method well known in the art (e.g., Nguyen et al. Sensors (Basel). 2015 May 5;15(5):10481-510). KD values ​​can be measured by SPR using a biosensor system such as the BIACORE® system. BIAcore kinetic analysis involves analyzing the binding and dissociation of an antigen from a chip on whose surface molecules (e.g., molecules containing epitope-binding domains) are immobilized. Another method well known in the art for determining the KD of a protein is by using Bio-Layer Interferometry (e.g., Shah et al. J Vis Exp. 2014;(84):51383). KD values ​​can be measured by Bio-Layer Interferometry using OCTET® technology (Octet QKe system, ForteBio). Alternatively or additionally, the KinExA® (Kinexclusion assay) assay, available from Sapidyne Instruments (Boise, Id.), can be used. Any method known in the art for assessing binding affinity between two binding partners is encompassed herein.

[0350] In some embodiments, the KD value is measured by surface plasmon resonance (SPR). The antigen (RANKL) can be immobilized, for example, on a solid surface. The antigen can be immobilized to a chip, for example, by covalent coupling (e.g., amine coupling). The chip can be a CM5 sensor chip. As the analyte binds to the ligand, accumulation of the protein on the sensor surface causes an increase in the refractive index. This change in refractive index is measured in real time (test sampling in a kinetic analysis experiment is performed every 0.1 s), and the results are plotted as response units (RU) versus time (called a sensorgram). A response (background response) also occurs when there is a difference in the refractive index of the running and sample buffers. To obtain the actual binding response, this background response must be subtracted from the sensorgram. The background response is recorded by injecting the analyte through a control or reference flow cell with no or an irrelevant ligand immobilized on the sensor surface. Real-time measurement of the association and dissociation of the binding interaction allows for the calculation of the association and dissociation rate constants and the corresponding affinity constants. 1 RU represents 1 pg of protein bound per square mm. Analyte binding of greater than 50 pg / mm 2 is generally required in practice to produce a good, reproducible response.

[0351] Dissociation of the antibody from the antigen can be monitored for about 3600 seconds. SPR analysis can be performed and data collected at about 15°C to about 37°C. SPR analysis can be performed and data collected at about 25°C to about 37°C. SPR analysis can be performed and data collected at about 37°C. SPR analysis can be performed and data collected at 37°C. D Values ​​can be measured by SPR using a BIAcore T200 instrument. SPR ratios and affinities can be determined by fitting the resulting sensorgram data to a 1:1 model in BIAcore T200 Evaluation software version 1.0. The collection rate can be approximately 1 Hz.

[0352] Another method for determining the KD of an antibody is by using Bio-Layer Interferometry (BLI), typically using OCTET® technology (Octet QKe system, ForteBio). In some embodiments, biosensor analysis is used. Typically, one reactive partner is immobilized on the surface of a biosensor (the "ligand," e.g., an antibody), while the other remains in solution (the "analyte," e.g., an antigen). The assay begins with an initial baseline or equilibrium phase using an assay buffer. Next, the ligand (e.g., an antibody) is immobilized (loaded) on the biosensor surface by either direct immobilization or a capture-based method. After ligand immobilization, the biosensor is immersed in a buffer solution for a baseline phase to assess assay drift and determine the ligand loading level. After the baseline phase, the biosensor is immersed in a solution containing the ligand's binding partner, the analyte (associated). In this phase, the binding interaction of the analyte to the immobilized ligand is measured. After analyte association, the biosensor is immersed in an analyte-free buffer solution, allowing the bound analyte to peel from the ligand (dissociation). The sequence of assay steps is then repeated on a new or regenerated biosensor for each analyte being tested. Each binding reaction is measured and reported in real time on a sensorgram trace. The instrument can be an Octet QKe system, an Octet RED96 system, an Octet QK384 system, or a RED384 system.

[0353] In certain embodiments, the second antibody binds to the same RANKL epitope as denosumab. Denosumab is known to bind to an epitope (DE epitope) comprising a portion of the amino acid sequence of the DE region of RANKL. The DE region of RANKL spans approximately the D and E β-sheet regions and the intervening loop sequence (DE loop). The DE region of human RANKL includes approximately amino acid residue 212 to approximately amino acid residue 250 (GFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIP, SEQ ID NO: 216). However, it is understood that the amino acid sequence and endpoints of the DE region of human RANKL are merely exemplary, and that the DE region may have a variable sequence and endpoint. In particular, within the DE loop of human RANKL, the amino acid sequence DLATE (SEQ ID NO: 215) has been shown to be important for denosumab binding. See, for example, International Publication No. WO 2001062932.

[0354] Thus, in some embodiments, the second anti-RANKL antibody binds to an epitope comprising the sequence "DLATE" (SEQ ID NO: 215). In some embodiments, the second anti-RANKL antibody binds to an epitope located within the DE loop of human RANKL.

[0355] "Epitope" refers to the region or area of ​​an antigen to which an antibody specifically binds, e.g., the region or area containing the residues that interact with the antibody. An epitope can be linear or conformational. The term "paratope" is derived from the above definition of "epitope" by reversing the perspective and refers to the region or area of ​​an antibody molecule involved in antigen binding, e.g., the region or area containing the residues that interact with the antigen. A paratope can be linear or conformational (e.g., discontinuous residues in a CDR). The epitope / paratope of a given antibody / antigen binding pair can be defined and characterized at different levels of detail using various experimental and computational epitope mapping methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (HX-MS), and various competitive binding methods. Because each method relies on unique principles, the description of an epitope is closely related to the method by which it was determined. Thus, the epitope / paratope of a given antibody / antigen pair is defined differently depending on the mapping method used.

[0356] The fact that epitope descriptions and definitions depend on the epitope mapping method used and can be obtained at different levels of detail means that comparisons of epitopes for different antibodies on the same antigen can be performed at different levels of detail as well. For example, epitopes described at the amino acid level, e.g., determined from X-ray structures, are said to be identical if they contain the same set of amino acid residues. Epitopes characterized by competitive binding are said to be overlapping if the binding of corresponding antibodies is mutually exclusive, i.e., binding of one antibody excludes simultaneous or sequential binding of the other antibody, and epitopes are said to be separate (unique) if the antigen can accommodate the binding of both corresponding antibodies simultaneously.

[0357] The epitope and paratope of a given antibody / antigen pair can be identified by routine methods, for example, the general location of the epitope can be determined by assessing the ability of the antibody to bind to different fragments of RANKL.

[0358] In certain embodiments, the second antibody competes with denosumab for RANKL binding. As used herein with respect to antibodies, the term "compete" means that the binding of a first antibody, or an antigen-binding portion thereof, to an antigen reduces subsequent binding of the same antigen by a second antibody, or an antigen-binding portion thereof. Generally, binding of a first antibody results in steric hindrance, a conformational change, or binding to a common epitope (or portion thereof), thereby reducing the binding of a second antibody to the same antigen. Standard competition assays can be used to determine whether two antibodies compete with each other. One suitable assay for antibody competition involves the use of BIAcore technology, which can measure the degree of interaction using surface plasmon resonance (SPR) technology, typically using a biosensor system (such as a BIACORE® system). For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody.

[0359] Another assay for measuring antibody competition uses an ELISA-based approach. Furthermore, a high-throughput process for "binning" antibodies based on their competition is described in International Patent Application WO 2003 / 48731. Competition exists when one antibody (or fragment) reduces the binding of another antibody (or its antigen-binding fragment) to RANKL. For example, a sequential binding competition assay can be used, in which different antibodies are added sequentially. The first antibody can be added to reach near-saturation binding. Then, the second antibody is added. Two antibodies are considered to compete with each other if the binding of the second antibody to RANKL is not detected or is significantly reduced (e.g., by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%) compared to a parallel assay in the absence of the first antibody (which can be set as 100%).

[0360] Exemplary anti-RANKL antibodies that can be used in the ADA assays disclosed herein are set forth in the Sequence Listing. Thus, in certain embodiments, the second antibody comprises (i) heavy chain CDR-H1, CDR-H2 and CDR3-H3 of SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 or 167, and (ii) light chain CDR-L1, CDR-L2 and CDR3 of SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168. As detailed above, CDRs may be identified according to the Kabat, Chothia, both Kabat and Chothia accumulation, AbM, contact, North, and / or conformation definitions, or any method of determining CDRs well known in the art. The CDR sequences shown in the sequence listing are based on the Kabat definition. However, other definitions of CDRs may also be used.

[0361] In some embodiments, the second antibody is (i) at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130, at least 131, at least 132, at least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, at least 140, at least 141, at least 142, at least 143, at least 144, at least 145, at least 146, at least 147, at least 148, at least 149, at least 150, at least 151, at least 152, at least 153, at least (ii) a CDR-H1 comprising a sequence that is at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116 or 122; (iii) a CDR-H1 comprising a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% identical to SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116 or 122; , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117 or 123; (iii) a CDR-H2 comprising a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least (iv) a CDR-H3 comprising a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118 or 124;(v) a CDR-L1 comprising a sequence that is at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119 or 125; and (vi) a CDR-L3 comprising a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 or 126.

[0362] In some embodiments, the second antibody comprises a heavy chain variable region (VH) that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 or 167. In some embodiments, the second antibody comprises a light chain variable region (VL) that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.

[0363] The constant region of the second antibody can be human or mouse. Examples of human and mouse constant region sequences are provided in the Sequence Listing. For performing assays with human samples, certain mouse constant region sequences may be preferred in some cases. This may reduce cross-reactivity or false positives. Endogenous antibodies in the sample do not interfere with proper detection of ADA molecules. Thus, in some embodiments, the third antibody comprises a human CH1 domain (such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1, or human IgG4 CH1), a human Fc domain (such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc), a mouse IgG1 CH1 (such as mouse IgG2a CH1, mouse IgG2b CH1, or mouse IgG3 CH1), a mouse IgG1 Fc (such as mouse IgG2a Fc, mouse IgG2b Fc, or mouse IgG3 Fc), a human CL domain (such as human kappa CL or human lambda CL), or a mouse CL domain (such as mouse kappa CL or mouse lambda CL).

[0364] Alternatively, osteoprotegerin (OPG) can be used to sequester soluble RANKL. OPG, also known as osteoclastogenesis inhibitory factor (OCIF) or tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), is a cytokine receptor of the tumor necrosis factor (TNF) receptor superfamily encoded by the TNFRSF11B gene. OPG is a decoy receptor for RANKL. An exemplary sequence of human OPG is shown in Sequence Table F.

[0365] In certain embodiments, the OPG is human OPG. In certain embodiments, the OPG comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 210 or 211.

[0366] The sample can be pretreated with a second anti-RANKL antibody or OPG disclosed herein to remove soluble RANKL, and then the first antibody or antibody conjugate and the third antibody can be added to complete the ADA assay. Alternatively, the first antibody or antibody conjugate, the second antibody or OPG, and the third antibody can be added simultaneously. This is particularly feasible when the second antibody competes or partially competes with the first antibody for RANKL binding (i.e., when the first antibody and the second antibody cannot simultaneously bind to RANKL). When the first antibody and the second antibody cannot simultaneously bind to RANKL, the presence of the second antibody prevents RANKL from cross-linking the immobilized first antibody and the detectable marker.

[0367] An excess of the second antibody or OPG may be added to ensure complete sequestration of soluble RANKL in the sample. In certain embodiments, an excess of the second antibody or OPG is added to the sample such that the molar ratio of the first antibody to the second antibody or OPG is at least 1:2, at least 1:3, at least 1:4, at least 1:5, at least 1:6, at least 1:7, at least 1:8, at least 1:9, at least 1:10, at least 1:15, at least 1:20, at least 1:30, at least 1:40, at least 1:50, at least 1:60, at least 1:70, at least 1:80, at least 1:90, at least 1:100, at least 1:200, at least 1:300, at least 1:400, at least 1:500, at least 1:600, at least 1:700, at least 1:800, at least 1:900, or at least 1:1000.

[0368] 3. Diagnostic kits In certain aspects, the present disclosure provides a kit adapted to determine the presence or absence of ADA to denosumab in a sample. The kit may include instructions and reagents for the ADA assay disclosed herein. For example, the kit may include: (a) a first antibody or antibody conjugate that binds to RANKL, the first antibody or antibody conjugate comprising a heavy chain (HC) CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 208, which can be bound to a solid substrate; (b) (i) a second antibody that binds to RANKL, the second antibody comprising an amino acid sequence that differs from the first antibody by at least one amino acid in one of its six CDR sequences, or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; (c) a third antibody that binds to the ADA and can be linked to a detectable marker; and (d) instructions for use.

[0369] The kit may also include an immobilization moiety that can be covalently or non-covalently linked to the first antibody. The kit may also include a binding partner that can bind to the solid substrate and the immobilization moiety. The binding partner may be already immobilized to the substrate, or reagents are provided that allow the user to bind the binding partner to the solid substrate.

[0370] The kit may also include a detectable marker. It may also include reagents that allow the user to covalently or non-covalently link a third antibody to the detectable marker. Alternatively, the kit may include a third antibody that is already covalently or non-covalently linked to a detectable marker.

[0371] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations will occur to those skilled in the art in light of them, and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. [Example]

[0372] Example 1. Background and Rationale In one denosumab bioequivalence study ("Study A"), an unusually high incidence of immunoreactivity was observed in the anti-drug antibody assay (ADA). Based on experience with previous denosumab clinical trials, this was unexpected, and an investigation was initiated. False-positive results due to target interference were suspected because positive samples gave uniform, low-magnitude signals consistent with treatment-induced shedding of receptor activator of nuclear factor-κB ligand (RANKL) rather than an anti-denosumab antibody response.

[0373] To explore this possibility, a subset of antibody-positive samples from Study A was treated with osteoprotegerin (OPG), a decoy receptor for RANKL, and retested in the antibody assay. The hypothesis was that if the antibody-positive results were due to soluble RANKL, OPG treatment would reduce the assay signal by competing with denosumab for binding. If the antibody-positive results were caused by anti-denosumab antibodies, OPG treatment would not affect the assay signal because OPG does not bind to ADA molecules. Specifically, as shown in Figures 1A-1B, in an exemplary MSD ADA assay, anti-drug antibodies can crosslink biotin-labeled denosumab and SULFO-TAG (ruthenium trisbipyridine)-labeled denosumab. This complex is immobilized using streptavidin-coated plates and detected by chemiluminescence (Figure 1A). Soluble RANKL trimers may crosslink labeled denosumab molecules (Figure 1B), potentially resulting in false-positive signals. In this case, pretreatment of samples with OPG can deplete soluble RANKL from the samples and eliminate false-positive signals. Indeed, OPG treatment resulted in a clear reduction in signal in most antibody-positive samples tested (data not shown), indicating that many of the positive results were due to target (RANKL) interference.

[0374] To address false-positive signals caused by soluble RANKL and develop a reliable ADA assay, an anti-RANKL monoclonal antibody that binds to human RANKL was incorporated into an improved ADA assay. Because the complementarity-determining regions (CDRs) of antibodies are the result of random mutations from the germline and subsequently undergo a selection process, the CDRs can be recognized as foreign by the immune system. Consequently, most anti-drug antibodies against monoclonal antibody therapeutics are CDR-directed. For this reason, screening and selection of anti-RANKL antibodies with CDRs different from those of denosumab was performed to ensure that the incorporated anti-RANKL antibody would not inadvertently bind to ADA and inhibit the signal.

[0375] Example 2. Materials and methods for denosumab ADA assay Mesoscale Discovery (MSD)-based bridging immunoassay method. Serum samples were diluted 1:20 with 300 mM acetic acid to allow dissociation of antibody complexes prior to analysis. The diluted samples were then incubated with a conjugate / anti-RANKL / neutralization mixture containing biotinylated denosumab, ruthenylated denosumab, anti-RANKL antibody, 1 M Tris, pH 9.5, and soluble drug (confirmatory assay only). During this incubation, the two antigen-binding sites of the anti-denosumab antibody formed a bridge between the conjugated denosumab molecules (Figure 1A). The sample mixture was then added to a blocked MSD streptavidin standard binding microtiter plate (MSD, #L15SA-1). The biotinylated molecules bound to the streptavidin-coated surface of the wells, resulting in immobilization of the crosslinked complexes. The plate was washed to remove any unbound conjugate, and tripropylamine Gold read buffer (MSD, #R92TG-2) was added to each well. An MSD SECTOR S 600 reader was used to pass a current across the plate-associated electrodes. The result was a series of electrically induced redox reactions involving ruthenium (from the captured conjugate) and tripropylamine. The subsequent luminescent signal was quantified as electrochemiluminescence (ECL) units. A screening assay was performed on the samples to detect the presence of bound antibodies to denosumab. Samples analyzed in the screening assay with a response below the screening assay cutpoint were considered negative, and responses equal to or greater than the screening assay cutpoint were then analyzed in the confirmatory assay. Samples tested in the confirmatory assay with a % inhibition result equal to or greater than the confirmatory assay cutpoint were considered positive for the presence of anti-denosumab antibodies.

[0376] Generation of biotinylated and ruthenylated denosumab molecules. Denosumab molecules were labeled with biotin (Thermo Scientific, #A39257) or ruthenium (MSD, #R91AO). The process included an initial buffer exchange to remove free label from the conjugated AMG 162 molecules, a conjugation reaction, and a post-conjugation buffer exchange.

[0377] Generation of an antibody against human RANKL. An anti-RANKL monoclonal antibody containing a murine IgG1 Fc was recombinantly expressed from HEK293-6E cells. This antibody is referred to as "anti-RANKL mAb" in Examples 3 to 6 below. FACS and ELISA assays using soluble RANKL confirmed that this anti-RANKL mAb binds to human RANKL. Binning assays showed that this anti-RANKL mAb binds to distinct epitope bins and does not compete with denosumab or OPG for RANKL binding.

[0378] Example 2B. Screening and selection of anti-RANKL molecules and RANKL controls Pooled normal human serum (PNHS) was spiked with low (10 ng / mL), medium (100 ng / mL), and high (1000 ng / mL) levels of a positive control rabbit anti-denosumab polyclonal antibody (control anti-drug antibody, or "ADA" for short). These control ADA samples were then evaluated in five different ways (using five different reaction buffers): 1) conjugate / neutralization mixture (untreated), 2) conjugate / neutralization mixture + denosumab (confirmatory assay), 3) conjugate / neutralization mixture + mouse anti-RANKL mAb (anti-RANKL1 treated), 4) conjugate / neutralization mixture + OPG (anti-RANKL2 treated), and 5) conjugate / neutralization mixture + mouse anti-RANKL mAb (anti-RANKL3 treated). Three different RANKL (1250 pg / mL) controls were also prepared (RC-1, RC-2, and RC-3) and included in the experiment for evaluation and selection of the best candidate to serve as a RANKL control. The evaluation helped to screen and select the optimal anti-RANKL treatment that inhibits specific target (RANKL) signaling while not affecting specific ADA signaling.

[0379] The results of the experiment are shown in Table 9. When the untreated conjugate / neutralization mixture was used, an increased (untreated) S / N ratio was observed with increasing ADA concentration. Various results were observed from the three different RANKL controls (RCs): RC-1 produced results near the assay cutpoint, RC-2 produced a weak response that was assay-negative, and RC-3 produced a strong response that was assay-positive. Based on the results produced by the three RANKL lots, RC-3 was selected for use as the target control. Results from a second evaluation in which the conjugate / neutralization mixture plus denosumab (ADA and target inhibition control) was used showed the expected inhibition (> / = 60%) of both the specific ADA signal and the specific target signal, indicating that the control condition performed as expected. Furthermore, results from the assay in which the conjugate / neutralization mixture plus anti-RANKL3 was used showed minimal inhibition of the specific ADA signal, indicating that the use of this anti-RANKL molecule does not affect the specific ADA signal. Because anti-RANKL3 treatment did not affect specific ADA signaling but inhibited specific target signaling, the anti-RANKL3 mouse antibody was considered to be the preferred agent among the three anti-RANKL treatment agents tested in this experiment.

[0380] [Table 2]

[0381] Example 3. Evaluation of the effect of anti-RANKL antibodies on specific ADA signals Pooled normal human serum (PNHS) was spiked with low (10 ng / mL), medium (100 ng / mL), and high (1000 ng / mL) levels of a positive control rabbit anti-denosumab polyclonal antibody (anti-drug antibody, or ADA). These ADA samples were then evaluated in three different ways (using three different reaction buffers): 1) conjugate / neutralization mixture (untreated), 2) conjugate / neutralization mixture plus anti-RANKL mAb (anti-RANKL mAb-treated), and 3) conjugate / neutralization mixture plus anti-RANKL mAb and denosumab (denosumab + anti-RANKL mAb-treated, confirmatory assay). A human RANKL (1250 pg / mL) control was also prepared and included in the experiment to confirm the effectiveness of the spiked anti-RANKL mAb in inhibiting the signal generated by RANKL (the target). Results generated by the RANKL control demonstrated that the assay worked as expected (data not shown). The results of the experiment are shown in Table 1A. When the untreated conjugate / neutralization mixture was used, an increased S / N ratio was observed with increasing ADA concentration. Results from assays in which the conjugate / neutralization mixture plus anti-RANKL mAb was used showed minimal inhibition (<20%) of the specific anti-denosumab ADA signal, indicating that the use of anti-RANKL mAb in the assay does not affect the specific anti-denosumab ADA signal. Results from assays in which the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab was used showed a greater than 20% inhibition of the S / N ratio.

[0382] [Table 3]

[0383] Additional experiments were performed in which six different positive control anti-denosumab antibodies, including polyclonal (pAb) and additional mouse and rat monoclonal antibodies, were added to the PNHS at low (10 ng / mL), medium (100 ng / mL), and high (1000 ng / mL) levels. These ADA samples were then evaluated in two different ways (using two different reaction buffers): 1) the conjugate / neutralization mixture (untreated), and 2) the conjugate / neutralization mixture plus anti-RANKL mAb (anti-RANKL mAb-treated). A human RANKL (1250 pg / mL) control was also prepared and included in the experiment to confirm the effectiveness of the added anti-RANKL mAb in inhibiting the signal generated by RANKL (the target). The results generated by the RANKL control demonstrated that the assay performed as expected (data not shown). The results of the experiment are shown in Table 1B. When using the untreated conjugate / neutralization mixture, increased S / N results with increasing ADA concentration were observed. Results from assays in which the conjugate / neutralization mix plus anti-RANKL mAb was used showed minimal inhibition (<20%) of the specific anti-denosumab ADA signal, indicating that the use of anti-RANKL mAb in the assay does not affect the specific anti-denosumab ADA signal.

[0384] [Table 4]

[0385] These results further demonstrated that the detection of ADA was not affected when anti-RANKL mAb was incorporated into the assay.

[0386] Example 4. Evaluation of the effect of anti-RANKL antibodies on specific RANKL signals PNHS were spiked with human RANKL at various levels, starting with low (1250 pg / mL), medium (2500 pg / mL), and high (5000 pg / mL) levels previously shown to generate low positivity. The spiked samples were then evaluated in three different ways (using three different reaction buffers): 1) conjugate / neutralization mixture (untreated), 2) conjugate / neutralization mixture plus anti-RANKL mAb, and 3) conjugate / neutralization mixture plus anti-RANKL mAb and denosumab (confirmatory assay).

[0387] The results of the experiment are shown in Table 2. When the untreated conjugate / neutralization mixture was used, an increased S / N ratio was observed with increasing RANKL concentration. Results from experiments in which the conjugate / neutralization mixture plus anti-RANKL mAb was used showed inhibition of the specific RANKL signal (>20%) at all concentrations tested, indicating that the use of anti-RANKL mAb in the assay inhibits the signal generated by the target. Results from assays in which the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab was used also showed a greater than 20% inhibition of the S / N ratio.

[0388] [Table 5]

[0389] These combined results indicated that the detection of RANKL was inhibited when anti-RANKL mAb was incorporated into the assay.

[0390] Example 5. Evaluation of the effect of anti-RANKL antibodies on samples containing both ADA and RANKL To simulate test samples that may contain both ADA and RANKL, PNHS was spiked with low (10 ng / mL) or medium (100 ng / mL) concentrations of ADA and low (1250 pg / mL), medium (2500 pg / mL), and high (5000 pg / mL) concentrations of RANKL. The combined samples were then evaluated in three different ways (using three different reaction buffers): 1) conjugate / neutralization mixture (untreated), 2) conjugate / neutralization mixture plus anti-RANKL mAb, and 3) conjugate / neutralization mixture plus anti-RANKL mAb and denosumab (confirmatory assay).

[0391] The results of the experiments are shown in Table 3A. When using the untreated conjugate / neutralization mixture, increasing S / N values ​​with increasing ADA and / or RANKL concentrations were observed. Results from experiments using the conjugate / neutralization mixture plus anti-RANKL mAb showed that some of the signal was inhibited, indicating that the specific ADA signal was not inhibited. Results from assays using the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab showed signal inhibition (>20%) at all concentrations tested.

[0392] [Table 6]

[0393] Table 3B shows the S / N results for the combination samples compared to those for samples containing only ADA (no RANKL) at the same concentrations. At both ADA concentrations (and increasing RANKL concentrations), an increase in S / N was observed. Results from experiments using the untreated conjugate / neutralization mix plus anti-RANKL mAb showed S / N results consistent with those produced by the ADA-only sample at the same concentrations, indicating that the anti-RANKL mAb successfully inhibited the RANKL-specific signal without affecting the specific ADA signal.

[0394] [Table 7]

[0395] These results indicated that the detection of ADA was not affected, but that of RANKL was inhibited when anti-RANKL mAb was incorporated into the assay.

[0396] Because denosumab binds to human RANKL with high affinity, soluble RANKL that may be present in human serum samples may interfere (causing false-negative results) or cross-react (causing false-positive results in the assay). To minimize possible interference or cross-reactivity by RANKL, a human-mouse chimeric anti-RANKL monoclonal antibody was incorporated into the assay, which competes with denosumab for binding to RANKL.

[0397] Example 6. Characterization of anti-RANKL antibodies ELISA assay. Soluble RANKL (sRANKL) was coated onto a Costar 3368 medium-binding 96-well plate at 10 μg / ml in 1× PBS / 0.05% azide (50 μl / well), and the samples were incubated overnight at 4°C. The plate was washed using a Titertek™ with reverse osmosis (RO) water for three wash cycles, then blocked with 250 μL of 1× PBS / 1% milk for three wash cycles, incubated at RT for at least 30 minutes, and then washed again using a Titertek™ with RO water for three wash cycles. The primary antibody was a purified mAb clone, titrated 1:3 from 1 μg / ml of 1× PBS / 1% milk (50 μL / well), and incubated at room temperature for 1 hour. Positive control 1 (dmab-IgG1) was a modified denosumab in which the original IgG2 Fc region of denosumab was replaced with the IgG1 Fc region. The IgG1 Fc region was more convenient for detection. Positive control 2 was OPG-Fc titrated 1:3 from 1 μg / ml. After incubation with the primary antibody, the plate was washed using a Titertek filter and three wash cycles with RO water, then incubated with the secondary antibody. The secondary antibody was goat anti-human IgG Fc (cross-adsorbed)-HRP (Thermo Fisher, 31413) at 0.4 μg / ml in 1x PBS / 1% milk (50 μL / well) and incubated for 1 hour at room temperature. Samples were then washed using a Titertek filter and three wash cycles with RO water. The substrate was treated with one step TMB (Neogen, 308177) (50 μl / well) and quenched with 1N HCl (50 μl / well) after 30 minutes at room temperature. Plates were read at 450 nm, and the results are shown in Table 4.

[0398] [Table 8]

[0399] FACS assay. The standard FACS protocol is as follows: Ren 218.9 stable cells (CHO / RANKL) were counted and seeded at 50,000 cells / well in a final volume of 50 μl / well in FACS buffer (1× PBS + 2% fetal bovine serum). Cells and primary antibodies at 5 μg / mL and 0.25 μg / mL were incubated for 1 hour at 4°C, followed by two washes with 200 μl / well of FACS buffer. Secondary antibodies mixed with dead cell stain 7-aminoactinomycin D (7AAD) (Sigma, A9400-5MG) were then added at 100 μl / well. Goat anti-human IgG Cy5 secondary antibody (Jackson, 109-175-098) and 7AAD, both at 5 μg / mL, were incubated with the samples for 15 minutes at 4°C, followed by one wash with 200 μl / well of FACS buffer. The cells were then resuspended in 100 ul of FACS buffer and read by flow cytometry using a high throughput screening (HTS) head.

[0400] [Table 9]

[0401] Additional ELISA assays using human, mouse, and rat soluble RANKL were performed using an ELISA protocol nearly identical to the above ELISA assay, except that human, mouse, and rat soluble RANKL were photobiotinylated (pb) and captured on neutravidin (NA). Tables 6-7 show the results of the ELISA assays using human, mouse, and rat RANKL.

[0402] [Table 10]

[0403] [Table 11]

[0404] Table 8 summarizes the characteristics of certain anti-RANKL antibodies disclosed herein.

[0405] [Table 12]

[0406] All references, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference in its entirety and set forth herein.

[0407] The use of the terms "a," "an," and "the," and similar referents in relation to the description of this disclosure (particularly with respect to the claims that follow) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprise," "have," "include," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise specified.

[0408] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each of the separate values ​​within that range, and each of the endpoints, unless otherwise indicated herein, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0409] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or representative language (e.g., "etc.") provided herein is intended merely to further clarify the disclosure and does not impose limitations on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0410] Preferred embodiments of the disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as appropriate, and the inventors intend the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or clearly contradicted by context.

[0411] [Table 13]

[0412] [Table 14]

[0413] [Table 15]

[0414] [Table 16]

[0415] [Table 17]

[0416] [Table 18]

[0417] [Table 19]

[0418] Table 20

[0419] Table 21

[0420] Table 22

[0421] Table 23

[0422] Table 24

[0423] Table 25

[0424] Table 26

[0425] Table 27

Claims

1. 1. A method for detecting anti-drug antibodies (ADA) in a sample, wherein the ADA binds to denosumab, the method comprising: (a) a first antibody or antibody conjugate that binds to RANKL, the first antibody or antibody conjugate comprising a heavy chain (HC) CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 208, and capable of being bound to a solid substrate; (b) (i) a second antibody that binds to RANKL, the second antibody comprising an amino acid sequence that differs from the first antibody by at least one amino acid in one of the six CDR sequences, or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; and (c) a third antibody that binds to the ADA and can be linked to a detectable marker. The method of claim 1, further comprising mixing the

2. 2. The method of claim 1, wherein the first antibody conjugate comprises an antibody molecule non-covalently or covalently linked to an immobilization moiety, the solid substrate comprises a binding partner, and the immobilization moiety can be bound to the solid substrate by binding to the binding partner.

3. 3. The method of claim 1 or 2, wherein the first antibody or antibody conjugate comprises CDR-H1 comprising SEQ ID NO:200, CDR-H2 comprising SEQ ID NO:201, and CDR-H3 comprising SEQ ID NO:202, and CDR-L1 comprising SEQ ID NO:205, CDR-L2 comprising SEQ ID NO:206, and CDR-L3 comprising SEQ ID NO:

207.

4. 4. The method of any one of claims 1 to 3, wherein the first antibody or antibody conjugate comprises a heavy chain variable region (VH) that is at least 90% identical to SEQ ID NO: 203 and a light chain variable region (VL) that is at least 90% identical to SEQ ID NO:

208.

5. The method of any one of claims 1 to 4, wherein the first antibody or antibody conjugate comprises a human heavy chain constant domain (CH) or a murine heavy chain constant domain (CH).

6. The method of any one of claims 1 to 5, wherein the first antibody or antibody conjugate comprises a human Fc domain or a mouse Fc domain.

7. The method of any one of claims 1 to 6, wherein the first antibody or antibody conjugate comprises a human light chain constant (CL) domain or a murine light chain constant (CL) domain.

8. The method of any one of claims 1 to 7, wherein the third antibody is covalently or non-covalently linked to a detectable marker.

9. The method of any one of claims 1 to 8, wherein the detectable marker is an isotope, an enzyme, a fluorescent moiety, a luminescent moiety, a chromogenic moiety, a metal, or an electric charge.

10. The method of any one of claims 1 to 9, wherein the third antibody comprises six CDR sequences identical to those of the first antibody.

11. The method of any one of claims 1 to 10, wherein the third antibody comprises a VH domain comprising the same sequence as the VH of the first antibody and a VL domain comprising the same sequence as the VL domain of the first antibody.

12. The method of any one of claims 1 to 11, wherein the third antibody comprises a heavy chain (HC) comprising the same sequence as the HC of the first antibody and a light chain (LC) comprising the same sequence as the LC of the first antibody.

13. The method of any one of claims 1 to 12, wherein the second antibody binds to the same epitope as denosumab.

14. The method of any one of claims 1 to 13, wherein the second antibody competes with denosumab for RANKL binding.

15. The method of any one of claims 1 to 14, wherein the second antibody comprises an amino acid sequence that differs from the first antibody by at least one amino acid in each of the six CDR sequences.

16. 16. The method of any one of claims 1 to 15, wherein the second antibody comprises (i) a heavy chain CDR-H1, CDR-H2, and CDR3-H3 of SEQ ID NO: 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167, and (ii) a light chain CDR-L1, CDR-L2, and CDR3 of SEQ ID NO: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, or 168.

17. The method of any one of claims 1 to 16, wherein the second antibody comprises a human heavy chain constant domain (CH) or a murine heavy chain constant domain (CH).

18. The method of any one of claims 1 to 17, wherein the second antibody comprises a human Fc domain or a mouse Fc domain.

19. The method of any one of claims 1 to 18, wherein the second antibody comprises a human CL domain or a mouse CL domain.

20. 1. A diagnostic kit for detecting anti-drug antibodies (ADA) in a sample, wherein the ADA binds to denosumab, the diagnostic kit comprising: (a) a first antibody or antibody conjugate that binds to RANKL, the first antibody or antibody conjugate comprising a heavy chain (HC) CDR-H1, CDR-H2, and CDR-H3 of SEQ ID NO: 203 and a light chain (LC) CDR-L1, CDR-L2, and CDR-L3 of SEQ ID NO: 208, and capable of being bound to a solid substrate; (b) (i) a second antibody that binds to RANKL, the second antibody comprising an amino acid sequence that differs from the first antibody by at least one amino acid in one of the six CDR sequences, or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; and (c) a third antibody that binds to the ADA and can be linked to a detectable marker; and (d) Instructions for use A diagnostic kit comprising:

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