CN117264071B

CN117264071B - Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof

Info

Publication number: CN117264071B
Application number: CN202311563001.2A
Authority: CN
Inventors: 方鹏; 曹雨霞; 游猛; 施磊; 冯庆君; 朱晓红
Original assignee: Jiangsu T Mab Biopharma Co ltd; Jiangsu Maiweikang New Drug Research And Development Co ltd
Current assignee: Jiangsu T Mab Biopharma Co ltd; Jiangsu Maiweikang New Drug Research And Development Co ltd
Priority date: 2023-11-22
Filing date: 2023-11-22
Publication date: 2024-03-22
Anticipated expiration: 2043-11-22
Also published as: CN117264071A

Abstract

The application relates to the field of antibody medicines, in particular to a binding agent of an anti-RANKL monoclonal antibody or a derivative thereof and application thereof. The anti-idiotype antibody has high binding force and high specificity of the anti-RANKL antibody, and can be used for detecting the content of the anti-RANKL antibody medicine in a sample, thereby playing an important role in the pharmacokinetic and pharmacodynamic analysis of the anti-RANKL antibody medicine.

Description

Binding agent of anti-RANKL monoclonal antibody or derivative thereof and application thereof

Technical Field

The application relates to the field of antibody medicines, in particular to a binding agent of an anti-RANKL monoclonal antibody or a derivative thereof and application thereof.

Background

The ligand of nuclear factor kappa-B receptor activator (Receptor activator of nuclear factor kappa-B ligand, RANKL) is a type II transmembrane protein and is a ligand of nuclear factor kappa-B receptor activator (Receptor activator of nuclear factor kappa-B, RANK). RANKL is an activator of NF- κb, which when bound to NF- κb induces differentiation of monocyte/macrophage lineage cells into osteoclasts and further leads to osteoclast precursor maturation. The RANKL/RANK/OGP system is a typical approach for RANKL to participate in bone remodeling, and RANKL expressed by osteoblasts binds to RANK of osteoclast precursor cells, and RANK binds to downstream tumor necrosis factor-related receptor 6 (TRAF 6) to initiate transcription of osteoclast production genes, inducing the osteoclast precursor cells into mature osteoclasts. RANKL is a member of tumor necrosis factor superfamily, is a key factor for osteoclast differentiation activation, has the effects of inducing osteoclast production activation and inhibiting osteoclast apoptosis, and can be used for treating osteoporosis.

An anti-Idiotype antibody (anti-idiotypic antibody) is an antibody directed against a specific epitope group (Idiotype) on the variable region of an antibody molecule, which is capable of specifically recognizing and binding to another unique site of the antibody (Idiotype). In the development of antibodies, two tests have been carried out using anti-idiotype antibodies as the main tool reagent: 1) The Anti-idiotype antibody can be used for detecting the content (free type, combined type and total amount) of various antibody drugs in blood because of different specific binding epitopes, thereby playing an important role in Pharmacokinetic (PK) and Pharmacodynamic (PD) analysis of the antibody drugs 2) the Anti-idiotype antibody plays an important role in immunogenicity (ADA) evaluation as a positive control for detection of the Anti-drug antibodies (ADA).

Therefore, development of an anti-idiotype antibody with high binding force and high specificity aiming at the RANKL target point is required to be applied to aspects such as antibody medicine development and immunogenicity evaluation.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide a binding agent for an anti-RANKL monoclonal antibody or a derivative thereof and use thereof, for solving the problems in the prior art.

In a first aspect, the present application provides a binding agent for an anti-RANKL monoclonal antibody or a derivative thereof, the binding agent comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 sequences;

wherein, the sequence of HCDR1 is shown as SEQ ID No. 1 or SEQ ID No. 2;

the sequence of HCDR2 is shown as SEQ ID No. 3 or SEQ ID No. 4;

the sequence of HCDR3 is shown as SEQ ID No. 5 or SEQ ID No. 6;

the sequence of LCDR1 is shown as SEQ ID No. 7 or SEQ ID No. 8;

the sequence of LCDR2 is shown as SEQ ID No. 9 or SEQ ID No. 10;

the sequence of LCDR3 is shown as SEQ ID No. 11 or SEQ ID No. 12.

The amino acid sequence is specifically as follows:

HCDR1：DYAMK（SEQ ID No:1）

HCDR1：SYKIE（SEQ ID No:2）

HCDR2：DINPNSGDSFYNWKFKD（SEQ ID No:3）

HCDR2：EISPEWNNTQYNEKFKG（SEQ ID No:4）

HCDR3：EQYGHIYSEYSWFAY（SEQ ID No:5）

HCDR3：EEDYCYDY（SEQ ID No:6）

LCDR1：KASQTVDTNVA（SEQ ID No:7）

LCDR1：RSGQSLKHSNGDTYLH（SEQ ID No:8）

LCDR2：SASRQSS（SEQ ID No:9）

LCDR2：QVLNRFS（SEQ ID No:10）

LCDR3：QQYNVYGFT（SEQ ID No:11）

LCDR3：SQSTVVPYT（SEQ ID No:12）。

the term "variable" as used herein means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called the Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are in a substantially p-folded configuration, connected by three CDRs forming the linker loop, and in some cases form part of a p-folded structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody, and the constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity.

In the binding agents provided herein, HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are selected from any one of the following:

(1) A sequence shown in SEQ ID No. 1 of HCDR1, a sequence shown in SEQ ID No. 3 of HCDR2, and a sequence shown in SEQ ID No. 5 of HCDR 3;

(2) HCDR1 is shown as SEQ ID No. 2, HCDR2 is shown as SEQ ID No. 4, and HCDR3 is shown as SEQ ID No. 6.

In the binding agents provided herein, LCDR1, LCDR2 and LCDR3 of the light chain variable region are selected from any one of:

(1) A sequence shown in SEQ ID No. 7 of LCDR1, a sequence shown in SEQ ID No. 9 of LCDR2, and a sequence shown in SEQ ID No. 11 of LCDR3;

(2) LCDR1 has the sequence shown in SEQ ID No. 8, LCDR2 has the sequence shown in SEQ ID No. 10, and LCDR3 has the sequence shown in SEQ ID No. 12.

In the binding agent provided herein, HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region are selected from any one of the following:

(1) A sequence shown in SEQ ID No. 1 for HCDR1, a sequence shown in SEQ ID No. 3 for HCDR2, a sequence shown in SEQ ID No. 5 for HCDR3, a sequence shown in SEQ ID No. 7 for LCDR1, a sequence shown in SEQ ID No. 9 for LCDR2, and a sequence shown in SEQ ID No. 11 for LCDR3;

(2) The sequence of HCDR1 is shown as SEQ ID No. 2, the sequence of HCDR2 is shown as SEQ ID No. 4, the sequence of HCDR3 is shown as SEQ ID No. 6, the sequence of LCDR1 is shown as SEQ ID No. 8, the sequence of LCDR2 is shown as SEQ ID No. 10, and the sequence of LCDR3 is shown as SEQ ID No. 12.

In the binding agents provided herein, the heavy chain variable region further comprises framework regions HFR1-HFR4; the amino acid sequence of HFR1 is shown as SEQ ID No. 13 or SEQ ID No. 14; the amino acid sequence of HFR2 is shown as SEQ ID No. 15 or SEQ ID No. 16; the amino acid sequence of HFR3 is shown as SEQ ID No. 17 or SEQ ID No. 18; the amino acid sequence of HFR4 is shown as SEQ ID No. 19 or SEQ ID No. 20, and the specific sequence is shown as follows:

HFR1：EVQLQQSGPELVKPGASVKMSCKASGYTFT（SEQ ID No:13）

HFR1：QVQLQQSGAELKKPGASVKISCKATGYTFI（SEQ ID No:14）

HFR2：WVRQSHGKSLEWIG（SEQ ID No:15）

HFR2：WIKQRPGHGLEWIG（SEQ ID No:16）

HFR3：RATLTVDKSSSTAYMQLNSLTSEDSAVYYCSR（SEQ ID No:17）

HFR3：KATFTADTSSNTVYLRLSSLTSEDSAVYYCAR（SEQ ID No:18）

HFR4：WGQGTLVTVSA（SEQ ID No:19）

HFR4：WGQGTTLTVSS（SEQ ID No:20）。

in the binding agents provided herein, the framework region of the heavy chain variable region is selected from any one of the following:

(1) A sequence shown in an amino acid sequence SEQ ID No. 13 of HFR1, a sequence shown in an amino acid sequence SEQ ID No. 15 of HFR2, a sequence shown in an amino acid sequence SEQ ID No. 17 of HFR3, and a sequence shown in an amino acid sequence SEQ ID No. 19 of HFR4;

(2) The sequence shown in the amino acid sequence SEQ ID No. 14 of HFR1, the sequence shown in the amino acid sequence SEQ ID No. 16 of HFR2, the sequence shown in the amino acid sequence SEQ ID No. 18 of HFR3, and the sequence shown in the amino acid sequence SEQ ID No. 20 of HFR 4.

In the binding agent provided by the application, the light chain variable region further comprises framework regions LFR1-LFR4; the amino acid sequence of LFR1 is shown as SEQ ID No. 21 or SEQ ID No. 22; the amino acid sequence of LFR2 is shown as SEQ ID No. 23 or SEQ ID No. 24; the amino acid sequence of LFR3 is shown as SEQ ID No. 25 or SEQ ID No. 26; the amino acid sequence of LFR4 is shown as SEQ ID No. 27 or SEQ ID No. 28, and the specific sequence is shown as follows:

LFR1：DIVMTQSQKFMSTSVGDRVSVTC（SEQ ID No:21）

LFR1：DVVMTQTPLSLPVSLGDQASISC（SEQ ID No:22）

LFR2：WYQQKPGQSPKTLIY（SEQ ID No:23）

LFR2：WYLQKPGQSPKVLIY（SEQ ID No:24）

LFR3：GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC（SEQ ID No:25）

LFR3：GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC（SEQ ID No:26）

LFR4：FGSGTKLEIK（SEQ ID No:27）

LFR4：FGGGTKLEIK（SEQ ID No:28）。

in the binding agents provided herein, the framework region of the light chain variable region is selected from any one of the following:

(1) The amino acid sequence of LFR1 is shown as a sequence of SEQ ID No. 21, the amino acid sequence of LFR2 is shown as a sequence of SEQ ID No. 23, the amino acid sequence of LFR3 is shown as a sequence of SEQ ID No. 25, and the amino acid sequence of LFR4 is shown as a sequence of SEQ ID No. 27;

(2) The amino acid sequence of LFR1 is shown as SEQ ID No. 22, the amino acid sequence of LFR2 is shown as SEQ ID No. 24, the amino acid sequence of LFR3 is shown as SEQ ID No. 26, and the amino acid sequence of LFR4 is shown as SEQ ID No. 28.

In the binding agent provided by the application, the amino acid sequence of the heavy chain variable region of the binding agent is shown as SEQ ID No. 29 or SEQ ID No. 30, and the heavy chain variable region is as follows, wherein the CDR regions are shown in bold:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYAMKWVRQSHGKSLEWIGDINPNSGDSFYNWKFKDRATLTVDKSSSTAYMQLNSLTSEDSAVYYCSREQYGHIYSEYSWFAYWGQGTLVTVSA （SEQ ID No:29）；

QVQLQQSGAELKKPGASVKISCKATGYTFISYKIEWIKQRPGHGLEWIGEISPEWNNTQYNEKFKGKATFTADTSSNTVYLRLSSLTSEDSAVYYCAREEDYCYDYWGQGTTLTVSS（SEQ ID No:30）。

in the binding agent provided by the application, the amino acid sequence of the light chain variable region of the binding agent is shown as SEQ ID No. 31 or SEQ ID No. 32, the light chain variable region is as follows, wherein the CDR regions are shown in bold:

DIVMTQSQKFMSTSVGDRVSVTCKASQTVDTNVAWYQQKPGQSPKTLIYSASRQSSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNVYGFTFGSGTKLEIK（SEQ ID No:31）；

DVVMTQTPLSLPVSLGDQASISCRSGQSLKHSNGDTYLHWYLQKPGQSPKVLIYQVLNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTVVPYTFGGGTKLEIK（SEQ ID No:32）。

in the binding agent provided herein, the amino acid sequences of the heavy chain variable region and the light chain variable region of the binding agent are selected from any one of the following:

(1) The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 29; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 31;

(2) The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 30; the amino acid sequence of the light chain variable region is shown as SEQ ID No. 32.

In the binding agent provided by the application, the amino acid sequence of the heavy chain of the binding agent is shown as SEQ ID No. 33 or SEQ ID No. 34, the variable region is shown in underline, the CDR region is shown in bold, and the specific sequence is as follows:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYAMKWVRQSHGKSLEWIGDINPNSGDSFYNWKFKDRA TLTVDKSSSTAYMQLNSLTSEDSAVYYCSREQYGHIYSEYSWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK*（SEQ ID No:33）；

QVQLQQSGAELKKPGASVKISCKATGYTFISYKIEWIKQRPGHGLEWIGEISPEWNNTQYNEKFKGKA TFTADTSSNTVYLRLSSLTSEDSAVYYCAREEDYCYDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK（SEQ ID No:34）。

in the binding agent provided by the application, the amino acid sequence of the light chain is shown as SEQ ID No. 35 or SEQ ID No. 36, the variable region is underlined, the CDR region is shown in bold, and the specific sequence is as follows:

DIVMTQSQKFMSTSVGDRVSVTCKASQTVDTNVAWYQQKPGQSPKTLIYSASRQSSGVPDRFTGSGSG TDFTLTISNVQSEDLAEYFCQQYNVYGFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*（SEQ ID No:35）；

DVVMTQTPLSLPVSLGDQASISCRSGQSLKHSNGDTYLHWYLQKPGQSPKVLIYQVLNRFSGVPDRFS GSGSGTDFTLKISRVEAEDLGVYFCSQSTVVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*（SEQ ID No:36）。

in the binding agent provided herein, the amino acid sequence of the binding agent is selected from any one of the following:

(1) The amino acid sequence of the heavy chain is shown as SEQ ID No. 33, and the amino acid sequence of the light chain is shown as SEQ ID No. 35, namely S-60-1 of the application;

(2) The amino acid sequence of the heavy chain is shown as SEQ ID No. 34, and the amino acid sequence of the light chain is shown as SEQ ID No. 36, namely S-320-1 of the present application.

The application aims at the RANKL target point, provides the amino acid sequences of the heavy chain variable region and the light chain variable region of the S-60-1 and S-320-1 antibodies, and the sequences are disclosed for the first time. The anti-RANKL anti-idiotype antibody has high binding force and high specificity.

In the binding agent provided by the application, the binding agent is a full-length antibody, a Fab fragment or F (ab) ₂ Fragments, fv fragments and scFv fragmentsA fragment or single domain antibody.

A "full length antibody" is an immunoglobulin molecule comprising at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable region is a domain in the heavy or light chain of an antibody that is involved in binding the antibody to its antigen. The constant region is not directly involved in binding of antibodies to antigens, but exhibits multiple effector functions. The light chains of antibodies can be classified into two types (called kappa (kappa) and lambda (lambda)) based on the amino acid sequences of their constant regions. The heavy chains of antibodies can be divided into mainly 5 different types based on the amino acid sequence of their constant regions: igA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses, e.g., igG1, igG2, igG3 and IgG4, igA1, and IgA2.

"Fab fragments" are monovalent fragments composed of VL, VH, CL and CH 1.

“F(ab) ₂ Fragments "are those having two antigen-binding F (ab) moieties, F (ab), linked together by disulfide bonds ₂ Fragment antibodies were obtained by pepsin digestion of whole IgG antibodies, removing most of the Fc region while leaving some hinge region intact.

"Fv fragments" consist of the antibody single arms VL and VH.

The two domains of an "scFv fragment" Fv fragment, VL and VH, can be joined by recombinant methods via a synthetic linker that makes both single protein chains, wherein the VL and VH regions pair to form a monovalent moiety, known as a single chain Fc (scFv).

A "single domain antibody" (sdAb), also known as a domain antibody, is an antibody fragment that contains only a single variable domain of the entire antibody. The first sdAb is derived from a camelid antibody, which is a dimer of two heavy chains, without light chains. Antibodies of this type are also found in cartilaginous fish, such as shark. To date, most of the resulting sdabs were engineered from camelids or cartilaginous fish, termed vh. In addition, sdabs from common IgG light chains have also been shown to be active in antigen binding. Although sdAb is much smaller (typically 12-15 KD) than other forms of antibodies (such as Fab and scFv), it has all the elements required for antigen binding.

These antibody fragments described above can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as the whole antibody.

In the binding agents provided herein, the binding agent is a monoclonal antibody, bispecific antibody, multispecific antibody, recombinant antibody, chimeric antibody, labeled antibody, anti-idiotype antibody, or fusion protein.

"monoclonal antibody" (monoclonal antibody, mAb) refers to an antibody that is highly homogeneous and directed against only a specific epitope, produced by cloning from a single B cell (the gene of which encodes only one antibody).

"bispecific antibody" (bispecific antibody, bsAb) is simply a diabody, which is an antibody-modified structure with two antigen-binding arms that is capable of recognizing two different antigens simultaneously, and is a specific form of artificial antibody. The bispecific antibody can specifically kill the podoma cells by combining tumor-related antigen sites and immune cell surface receptors (mostly T-gono E cell receptors) and simultaneously connecting tumor cells and T-gono-hexoses to trigger immune response with guidance.

"multispecific antibodies" are artificial antibodies that bind specifically to multiple antigens or epitopes simultaneously. Multispecific antibodies do not exist under natural conditions but are made by cell fusion or recombinant DNA techniques. Because of the specificity and the difunctional, the antibody has become a research hotspot in the field of antibody engineering, and has wide application prospect in the fields of tumor treatment, autoimmune diseases and the like.

"recombinant antibodies" are antibodies made by inserting a fragment of DNA into yeast, viruses or bacteria using recombinant DNA technology, from which researchers can extract antibodies for medical experiments and research in which medical scientists can use recombinant DNA to treat and treat diseases in patients. Recombinant DNA technology requires a laboratory environment in which researchers can work with a variety of organisms. The vector is a vector for the DNA of interest, and researchers can choose the most appropriate organism and vector based on genetic material and past success or failure, and carefully insert the DNA into the organism, forcing it to clone and express the antibody. Under controlled conditions she can develop multiple generations of organisms, all of which produce recombinant antibodies.

The "chimeric antibody" is an antibody molecule expressed by a mouse-derived antibody V region gene and a human antibody C region gene spliced into a chimeric gene, and then inserted into a vector to transfect myeloma tissues.

The "labeled antibody" is an antibody obtained by enzymatic labeling, ferritin labeling or by colloidal gold labeling.

A "fusion protein" is a protein consisting of at least two domains, which are encoded by separate genes that have been linked together so that they are transcribed and translated as a unit to produce a polypeptide.

Among the binding agents provided herein, the binding agent is selected from anti-idiotype antibodies, suitable for specifically binding to anti-RANKL antibody drugs or derivatives thereof.

"anti-idiotype antibody" refers to an antibody that specifically recognizes/binds to the idiotype of the antibody used to make them (i.e., has the idiotype of the variable region of the antibody used to make them as an epitope), which is capable of mimicking/rebuilding the epitope recognized by the antibody used to make them. The binding agent of the anti-RANKL monoclonal antibody drug or the derivative thereof is an anti-idiotype antibody of the anti-RANKL monoclonal antibody, can identify specific antigen epitope on a variable region of the anti-RANKL monoclonal antibody molecule, and has high binding force and high specificity to the RANKL monoclonal antibody.

An "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "epitopes" are also known in the art as "antigenic determinants". Epitopes or antigenic determinants are generally composed of chemically active surface groupings of molecules such as amino acids or carbohydrates or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics.

By "suitable" is meant that the binding agent of the present application can specifically bind to an anti-RANKL antibody drug or derivative thereof under suitable conditions, specifically to an epitope population on the variable region of an anti-RANKL monoclonal antibody. The anti-RANKL antibody drug or derivative thereof may specifically bind RANKL. The anti-RANKL antibody drug may be, for example, desiumab. Deshu mab is a humanized IgG2 monoclonal antibody that specifically binds to RANKL, a transmembrane or soluble protein necessary for osteoclast formation, function and survival; osteoclasts are responsible for bone resorption and thus regulate bone calcium release.

anti-RANKL monoclonal antibody drug derivatives include antibodies modified by methods such as glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by known techniques including, but not limited to: specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. The derivative may contain one or more non-classical amino acids. The anti-RANKL monoclonal antibody drug derivative may also be an antibody-drug conjugate (ADC). "drug" encompasses any substance that is effective in preventing or treating a tumor, such as a cancer, including a chemotherapeutic agent, cytokine, angiogenesis inhibitor, cytotoxic agent, other antibody, small molecule drug, or immunomodulator (e.g., immunosuppressant).

In the binding agents provided herein, the anti-idiotype antibody is a non-blocking antibody. By "non-blocking antibody" is meant an antibody that recognizes an anti-RANKL antibody by ELISA, does not recognize control human IgG, and does not block binding of the anti-RANKL antibody to RANKL (ELISA plate coated antigen, anti-RANKL antibody incubated with the prepared anti-idiotype antibody followed by addition to ELISA plate, detection with anti-human IgG secondary antibody, signal values were not reduced compared to the non-idiotype antibody added incubation group (PBS group).

In another aspect, the present application provides biological materials associated with the foregoing binding agents, including one or more of the following:

1) A nucleotide molecule encoding the aforementioned binding agent. The full-length nucleotide sequence of the binding agent or fragment thereof of the present application can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One approach is to synthesize the sequence of interest by synthetic means, especially when the fragment length is short. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the coding sequences for the light and heavy chains may be fused together to form an anti-idiotype antibody.

2) Recombinant expression vector comprising the nucleotide of 1). A "recombinant expression vector" has convenient restriction sites located near the promoter sequence to provide for insertion of a nucleic acid sequence encoding a heterologous protein. A selectable marker that is effective in the expression host may be present. Suitable expression vectors include, but are not limited to, bacterial plasmids, phages, yeast plasmids, viral vectors or other vectors. Examples of viral vectors include, but are not limited to, viral vectors based on: vaccinia virus; poliovirus; adenovirus, herpes simplex virus; retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, hawy sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and breast tumor), and the like.

3) Host cells comprising the recombinant expression vector of 2) or the nucleotide of 1) integrated into the genome. Suitable host cells include eukaryotic host cells, such as mammalian cells, insect host cells, yeast cells; and prokaryotic cells, such as bacterial cells. Introduction of the gene of interest into the host cell may be accomplished, for example, by calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods.

4) A kit comprising the aforementioned binding agent, a nucleotide of 1), a recombinant expression vector of 2), or a host cell of 3). The anti-idiotype antibody contained in the kit can bind to the anti-RANKL antibody and thus can be used for directly detecting the anti-RANKL antibody. The anti-idiotype antibody in the anti-RANKL antibody immunogenicity detection kit can be combined with the anti-RANKL antibody, and can be used as a positive control for detecting the anti-RANKL antibody. The kit of the application can also comprise reagents such as conventional buffers, washing solutions, coating solutions and the like which are necessary for detection. The kit can be a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme linked immunoassay kit or a fluorescent immunoassay kit. The kit can directly use the anti-idiotype antibody and the biological material contained in the kit.

In another aspect, the application provides the use of the binding agent and the biological material in preparing an anti-RANKL monoclonal antibody detection product or a positive control of an anti-RANKL antibody drug immunogenicity detection product.

In the uses provided herein, anti-RANKL monoclonal antibody detection products include qualitative or quantitative analysis for anti-RANKL monoclonal antibodies or derivatives thereof; further, the anti-RANKL monoclonal antibody detection product is: the product of the pharmacokinetics analysis or pharmacodynamics analysis of the antibody.

An anti-RANKL antibody derivative may refer to an anti-RANKL antibody modified with a functional group having an antigen-specific recognition ability and binding ability similar to those of an unmodified antibody or higher reactivity.

Pharmacodynamics and pharmacokinetics share common influencing factors such as dosage, pharmaceutical benefits and adverse reactions. The anti-idiotype antibody can be used for detecting the change rule and the metabolic condition of the anti-RANKL antibody in a pharmacokinetics analysis test and a pharmacodynamics analysis of the anti-RANKL antibody.

In another aspect, the present application provides a method for detecting an anti-RANKL monoclonal antibody drug or derivative thereof in a sample for non-disease diagnostic or therapeutic purposes, comprising:

(a) Contacting the sample with the binding agent;

(b) Detecting the presence or absence of an immune complex of the binding agent bound to the anti-RANKL monoclonal antibody drug or the derivative thereof, so as to qualitatively analyze whether the anti-RANKL monoclonal antibody drug or the derivative thereof is present in the sample; or detecting the amount of the immune complex to quantitatively analyze the sample for the anti-RANKL monoclonal antibody drug or derivative thereof.

In step (a), the binding agent is immobilized directly or indirectly to the solid support. In some embodiments, the solid support is selected from the group consisting of acetate membranes, test tubes, EP tubes, multiwell plates, microplate wells, and microspheres.

In the method provided herein, in step (b), detecting the presence of an immune complex or detecting the amount of an immune complex is accomplished using a method selected from the group consisting of:

i. via a detectable label directly or indirectly linked to a binding agent;

the binding agent or the anti-RANKL monoclonal antibody drug or the derivative thereof, and the detectable label is directly or indirectly connected to the other reagent;

via another of the foregoing binding agents, the other binding agent having a detectable label directly or indirectly attached thereto and recognizing the additional idiotype;

implementation via an additionally added anti-RANKL monoclonal antibody drug or derivative thereof, to which a detectable label is directly or indirectly attached.

In some embodiments, i.is that when the binding agent binds to an anti-RANKL monoclonal antibody drug or derivative thereof to form an immune complex, the presence of the immune complex can be recognized by detection of the detectable label due to the detectable label carried directly or indirectly by the binding agent.

In some embodiments, the other agent in ii. May be, for example, a secondary antibody that is homologous to the binding agent, is different from the anti-RANKL monoclonal antibody drug or derivative thereof, and thus may bind to the binding agent, not to the anti-RANKL monoclonal antibody drug or derivative thereof. The secondary antibody can be used as a detection antibody for detecting the combination of a binding agent and an anti-RANKL monoclonal antibody drug or a derivative thereof, if the combination generates an immune complex, a detectable label carried by the secondary antibody can be identified, and if the binding agent does not combine with the anti-RANKL monoclonal antibody drug or the derivative thereof, the secondary antibody can not detect the existence of the immune complex.

In some embodiments, iii.and iv.can be, for example, a double antibody sandwich method.

The principle of the double antibody sandwich method is that a binding agent is bound on a solid phase carrier to form a solid phase antibody, then the solid phase antibody is bound with a corresponding anti-RANKL monoclonal antibody drug or a derivative thereof in a sample to form an immune complex, the immune complex is washed, then the binding agent is additionally added, a detectable label is directly or indirectly connected with the additionally added binding agent, the binding agent is bound with the immune complex to form a binding agent-anti-RANKL monoclonal antibody drug or a derivative thereof-binding agent complex, the substrate is added for color development, and the situation of the anti-RANKL monoclonal antibody drug or the derivative thereof in the sample is judged.

The principle of the medium double antibody sandwich method is that an anti-RANKL monoclonal antibody drug or a derivative thereof is combined on a solid phase carrier to form a solid phase antibody, then the solid phase antibody is combined with a binding agent to form an immune complex, the anti-RANKL monoclonal antibody drug or the derivative thereof is additionally added after washing, a detectable marker is directly or indirectly connected with the additionally added anti-RANKL monoclonal antibody drug or the derivative thereof, the anti-RANKL monoclonal antibody drug or the derivative thereof is combined with the immune complex to form an anti-RANKL monoclonal antibody drug or the derivative thereof-binding agent-anti-RANKL monoclonal antibody drug or the derivative complex thereof, the color of the added matrix is developed, and the condition of the binding agent is judged.

When the quantitative detection is carried out on the sample, the concentration of the binding agent added in the reaction is in a functional relation with the concentration of the immune complex, and the content of the anti-RANKL monoclonal antibody drug or the derivative thereof in the sample can be estimated according to the generation amount of the immune complex: under certain reaction conditions, the concentration of the added binding agent is certain, and the amount of immune complex generated by the reaction is in direct proportion to the amount of the corresponding anti-RANKL monoclonal antibody drug or the derivative thereof contained in the sample to be detected. The amount of the anti-RANKL monoclonal antibody drug or derivative thereof may be determined by detecting the amount of the label. The content of the anti-RANKL monoclonal antibody drug or the derivative thereof in the sample can be calculated by using an experimental standard curve. Such as immune one-way diffusion test, immune turbidimetry test and enzyme-linked immunosorbent assay. Thus, pharmacokinetic analysis of anti-RANKL monoclonal antibody drugs or derivatives thereof, e.g. the law of the in vivo drug concentration over time, may be performed.

In some embodiments, the detectable label comprises: colloidal gold, radiolabels, luminescent substances, colored substances, enzymes, such as fluorescent labels, chromophore labels, electron dense labels, such as radioisotopes, fluorophores, rhodamine and derivatives thereof, luciferases, luciferins, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labels, and the like.

In some embodiments, the effects of antibodies of the present disclosure, such as binding activity and/or cross-reactivity, can be detected using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, and the like. In some embodiments, the assay may include, for example, ELISA (enzyme linked immunosorbent assay), FACS (flow cytometry fluorescence sorting technique) binding assay, biacore, competitive binding assay, and the like. In some embodiments, the reactivity of the antibodies of the present disclosure with antigen (antigenic peptide) binding is characterized, for example, in ELISA, e.g., reading a reaction value ∈ 0.5 at 450nm by peroxidase-labeled ELISA is determined to be better reactivity and useful in immunoassays.

Compared with the prior art, the beneficial effects of this application are:

1. the anti-idiotype antibody has high binding force and high specificity of the anti-RANKL antibody, and can be used for detecting the content of the anti-RANKL antibody medicine in a sample, thereby playing an important role in the pharmacokinetic and pharmacodynamic analysis of the anti-RANKL antibody medicine.

2. The anti-idiotype antibody has good performance test, can be used as positive control for immunogenicity ADA analysis, and plays an important role in immunogenicity evaluation.

Drawings

FIG. 1 is a standard graph of quantitative analysis of anti-RANKL humanized monoclonal antibodies.

Fig. 2 is a graph of anti-idiotype antibody ADA analysis.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application is further described below with reference to examples. It should be understood that the examples are presented by way of illustration only and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, unless otherwise indicated, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventors of the present application have found a binding agent for an anti-RANKL monoclonal antibody drug or a derivative thereof and an application thereof through a great deal of research and study, and completed the present application on the basis of this.

The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Example 1

Preparation of anti-idiotype antibodies

1. Animal immunization and antibody production

Pepsin (Sigma, cat# P6887) enzyme-cleaved anti-RANKL humanized monoclonal antibody (namely Deshu monoclonal antibody, manufactured by Miwei (Shanghai) Biotech Co., ltd.) and the Fc region of the antibody was removed to prepare F (ab) ₂ And (3) a sample. F (ab) to be obtained ₂ The samples were used as immunogens for immunization of animals, and 5 Balb/c mice were immunized 3 times each for 3 weeks. The serum of the immunized mice is required to be specifically combined with the corresponding immunogen, and the titer of the antiserum is more than 1:10,000.

Spleen cells of 1-2 mice with highest titers among immunized mice are selected for fusion with SP2/0 myeloma cells. Amplifying and culturing hybridoma cells, taking supernatant, detecting titer by ELISA, and detecting antibody subtype; cells positive for anti-RANKL humanized monoclonal antibodies and negative for irrelevant human IgG were selected for cloning. To ensure the positive rate of hybridoma cell strain and the stable production of antibody, 3-4 clones were carried out, each clone was separated by 1-2 weeks, and after 3-4 times of 100% positive clones, the cells were inoculated into mice after expanded culture to prepare ascites, thus obtaining the anti-idiotype antibody. In the cloning process, the specificity of hybridoma supernatant is detected by an indirect ELISA method, namely, the murine monoclonal antibodies in the supernatant only bind to the anti-RANKL humanized monoclonal antibodies, but not to irrelevant human IgG, and the result shows that the antibody of the strain S-66-3 has poor specificity and cross reaction with human IgG, and finally, the monoclonal antibodies with good specificity of the strain S-60-1 and the strain S-320-1, namely 2, are obtained.

TABLE 1 hybridoma supernatant specificity characterization results

2. Target competitive and noncompetitive testing

For detection of 2 antibodies produced by hybridoma cells, the following conditions should be satisfied:

1) Target non-blocking antibodies: detecting by ELISA method, recognizing anti-RANKL antibody, not recognizing control human IgG, and not blocking the binding of anti-RANKL antibody and RANKL (ELISA plate coated antigen, adding ELISA plate after incubation of anti-RANKL antibody and prepared anti-idiotype antibody, detecting by anti-human IgG secondary antibody, and signal value is not reduced compared with the incubation group without anti-idiotype antibody (PBS group);

2) Target blocking antibodies: the ELISA method is used for detection, the anti-RANKL antibody is identified, the control human IgG is not identified, the binding between the anti-RANKL antibody and the RANKL can be blocked (ELISA plate coated antigen, the anti-RANKL antibody is added into the ELISA plate after being incubated with the prepared anti-idiotype antibody, the anti-human IgG secondary antibody is used for detection, and the signal value is obviously reduced compared with that of an incubation group (PBS) without the anti-idiotype antibody).

The results are shown in the following table, and the target blocking property of the anti-idiotype antibody is judged by comparing the difference of OD values of the added antibody group and the non-added antibody group. The results show that the signal values after addition of S-60-1 and S-320-1 are similar to those of the PBS group, and thus are target non-blocking antibodies.

Example 2

Sequencing of anti-idiotype antibodies

1. Experimental procedure

And (3) amplifying and culturing the hybridoma cells, extracting total RNA of the cells, and carrying out reverse transcription to obtain cDNA. And (3) PCR amplification to obtain heavy chain and light chain variable region genes of the antibody, sequencing and performing bioinformatic analysis of the sequence, and eliminating nonfunctional antibody genes. Antibodies are named according to their hybridoma cell clone of origin, and the amino acid sequences of the heavy and light chains are shown below. The variable regions of the heavy and light chains are shown underlined and the CDR regions (complementarity determining regions) of the heavy and light chains, defined according to the KABAT naming system, are shown bolded.

2. Sequencing results

S-60-1 complete antibody sequences

Heavy chain sequence:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYAMKWVRQSHGKSLEWIGDINPNSGDSFYNWKFKDRA TLTVDKSSSTAYMQLNSLTSEDSAVYYCSREQYGHIYSEYSWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK*（SEQ ID No:33）。

light chain sequence:

DIVMTQSQKFMSTSVGDRVSVTCKASQTVDTNVAWYQQKPGQSPKTLIYSASRQSSGVPDRFTGSGSG TDFTLTISNVQSEDLAEYFCQQYNVYGFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*（SEQ ID No:35）。

s-320-1 complete antibody sequences

Heavy chain sequence:

light chain sequence:

the antibody can be obtained by conventional genetic engineering to obtain an isolated monoclonal antibody, for example, by synthesizing the gene of the antibody, constructing a recombinant vector, transfecting 293 cells, carrying out recombinant expression, and purifying.

Example 3

Application of Anti-idiotype antibody of Anti-RANKL antibody in non-clinical and clinical drug analysis

1. Use of anti-idiotype antibodies in non-clinical drug generations

Enzyme-linked immunosorbent assays (ELISA) are often used in antibody drug clinical and non-clinical pharmacokinetic studies. In the embodiment, the cynomolgus monkey serum drug-substitution analysis is taken as an example, the target protein recombinant human RANKL extracellular protein is taken as a capture reagent, and the anti-uniqueness antibody is taken as a detection reagent, so that the application of the two anti-uniqueness antibodies in the invention in non-clinical drug-substitution analysis is examined.

1. Test method

1.1 Coating and sealing: diluting recombinant human RANKL extracellular protein (ACRO, product number RAL-H5240) to 2 mug/mL with coating liquid, and standing overnight at 2-8 ℃; the ELISA plate is patted dry, and the ELISA plate is washed 3 times by a washing buffer solution; the ELISA plate is dried, 3% BSA/PBST is added, and the ELISA plate is incubated for 2 hours at room temperature; the blocked ELISA plates were then dried by pipetting, and the ELISA plates were rinsed 3 times with wash buffer.

1.2 Standard curve and quality control dilution: anti-RANKL humanized mab (manufactured by Jiangsu meweikang new drug development limited) was diluted with 100% pooled monkey serum (pooled monkey serum, PMS) to prepare standard curves with the following concentrations: 10000ng/ml (ULOQ, upper limit of quantitation), 5000ng/ml, 2500ng/ml, 1250ng/ml, 625ng/ml, 312.5ng/ml, 156.25ng/ml (LLOQ, lower limit of quantitation), 78.12ng/ml (anchor point). The blank (0 ng/ml) was used as part of the standard curve, but was not involved in the standard curve fitting. The 5 quality control concentrations were also prepared with 100% pooled monkey serum: 10000ng/ml (ULOQ, upper limit of quantitation), 8000ng/ml (HQC, high concentration quality control), 2000ng/ml (MQC, medium concentration quality control), 400ng/ml (LQC, low concentration quality control), 156.25ng/ml (LLOQ, lower limit of quantitation).

1.3 Diluting and adding a sample to be tested: after 50 times of pre-dilution of the monkey serum sample to be tested, the standard curve sample and the quality control product with 1% BSA/PBST respectively, adding the samples into an ELISA plate according to 100 μl/well, providing a multiplex well, standing at room temperature and incubating for 2h.

1.4 Adding a detection antibody: after incubation, the ELISA plates were patted dry and washed 6 times with wash buffer; 2 anti-idiotype antibodies (S-320-1, S-60-1) were diluted to 2. Mu.g/ml with 1% BSA/PBST, respectively, and then added to ELISA plates, and incubated at room temperature for 1h.

1.5 Adding a detection secondary antibody: after incubation, the ELISA plates were patted dry and washed 6 times with wash buffer; goat anti-mouse IgG HRP-labeled secondary antibody (Jackson ImmunoResearch, cat. No. 115-035-003) was diluted 20000 times with 1% BSA/PBST and added to ELISA plate, and incubated at room temperature for 1h.

1.6 Color development: after incubation, the ELISA plates were patted dry and washed 6 times with wash buffer; adding TMB substrate (KPL, cat. No. 5120-0047) equilibrated to room temperature into ELISA plate, and incubating at room temperature in dark place for about 10 min; 1M H is added ₃ PO ₄ The reaction was terminated.

1.7 Reading a plate: after the end of the color reaction, the OD value at the wavelength of 450nm was read with 650nm as a reference wavelength.

1.8 Concentration calculation: and performing four-parameter fitting on the standard curve concentration and the OD value, and calculating the drug concentration of the sample to be detected by using a fitting equation.

1.9 The method is used for detecting the concentration of the anti-RANKL humanized monoclonal antibody in monkey serum, a standard curve is shown in figure 1, two anti-idiotype antibodies, namely S-60-1 and S-320-1, can be used as detection antibodies for drug generation analysis, and the antibody S-60-1 has higher detection sensitivity and detection signal intensity. The results are shown in Table 2, and the drug generation analysis method established by the two antibodies has good accuracy and precision, and can accurately quantify the drug concentration in the serum of the cynomolgus monkey.

TABLE 2 accuracy and precision

2. Application of anti-idiotype antibody in detection of drug immunogenicity ADA

The immune response of a therapeutic protein drug may affect the pharmacokinetics, pharmacodynamics, safety and effectiveness of the drug. The adverse reactions of most drugs are caused by humoral immune mechanisms-mediated immune responses, and detection of drug-resistant antibodies (ADA) is a major method for assessing drug immunogenicity. This example mainly examined the use of anti-idiomatic antibodies in the detection of immunogenic ADA.

1. Test procedure

1.1 Coating and sealing: diluting the anti-RANKL humanized monoclonal antibody (manufactured by Jiangsu Meikang New drug development Co., ltd.) to 1 mug/ml with a coating solution, and standing at 2-8 ℃ overnight; the ELISA plate is patted dry, and the ELISA plate is washed 3 times by a washing buffer solution; the ELISA plate is dried, 3% BSA/PBST is added, and the ELISA plate is incubated for 2 hours at room temperature; the blocked ELISA plates were then dried by pipetting, and the ELISA plates were rinsed 3 times with wash buffer.

1.2 Preparing a sample to be tested: 2 anti-idiotype antibodies (S-320-1, S-60-1) were diluted with 10% pooled monkey serum (10% PMS) to 10. Mu.g/ml as starting concentration, 3-fold gradient dilution for 7 spots and 10% PMS as 0 spot.

1.3 Sample adding: anti-RANKL humanized monoclonal antibody was labeled according to the instructions with EZ-LINK NHS-LC-LC-Biotin (Thermo, cat # 21343) to give anti-RANKL antibody-Biotin, which was diluted to 50ng/ml with 1% BSA/PBST. Each of the above diluted anti-idiotype antibody samples and anti-RANKL antibody-Biotin was added to ELISA plates at 50. Mu.l/well, multiplexed wells were set, and incubated at room temperature for 2h.

1.4 Adding SA-HRP: after incubation, the ELISA plates were dried by pipetting, and washed 6 times with wash buffer. SA-HRP (southern Biotech, cat. No. 7105-05) was diluted 1:5000 with 1% BSA/PBST and added to ELISA plates and incubated for 1h at room temperature.

1.5 Color development: after incubation, the ELISA plates were dried by pipetting, and washed 6 times with wash buffer. TMB substrate (KPL, cat. No. 5120-0047) equilibrated beforehand to room temperature was added to ELISA plates and incubated at room temperature for about 3min in the absence of light. 1M H is added ₃ PO ₄ The reaction was terminated.

1.6 Reading a plate: after the end of the color reaction, the OD value at the wavelength of 450nm was read with 650nm as a reference wavelength.

2. Test results

As shown in FIG. 2, the anti-idiotype antibody S-60-1 has good performance test, and the antibody concentration and the signal value have good dose-response relationship, and can be used as positive control for ADA analysis.

In conclusion, the anti-idiotype monoclonal antibody capable of specifically recognizing the anti-RANKL humanized monoclonal antibody has the advantages of good uniformity and strong binding force, can be widely applied to the field of drug research and development, and is mainly applied to immunogenicity analysis, pharmacokinetic analysis and the like of antibody drugs.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.

Claims

1. A binding agent for an anti-RANKL monoclonal antibody, the binding agent comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3; wherein the HCDR1, HCDR2, HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, LCDR3 of the light chain variable region are selected from any one of: (1) The sequence of the HCDR1 is shown as SEQ ID No. 1, the sequence of the HCDR2 is shown as SEQ ID No. 3, the sequence of the HCDR3 is shown as SEQ ID No. 5, the sequence of the LCDR1 is shown as SEQ ID No. 7, the sequence of the LCDR2 is shown as SEQ ID No. 9, and the sequence of the LCDR3 is shown as SEQ ID No. 11; (2) The sequence of the HCDR1 is shown as SEQ ID No. 2, the sequence of the HCDR2 is shown as SEQ ID No. 4, the sequence of the HCDR3 is shown as SEQ ID No. 6, the sequence of the LCDR1 is shown as SEQ ID No. 8, the sequence of the LCDR2 is shown as SEQ ID No. 10, and the sequence of the LCDR3 is shown as SEQ ID No. 12.

2. The binding agent of claim 1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the binding agent are selected from any one of the following: (1) The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 29; and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 31; (2) The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 30; and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 32.

3. The binding agent of claim 1, wherein the amino acid sequence of the binding agent is selected from any one of the following:

(1) The amino acid sequence of the heavy chain is shown as SEQ ID No. 33, and the amino acid sequence of the light chain is shown as SEQ ID No. 35; (2) The amino acid sequence of the heavy chain is shown as SEQ ID No. 34, and the amino acid sequence of the light chain is shown as SEQ ID No. 36.

4. The binding agent of claim 1, wherein the binding agent is a full-length antibody, fab fragment, F (ab) ₂ Fragments, fv fragments or scFv fragments.

5. The binding agent of claim 1, wherein the binding agent is a monoclonal antibody or an anti-idiotype antibody.

6. The binding agent of claim 5, wherein the binding agent is an anti-idiotype antibody adapted to specifically bind to an anti-RANKL antibody drug.

7. The binding agent of claim 6, wherein the anti-RANKL antibody drug is desquamation; and/or, the anti-idiotype antibody is a non-blocking antibody.

8. A biological material associated with the binding agent of any one of claims 1 to 7, wherein the biological material comprises one or more of the following:

1) A polynucleotide encoding a binding agent according to any one of claims 1 to 7;

2) A recombinant expression vector comprising the polynucleotide of 1);

3) A host cell comprising 2) the recombinant expression vector or the polynucleotide of 1) integrated into the genome;

4) A kit comprising the binding agent of any one of claims 1-7, the polynucleotide of 1), the recombinant expression vector of 2), or the host cell of 3).

9. Use of the binding agent according to any one of claims 1 to 7 or the biomaterial according to claim 8 for the preparation of an anti-RANKL monoclonal antibody detection product or a positive control for an anti-RANKL antibody drug immunogenicity detection product.

10. The use according to claim 9, wherein the anti-RANKL monoclonal antibody detection product comprises a qualitative or quantitative analysis for anti-RANKL monoclonal antibodies.

11. The use according to claim 10, wherein the anti-RANKL monoclonal antibody detection product is: the product of the pharmacokinetics analysis or pharmacodynamics analysis of the antibody.

12. A method for detecting an anti-RANKL monoclonal antibody drug in a sample for non-disease diagnostic or therapeutic purposes, the method comprising:

(a) Contacting a sample with the binding agent of any one of claims 1 to 7;

(b) Detecting the presence or absence of an immunocomplex of the binding agent according to any of claims 1 to 7 bound to an anti-RANKL monoclonal antibody drug, to qualitatively analyze the presence or absence of the anti-RANKL monoclonal antibody drug in the sample; or detecting the amount of the immune complex to quantitatively analyze the anti-RANKL monoclonal antibody drug in the sample.

13. The method of claim 12, characterized by one or more of the following features:

in step (a), the binding agent or anti-RANKL monoclonal antibody drug is immobilized directly or indirectly to a solid support; in step (b), detecting the presence of said immune complex or detecting the amount of said immune complex is accomplished using a method selected from any one of the following:

i. via a detectable label directly or indirectly attached to the binding agent;

realization via a secondary antibody binding to the binding agent or the anti-RANKL monoclonal antibody drug, said secondary antibody being directly or indirectly linked to a detectable label;

realization via the additionally added anti-RANKL monoclonal antibody drug, which is directly or indirectly connected with a detectable label.