Anti-drug antibody assay

Abstract

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

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 373,426, filed on August 24, 2022, under 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety.

[0003] Incorporation by Reference of Electronically Submitted Materials

[0004] Incorporated by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing filed concurrently with this document and identified as follows: 280,159-byte XML file named "10200-US01-PRI_Sequence_listing", created on August 24, 2022. Technical Field

[0005] The present invention relates to methods for detecting anti-drug antibodies and kits for using such assays. Background Art

[0006] Assays for detecting anti-drug antibodies (ADA) help understand potential immune responses to biopharmaceutical candidates. Determining the presence of ADA and evaluating its clinical impact is an essential part of any large molecule development program.

[0007] All biopharmaceuticals are potentially immunogenic and may induce ADA. The clinical impact of ADA formation can be highly variable and may result in serious adverse events that put patients at risk. Therefore, it is important to develop and validate ADA assays with appropriate sensitivity, specificity, and selectivity for detection 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 can mediate hypersensitivity responses or have the ability to interfere with the interaction between the therapeutic and its target to produce a neutralizing effect.

[0008] In 2019, the FDA issued an industry guidance titled “Immunogenicity Testing of Therapeutic Protein Products–Developing and Validating Assays for Anti-Drug Antibody Detection.” The guidance provides recommendations to facilitate industry development and validation of assays for evaluating the immunogenicity of therapeutic protein products during clinical trials.

[0009] There is a need to develop ADA assays that address issues of accuracy, target interference, and simultaneously meet the requirements of the 2019 FDA guidance. Summary of the invention

[0010] Based on the disclosure provided herein, those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following Example (E).

[0011] E1. A method for detecting anti-drug antibodies (ADA) in a sample, wherein the ADA binds to denosumab, the method comprising mixing the sample with:

[0012] (a) a first antibody or antibody conjugate that binds to RANKL, comprising heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208; wherein the first antibody or antibody conjugate can be attached to a solid substrate;

[0013] (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 the six CDR sequences; or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; and

[0014] (c) a third antibody that binds to the ADA and can be linked to a detectable marker.

[0015] E2. The method of E1, wherein the sample comprises soluble RANKL that interferes with the detection of the ADA.

[0016] E3. A method of reducing false signals in an anti-drug antibody (ADA) assay, wherein the ADA assay detects the presence of anti-denosumab antibodies in a sample, the method comprising mixing the sample with:

[0017] (a) a first antibody or antibody conjugate that binds to RANKL, comprising heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208; wherein the first antibody or antibody conjugate can be attached to a solid substrate;

[0018] (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 the six CDR sequences; or (ii) an osteoprotegerin (OPG) protein that binds to RANKL; and

[0019] (c) a third antibody that binds to the ADA and can be linked to a detectable marker.

[0020] E4. A method as described in E3, wherein the false signal is caused by the presence of soluble RANKL that interferes with the detection of the ADA.

[0021] E5. The method of any one of E1-E4, wherein the first antibody conjugate comprises an antibody molecule covalently linked to a fixed moiety.

[0022] E6. The method of any one of E1-E4, wherein the first antibody conjugate comprises an antibody molecule non-covalently linked to a fixed moiety.

[0023] E7. The method of E5 or E6, wherein the solid substrate comprises a binding partner, and the immobilization moiety can be attached to the solid substrate by binding to the binding partner.

[0024] E8. A method as described in E7, wherein the fixed part is biotin and the binding partner is streptavidin or avidin; or the fixed part is avidin or streptavidin and the binding partner is biotin.

[0025] E9. A method as described in E7, wherein the fixed portion 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 of the enzyme, IgG and protein A, IgG and protein G, or a ligand and a receptor for the ligand.

[0026] E10. The method of any one of E1-E4, wherein the first antibody is immobilized on a solid matrix comprising protein A or protein G.

[0027] E11. A method as described in any of E1-E10, wherein the first antibody or antibody conjugate comprises a CDR-H1 comprising SEQ ID NO: 200, a CDR-H2 comprising SEQ ID NO: 201, and a CDR-H3 comprising SEQ ID NO: 202, and a CDR-L1 comprising SEQ ID NO: 205, a CDR-L2 comprising SEQ ID NO: 206, and a CDR-L3 comprising SEQ ID NO: 207.

[0028] E12. A method as described in any 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.

[0029] E13. A method as described in any 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.

[0030] 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.

[0031] E15. A method as described in E14, wherein the 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.

[0032] E16. The method of E14, wherein:

[0033] the human IgG2 CH1 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 the CH1 domain of SEQ ID NO: 194;

[0034] the human IgG3 CHI 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 the CHI domain of SEQ ID NO: 190; or

[0035] 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.

[0036] E17. The method of any one of E1-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.

[0037] E18. A method as described in E17, wherein the 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.

[0038] E19. The method of E17, wherein:

[0039] the human IgG2 Fc 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: 196;

[0040] the human IgG3 Fc 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: 192; or

[0041] 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.

[0042] E20. The method of any one of E1-E13, wherein the first antibody or antibody conjugate comprises a murine CH1 domain, such as murine IgG1 CH1, murine IgG2a CH1, murine IgG2b CH1, or murine IgG3 CH1.

[0043] E21. A method as described in E20, wherein the 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.

[0044] E22. A method as described in E20, wherein the 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.

[0045] E23. A method as described in E20, 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.

[0046] E24. A method as described in E20, wherein the 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.

[0047] E25. The method of any one of E1-E13 and E20-E24, wherein the first antibody or antibody conjugate comprises a murine Fc domain, such as murine IgG1 Fc, murine IgG2a Fc, murine IgG2b Fc, or murine IgG3 Fc.

[0048] E26. A method as described in E25, 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.

[0049] E27. A method as described in E25, 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.

[0050] E28. A method as described in E25, 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.

[0051] E29. A method as described in E25, 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.

[0052] E30. The method of any one of E1-E29, wherein the first antibody or antibody conjugate comprises a human CL domain, such as human κCL or human λCL.

[0053] E31. A method as described in E30, wherein the human κCL 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.

[0054] E32. A method as described in E30, wherein the human λCL 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.

[0055] E33. The method of any one of E1-E29, wherein the first antibody or antibody conjugate comprises a murine CL domain, such as murine κCL or murine λCL.

[0056] E34. A method as described in E33, wherein the mouse κCL 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.

[0057] E35. A method as described in E33, wherein the mouse λCL 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.

[0058] E36. The method of any one of E1-E35, wherein the third antibody is covalently linked to a detectable marker.

[0059] E37. The method of any one of E1-E35, wherein the third antibody is non-covalently linked to a detectable marker.

[0060] 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 colored moiety, a metal or a charge.

[0061] E39. The method of any one of E1-E38, wherein the third antibody comprises six CDR sequences that are identical to the six CDR sequences of the first antibody.

[0062] E40. The method of any one of E1-E39, wherein the third antibody comprises:

[0063] (i) the heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203, and the light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208; or

[0064] (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.

[0065] E41. The method of any one of E1-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.

[0066] 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.

[0067] E43. The method of any one of E1-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.

[0068] E44. A method as described in E43, wherein the 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.

[0069] E45. The method of E43, wherein:

[0070] the human IgG2 CH1 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: 194;

[0071] the human IgG3 CH1 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: 190; or

[0072] 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.

[0073] E46. The method of any one of E1-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.

[0074] E47. A method as described in E46, wherein the 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.

[0075] E48. The method of E46, wherein

[0076] the human IgG2 Fc 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: 196;

[0077] the human IgG3 Fc 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: 192; or

[0078] 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.

[0079] E49. The method of any one of E1-E42, wherein the third antibody comprises a murine CH1 domain, such as murine IgG1 CH1, murine IgG2a CH1, murine IgG2b CH1, or murine IgG3 CH1.

[0080] E50. A method as described in E49, wherein the 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.

[0081] E51. A method as described in E49, wherein the 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.

[0082] E52. A method as described in E49, 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.

[0083] E53. A method as described in E49, wherein the 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.

[0084] E54. The method of any one of E1-E42 and E49-E53, wherein the third antibody comprises a murine Fc domain, such as murine IgG1 Fc, murine IgG2a Fc, murine IgG2b Fc or murine IgG3 Fc.

[0085] E55. A method as described in E54, 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.

[0086] E56. A method as described in E54, 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.

[0087] E57. A method as described in E54, 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.

[0088] E58. A method as described in E54, 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.

[0089] E59. The method of any one of E1-E58, wherein the third antibody comprises a human CL domain, such as human κCL or human λCL.

[0090] E60. A method as described in E59, wherein the human κCL 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.

[0091] E61. A method as described in E59, wherein the human λCL 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.

[0092] E62. The method of any one of E1-E58, wherein the third antibody comprises a murine CL domain, such as murine κCL or murine λCL.

[0093] E63. A method as described in E62, wherein the mouse κCL 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.

[0094] E64. A method as described in E62, wherein the mouse λCL 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.

[0095] E65. The method of any one of E1-E64, wherein the third antibody comprises a VH domain and a VL domain, the VH domain comprising the same sequence as the VH of the first antibody, and the VL domain comprising the same sequence as the VL domain of the first antibody.

[0096] E66. The method of any one of E1-E65, wherein the third antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising the same sequence as the HC of the first antibody, and the LC comprising the same sequence as the LC of the first antibody.

[0097] 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.

[0098] 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.

[0099] E69. The method of any one of E1-E66, wherein the third antibody is covalently linked to a fluorescent moiety, a luminescent moiety, or a colored moiety.

[0100] E70. The method of any one of E1-E69, wherein the second antibody binds to the same epitope as denosumab.

[0101] E71. The method of any one of E1-E70, wherein the second antibody competes with denosumab for binding to RANKL.

[0102] 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 the six CDR sequences.

[0103] E73. The method of any one of E1-E72, wherein the second antibody comprises an amino acid sequence that differs from the first antibody in CDR sequences by at least two, at least three, at least four, at least five, at least six amino acids.

[0104] E74. The method of any one of E1-E73, wherein the ADA does not bind to the second antibody.

[0105] E75. The method of any one of E1-E74, 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.

[0106] E76. The method of any one of E1-E75, wherein the second antibody comprises:

[0107] (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;

[0108] (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;

[0109] (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;

[0110] (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;

[0111] (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; and

[0112] (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.

[0113] E77. The method of any one of E1-E76, wherein the second antibody comprises:

[0114] (1) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 1-6;

[0115] (2) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 6-12;

[0116] (3) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 13-18;

[0117] (4) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 19-24;

[0118] (5) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 25-30;

[0119] (6) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 31-36;

[0120] (7) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 37-42;

[0121] (8) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 43-48;

[0122] (9) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 49-54;

[0123] (10) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 55-60;

[0124] (11) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 61-66;

[0125] (12) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 67-72;

[0126] (13) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 73-78;

[0127] (14) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 79-84;

[0128] (15) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 85-90;

[0129] (16) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 91-96;

[0130] (17) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 97-102;

[0131] (18) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 103-108;

[0132] (19) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 109-114;

[0133] (20) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 115-120; or

[0134] (21) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 121-126.

[0135] E78. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 1-6, respectively.

[0136] E79. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 6-12, respectively.

[0137] E80. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 13-18, respectively.

[0138] E81. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 19-24, respectively.

[0139] E82. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 25-30, respectively.

[0140] E83. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 31-36, respectively.

[0141] E84. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 37-42, respectively.

[0142] E85. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 43-48, respectively.

[0143] E86. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 49-54, respectively.

[0144] E87. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 55-60, respectively.

[0145] E88. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 61-66, respectively.

[0146] E89. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 67-72, respectively.

[0147] E90. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 73-78, respectively.

[0148] E91. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 79-84, respectively.

[0149] E92. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 85-90, respectively.

[0150] E93. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 91-96, respectively.

[0151] E94. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 97-102, respectively.

[0152] E95. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 103-108, respectively.

[0153] E96. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 109-114, respectively.

[0154] E97. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 115-120, respectively.

[0155] E98. The method of E77, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 121-126, respectively.

[0156] E99. A method as described in any of E1-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.

[0157] E100. The method of any one of E1-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.

[0158] E101. The method of any one of E1-E100, wherein the second antibody comprises:

[0159] (1) a VH and a VL, the 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 the 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;

[0160] (2) a VH and a VL, the 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 the 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;

[0161] (3) a VH and a VL, the 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 the 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;

[0162] (4) a VH and a VL, the 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 the 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;

[0163] (5) a VH and a VL, the 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 the 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;

[0164] (6) a VH and a VL, the 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 the 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;

[0165] (7) a VH and a VL, the 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 the 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;

[0166] (8) a VH and a VL, the 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 the 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;

[0167] (9) a VH and a VL, the 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 the 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;

[0168] (10) a VH and a VL, the 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 the 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;

[0169] (11) a VH and a VL, the 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 the 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;

[0170] (12) a VH and a VL, the 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 the 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;

[0171] (13) a VH and a VL, the 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 the 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;

[0172] (14) a VH and a VL, the 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 the 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;

[0173] (15) a VH and a VL, the 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 the 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;

[0174] (16) a VH and a VL, the 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 the 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;

[0175] (17) a VH and a VL, the 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 the 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;

[0176] (18) a VH and a VL, the 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 the 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;

[0177] (19) a VH and a VL, the 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 the 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;

[0178] (20) a VH and a VL, the 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 the 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

[0179] (21) VH and VL, wherein the VH comprises 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 the VL comprises 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.

[0180] E102. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0181] E103. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0182] E104. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0183] E105. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0184] E106. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0185] E107. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0186] E108. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0187] E109. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0188] E110. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0189] E111. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0190] E112. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0191] E113. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0192] E114. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0193] E115. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0194] E116. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0195] E117. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0196] E118. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0197] E119. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0198] E120. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0199] E121. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0200] E122. A method as described in E101, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0201] E123. The method of any one of E1-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.

[0202] E124. A method as described in E123, wherein the 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] E125. The method of E123, wherein:

[0204] the human IgG2 CH1 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: 194;

[0205] the human IgG3 CH1 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: 190; or

[0206] 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.

[0207] E126. The method of any one of E1-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.

[0208] E127. A method as described in E126, wherein the 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.

[0209] E128. The method of E126, wherein

[0210] the human IgG2 Fc 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: 196;

[0211] the human IgG3 Fc 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: 192; or

[0212] 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.

[0213] E129. The method of any one of E1-E122, wherein the second antibody comprises a murine CH1 domain, such as murine IgG1 CH1, murine IgG2a CH1, murine IgG2b CH1, or murine IgG3 CH1.

[0214] E130. A method as described in E129, wherein the 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.

[0215] E131. A method as described in E129, wherein the 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.

[0216] E132. A method as described in E129, 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.

[0217] E133. A method as described in E129, wherein the 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.

[0218] E134. The method of any one of E1-E122 and E129-E133, wherein the second antibody comprises a murine Fc domain, such as murine IgG1 Fc, murine IgG2a Fc, murine IgG2b Fc, or murine IgG3 Fc.

[0219] E135. A method as described in E134, 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.

[0220] E136. A method as described in E134, 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.

[0221] E137. A method as described in E134, 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.

[0222] E138. A method as described in E134, 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.

[0223] E139. The method of any one of E1-E138, wherein the second antibody comprises a human CL domain, such as human κCL or human λCL.

[0224] E140. A method as described in E139, wherein the human κCL 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.

[0225] E141. A method as described in E139, wherein the human λCL 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.

[0226] E142. The method of any one of E1-E138, wherein the second antibody comprises a murine CL domain, such as murine κCL or murine λCL.

[0227] E143. A method as described in E142, wherein the mouse κCL 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.

[0228] E144. A method as described in E142, wherein the mouse λCL 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.

[0229] E145. The method as described in any one of E1-E144, wherein 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 KD values ​​of M or less bind to RANKL.

[0230] E146. The method of any one of E1-E145, wherein the sample is pre-treated with the second antibody when mixed with the first antibody or antibody conjugate and the third antibody.

[0231] E147. The method of any one of E1-E145, wherein the sample is pre-treated with the OPG when mixed with the first antibody or antibody conjugate and the third antibody.

[0232] E148. The method of any one of E1-E145, wherein the sample is mixed simultaneously with the first antibody or antibody conjugate, the second antibody and the third antibody.

[0233] E149. The method of any one of E1-E145, wherein the sample is mixed simultaneously with the first antibody or antibody conjugate, the OPG and the third antibody complex.

[0234] E150. The method of any one of E1-E149, wherein the RANKL is human RANKL.

[0235] E151. The method of any one of E1-E150, wherein both the first antibody and the second antibody bind to a human RANKL epitope (DLATE) comprising SEQ ID NO:214.

[0236] E152. The method of any one of E1-E150, wherein both the first antibody and the second antibody bind to an epitope of human RANKL comprising one or more residues from SEQ ID NO:215.

[0237] E153. A diagnostic kit for detecting anti-drug antibodies (ADA) in a sample, wherein the ADA binds to denosumab, the diagnostic kit comprising:

[0238] (a) a first antibody or antibody conjugate that binds to RANKL, comprising heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208; wherein the first antibody or antibody conjugate can be attached to a solid substrate;

[0239] (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 the six CDR sequences; or (ii) an osteoprotegerin (OPG) protein that binds to RANKL;

[0240] (c) a third antibody that binds to the ADA and can be linked to a detectable marker; and

[0241] (d) Instructions for use.

[0242] E154. The diagnostic kit of E153, wherein the first antibody conjugate comprises an antibody molecule covalently linked to an immobilization moiety.

[0243] E155. The diagnostic kit of E153 or E154, wherein the first antibody conjugate comprises an antibody molecule non-covalently linked to an immobilization moiety.

[0244] E156. The diagnostic kit of E154 or E155, wherein said solid substrate comprises a binding partner, and said immobilization moiety can be attached to the solid substrate by binding to said binding partner.

[0245] E157. A diagnostic kit as described in E156, wherein the immobilization part is biotin and the binding partner is streptavidin or avidin; or the immobilization part is avidin or streptavidin and the binding partner is biotin.

[0246] E158. A diagnostic kit as described in E156, wherein the fixed part and the binding partner are one of the following binding pairs: an antigen and an antibody bound 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 of the enzyme, IgG and protein A, IgG and protein G, or a ligand and a receptor for the ligand.

[0247] E159. The diagnostic kit as described in E153, wherein the first antibody is immobilized on a solid matrix comprising protein A or protein G.

[0248] E160. A diagnostic kit as described in any one of E153-E159, wherein the first antibody or antibody conjugate comprises a CDR-H1 comprising SEQ ID NO: 200, a CDR-H2 comprising SEQ ID NO: 201, and a CDR-H3 comprising SEQ ID NO: 202, and a CDR-L1 comprising SEQ ID NO: 205, a CDR-L2 comprising SEQ ID NO: 206, and a CDR-L3 comprising SEQ ID NO: 207.

[0249] E161. A diagnostic kit as described in any of E153-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.

[0250] E162. A diagnostic kit as described in any of E153-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.

[0251] E163. The diagnostic kit of any one of E153-E162, 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.

[0252] E164. A diagnostic kit as described in E163, wherein the 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.

[0253] E165. The diagnostic kit as described in E163, wherein:

[0254] the human IgG2 CH1 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 the CH1 domain of SEQ ID NO: 194;

[0255] the human IgG3 CHI 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 the CHI domain of SEQ ID NO: 190; or

[0256] 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.

[0257] E166. The diagnostic kit of any one of E153-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.

[0258] E167. A diagnostic kit as described in E166, wherein the 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.

[0259] E168. The diagnostic kit as described in E166, wherein:

[0260] the human IgG2 Fc 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: 196;

[0261] the human IgG3 Fc 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: 192; or

[0262] 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.

[0263] E169. The diagnostic kit of any one of E153-E162, wherein the first antibody or antibody conjugate comprises a murine CH1 domain, such as murine IgG1 CH1, murine IgG2a CH1, murine IgG2b CH1 or murine IgG3 CH1.

[0264] E170. A diagnostic kit as described in E169, wherein the 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.

[0265] E171. A diagnostic kit as described in E169, wherein the 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.

[0266] E172. A diagnostic kit as described in 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.

[0267] E173. A diagnostic kit as described in E169, wherein the 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.

[0268] E174. The diagnostic kit of any one of E153-E162 and E169-E173, wherein the first antibody or antibody conjugate comprises a murine Fc domain, such as murine IgG1 Fc, murine IgG2a Fc, murine IgG2b Fc or murine IgG3 Fc.

[0269] E175. A diagnostic kit as described in 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.

[0270] E176. A diagnostic kit as described in 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.

[0271] E177. A diagnostic kit as described in 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.

[0272] E178. A diagnostic kit as described in 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.

[0273] E179. The diagnostic kit of any one of E153-E178, wherein the first antibody or antibody conjugate comprises a human CL domain, such as human κCL or human λCL.

[0274] E180. A diagnostic kit as described in E179, wherein the human κCL 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.

[0275] E181. A diagnostic kit as described in E179, wherein the human λCL 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.

[0276] E182. The diagnostic kit of any one of E153-E178, wherein the first antibody or antibody conjugate comprises a murine CL domain, such as murine κCL or murine λCL.

[0277] E183. A diagnostic kit as described in E182, wherein the mouse κCL 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.

[0278] E184. A diagnostic kit as described in E182, wherein the mouse λCL 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.

[0279] E185. The diagnostic kit of any one of E153-E184, wherein said third antibody is covalently linked to a detectable marker.

[0280] E186. The diagnostic kit of any one of E153-E184, wherein said third antibody is non-covalently linked to a detectable marker.

[0281] E187. A diagnostic kit as described in any of E153-E186, wherein the detectable marker is an isotope, an enzyme, a fluorescent moiety, a luminescent moiety, a colored moiety, a metal or a charge.

[0282] E188. The diagnostic kit of any one of E153-E187, wherein the third antibody comprises six CDR sequences identical to the six CDR sequences of the first antibody.

[0283] E189. The diagnostic kit of any one of E153-E188, wherein the third antibody comprises:

[0284] (i) the heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203, and the light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208; or

[0285] (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.

[0286] E190. A diagnostic kit as described in any of E153-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.

[0287] E191. A diagnostic kit as described in any of E153-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.

[0288] E192. The diagnostic kit of any one of E153-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.

[0289] E193. A diagnostic kit as described in E192, wherein the 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.

[0290] E194. The diagnostic kit as described in E192, wherein:

[0291] the human IgG2 CH1 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: 194;

[0292] the human IgG3 CH1 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: 190; or

[0293] 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.

[0294] E195. The diagnostic kit of any one of E192-E194, wherein said third antibody comprises a human Fc domain, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc.

[0295] E196. A diagnostic kit as described in E195, wherein the 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.

[0296] E197. The diagnostic kit as described in E195, wherein

[0297] the human IgG2 Fc 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: 196;

[0298] the human IgG3 Fc 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: 192; or

[0299] 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.

[0300] E198. The diagnostic kit of any one of E153-E191, wherein the third antibody comprises a murine CH1 domain, such as murine IgG1 CH1, murine IgG2a CH1, murine IgG2b CH1 or murine IgG3 CH1.

[0301] E199. A diagnostic kit as described in E198, wherein the 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.

[0302] E200. The diagnostic kit of E198, wherein the 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.

[0303] E201. A diagnostic kit as described in E198, 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.

[0304] E202. A diagnostic kit as described in E198, wherein the 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.

[0305] E203. The diagnostic kit of any one of E153-E191 and E198-E202, wherein the third antibody comprises a murine Fc domain, such as murine IgG1 Fc, murine IgG2a Fc, murine IgG2b Fc or murine IgG3 Fc.

[0306] E204. A diagnostic kit as described in E203, 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.

[0307] E205. A diagnostic kit as described in 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.

[0308] E206. A diagnostic kit as described in 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.

[0309] E207. A diagnostic kit as described in 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.

[0310] E208. The diagnostic kit of any one of E153-E207, wherein the third antibody comprises a human CL domain, such as human κCL or human λCL.

[0311] E209. A diagnostic kit as described in E208, wherein the human κCL 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.

[0312] E210. A diagnostic kit as described in E208, wherein the human λCL 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.

[0313] E211. The diagnostic kit of any one of E153-E207, wherein the third antibody comprises a murine CL domain, such as murine κCL or murine λCL.

[0314] E212. A diagnostic kit as described in E211, wherein the mouse κCL 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.

[0315] E213. A diagnostic kit as described in E211, wherein the mouse λCL 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.

[0316] E214. The diagnostic kit of any one of E153-E213, wherein the third antibody comprises a VH domain and a VL domain, the VH domain comprising the same sequence as the VH of the first antibody, the VL domain comprising the same sequence as the VL domain of the first antibody.

[0317] E215. A diagnostic kit as described in any one of E153-E214, wherein the third antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising the same sequence as the HC of the first antibody, and the LC comprising the same sequence as the LC of the first antibody.

[0318] E216. The diagnostic kit of any one of E153-E215, wherein the detectable marker comprises a secondary antibody that binds to the constant domain of the third antibody.

[0319] E217. The diagnostic kit of any one of E153-E216, wherein the detectable marker comprises a secondary antibody that binds to the Fc domain of the third antibody.

[0320] E218. The diagnostic kit of any one of E153-E215, wherein the third antibody is covalently linked to a fluorescent moiety, a luminescent moiety or a colored moiety.

[0321] E219. The diagnostic kit of any one of E153-E218, wherein the second antibody binds to the same epitope as denosumab.

[0322] E220. The diagnostic kit of any one of E153-E219, wherein the second antibody competes with denosumab for binding to RANKL.

[0323] E221. The diagnostic kit of any one of E153-E220, 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.

[0324] E222. The diagnostic kit of any one of E153-E221, wherein the second antibody comprises an amino acid sequence that differs from the first antibody in CDR sequences by at least two, at least three, at least four, at least five, at least six amino acids.

[0325] E223. A diagnostic kit as described in any one of E153-E222, wherein the ADA does not bind to the second antibody.

[0326] E224. A diagnostic kit as described in any one of E153-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) heavy chain CDR-H2, CDR-H3 and CDR-H4 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; NO:128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168 light chain CDR-L1, CDR-L2 and CDR-L3.

[0327] E225. The diagnostic kit of any one of E153-E224, wherein the second antibody comprises:

[0328] (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;

[0329] (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;

[0330] (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;

[0331] (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;

[0332] (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; and

[0333] (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.

[0334] E226. A diagnostic kit as described in any one of E153-E225, wherein the second antibody comprises:

[0335] (1) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 1-6;

[0336] (2) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 6-12;

[0337] (3) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 13-18;

[0338] (4) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 19-24;

[0339] (5) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 25-30;

[0340] (6) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 31-36;

[0341] (7) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 37-42;

[0342] (8) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 43-48;

[0343] (9) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 49-54;

[0344] (10) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 55-60;

[0345] (11) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 61-66;

[0346] (12) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 67-72;

[0347] (13) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 73-78;

[0348] (14) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 79-84;

[0349] (15) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 85-90;

[0350] (16) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 91-96;

[0351] (17) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 97-102;

[0352] (18) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 103-108;

[0353] (19) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 109-114;

[0354] (20) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively comprise SEQ ID NOs. 115-120; or

[0355] (21) CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, which respectively contain SEQ ID NOs. 121-126.

[0356] E227. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NO. 1-6, respectively.

[0357] E228. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 6-12, respectively.

[0358] E229. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 13-18, respectively.

[0359] E230. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 19-24, respectively.

[0360] E231. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 25-30, respectively.

[0361] E232. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 31-36, respectively.

[0362] E233. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 37-42, respectively.

[0363] E234. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 43-48, respectively.

[0364] E235. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 49-54, respectively.

[0365] E236. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 55-60, respectively.

[0366] E237. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 61-66, respectively.

[0367] E238. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 67-72, respectively.

[0368] E239. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 73-78, respectively.

[0369] E240. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 79-84, respectively.

[0370] E241. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 85-90, respectively.

[0371] E242. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 91-96, respectively.

[0372] E243. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 97-102, respectively.

[0373] E244. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 103-108, respectively.

[0374] E245. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 109-114, respectively.

[0375] E246. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 115-120, respectively.

[0376] E247. The diagnostic kit of E226, wherein the second antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 comprising SEQ ID NOs. 121-126, respectively.

[0377] E248. A diagnostic kit as described in any of E153-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.

[0378] E249. A diagnostic kit as described in any of E153-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.

[0379] E250. A diagnostic kit as described in any one of E153-E249, wherein the second antibody comprises:

[0380] (1) a VH and a VL, the 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 the 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;

[0381] (2) a VH and a VL, the 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 the 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;

[0382] (3) a VH and a VL, the 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 the 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;

[0383] (4) a VH and a VL, the 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 the 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;

[0384] (5) a VH and a VL, the 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 the 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;

[0385] (6) a VH and a VL, the 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 the 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;

[0386] (7) a VH and a VL, the 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 the 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;

[0387] (8) a VH and a VL, the 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 the 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;

[0388] (9) a VH and a VL, the 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 the 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;

[0389] (10) a VH and a VL, the 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 the 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;

[0390] (11) a VH and a VL, the 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 the 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;

[0391] (12) a VH and a VL, the 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 the 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;

[0392] (13) a VH and a VL, the 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 the 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;

[0393] (14) a VH and a VL, the 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 the 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;

[0394] (15) a VH and a VL, the 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 the 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;

[0395] (16) a VH and a VL, the 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 the 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;

[0396] (17) a VH and a VL, the 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 the 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;

[0397] (18) a VH and a VL, the 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 the 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;

[0398] (19) a VH and a VL, the 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 the 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;

[0399] (20) a VH and a VL, the 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 the 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

[0400] (21) VH and VL, wherein the VH comprises 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 the VL comprises 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.

[0401] E251. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0402] E252. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0403] E253. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0404] E254. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0405] E255. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0406] E256. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0407] E257. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0408] E258. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0409] E259. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0410] E260. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0411] E261. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0412] E262. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0413] E263. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0414] E264. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0415] E265. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0416] E266. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0417] E267. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0418] E268. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0419] E269. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0420] E270. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0421] E271. A diagnostic kit as described in E250, wherein the second antibody comprises a VH and a VL, the 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 the 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.

[0422] E272. The diagnostic kit of any one of E153-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.

[0423] E273. A diagnostic kit as described in E272, wherein the 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.

[0424] E274. The diagnostic kit as described in E272, wherein:

[0425] the human IgG2 CH1 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: 194;

[0426] the human IgG3 CH1 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: 190; or

[0427] 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.

[0428] E275. The diagnostic kit of any one of E153-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.

[0429] E276. A diagnostic kit as described in E275, wherein the 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.

[0430] E277. A diagnostic kit as described in E275, wherein

[0431] the human IgG2 Fc 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: 196;

[0432] the human IgG3 Fc 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: 192; or

[0433] 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.

[0434] E278. The diagnostic kit of any one of E153-E271, wherein the second antibody comprises a murine CH1 domain, such as murine IgG1 CH1, murine IgG2a CH1, murine IgG2b CH1 or murine IgG3 CH1.

[0435] E279. A diagnostic kit as described in E278, wherein the 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.

[0436] E280. A diagnostic kit as described in E278, wherein the 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.

[0437] E281. A diagnostic kit as described in E278, 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.

[0438] E282. A diagnostic kit as described in E278, wherein the 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.

[0439] E283. The diagnostic kit of any one of E153-E271 and E278-E282, wherein the second antibody comprises a murine Fc domain, such as murine IgG1 Fc, murine IgG2a Fc, murine IgG2b Fc or murine IgG3 Fc.

[0440] E284. A diagnostic kit as described in E283, 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.

[0441] E285. A diagnostic kit as described in 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.

[0442] E286. A diagnostic kit as described in 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.

[0443] E287. A diagnostic kit as described in 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.

[0444] E288. The diagnostic kit of any one of E153-E287, wherein the second antibody comprises a human CL domain, such as human κCL or human λCL.

[0445] E289. A diagnostic kit as described in E288, wherein the human κCL 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.

[0446] E290. A diagnostic kit as described in E288, wherein the human λCL 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.

[0447] E291. The diagnostic kit of any one of E153-E287, wherein the second antibody comprises a murine CL domain, such as murine κCL or murine λCL.

[0448] E292. A diagnostic kit as described in E291, wherein the mouse κCL 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.

[0449] E293. A diagnostic kit as described in E291, wherein the mouse λCL 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.

[0450] E294. A kit as described in any one of E153-E293, wherein 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 KD values ​​of M or less bind to RANKL.

[0451] E295. The diagnostic kit of any one of E153-E294, wherein the RANKL is human RANKL.

[0452] E296. A diagnostic kit as described in any one of E153-E295, wherein both the first antibody and the second antibody bind to a human RANKL epitope (DLATE) comprising SEQ ID NO: 214.

[0453] E297. A diagnostic kit as described in any one of E153-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.

[0454] E298. A diagnostic kit for use in the method described in any one of E1-E152. BRIEF DESCRIPTION OF THE DRAWINGS

[0455] Figure 1A An exemplary ADA assay is shown. Figure 1B Soluble RANKL was shown to interfere with the ADA assay.

[0456] Figure 2A and 2B It was demonstrated that a secondary anti-RANKL antibody or OPG could reduce signal interference by sequestering soluble RANKL. DETAILED DESCRIPTION

[0457] 1. Overview of Anti-Drug Antibody (ADA) Assay

[0458] In one aspect, the invention provides a method for detecting anti-drug antibodies (ADA) to denosumab in a sample. Typically, the sample is obtained from a patient who has been treated with denosumab.

[0459] Denosumab (trade name and ) is a human monoclonal antibody targeting RANKL. It 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, the treatment of men with osteoporosis at high risk of fracture to increase bone mass, the treatment of glucocorticoid-induced osteoporosis in men and women at high risk of fracture, the treatment of men at high risk of fracture receiving androgen deprivation therapy for non-metastatic prostate cancer to increase bone mass, and the treatment of women at high risk of fracture receiving adjuvant aromatase inhibitor therapy for breast cancer to increase bone mass. Xgeva is a prescription drug used to prevent fractures, spinal cord compression, or the need for radiation or surgery to the bone in patients with multiple myeloma and patients with bone metastases from solid tumors.

[0460] As taught and exemplified in the Examples, in one study, an abnormally high incidence of immune responses was observed in an anti-drug antibody (ADA) assay for denosumab. Based on past experience with clinical studies of denosumab, this was considered a false positive and was likely due to interference of nuclear factor-κB ligand (RANKL) with the ADA assay ( Figure 1A and Figure 1B ).

[0461] In order to solve the problem of false positive signals caused by soluble RANKL and develop a reliable ADA assay, an exemplary anti-RANKL monoclonal antibody that competes with denosumab for binding to RANKL was incorporated into the improved ADA assay. Because the complementary determining region (CDR) of an antibody is the result of a selection process after random mutations in the germline, the CDR is the domain most likely to be recognized as foreign by the immune system. Therefore, most anti-drug antibodies of monoclonal antibody therapeutics are directed against the CDR. Based on these data, it is believed that to reduce the interference of RANKL ligands in ADA assays, two strategies can be used: (i) using OPG (which is a decoy receptor for RANKL) to isolate soluble RANKL; or (ii) using an antibody that binds to RANKL to isolate RANKL; the antibody should have a CDR sequence different from that of denosumab so that the antibody itself does not bind to ADA ( Figure 2A and Figure 2B ).

[0462] 2. Immunoassay for Detection of ADA

[0463] The most commonly used ADA assay is based on solid phase immunoassay. Standard solid phase immunoassay using monoclonal antibodies involves the formation of a complex between an antibody adsorbed on a solid matrix ("capture" molecule), a target molecule (in this case, ADA for denosumab) and an antibody connected to a detectable marker ("tracer" molecule). Thus, a sandwich is formed: solid matrix-capture molecule-target molecule-tracer molecule. In this sandwich, the amount of the detectable marker is typically proportional to the concentration of ADA in the sample. A sandwich assay is a double antigen bridging immunoassay, in which the capture antibody and the tracer antibody bind to different epitopes of the antigen. Hoesel, W. et al. reported an anti-EPO double antigen bridging assay in J. Immunol. Methods [Journal of Immunology Methods] 294 (2004) 101-110, in which a mixture of immobilized rhEPO coupled to an amino group and to a carbohydrate group was used. Immunoassays (such as double antigen bridging ELISA) are common assay types for studying the immunogenicity response of patients to antibody drugs. Mire-Sluis, AR et al., J. Immunol. Methods 289 (2004) 1-16, summarize recommendations for designing and optimizing immunoassays using detection of host antibodies against biotechnology products.

[0464] Capture molecules

[0465] The "capture" molecule is typically an antibody covalently or non-covalently linked to an immobilized portion, and the solid matrix comprises a binding partner bound to the immobilized portion. The capture molecule can be immobilized on the solid matrix through the interaction between the immobilized portion and its binding partner. The capture molecule is also referred to herein as a "first antibody or antibody conjugate".

[0466] Exemplary binding pairs that can be used as immobilization moieties and their binding partners include, for example (first component-second component): streptavidin-biotin, avidin-biotin, antibody-antigen (see, e.g., Hermanson, GT 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 attached to the surface of a solid substrate.

[0467] In certain embodiments, conjugation of the antibody to the fixed moiety is by covalent conjugation via the N-terminus and / or the ε-amino group (lysine), the ε-amino group of a different lysine, the carboxyl, thiol, hydroxyl and / or phenolic functional groups of the amino acid backbone of the antibody and / or the sugar alcohol groups of the carbohydrate structure of the antibody.

[0468] Since it is believed that ADA is usually caused by CDR sequences, in order to detect ADA against denosumab, the capture molecule should contain an antibody with 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 complementary determining region (CDR-H1, CDR-H2, CDR-H3) and light chain complementary determining region (CDR-L1, CDR-L2, CDR-L3) of denosumab are shown in Table F of the sequence table.

[0469] As used herein, "complementarity determining regions" (CDRs) can be identified according to the cumulative, AbM, contact, North and / or conformational definitions of Kabat, Chothia, both Kabat and Chothia, or any CDR determination method 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 properties of the amino acid residues that make up the CDRs in a particular antibody can be determined using methods well known in the art. The AbM definition of CDRs is a compromise between Kabat and Chothia, and uses the AbM antibody modeling software from Oxford Molecular. The "contact" definition of CDRs is based on observed antigen contacts and is described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. The "conformational" definition of CDRs is based on residues that make an enthalpic contribution to antigen binding (see, e.g., Makabe et al., 2008, J. Biol. Chem., 283:1 156-1 166). North has identified classical CDR conformations using a different set of preferred CDR definitions (North et al., 2011, J. Mol. Biol. 406:228-256). In another approach, referred to herein as the "conformational definition" of CDRs, positions of CDRs can be identified as residues that make an enthalpic contribution to antigen binding (Makabe et al., 2008, J Biol. Chem. 283:1 156-1 166). Other CDR boundary definitions may not strictly follow one of the above methods, but will still overlap at least a portion of the Kabat CDRs, although they may be shortened or extended without significantly affecting antigen binding based on predictions or experimental findings of specific residues or groups of residues or even entire CDRs. As used herein, CDRs may refer to CDRs defined by any method known in the art (including combinations of methods). The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDRs (or other residues of the antibody) may be defined according to any of the Kabat, Chothia, North, extension, AbM, contact and / or conformational definitions.

[0470] For example, the following table shows several commonly used definitions of CDR:

[0471]

[0472] 1. Some of these definitions (particularly for the Chothia loops) vary depending on the individual publications examined. Some papers describe the Chothia CDRs as: CDR-L1: L24-34; CDR-L2: L50-56; CDR-L3: L89-97; CDR-H1: H26-32; CDR-H2: H52-56; CDR-H3: H95-102.

[0473] 2. Any numbering scheme may be used for these CDR definitions, except that contact definitions use the Chothia or Martin (enhanced Chothia) definitions.

[0474] 3. When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop. (This is because the Kabat numbering scheme places the insertion at H35A and H35B.)

[0475] The CDR sequences provided in the sequence table are based on the Kabat definition. However, other definitions of CDRs may also be used. Therefore, in some embodiments, the first antibody comprises CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO:203, and CDR-L1, CDR-L2 and CDR-L3 sequences of SEQ ID NO:208. In some embodiments, the first antibody comprises CDR-H1 containing SEQ ID NO:200, CDR-H2 containing SEQ ID NO:201 and CDR-H3 containing SEQ ID NO:202, and CDR-L1 containing SEQ ID NO:205, CDR-L2 containing SEQ ID NO:206 and CDR-L3 containing SEQ ID NO:207.

[0476] Outside the CDR regions, the sequence of the first antibody may differ from that of denosumab; however, for convenience, the VH and / or VL sequences of denosumab may be used. Thus, in some embodiments, the first antibody or antibody conjugate comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH has 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% identity to SEQ ID NO: 203, and the VL has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 203. NO:208 has 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% identity.

[0477] The constant region of the first antibody (or capture antibody) can be human or mouse. The example of human and mouse constant region sequence is provided in Sequence Table E. For the purpose of using human sample to measure, in some cases, some mouse constant region sequences can be preferred. This can reduce cross reaction or false positive sometimes. The endogenous human antibody in the sample will not interfere with the appropriate detection of ADA molecule. Therefore, in some embodiments, the first antibody or antibody conjugate comprises human CH1 domain (such as human IgG1CH1, human IgG2CH1, human IgG3CH1 or human IgG4CH1), human Fc domain (such as human IgG1Fc, human IgG2Fc, human IgG3Fc or human IgG4Fc), mouse IgG1CH1 (such as mouse IgG2aCH1, mouse IgG2bCH1 or mouse IgG3CH1), mouse IgG1Fc (such as mouse IgG2aFc, mouse IgG2bFc or mouse IgG3Fc), human CL domain (such as human κCL or human λCL) or mouse CL domain (such as mouse κCL or mouse λCL).

[0478] In an exemplary embodiment, the first antibody or antibody conjugate comprises denosumab conjugated to biotin.

[0479] The heavy chain sequence and light chain sequence of denosumab are provided as SEQ ID NO.198 and 199, respectively. It should be noted that, generally, during the recombinant production process, the terminal lysine and glycine of the heavy chain may be sheared. C-terminal lysine shearing is a common phenomenon that occurs during the biological production of monoclonal antibodies. Generally, lysine residues are removed via carboxypeptidase D (CpD), which results in the production of a mixture of antibody isotypes with zero or one C-terminal lysine residue on each heavy chain. In addition, after the C-terminal lysine cleavage, peptidyl glycine α-amidation monooxygenase (PAM) catalyzes the hydroxylation of glycine and removes glyoxylic acid from the glycine residue, thereby leaving the amidated C-terminal proline. Therefore, during the recombinant production of monoclonal antibodies, the product is generally a mixture of C-terminal processing variants at the heavy chain C-terminal end (amidation) proline, glycine or lysine. Therefore, in some embodiments, the terminal lysine of SEQ ID NO: 198 may not be present; 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 not be present; in some embodiments, the terminal glycine-lysine of SEQ ID NO: 198 may be present.

[0480] "Solid matrix" refers to a non-fluid substance, and includes particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass and ceramics; gel substances such as silica, alumina and polymer gels; capillaries, which can be made of polymers, metals, glass and / or ceramics; zeolites and other porous substances; electrodes; microtiter plates; solid strips; cuvettes, tubes or other spectrometer sample containers. In ADA assays, solid matrix refers to an inert solid surface, which usually contains a "binding partner" (e.g., avidin or streptavidin) on its surface, intended to interact with the "capture" antibody; in this case, the first antibody or antibody conjugate contains a fixed portion (e.g., biotin) recognized by the "binding partner". The solid phase can be a fixed component, such as a tube, strip, cuvette or microtiter plate, or can be a non-fixed component, such as beads and microparticles. Microparticles can also be used as solid phases for uniform assay formats. A variety of microparticles that allow non-covalent or covalent attachment of proteins and other substances can be used. 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, for example, Martin, CR et al., Analytical Chemistry-News & Features 70 (1998) 322A-327A.

[0481] Typically, the first antibody or antibody conjugate is immobilized to a solid matrix by passive adsorption and is therefore attached to the solid phase at a site that does not interfere with its binding to the ADA. Passive adsorption is described, for example, by Butler, JE Solid Phases in Immunoassay, in Immunoassays, Diamandis, EP and Christopoulos, TK (eds.) Academic Press San Diego (1996), pp. 205-225.

[0482] Tracer molecules

[0483] In a typical ADA assay, a "tracer" molecule (also referred to herein as a "third antibody") needs to be able to bind to the ADA molecule to form a sandwich (see, e.g. Figure 1A ). The most convenient way is to have the same CDR sequence as the first antibody, since ADA is typically caused by the CDR sequence. The tracer molecule is also linked to a detectable marker to facilitate the detection of ADA.

[0484] Therefore, in certain embodiments, the third antibody comprises the same six CDRs as denosumab. As described in detail above, CDRs can be identified according to the accumulation, AbM, contact, North and / or conformational definitions of Kabat, Chothia, Kabat and Chothia or any CDR determination method well known in the art. The CDR sequences provided in the sequence table are based on the Kabat definition. However, other definitions of CDR can also be used. Therefore, in some embodiments, the third antibody comprises antibodies containing CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO:203 and CDR-L1, CDR-L2 and CDR-L3 sequences of SEQ ID NO:208. In some embodiments, the third antibody comprises an antibody comprising a CDR-H1 comprising SEQ ID NO:200, a CDR-H2 comprising SEQ ID NO:201, and a CDR-H3 comprising SEQ ID NO:202, and a CDR-L1 comprising SEQ ID NO:205, a CDR-L2 comprising SEQ ID NO:206, and a CDR-L3 comprising SEQ ID NO:207.

[0485] Outside the CDR regions, the sequence of the third antibody may differ from that of denosumab; however, for convenience, the VH and / or VL sequences of denosumab may be used. Thus, in some embodiments, the third antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the VH has 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% identity to SEQ ID NO: 203, and the VL has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 203. NO:208 has 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% identity.

[0486] The constant region of the third antibody (or missing agent antibody) can be human or mouse. The example of human and mouse constant region sequence is provided in Sequence Table E. For the purpose of using human sample to measure, in some cases, some mouse constant region sequences can be preferred. This can reduce cross reaction or false positive sometimes. The endogenous human antibody in the sample will not interfere with the appropriate detection of ADA molecule. Therefore, in some embodiments, the third antibody comprises human CH1 domain (such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1 or human IgG4 CH1), human Fc domain (such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc), mouse IgG1 CH1 (such as mouse IgG2a CH1, mouse IgG2b CH1 or mouse IgG3 CH1), mouse IgG1 Fc (such as mouse IgG2a Fc, mouse IgG2b Fc or mouse IgG3 Fc), human CL domain (such as human κ CL or human λ CL) or mouse CL domain (such as mouse κ CL or mouse λ CL).

[0487] In some embodiments, the third antibody comprises a VH domain and a VL domain, the VH domain comprising the same sequence as the VH of the first antibody, and the VL domain comprising the same sequence as the VL domain of the first antibody. In some embodiments, the third antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising the same sequence as the HC of the first antibody, and the LC comprising the same sequence as the LC of the first antibody.

[0488] Typically, the "tracer" molecule (or "tertiary antibody" herein) is covalently or non-covalently linked to a detectable marker. The detectable marker can be an isotope, an enzyme, a fluorescent moiety, a luminescent moiety, a colored moiety, a metal or a charge. For example, the detectable marker can be an electrochemiluminescent label, such as a ruthenium bipyridyl complex. Developers (fluorescent or luminescent groups and dyes), enzymes, NMR active groups or metal particles, haptens (such as digoxin) are also examples of detectable markers. The detectable marker can also be a photoactivated cross-linking group, such as an azido or aziridine group. Metal chelates that can be detected by electrochemiluminescence are also preferred signaling groups, particularly preferably ruthenium chelates, such as ruthenium (bipyridyl) 32+ chelates. Suitable ruthenium labeling groups are described in, for example, EP 0 580 979, WO 90 / 05301, WO 90 / 11511 and WO92 / 14138. Furthermore, if desired, almost any antibody can be labeled with biotin, HRP enzyme, or one of several fluorophores.

[0489] Sometimes, an indirect detection method is used. In this case, the "tracer" molecule is considered to be a "primary antibody" combined with ADA, and the primary antibody is not labeled for direct detection. On the contrary, in the second step, a "secondary antibody" labeled with a detectable label is used to detect the primary antibody-ADA complex. Thus, ADA is detected indirectly. Another form of indirect detection involves the use of primary or secondary antibodies labeled with affinity tags (such as biotin). Then, secondary (or tertiary) probes (such as streptavidin labeled with detectable enzymes or fluorophore labels) can be used to detect biotin labels to produce detectable signals. There are several variants of these detection and detection strategies. Generally, this strategy depends on a specific probe (e.g., a primary antibody), which is directly or indirectly connected to a certain class of detectable markers (e.g., an enzyme whose activity can produce a colored product when reacting with its substrate).

[0490] Most primary antibodies can be produced or manipulated to have one of mouse, rabbit or several other non-human constant domains. Many of these are antibodies of IgG classification (particularly 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 selection of secondary antibodies depends on the animal species (host species) that produces the primary antibodies. 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 mice. The ready-made secondary antibodies combined with the constant domains or Fc domains of the primary antibodies are usually easy to obtain.

[0491] Biotin-based systems are commonly used as detectable markers. Using biotin-binding proteins as probes, the high-specific affinity interactions between biotin and avidin or streptavidin are the basis of multiple detection and affinity purification methods. Biotin is very small (244 daltons), so its covalent attachment to antibodies or other probes rarely interferes with the function of these antibodies or other probes. However, its presence as a label on the probe allows efficient and specific secondary detection using avidin or streptavidin. Both biotin-binding proteins can be obtained in pure form, and these forms are labeled using enzymes or fluorescent tags so that they can be detected in various types of assay systems.

[0492] Enzyme labels are commonly used as secondary antibody (or streptavidin) tags for detection in blotting and immunoassays. Enzymes provide detectable signals via their activity; reacting with specific substrate chemicals to produce colored, luminescent or fluorescent products. Although reporter enzymes like β-galactosidase and luciferase have been successfully used to make probes, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes most widely used as labels for protein detection. A range of chromogenic, fluorescent and chemiluminescent substrates are available for use with either enzyme.

[0493] Alkaline phosphatase (AP), commonly isolated from calf intestine, is a large (140 kDa) protein that catalyzes the hydrolysis of phosphate groups from substrate molecules, resulting in colored or fluorescent products, or the release of light as a byproduct of the reaction. AP has optimal enzymatic activity at alkaline pH (pH 8-10) and can be inhibited by cyanide, arsenate, inorganic phosphates, and divalent cation chelators such as EDTA. AP offers certain advantages as a detectable marker. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing the reaction to proceed for a longer period of time.

[0494] Horseradish peroxidase (HRP) is a 40 kDa protein that catalyzes the oxidation of substrates by hydrogen peroxide, thereby producing colored or fluorescent products or releasing light as a byproduct of the reaction. HRP works best 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 specific activity of both enzymes and antibodies. In addition, 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 increases in sensitivity can be achieved by using poly-HRP-conjugated secondary antibodies.

[0495] Two common fluorophores used to label probes are fluorescein (fluorescein isothiocyanate, FITC) and rhodamine (tetramethylrhodamine isothiocyanate, TRITC).

[0496] Other detectable markers include fluorescent proteins, such as various forms of green fluorescent protein (GFP), and phycobiliproteins (allophycocyanin, phycocyanin, phycoerythrin, and phycoerythrocyanin).

[0497] Reduce signal interference

[0498] As disclosed herein, in an exemplary ADA assay for detecting denosumab ADA, a large number of samples showed false positive signals and are believed to be caused by RANKL interfering with the ADA assay ( Figure 1A and Figure 1B ).

[0499] 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 comprises residues 69-317 of the membrane-bound form (as numbered in WO 98 / 46757), as shown in SEQ ID NOs: 212 and 213. Proteolytic cleavage between residues 139 and 140 of membrane-bound RANKL also produces a soluble form as shown in SEQ ID NO: 214.

[0500] To address the problem of false positive signals caused by soluble RANKL, it is important to include molecules in the ADA assay that can sequester soluble RANKL. Two strategies are provided herein, one using a secondary anti-RANKL antibody and the other using the decoy receptor OPG.

[0501] In order to effectively isolate RANKL without interfering with the detection of ADA, the second antibody should have the following characteristics: (1) it should have a different CDR sequence from denosumab (since ADA is usually directed against CDRs, having different CDRs usually ensures that there is no cross-reaction between the second antibody and ADA); and (2) it should bind to RANKL. Preferably, the second antibody binds to the same RANKL epitope as denosumab; or the second antibody competes (or at least partially competes) with denosumab for binding to RANKL. When the second antibody and denosumab cannot bind to RANKL at the same time, the signal interference caused by RANKL can be most effectively reduced. However, competing (or at least partially competing) with denosumab for binding to RANKL is not an absolute requirement. For example, in Examples 3-6, it was found that the exemplary anti-RANKL mAb did not compete with denosumab for binding to RANKL. Epitope grouping shows that the anti-RANKL mAb binds to different epitope groups and does not block the binding of denosumab or OPG to RANKL. Those skilled in the art will be able to readily evaluate the binding behavior of the secondary antibody and design the ADA assay accordingly based on common sense. Example 2B also provides examples on how to evaluate and select a suitable secondary antibody.

[0502] Additionally, although it is preferred that the second antibody competes (or at least partially competes) with denosumab for binding to RANKL, the second antibody does not have to bind to RANKL with a similar affinity (KD value) as denosumab. Even a poor competitor with low binding affinity for RANKL may be sufficient to reduce / eliminate interference. In this case, the competing antibody can be added to the serum sample in large amounts, and only a small amount of RANKL needs to be isolated.

[0503] KD is the equilibrium dissociation constant between an antigen binding protein and its target or antigen, k off / k onThe ratio of KD and KA is inversely related. The KD value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), so the lower the KD value (the lower the required concentration), the higher the affinity of the antibody. In an exemplary aspect, the KD value of the second antibody for 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 to about 10 -15 M, about 10 -6 M to about 10 -15 M, about 10 -7 M to about 10 -15 M, about 10 -8 M to about 10 -15 M, about 10 -9 M to about 10 -15 M, about 10 -10 M to about 10 -15 M, about 10 -5 M to about 10 -14 M, about 10 -6 M to about 10 -14 M, about 10 -7 M to about 10 -14 M, about 10 -8 M to about 10 -14 M, about 10 -9 M to about 10 -14 M, about 10 -10 M to about 10 -14 M, about 10 -5 M to about 10 -13 M, about 10 -6 M to about 10 -13 M, about 10 -7 M to about 10 -13 M, about 10 -8 M to about 10 -13 M, about 10 -9 M to about 10 -13 M, or about 10-10 M to about 10 -13 M.

[0504] In exemplary aspects, the KD of the second anti-RANKL antibody is micromolar, nanomolar, picomolar, or femtomolar. In exemplary aspects, the KD is about 10 -4 Up to 10 -6 M, or 10 -7 Up to 10 -9 M, or 10 -10 Up to 10 -12 , or 10 -13 Up to 10 -15 In exemplary aspects, the second antibody binds to human RANKL with a KD value 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. Or less, about 2nM or less, about 1nM or less, about 900pM or less, about 800pM or less, about 700pM or less, about 600pM or less, about 500pM or less, about 400pM or less, about 300pM or less, about 250pM or less, about 200pM or less, about 150pM or less, about 100pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 25pM or less, about 20pM or less, about 15pM or less, about 10pM or less, about 5pM or less, or about 1pM or less.

[0505] KD values ​​can be determined using methods established in the art. D An exemplary method is surface plasmon resonance (SPR), which is well known in the art (e.g., Nguyen et al. Sensors (Basel). 2015 May 5;15(5):10481-510). Biosensor systems (e.g., The KD value is measured by SPR using a BIAcore system. BIAcore kinetic analysis involves analyzing the binding and dissociation of an antigen to a chip having an immobilized molecule (e.g., a molecule comprising an epitope binding domain) on its surface. Another well-known method in the art for determining the KD of a protein is by using biolayer interferometry (e.g., Shah et al. J Vis Exp. 2014;(84):51383). The KD value can be measured using KD values ​​were measured by biolayer interferometry using the Octet QKe system, ForteBio. Alternatively or additionally, the KD values ​​may be measured using a 100 nm optical density spectroscopy (OD 100 nm) instrument available from Sapidyne Instruments (Boise, Id.). (Kinetic Exclusion Assay) Assay. Any method known in the art for assessing the binding affinity between two binding partners is contemplated herein.

[0506] In some aspects, KD value is measured by surface plasmon resonance (SPR). Antigen (RANKL) can be fixed on, for example, a solid surface. Antigen can be fixed to a chip, for example, by covalent coupling (such as amine coupling). The chip can be a CM5 sensor chip. As analyte binds to ligand, the accumulation of protein on the sensor surface causes an increase in refractive index. The refractive index change is measured in real time (sampling is performed every 0.1s in a kinetic analysis experiment), and the result is plotted as a response unit (RU) compared to time (called a sensorgram). If there is a difference in the refractive index of the running buffer and the sample buffer, a response (background response) will also be generated. The background response must be subtracted from the sensorgram to obtain an actual binding response. The background response is recorded by injecting the analyte through a control or reference flow cell, which does not have a ligand or an unrelated ligand fixed on the sensor surface. The real-time measurement of the association and dissociation of the binding interaction allows the calculation of association and dissociation rate constants and corresponding affinity constants. One RU represents a protein bound to 1pg per square mm. In practice, analyte binding of more than 50 pg per square mm is usually required to produce a good, reproducible response.

[0507] The dissociation of the antibody from the antigen can be monitored for about 3600 seconds. The SPR analysis can be performed and data collected at about 15°C to about 37°C. The SPR analysis can be performed and data collected at about 25°C to 37°C. The SPR analysis can be performed and data collected at about 37°C. The SPR analysis can be performed and data collected at 37°C. DThe values ​​can be measured by SPR using a BIAcore T200 instrument. The SPR rate and affinity can be determined by fitting the resulting sensorgram data to a 1:1 model in the BIAcore T200 Evaluation Software Version 1.0. The acquisition rate can be about 1 Hz.

[0508] Another method for determining antibody KD is to use biolayer interferometry (BLI), typically using Technology (Octet QKe system, Pall Forte Biotech). In some embodiments, biosensor analysis is used. Typically, one interactor is immobilized on the surface of the biosensor ("ligand", such as an antibody) and the other is retained in the solution ("analyte", such as an antigen). The assay begins with an initial baseline or equilibrium step using an assay buffer. Next, the ligand (such as an antibody) is immobilized on the surface of the biosensor (loading) by direct immobilization or a capture-based method. After ligand immobilization, the biosensor is immersed in a buffer solution for a baseline step to assess assay drift and determine the loading level of the ligand. After the baseline step, the biosensor is immersed in a solution containing the binding partner of the ligand (analyte) (association). In this step, the binding interaction of the analyte with the immobilized ligand is measured. After the analyte is associated, the biosensor is immersed in a buffer solution without the analyte, and the bound analyte is allowed to dissociate from the ligand (dissociation). A series of assay steps are then repeated for each analyte being tested on a new or regenerated biosensor. Each binding response is measured and reported in real time on the sensor trace. The instrument may be an Octet QKe system, an Octet RED96 system, an Octet QK384 system or a RED384 system.

[0509] In some aspects, the second antibody binds to the same RANKL epitope as denosumab. It is known that denosumab binds to an epitope (DE epitope) of a part of the amino acid sequence of the DE district comprising RANKL. The DE district of RANKL roughly spans D and E β sheets and a loop sequence (DE loop) therebetween. The DE district in human RANKL comprises about amino acid residues 212 to about amino acid residues 250 (GFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIP, SEQ ID NO: 216). However, the amino acid sequence and endpoints in the DE district of human RANKL are only exemplary, and it should be understood that the DE district may have a variable sequence and endpoints. In particular, it has been shown that in the DE ring of human RANKL, the amino acid sequence DLATE (SEQ ID NO: 215) is important for denosumab to bind. See, for example, WO 2001062932.

[0510] Thus, in some embodiments, the second anti-RANKL antibody binds to an epitope comprising the sequence of "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.

[0511] "Epitope" refers to the range or region of the antigen to which the antibody specifically binds, such as the range or region containing residues that interact with the antibody. The epitope can be linear or conformational. The term "paratope" is derived from the definition of "epitope" above by inverting the angle, and refers to the range or region in the antibody molecule that participates in antigen binding, such as the range or region containing residues that interact with the antigen. The paratope can be linear or conformational (such as discontinuous residues in CDR). The epitope / paratope of a given antibody / antigen binding pair can be defined and characterized at different levels of detail using a variety of 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. Since each method relies on a unique principle, the description of the epitope is closely related to the method for determining the epitope. Therefore, the epitope / paratope of a given antibody / antigen pair will be defined differently depending on the mapping method used.

[0512] It follows from the fact that the description and definition of epitopes depends on the epitope mapping method used and is obtained at different levels of detail that the comparison of epitopes of different antibodies on the same antigen can be similarly performed at different levels of detail. For example, epitopes described at the amino acid level (e.g., determined from an X-ray structure) are considered to be identical if they contain the same set of amino acid residues. Epitopes characterized by competitive binding are considered to overlap if the binding of the corresponding antibodies is mutually exclusive, i.e., binding of one antibody excludes simultaneous or sequential binding of another antibody; and epitopes are considered to be separate (unique) if the antigen can accommodate binding of two corresponding antibodies simultaneously.

[0513] The epitope and paratope of a given antibody / antigen pair can be identified by conventional methods. For example, the general location of the epitope can be determined by evaluating the ability of an antibody to bind to different fragments of RANKL.

[0514] In some aspects, the second antibody competes with denosumab for binding to RANKL. As used herein, the term "competition" with respect to antibodies means that the binding of the first antibody or its antigen-binding portion to the antigen reduces the subsequent binding of the second antibody or its antibody-binding portion to the same antigen. Typically, the binding of the first antibody produces steric hindrance, conformational changes, or binding to a common epitope (or portion thereof), such that the binding of the second antibody to the same antigen is reduced. Standard competition assays can be used to determine whether two antibodies compete with each other. A suitable assay for antibody competition involves the use of Biacore technology, which can use surface plasmon resonance (SPR) technology, typically using a biosensor system (such as 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.

[0515] Another assay for measuring antibody competition uses an ELISA-based method. In addition, a high-throughput method for "grouping" these antibodies based on antibody competition is described in International Patent Application No. WO2003 / 48731. If one antibody (or fragment) reduces the combination of another antibody (or its antigen-binding fragment) with RANKL, there is competition. For example, sequential binding competition assays can be used, in which different antibodies are added sequentially. The first antibody can be added to reach a near-saturated combination. Then, a second antibody is added. If the combination of the second antibody with RANKL is not detected, or compared with a parallel assay (its value can be set to 100%) in the absence of the first antibody, it is significantly reduced (for example, reduced 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%), it is considered that the two antibodies compete with each other.

[0516] Exemplary anti-RANKL antibodies that can be used in the ADA assays disclosed herein are shown 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 described in detail above, CDRs can be identified according to Kabat, Chothia, cumulative, AbM, contact, Northern and / or conformational definitions of both Kabat and Chothia, or any CDR determination method well known in the art. The CDR sequences provided in the sequence table are based on the Kabat definition. However, other definitions of CDRs may also be used.

[0517] In some embodiments, the second antibody comprises (i) 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%, at 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 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: NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116 or 122 having 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; (iii) a CDR-H3 comprising a sequence that is 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: 2, 8, 14, 20, 26, 32, 38, 44, 50 NO:3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111, 117 or 123 having 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; (iv) a CDR-L1 comprising a sequence that is 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:3, 9, 15, 21, 27, 33, 39, NO:4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112, 118 or 124 having 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;(v) a CDR-L2 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: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:5, 11, 17, 23, 29, 35, 41, NO:6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120 or 126 having 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.;

[0518] 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.

[0519] 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 table. For the purpose of using human samples for determination, in some cases, certain mouse constant region sequences may be preferred. This can sometimes reduce cross-reactions or false positives. Endogenous antibodies in the sample will not interfere with the appropriate detection of ADA molecules. Therefore, in some embodiments, the third antibody comprises human CH1 domains (such as human IgG1 CH1, human IgG2 CH1, human IgG3 CH1 or human IgG4 CH1), human Fc domains (such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc), mouse IgG1 CH1 (such as mouse IgG2a CH1, mouse IgG2b CH1 or mouse IgG3 CH1), mouse IgG1 Fc (such as mouse IgG2a Fc, mouse IgG2b Fc or mouse IgG3 Fc), human CL domains (such as human κ CL or human λ CL) or mouse CL domains (such as mouse κ CL or mouse λ CL).

[0520] Alternatively, osteoprotegerin (OPG) can be used to isolate soluble RANKL. OPG is also known as osteoclastogenesis inhibitory factor (OCIF) or tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), which 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.

[0521] In certain embodiments, OPG is human OPG. In certain embodiments, 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.

[0522] The sample can be pretreated with the 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 ADA determination. Alternatively, the first antibody or antibody conjugate, the second antibody or OPG and the third antibody can be added simultaneously. When the second antibody competes with the first antibody or partially competes for RANKL binding (that is, when the first antibody and the second antibody cannot be combined with RANKL at the same time), this is particularly feasible. When the first antibody and the second antibody cannot be combined with RANKL at the same time, the presence of the second antibody will prevent RANKL from bridging the fixed first antibody and detectable marker.

[0523] Excessive second antibody or OPG can be added to ensure complete isolation of soluble RANKL in the sample. In certain embodiments, excessive second antibody or OPG is added to the sample so 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.

[0524] 3. Diagnostic kits

[0525] In certain aspects, the present disclosure provides a kit suitable for determining whether there is an ADA for denosumab in a sample. The kit may include instructions and reagents for ADA determination disclosed herein. For example, the kit may include: (a) a first antibody or antibody conjugate bound to RANKL, comprising SEQ ID NO:203 heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 and SEQ ID NO:208 light chain (LC) CDR-L1, CDR-L2 and CDR-L3; wherein the first antibody or antibody conjugate may be attached to a solid matrix; (b) (i) a second antibody bound to RANKL, wherein the second antibody is included in one of the six CDR sequences and the first antibody difference of at least one amino acid amino acid sequence; or (ii) osteoprotegerin (OPG) protein bound to RANKL; (c) a third antibody bound to the ADA and connectable to a detectable marker; and (d) instructions for use.

[0526] The kit may also include an immobilization portion that can be covalently or non-covalently attached to the first antibody. The kit may also include a solid matrix and a binding partner that can be bound to the immobilization portion. The binding partner may have been fixed to the matrix, or reagents may be provided to allow the user to attach the binding partner to the solid matrix.

[0527] The kit may also include a detectable marker. A reagent that allows the user to covalently or non-covalently couple the third antibody to the detectable marker may also be included. Alternatively, the kit may include a third antibody that has been covalently or non-covalently attached to the detectable marker.

[0528] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes made therefrom will be proposed to those skilled in the art and will be included within the spirit and scope of the present application and within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0529] Examples

[0530] Example 1. Background and rationale

[0531] In a bioequivalence study of denosumab (“Study A”), an unusually high incidence of immune responses was observed in the anti-drug antibody assay (ADA). This was unexpected based on experience from past clinical studies of denosumab and initiated an investigation. Because the signals from positive samples were uniform and low in amplitude, more consistent with treatment-induced shedding of receptor activator of nuclear factor-κB ligand (RANKL) rather than anti-denosumab antibody responses, target interference was suspected to have resulted in false-positive results.

[0532] To investigate this possibility, a subset of antibody-positive samples from Study A were 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, then OPG treatment would reduce the assay signal by competing with denosumab for binding. If the antibody-positive results were due to anti-denosumab antibodies, then OPG treatment would have no effect on the assay signal because OPG does not bind to ADA molecules. In particular, if Figure 1A-1B As shown, in an exemplary MSD ADA assay, the anti-drug antibody can bridge biotin-labeled denosumab and SULFO-TAG (ruthenium terpyridine)-labeled denosumab. The complex is immobilized using a streptavidin-coated plate and detected by chemiluminescence ( Figure 1A It is possible that soluble RANKL trimers can also bridge the labeled denosumab molecules ( Figure 1B ) and lead to false positive signals. If this is the case, pre-treating the sample with OPG can deplete the soluble RANKL from the sample and eliminate the false positive signals. Indeed, it was confirmed that in most of the antibody positive samples tested, OPG treatment resulted in a significant reduction in signal (data not shown), which suggests that many positive results are due to target (RANKL) interference.

[0533] In order to solve the problem of false positive signals caused by soluble RANKL and develop a reliable ADA assay, an anti-RANKL monoclonal antibody that binds to human RANKL is incorporated into an improved ADA assay. Because the complementary determining region (CDR) of an antibody is the result of a selection process after random mutations in the germline, the CDR can be recognized as foreign by the immune system. Therefore, most anti-drug antibodies of monoclonal antibody therapeutics are directed against CDR. For this reason, anti-RANKL antibodies with different CDRs compared to denosumab are screened and selected to ensure that the incorporated anti-RANKL antibodies do not inadvertently bind to ADA, thereby inhibiting the signal. Example 2. Materials and methods for ADA assays for denosumab

[0534] Based on the bridging immunoassay of Merck (Meso Scale Discovery, MSD). Serum samples were diluted 1:20 in 300mM acetic acid to allow the antibody complex to dissociate before analysis. The diluted samples were then incubated with a conjugate / anti-RANKL / neutralization mixture containing biotinylated denosumab, ruthenium denosumab, anti-RANKL antibody, 1M Tris (pH 9.5) and soluble drug (confirmatory assay only). During this incubation period, the two antigen-binding sites of the anti-denosumab antibody form a bridge between the conjugated denosumab molecules ( Figure 1A ). The sample mixture is then added to a closed Merck & Co. (MSD) streptavidin standard-bound microtiter plate (Merck & Co. (MSD), No. L15SA-1). The biotinylated molecule binds to the streptavidin-coated well surface, resulting in the fixation of the bridged complex. The plate is washed to remove any unbound complex, and tripropylamine GOLD read buffer (Merck & Co. (MSD), No. R92TG-2) is added to each well. Using the MSD SECTOR S600 reader, a current is applied to the electrode associated with the plate. The result is a series of electrically induced oxidation-reduction reactions involving ruthenium (from the captured complex) and tripropylamine. The subsequent luminescent signal is quantified as electrochemiluminescence (ECL) units. The sample is subjected to a screening assay to detect the presence of binding antibodies to denosumab. Samples with responses below the screening assay cutoff point analyzed in the screening assay are considered negative, and responses equal to or above the screening assay cutoff point are then analyzed in the confirmatory assay. Samples tested in the confirmatory assay with % inhibition results equal to or above the confirmatory assay cut-point were considered positive for the presence of anti-denosumab antibodies.

[0535] Generation of biotinylated and ruthenated denosumab molecules. Denosumab molecules were labeled with biotin (ThermoScientific, No. A39257) or ruthenium (MSD, No. R91AO). The process included an initial buffer exchange, a conjugation reaction, and a post-conjugation buffer exchange to remove free label from the conjugated AMG 162 molecules.

[0536] Generation of antibodies against human RANKL. An anti-RANKL monoclonal antibody containing mouse IgG1 Fc was recombinantly expressed from HEK293-6E cells. The antibody was named "anti-RANKL mAb" in Examples 3-6 described below. FACS and ELISA assays using soluble RANKL confirmed that the anti-RANKL mAb binds to human RANKL. Grouping assays showed that the anti-RANKL mAb binds to different epitope groups and does not compete with denosumab or OPG for binding to RANKL.

[0537] Example 2B. Screening and selection of anti-RANKL molecules and RANKL controls

[0538] Positive control rabbit anti-denosumab polyclonal antibody (control anti-drug antibody or "ADA") at low (10 ng / mL), medium (100 ng / mL) and high (1000 ng / mL) levels were added to pooled normal human serum (PNHS). These control ADA samples were then evaluated in 5 different ways (using 5 different reaction buffers): 1) using the conjugate / neutralization mixture (untreated); 2) using the conjugate / neutralization mixture plus denosumab (confirmatory assay); 3) using the conjugate / neutralization mixture plus mouse anti-RANKL mAb (anti-RANKL1 treated); 4) using the conjugate / neutralization mixture plus OPG (anti-RANKL2 treated); and 5) using the conjugate / neutralization mixture plus mouse anti-RANKL mAb (anti-RANKL3 treated). Three different RANKL (1250 pg / mL) controls (RC-1, RC-2 and RC-3) were also prepared and included in the experiment for evaluation and selection of the best candidates for use as RANKL controls. This evaluation was used to screen and select the best anti-RANKL treatment that does not affect specific ADA signaling but inhibits specific target (RANKL) signaling.

[0539] The results of the experiment are shown in Table 9. When using untreated conjugate / neutralization mixture, it is observed that as the concentration of ADA increases, the results of (untreated) S / N increase. Different results were observed from 3 different RANKL controls (RCs), in which RC-1 produced results around the assay cutoff point, RC-2 produced a negative weak response in the assay, and RC-3 produced a positive strong response in the assay. Based on the results produced by 3 RANKL batches, RC-3 was selected as a target control. The results of the second evaluation using conjugate / neutralization mixture plus denosumab (ADA and target inhibition control) showed the expected inhibition of both specific ADA signals and specific target signals (> / =60%), which shows that the control conditions are as effective as expected. In addition, the results of the assay using conjugate / neutralization mixture plus anti-RANKL3 showed minimal inhibition of specific ADA signals, which shows that the use of the anti-RANKL molecule does not affect the specific ADA signal. Because anti-RANKL3 treatment does not affect specific ADA signaling but inhibits specific target signaling, anti-RANKL3 murine antibody is considered the preferred agent among the three anti-RANKL treatment agents tested in this experiment.

[0540] Table 9. Summary of results of anti-RANKL molecular screening

[0541]

[0542] Example 3. Evaluation of the effect of anti-RANKL antibodies on specific ADA signals

[0543] Positive control rabbit anti-denosumab polyclonal antibody (anti-drug antibody or ADA) at low (10ng / mL), medium (100ng / mL) and high (1000ng / mL) levels was added to the normal human serum (PNHS) pooled. These ADA samples were then evaluated in 3 different ways (using 3 different reaction buffers): 1) using conjugate / neutralization mixture (untreated); 2) using conjugate / neutralization mixture plus anti-RANKL mAb (anti-RANKL mAb processed); and 3) using conjugate / neutralization mixture plus anti-RANKL mAb and denosumab (denosumab+anti-RANKL mAb processed, confirmatory assay). Human RANKL (1250pg / mL) control was also prepared and included in the experiment to confirm the efficacy of the added anti-RANKL mAb in inhibiting the signal generated by RANKL (target). The results produced by the RANKL control showed 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, it was observed that the S / N results increased with increasing ADA concentrations. The results of the assay using the conjugate / neutralization mixture plus anti-RANKL mAb showed minimal inhibition (<20%) of the specific anti-denosumab ADA signal, indicating that the use of anti-RANKL mAb in the assay did not affect the specific anti-denosumab ADA signal. The results of the assay using the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab showed inhibition of the S / N results by greater than 20%.

[0544] Table 1A. Effect of anti-RANKL antibodies on ADA signaling

[0545]

[0546] S / N suppression % = (untreated S / N – treated S / N) / untreated S / N * 100

[0547] Additional experiments were performed in which six different positive control anti-denosumab antibodies, including polyclonal antibodies (pAbs) and additional mouse and rat monoclonal antibodies, at low (10 ng / mL), medium (100 ng / mL) and high (1000 ng / mL) levels were added to PNHS. These ADA samples were then evaluated in 2 different ways (using 2 different reaction buffers): 1) using conjugate / neutralization mixture (untreated); and 2) using conjugate / neutralization mixture plus anti-RANKLmAb (anti-RANKL mAb treatment). Human RANKL (1250 pg / mL) controls were also prepared and included in the experiment to confirm the efficacy of the added anti-RANKL mAb in inhibiting the signal generated by RANKL (target). The results produced by the RANKL control showed that the assay worked as expected (data not shown). The results of the experiment are shown in Table 1B. When using untreated conjugate / neutralization mixture, it was observed that the results of S / N increased with the increase of ADA concentration. The assay results using the conjugate / neutralization mixture plus anti-RANKL mAb showed minimal inhibition (<20%) of the specific anti-denosumab ADA signal, indicating that the use of anti-RANKL mAb in the assay did not affect the specific anti-denosumab ADA signal.

[0548] Table 1B. Effects of anti-RANKL antibodies on ADA signaling

[0549]

[0550]

[0551] S / N suppression % = (untreated S / N – treated S / N) / untreated S / N * 100

[0552] These results further indicated that the detection of ADA was not affected when anti-RANKL mAb was incorporated into the assay.

[0553] Example 4. Evaluation of the effect of anti-RANKL antibodies on specific RANKL signaling

[0554] Different levels of human RANKL were added to PNHS starting at low levels, which were previously shown to produce low positive (1250pg / mL), medium (2500pg / mL), and high (5000pg / mL) levels. The samples with added RANKL were then evaluated in 3 different ways (using 3 different reaction buffers): 1) using the conjugate / neutralization mixture (untreated); 2) using the conjugate / neutralization mixture plus anti-RANKL mAb; and 3) using the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab (confirmatory assay).

[0555] The results of the experiment are shown in Table 2. When the untreated conjugate / neutralization mixture was used, it was observed that the S / N results increased with increasing RANKL concentrations. The experimental results using the conjugate / neutralization mixture plus anti-RANKL mAb showed inhibition of specific RANKL signals (>20%) at all tested concentrations, indicating that the use of anti-RANKL mAb in the assay inhibited the signal generated by the target. The assay results using the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab also showed inhibition of the S / N results by greater than 20%.

[0556] Table 2. Effects of anti-RANKL antibodies on RANKL signaling

[0557]

[0558] S / N suppression % = (untreated S / N – treated S / N) / untreated S / N * 100

[0559] These combined results indicate that detection of RANKL is inhibited when anti-RANKL mAb is incorporated into the assay.

[0560] Example 5. Evaluation of the effect of anti-RANKL antibodies on samples containing both ADA and RANKL

[0561] To simulate research samples that may contain both ADA and RANKL, 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 were added to PNHS. The combined samples were then evaluated in 3 different ways (using 3 different reaction buffers): 1) using the conjugate / neutralization mixture (untreated); 2) using the conjugate / neutralization mixture plus anti-RANKL mAb; and 3) using the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab (confirmatory assay).

[0562] The results of the experiment are shown in Table 3A. When the untreated conjugate / neutralization mixture was used, it was observed that the S / N value increased with increasing concentrations of ADA and / or RANKL. The experimental results using the conjugate / neutralization mixture plus anti-RANKL mAb showed that a portion of the signal was inhibited, indicating that the specific ADA signal was not inhibited. The results of the assay using the conjugate / neutralization mixture plus anti-RANKL mAb and denosumab showed inhibition of the signal at all tested concentrations (>20%).

[0563] Table 3A. Effect of anti-RANKL antibodies on samples containing both ADA and RANKL

[0564]

[0565] S / N suppression % = (untreated S / N – treated S / N) / untreated S / N * 100

[0566] Table 3B shows the S / N results for the combined samples compared to the S / N results for samples containing only the same concentration of ADA (without RANKL). At both ADA concentrations (and increased RANKL concentrations), increases in S / N were observed. The results of the experiment using the untreated conjugate / neutralization mixture plus anti-RANKL mAb showed that the S / N results were consistent with those produced by samples with only the same concentration of ADA, indicating that the anti-RANKL mAb successfully inhibited the RANKL-specific signal without affecting the specific ADA signal. Table 3B. Effect of anti-RANKL antibodies on samples containing both ADA and RANKL compared to samples containing only ADA

[0567]

[0568] Recovery % = (untreated S / N ÷ treated S / N) * 100

[0569] These results indicate that when anti-RANKL mAb was incorporated into the assay, the detection of ADA was not affected, whereas the detection of RANKL was inhibited.

[0570] Since 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 RANKL interference or cross-reaction, a human-mouse chimeric anti-RANKL monoclonal antibody that competes with denosumab for binding to RANKL was incorporated into the assay.

[0571] Example 6. Characteristics of anti-RANKL antibodies

[0572] ELISA assay. Costar 3368 medium-bound 96-well plates were coated with 10ug / ml soluble RANKL (sRANKL) in 1XPBS / 0.05% azide (50ul / well); samples were incubated overnight at 4°C. Plates were washed with RO (reverse osmosis) water using Titertek for 3 cycles; then blocked with 250ul of 1XPBS / 1% milk and incubated at RT for at least 30min; then washed with RO water using Titertek for another 3 cycles. The primary antibody was a purified mAb clone, which was titrated 1:3 from 1ug / ml 1XPBS / 1% milk (50ul / well) and incubated for 1 hour at RT. 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. IgG1 Fc is more convenient for detection. Positive control 2 is OPG-Fc titrated from 1ug / ml at 1:3. After incubation with primary antibody, the plate was washed with RO water using Titertek, washed for 3 cycles, and then incubated with secondary antibody. The secondary antibody was goat anti-human IgG Fc (cross-adsorbed)-HRP (Thermo Fisher, 31413) at 0.4ug / ml in 1XPBS / 1% milk (50ul / well), which was incubated for 1 hour at RT. The sample was then washed with RO water using Titertek, washed for 3 cycles. The matrix was treated with 1 step TMB (Neogen, 308177) (50ul / well), and quenched with 1N hydrochloric acid (50ul / well) after 30 minutes at RT. The plate was read at 450nm, and the results are shown in Table 4.

[0573] Table 4. Soluble human RANKL binding data

[0574]

[0575] FACS assay. The standard FACS protocol was as follows: Ren 218.9 stable cells (CHO / RANKL) were counted and seeded into FACS buffer (1X PBS + 2% fetal bovine serum) to give 50,000 cells / well in a final volume of 50ul / well. The cells were incubated with 5ug / mL and 0.25ug / mL of primary antibodies at 4°C for 1 hour, then washed twice with 200ul / well of FACS buffer. Then 100ul / well of secondary antibodies mixed with dead cell stain 7-aminoactinomycin D (7AAD) (Sigma, A9400-5MG) were added. Goat anti-human IgG Cy5 secondary antibody (Jackson, 109-175-098) and 7AAD were both used at 5.0ug / mL and incubated with the samples at 4°C for 15 minutes, then washed once with 200ul / 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.

[0576] Table 5 FACS binding data

[0577]

[0578] Other ELISA assays performed with human mouse and rat soluble RANKL.Except that human, mouse and rat photobiotinylation (pb) and capture on neutravidin (NA), an ELISA protocol almost identical to that described above was used.Tables 6-7 show the results of ELISA assays performed using human, mouse and rat RANKL.

[0579] Table 6. Soluble human and mouse RANKL binding data

[0580]

[0581] Table 7. Soluble rat RANKL binding data

[0582]

[0583] Table 8 summarizes the characteristics of certain anti-RANKL antibodies disclosed herein.

[0584] Table 8 Characteristics of antibodies against human RANKL

[0585]

[0586]

[0587] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0588] Unless otherwise indicated herein or clearly contradicted by context, the use of the terms "a and an," "the," and similar referents in the context of describing the present disclosure (especially in the context of the following claims) will be construed to cover both the singular and the plural. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0589] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value and endpoint falling within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0590] Unless otherwise indicated herein or otherwise clearly contradictory to the context, all methods described herein may be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better describe the present disclosure, rather than to impose limitations on the scope of the present disclosure. The language in the specification should not be interpreted as indicating that any unclaimed element is necessary for practicing the present disclosure.

[0591] Preferred embodiments of the disclosure are described herein, including the best modes known to the inventors for implementing the disclosure. After reading the above description, the variations of those preferred embodiments may become apparent to those of ordinary skill in the art. The inventors expect that skilled artisans will adopt such variations as appropriate, and the inventors intend to implement the disclosure in a manner other than that specifically described herein. Therefore, the disclosure includes all modifications and equivalents allowed by applicable law of the subject matter recited in the appended claims. In addition, unless otherwise indicated herein or the context is otherwise clearly contradictory, the disclosure encompasses any combination of the above-mentioned elements with all possible variations thereof.

[0592] Sequence Table

[0593] Table A: CDR sequence ID number (AA)

[0594]

[0595] Table B: Sequence ID number of variable region (AA)

[0596] Antibody Name HC variable (VH) LC variable (VL) A1 127 128 B1 129 130 C1 131 132 D1 133 134 E1 135 136 F1 137 138 G1 139 140 H1 141 142 I1 143 144 J1 145 146 K1 147 148 L1 149 150 M1 151 152 N1 153 154 O1 155 156 P1 157 158 Q1 159 160 R1 161 162 S1 163 164 T1 165 166 U1 167 168

[0597] Table C: CDR sequences

[0598]

[0599]

[0600]

[0601]

[0602]

[0603] Table D: VH VL sequences

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610] Table E: Constant region sequences

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618] Table F: Other sequences

[0619]

[0620]

[0621]

[0622]

[0623]

Claims

1. A method for detecting anti-drug antibodies (ADA) in a sample, wherein the ADA binds to denosumab, the method comprising mixing the sample with: (a) a first antibody or antibody conjugate that binds to RANKL, comprising heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208; wherein the first antibody or antibody conjugate can be attached 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 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.

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

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

207.

4. The method of any one of claims 1-3, 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 1-5, wherein the first antibody or antibody conjugate comprises a human Fc domain or a murine Fc domain.

7. The method of any one of claims 1-6, 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 1-7, wherein the third antibody is covalently or non-covalently linked to a detectable marker.

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

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

11. The method of any one of claims 1-10, 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 1-11, 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), the HC comprising the same sequence as the LC of the first antibody.

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

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

15. The method of any one of claims 1-14, 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. The method of any one of claims 1-15, wherein 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.

17. The method of any one of claims 1-16, 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 1-17, wherein the second antibody comprises a human Fc domain or a murine Fc domain.

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

20. 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, comprising heavy chain (HC) CDR-H1, CDR-H2 and CDR-H3 of SEQ ID NO: 203 and light chain (LC) CDR-L1, CDR-L2 and CDR-L3 of SEQ ID NO: 208; wherein the first antibody or antibody conjugate can be attached 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 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.

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