Anti-idiotype antibodies and uses thereof

Abstract

The invention relates to the field of antibody drugs, in particular to an anti-idiotype antibody and application thereof. The present invention provides anti-idiotypic antibodies and uses thereof. The invention provides an anti-idiotype mouse monoclonal antibody of a B7-H3 humanized monoclonal antibody with high binding force and high specificity. The antibody disclosed by the invention is an anti-idiotype monoclonal antibody for specifically recognizing the anti-B7-H3 humanized monoclonal antibody, is good in uniformity and strong in binding force, is 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.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on June 30, 2023, with application number 202310813856X and invention name “Anti-idiotypic antibodies and their applications”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of antibody drugs, and in particular to anti-idiotypic antibodies and applications thereof. Background Art

[0003] B7-H3 (also known as CD276) is a type I transmembrane protein and a member of the B7 ligand family. It is overexpressed in most cancer types, but expressed at low levels in normal tissues. B7-H3 is mainly expressed on the surface of non-immune cells such as fibroblasts, endothelial cells, and osteoblasts, as well as activated APCs and NK cells. Studies have shown that B7-H3 is overexpressed on the surface of various tumors such as melanoma, leukemia, breast cancer, and prostate cancer, and is closely related to tumor growth, metastasis, recurrence, and poor prognosis. More and more people are beginning to pay attention to the application value of this target in tumor immunotherapy.

[0004] Anti-idiotypic antibodies are antibodies that target specific antigenic epitope groups (idiotypes) on the variable region of antibody molecules and can specifically recognize and bind to another unique site (Idiotype) of an antibody. In the development of antibody drugs, there are two tests that use anti-idiotypic antibodies as the main tool reagents: 1) Because anti-idiotypic antibodies have different specific binding epitopes, they can be used to detect the content of various types of antibody drugs in the blood (free, bound and total amount), thus playing an important role in the pharmacokinetic (PK) and pharmacodynamic (PD) analysis of antibody drugs; 2) Anti-idiotypic antibodies, as positive controls for anti-drug antibodies (ADA) detection, also play an important role in the evaluation of immunogenicity.

[0005] At present, there are no anti-idiotypic antibodies with good uniformity, strong binding ability and high specificity used in the field of drug research and development. Summary of the invention

[0006] In view of this, the present invention provides anti-idiotypic antibodies and uses thereof.

[0007] The present invention provides an anti-idiotypic antibody and its application. The present invention provides a B7-H3 humanized monoclonal antibody anti-idiotypic mouse monoclonal antibody with high binding force and high specificity. The present invention is an anti-idiotypic monoclonal antibody that specifically recognizes the anti-B7-H3 humanized monoclonal antibody, has good uniformity and strong binding force, and is widely used in the field of drug research and development, mainly in the immunogenicity analysis and pharmacokinetic analysis of antibody drugs.

[0008] The anti-B7-H3 humanized monoclonal antibody involved in the present invention has:

[0009] (I), CDR1, CDR2 and CDR3 of the heavy chain of the anti-B7-H3 humanized monoclonal antibody have the amino acid sequences shown in SEQ ID No. 81, 82 and 83 respectively; and

[0010] (II), CDR1, CDR2 and CDR3 of the light chain of the anti-B7-H3 humanized monoclonal antibody have the amino acid sequences shown in SEQ ID No. 84, 85 and 86 respectively; or

[0011] (III) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I) or (II); or

[0012] (IV) A sequence having 80% or more homology with the amino acid sequence shown in any one of (I) to (III).

[0013] Furthermore, the anti-B7-H3 humanized monoclonal antibody involved in the present invention has:

[0014] (I), the variable region of the heavy chain of the anti-B7-H3 humanized monoclonal antibody has the amino acid sequence shown in SEQ ID No.87; and

[0015] (II), the variable region of the light chain of the anti-B7-H3 humanized monoclonal antibody has the amino acid sequence shown in SEQ ID No.88; or

[0016] (III) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I) or (II); or

[0017] (IV) A sequence having 80% or more homology with the amino acid sequence shown in any one of (I) to (III).

[0018] Furthermore, the anti-B7-H3 humanized monoclonal antibody involved in the present invention has:

[0019] (I), the heavy chain of the anti-B7-H3 humanized monoclonal antibody has the amino acid sequence shown in SEQ ID No.89; and

[0020] (II), the light chain of the anti-B7-H3 humanized monoclonal antibody has the amino acid sequence shown in SEQ ID No.90; or

[0021] (III) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I) or (II); or

[0022] (IV) A sequence having 80% or more homology with the amino acid sequence shown in any one of (I) to (III).

[0023] Specifically, the anti-B7-H3 humanized monoclonal antibody involved in the present invention has:

[0024] Heavy chain amino acid sequence (as shown in SEQ ID No.89)

[0025] EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYIMHWVRQAPGQGLEWMGGINPASGG

[0026] TTYNQKFKGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARKGKDYFDWYFDVWGQG

[0027] TTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF

[0028] PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP

[0029] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

[0030] KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[0031] VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[0032] YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0033] Light chain amino acid sequence (as shown in SEQ ID No.90)

[0034] DIQLTQSPSSSLSASVGDRVTMTCSASSSVSYMHWYQQKPGKAPKLLIYDTSKLASGVPSR

[0035] FSSGSGSGTDFTLTISSLQPEDFATYYCQQWSSAPLTFGGGTKVEIK RTVAAPSVFIFPPSDE

[0036] QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

[0037] KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0038] Therein, the variable regions are underlined and the CDRs are further indicated in bold italics (defined according to the KABAT nomenclature system).

[0039] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0040] The present invention provides anti-idiotypic antibodies, including a first anti-idiotypic antibody, a second anti-idiotypic antibody, a third anti-idiotypic antibody and / or a fourth anti-idiotypic antibody;

[0041] (I), CDR1, CDR2 and CDR3 of the heavy chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 1, 2 and 3, respectively; and

[0042] The CDR1, CDR2 and CDR3 of the light chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 11, 12 and 13, respectively; or

[0043] (II), CDR1, CDR2 and CDR3 of the heavy chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 21, 22 and 23, respectively; and

[0044] The CDR1, CDR2 and CDR3 of the light chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 31, 32 and 33, respectively; or

[0045] (III), CDR1, CDR2 and CDR3 of the heavy chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 41, 42 and 43, respectively; and

[0046] The CDR1, CDR2 and CDR3 of the light chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No.51, 52 and 53, respectively; or

[0047] (IV), CDR1, CDR2 and CDR3 of the heavy chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 61, 62 and 63, respectively; and

[0048] The CDR1, CDR2 and CDR3 of the light chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No.71, 72 and 73, respectively; or

[0049] (V) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (I) to (IV); or

[0050] (VI) A sequence having an identity of 80% or more to the amino acid sequence shown in any one of (I) to (V).

[0051] Preferably, the homology with the amino acid sequence shown in any one of (I) to (V) is 85%, 90%, 95%, 98% or 99%.

[0052] In some specific embodiments of the present invention,

[0053] (i) FR1, FR2, FR3 and FR4 of the heavy chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 4, 5, 6 and 7, respectively; and

[0054] FR1, FR2, FR3 and FR4 of the light chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 14, 15, 16 and 17, respectively; or

[0055] (ii) FR1, FR2, FR3 and FR4 of the heavy chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 24, 25, 26 and 27, respectively; and

[0056] FR1, FR2, FR3 and FR4 of the light chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 34, 35, 36 and 37, respectively; or

[0057] (iii) FR1, FR2, FR3 and FR4 of the heavy chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 44, 45, 46 and 47, respectively; and

[0058] FR1, FR2, FR3 and FR4 of the light chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 54, 55, 56 and 57, respectively; or

[0059] (iv) FR1, FR2, FR3 and FR4 of the heavy chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 64, 65, 66 and 67, respectively; and

[0060] FR1, FR2, FR3 and FR4 of the light chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No.74, 75, 76 and 77, respectively; or

[0061] (v) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (i) to (iv); or

[0062] (vi) A sequence having 80% or more homology with the amino acid sequence shown in any one of (i) to (v).

[0063] Preferably, the homology with the amino acid sequence shown in any one of (i) to (v) is 85%, 90%, 95%, 98% or 99%.

[0064] In some specific embodiments of the present invention,

[0065] (A), the variable region of the heavy chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 9; and

[0066] The variable region of the light chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 19; or

[0067] (B), the variable region of the heavy chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 29; and

[0068] The variable region of the light chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.39; or

[0069] (C) the variable region of the heavy chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 49; and

[0070] The variable region of the light chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.59; or

[0071] (D), the variable region of the heavy chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.69; and

[0072] The variable region of the light chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.79; or

[0073] (E) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (A) to (D); or

[0074] (F) A sequence having 80% or more homology with the amino acid sequence shown in any one of (A) to (E).

[0075] Preferably, the homology with the amino acid sequence shown in any one of (A) to (E) is 85%, 90%, 95%, 98% or 99%.

[0076] In some specific embodiments of the present invention,

[0077] (a) the signal peptide of the heavy chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 8; and

[0078] The signal peptide of the light chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.18; or

[0079] (b) the signal peptide of the heavy chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 28; and

[0080] The signal peptide of the light chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.38; or

[0081] (c) the signal peptide of the heavy chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.48; and

[0082] The signal peptide of the light chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.58; or

[0083] (d) the signal peptide of the heavy chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.68; and

[0084] The signal peptide of the light chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.78; or

[0085] (e) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (a) to (d); or

[0086] (f) A sequence having 80% or more homology with the amino acid sequence shown in any one of (a) to (e).

[0087] Preferably, the homology with the amino acid sequence shown in any one of (a) to (e) is 85%, 90%, 95%, 98% or 99%.

[0088] In some specific embodiments of the present invention,

[0089] ①, the heavy chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.10; and

[0090] The light chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 20; or

[0091] ②, the heavy chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.30; and

[0092] The light chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.40; or

[0093] ③, the heavy chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.50; and

[0094] The light chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.60; or

[0095] ④, the heavy chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.70; and

[0096] The light chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.80; or

[0097] ⑤. A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of ① to ④; or

[0098] ⑥. A sequence having an amino acid sequence homology of 80% or more to any one of ① to ⑤.

[0099] Preferably, the homology with the amino acid sequence shown in any one of ① to ⑤ is 85%, 90%, 95%, 98% or 99%.

[0100] In some specific embodiments of the present invention, the anti-idiotypic antibodies include target blocking antibodies and target non-blocking antibodies;

[0101] The target blocking antibody comprises a first anti-idiotypic antibody and a third anti-idiotypic antibody;

[0102] The target non-blocking antibodies include a second anti-idiotypic antibody and a fourth anti-idiotypic antibody.

[0103] In some specific embodiments of the present invention, the first anti-idiotypic antibody is S-412-8; the second anti-idiotypic antibody is S-322-4; the third anti-idiotypic antibody is S-401-2; and the fourth anti-idiotypic antibody is S-255-11.

[0104] The present invention also provides a biomaterial comprising any of the following:

[0105] (I), a nucleic acid encoding the anti-idiotypic antibody; and / or

[0106] (II), a nucleic acid encoding the anti-idiotypic antibody and an acceptable carrier; and / or

[0107] (III), a host that secretes the anti-idiotypic antibody; and / or

[0108] (IV), chemically labeled or biologically labeled anti-idiotypic antibodies; and / or

[0109] (V), the anti-idiotypic antibody coupled to a carrier.

[0110] Based on the above research, the present invention also provides the use of any of the following items in blocking the binding between antibodies and antigens:

[0111] (I), the anti-idiotypic antibody; and / or

[0112] (II) the biological material.

[0113] In some specific embodiments of the present invention, the antibody comprises an anti-B7-H3 humanized monoclonal antibody; and the antigen comprises a B7-H3 extracellular protein.

[0114] The present invention also provides the use of any of the following items in detecting the immunogenicity of a drug:

[0115] (I), the anti-idiotypic antibody; and / or

[0116] (II) the biological material.

[0117] In some specific embodiments of the present invention, the immunogenicity of the test drug is based on ADA in the test sample;

[0118] The drug includes an anti-B7-H3 humanized monoclonal antibody drug.

[0119] In some specific embodiments of the present invention, the sample comprises serum; and the serum is mouse serum.

[0120] The present invention also provides the use of any of the following items in non-clinical pharmacokinetic analysis:

[0121] (I), the anti-idiotypic antibody; and / or

[0122] (II) the biological material.

[0123] In some specific embodiments of the present invention, the nonclinical pharmacokinetic analysis includes detecting the concentration of the drug in the blood;

[0124] The drug includes an anti-B7-H3 humanized monoclonal antibody drug.

[0125] The present invention also provides the use of the anti-idiotypic antibody or the biomaterial in preparing any of the following neutralizing antibodies, reagents and / or kits:

[0126] (I), anti-B7-H3 humanized monoclonal antibody; and / or

[0127] (II) Detection of ADA; and / or

[0128] (III) Detect drug concentration.

[0129] In some specific embodiments of the present invention, the drug comprises an anti-B7-H3 humanized monoclonal antibody drug.

[0130] The present invention also provides a reagent and / or a kit, comprising any of the following:

[0131] (I), the anti-idiotypic antibody; and / or

[0132] (II) the biological material.

[0133] The present invention provides a B7-H3 humanized monoclonal antibody anti-idiotypic mouse monoclonal antibody with high binding force and high specificity. The present invention is an anti-idiotypic monoclonal antibody that specifically recognizes the anti-B7-H3 humanized monoclonal antibody, has good uniformity and strong binding force, and is widely used in the field of drug research and development, mainly in the immunogenicity analysis and pharmacokinetic analysis of antibody drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0135] Figure 1 The binding force curve of the anti-idiotypic antibody and the anti-B7-H3 humanized monoclonal antibody in Example 1 is shown;

[0136] Figure 2 The anti-idiotypic antibody ADA analysis curve diagram in Example 2 is shown;

[0137] Figure 3 The anti-idiotypic antibody and neutralizing antibody analysis curve diagram in Example 3 is shown;

[0138] Figure 4The standard curve of the anti-B7-H3 humanized monoclonal antibody drug concentration-time variation in Example 4 is shown;

[0139] Figure 5 The anti-B7-H3 humanized antibody serum drug concentration-time change curve in Example 5 is shown. DETAILED DESCRIPTION

[0140] The present invention discloses anti-idiotypic antibodies and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve them. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0141] 1. Preparation of anti-idiotypic antibodies

[0142] 1. Animal immunization and antibody preparation

[0143] Pepsin (Sigma, catalog number P6887) was used to enzymatically digest the anti-B7-H3 humanized monoclonal antibody (produced by Maiwei (Shanghai) Biotechnology Co., Ltd., whose heavy chain has the amino acid sequence shown in SEQ ID No.89 and whose light chain has the amino acid sequence shown in SEQ ID No.90), remove the Fc region of the antibody, and prepare the F(ab)2 sample. The obtained F(ab)2 sample was used as the immunogen for immunizing animals, and 5 Balb / c mice were immunized, each immunized 3 times, and each cycle was 3 weeks. The immunized mouse serum should have specific binding with the corresponding immunogen, and the antiserum titer should be above 1:10,000.

[0144] The spleen cells of 1-2 mice with the highest titer among the immunized mice were selected for fusion with SP2 / 0 myeloma cells. The hybridoma cells were amplified and cultured, and the supernatant was taken to detect the titer by ELISA; cells that were positive for anti-B7-H3 humanized monoclonal antibody and negative for irrelevant human IgG were selected for cloning. To ensure the positive rate of the hybridoma cell line and the stable production of antibodies, 3-4 clones were performed, each clone was separated by 1-2 weeks, and after 3-4 clones were 100% positive, the cells were expanded and cultured and inoculated into mice to prepare ascites to obtain anti-idiotypic antibodies. During the cloning process, the specificity of the hybridoma supernatant was detected by the indirect ELISA method, that is, the mouse monoclonal antibody in the supernatant only bound to the anti-B7-H3 humanized monoclonal antibody, but not to irrelevant human IgG, and finally 4 unique monoclonal antibodies were obtained. The results are shown in Table 1.

[0145] Table 1 Specificity identification results of hybridoma supernatants

[0146]

[0147] 2. Target competitive and non-competitive testing

[0148] The detection of the four unique monoclonal antibodies produced by hybridoma cells should meet the following conditions:

[0149] 1) Target non-blocking antibody: detected by ELISA method, recognizes anti-B7-H3 antibody, does not recognize control human IgG, and cannot block the binding of anti-B7-H3 antibody to B7-H3 (ELISA plate is coated with antigen, anti-B7-H3 antibody is incubated with the prepared anti-idiotypic antibody and then added to the ELISA plate, and detected using anti-human IgG secondary antibody, and the signal value is not reduced compared with the group without anti-idiotypic antibody incubation (PBS group)); or

[0150] 2) Target blocking antibody: Detected by ELISA method, it recognizes anti-B7-H3 antibody, does not recognize control human IgG, and can block the binding of anti-B7-H3 antibody to B7-H3 (ELISA plate is coated with antigen, anti-B7-H3 antibody is incubated with the prepared anti-idiotypic antibody and then added to the ELISA plate, and detected using anti-human IgG secondary antibody, the signal value is significantly lower than that of the group not incubated with anti-idiotypic antibody (PBS group)).

[0151] The results are shown in Table 2. The target blocking properties of the anti-idiotypic antibodies were determined by comparing the OD values ​​of the antibody group and the non-antibody group. S-412-8 and S-401-2 are target blocking antibodies, and S-322-4 and S-255-11 are target non-blocking antibodies.

[0152] Table 2 Specificity identification results of hybridoma supernatants

[0153]

[0154]

[0155] 2. Sequencing of anti-idiotypic antibodies

[0156] 1. Experimental process

[0157] The hybridoma cells were expanded and cultured, total cell RNA was extracted, and cDNA was obtained by reverse transcription. The heavy and light chain variable region genes of the antibody were obtained by PCR amplification, sequenced and subjected to bioinformatics analysis of the sequence, and non-functional antibody genes were eliminated. The antibodies were named according to the hybridoma cell clones from which they were derived, and the amino acid sequences of the heavy and light chains are shown below. The variable regions of the heavy and light chains are underlined, and the CDR regions (complementarity determining regions) of the heavy and light chains defined according to the KABAT naming system are highlighted.

[0158] 2. Sequencing results

[0159] 1) S-412-8 complete antibody sequence

[0160] CDR region of the S-412-8 antibody heavy chain sequence:

[0161] CDR1 (SEQ ID No. 1): NYGMN;

[0162] CDR2 (SEQ ID No. 2): WINPYTGEPTYADDFKG;

[0163] CDR3(SEQ ID No.3): RKYGNYYVMDY;

[0164] FR region of the S-412-8 antibody heavy chain sequence:

[0165] FR1(SEQ ID No.4):QIQLVQSGPELKKPGETVKISCKASGYTFT

[0166] FR2(SEQ ID No.5):WVRQTPGKGLKWMG

[0167] FR3(SEQ ID No.6):RFAFSLDTSASTAFLQINNLTNEDMTTYFCAR

[0168] FR4(SEQ ID No.7):WGQGTSVTVSS

[0169] Signal peptide (SEQ ID No. 8): MDWLWNLLFLMAAAQSAQA

[0170] Variable region of the heavy chain sequence of S-412-8 antibody (SEQ ID No. 9):

[0171] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVRQTPGKGLKWMGWINPYTGEPTYADDFKGRFAFSLDTSASTAFLQINNLTNEDMTTYFCARRKYGNYYVMDYWGQGTSVTVSS

[0172] S-412-8 heavy chain sequence (SEQ ID No. 10):

[0173] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVRQTPGKGLKWMGWINPYTGEPTYADDFKGRFAFSLDTSASTAFLQINNLTNEDMTTYFCARRKYGNYYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0174] CDR regions of the S-412-8 antibody light chain sequence:

[0175] CDR1 (SEQ ID No.11): RASESVEYYGTSVLQ;

[0176] CDR2 (SEQ ID No.12): GASNVES;

[0177] CDR3 (SEQ ID No.13): QQSGKVPWT;

[0178] FR regions of the S-412-8 antibody light chain sequence:

[0179] FR1 (SEQ ID No.14): DIVLTQSPASLAVSLGQRATISC

[0180] FR2 (SEQ ID No.15): WYQQKPGQPPKLLIY

[0181] FR3 (SEQ ID No.16): GVPARFSGSGSGTDFSLNIHPVEEDDIAMYFC

[0182] FR4 (SEQ ID No.17): FGGGTKLAIK

[0183] Signal peptide (SEQ ID No. 18): MESDTLLLWVLLLWVPGSTG

[0184] Variable region of the light chain sequence of S-412-8 antibody (SEQ ID No. 19):

[0185] DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSVLQWYQQKPGQPPKLLIYGASN VESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSGKVPWTFGGGTKLAIK

[0186] S-412-8 light chain sequence (SEQ ID No. 20):

[0187] DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSVLQWYQQKPGQPPKLLIYGASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSGKVPWTFGGGTKLA IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0188] 2) S-322-4 complete antibody sequence

[0189] CDR region of the S-322-4 antibody heavy chain sequence:

[0190] CDR1 (SEQ ID No. 21): HTYIH

[0191] CDR2(SEQ ID No.22):RIDPVNDNSDYDPKFQG

[0192] CDR3(SEQ ID No.23):LSGGYYVYAMD

[0193] FR region of the S-322-4 antibody heavy chain sequence:

[0194] FR1(SEQ ID No.24):EVPLQQSGAELVKPGASVKLSCTVSGFNIQ

[0195] FR2(SEQ ID No.25):WMKQRPEQGLEWIG

[0196] FR3 (SEQ ID No.26): KATITADMSSNTAYLQFNSLTAEDTAVYYCAR

[0197] FR4 (SEQ ID No.27): YWGQGTSVTVSS

[0198] Signal peptide (SEQ ID No.28): MKCSWVIFFLMAMLPGVNS

[0199] Variable region of the S-322-4 antibody heavy chain sequence (SEQ ID No.29):

[0200] EVPLQQSGAELVKPGASVKLSCTVSGFNIQHTYIHWMKQRPEQGLEWIGRIDPVNDNSDYDPKFQGKATITADMSSNTAYLQFNSLTAEDTAVYYCARLSGGYYVYAMDYWGQGTSVTVSS

[0201] S-322-4 heavy chain sequence (SEQ ID No.30):

[0202] EVPLQQSGAELVKPGASVKLSCTVSGFNIQHTYIHWMKQRPEQGLEWIGRIDPVNDNSDYDPKFQGKATITADMSSNTAYLQFNSLTAEDTAVYYCARLSGGYYVYAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0203] CDR region of the S-322-4 antibody light chain sequence:

[0204] CDR1 (SEQ ID No.31): KSSQSLLYSYNQKNYLA

[0205] CDR2 (SEQ ID No.32): WASTRES

[0206] CDR3 (SEQ ID No.33): QQYHTYVT

[0207] FR regions of the light chain sequence of S-322-4 antibody:

[0208] FR1 (SEQ ID No.34) DIVMSQSPSSLAVSVGEKVTMSC

[0209] FR2 (SEQ ID No.35): WYQQKPGQSPKLLIY

[0210] FR3 (SEQ ID No.36): GVPDRFTGSGSGTDFTLTIISVKAEDLAVYYC

[0211] FR4 (SEQ ID No.37): FGAGTKLELK

[0212] Signal peptide (SEQ ID No.38): MDSQAQVLMLLLLWVSGTCG

[0213] Variable region of the light chain sequence of S-322-4 antibody (SEQ ID No.39):

[0214] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSYNQKNYLAWYQQKPGQSPKLLIY WASTRESGVPDRFTGSGSGTDFTLTIISVKAEDLAVYYCQQYHTYVTFGAGTKLELK

[0215] S-322-4 light chain sequence (SEQ ID No.40):

[0216] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSYNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIISVKAEDLAVYYCQQYHTYVTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0217] 3) S-401-2 complete antibody sequence

[0218] CDR region of the S-401-2 antibody heavy chain sequence:

[0219] CDR1 (SEQ ID No. 41): NYGMN

[0220] CDR2(SEQ ID No.42):WINPYTGEPTYADDFKG

[0221] CDR3(SEQ ID No.43):REYGNYYVMDY

[0222] FR region of the S-401-2 antibody heavy chain sequence:

[0223] FR1(SEQ ID No.44):QIQLVQSGPELKKPGETVKISCKASGYTFT

[0224] FR2(SEQ ID No.45):WVKQAPGKGLKWMG

[0225] FR3(SEQ ID No.46):RFAFSLETSASTAYLQINNLKNEDMATYFCAR

[0226] FR4(SEQ ID No.47):WGQGTSVTVSS

[0227] Signal peptide (SEQ ID No. 48): MDWLWNLLFLMAAAQSAQA

[0228] Variable region of the heavy chain sequence of S-401-2 antibody (SEQ ID No. 49):

[0229] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINPYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARREYGNYYVMDYWGQGTSVTVSS

[0230] S-401-2 heavy chain sequence (SEQ ID No.50):

[0231] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINPYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARREYGNYYVMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0232] CDR regions of the light chain sequence of S-401-2 antibody:

[0233] CDR1 (SEQ ID No.51): RASESVEYYGTSVMQ

[0234] CDR2 (SEQ ID No.52): AASNVES

[0235] CDR3 (SEQ ID No.53): QQSGKVPWT

[0236] FR regions of the light chain sequence of S-401-2 antibody:

[0237] FR1 (SEQ ID No.54): DIVLTQSPASLAVSLGQRATISC

[0238] FR2 (SEQ ID No.55): WYQQKPGQPPKLLIY

[0239] FR3 (SEQ ID No.56): GVPARFSGSGSGTDFSLNIHPVEEDDIAMYFC

[0240] FR4 (SEQ ID No.57): FGGGTKLEIK

[0241] Signal peptide (SEQ ID No. 58): MESDTLLLWVLLLWVPGSTG

[0242] Variable region of the light chain sequence of S-401-2 antibody (SEQ ID No.59):

[0243] DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSVMQWYQQKPGQPPKLLIYAAS NVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSGKVPWTFGGGTKLEIK

[0244] S-401-2 light chain sequence (SEQ ID No.60):

[0245] DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSVMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSGKVPWTFGGGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0246] 4) S-255-11 complete antibody sequence

[0247] CDR region of the S-255-11 antibody heavy chain sequence:

[0248] CDR1 (SEQ ID No. 61): SDYAWN

[0249] CDR2(SEQ ID No.62):YITYSGTTAFNPSLIS

[0250] CDR3(SEQ ID No.63):GPYSGTNYAMDY

[0251] FR region of the S-255-11 antibody heavy chain sequence:

[0252] FR1(SEQ ID No.64):DVQLQESGPGLVKPSQSLSLTCTVTGYSIT

[0253] FR2(SEQ ID No.65):WIRQFPGNKLEWMG

[0254] FR3 (SEQ ID No.66): RISLTRDTSKNQFFLQLSSVTTEDTATFYCAR

[0255] FR4 (SEQ ID No.67): WGQGTSVTVSS

[0256] Signal peptide (SEQ ID No.68): MRMLILLWLFTAFPGILS

[0257] Variable region of the S-255-11 antibody heavy chain sequence (SEQ ID No.69):

[0258] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGTTAFNPSLISRISLTRDTSKNQFFLQLSSVTTEDTATFYCARGPYSGTNYAMDYWGQGTSVTVSS

[0259] S-255-11 heavy chain sequence (SEQ ID No.70):

[0260] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGTTAFNPSLISRISLTRDTSKNQFFLQLSSVTTEDTATFYCARGPYSGTNYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0261] CDR region of the S-255-11 antibody light chain sequence:

[0262] CDR1(SEQ ID No.71):RASESVDDYGVSFMN

[0263] CDR2 (SEQ ID No. 72): AAFKQGS

[0264] CDR3(SEQ ID No.73):QQSKEVPWT

[0265] FR region of the S-255-11 antibody light chain sequence:

[0266] FR1(SEQ ID No.74):DIVLTQSPASLAVSLGQRATISC

[0267] FR2(SEQ ID No.75):WFQQKPGQPPKLLIY

[0268] FR3(SEQ ID No.76):GVPARISGSGSYTDFSLNIHPMEEDDTAMYFC

[0269] FR4(SEQ ID No.77):FGGGTKLEVK

[0270] Signal peptide (SEQ ID No. 78): MEKDTLLLWVLLLWVPGSTG

[0271] Variable region of the light chain sequence of the S-255-11 antibody (SEQ ID No. 79):

[0272] DIVLTQSPASLAVSLGQRATISCRASESVDDYGVSFMNWFQQKPGQPPKLLIYAAF KQGSGVPARISGSGSYTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEVK

[0273] S-255-11 light chain sequence (SEQ ID No.80):

[0274] DIVLTQSPASLAVSLGQRATISCRASESVDDYGVSFMNWFQQKPGQPPKLLIYAAFKQGSGVPARISGSGSYTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLE VKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0275] The present invention is an anti-idiotypic monoclonal antibody that specifically recognizes anti-B7-H3 humanized monoclonal antibody, has good uniformity and strong binding ability, and is widely used in the field of drug research and development, and is mainly used in immunogenicity analysis and pharmacokinetic analysis of antibody drugs.

[0276] The present invention provides a B7-H3 humanized monoclonal antibody anti-idiotypic mouse monoclonal antibody with high binding force and high specificity.

[0277] The anti-idiotypic antibody provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0278] The present invention will be further described below in conjunction with embodiments:

[0279] Example 1: Binding of anti-idiotypic antibodies to anti-B7-H3 humanized mAbs

[0280] 1. Test methods

[0281] 1.1 Coating and blocking: dilute the anti-B7-H3 humanized monoclonal antibody (produced by Maiwei (Shanghai) Biotechnology Co., Ltd.) with coating solution to 2 μg / mL and incubate at 2-8°C overnight; pat the ELISA plate dry and rinse the ELISA plate 3 times with washing buffer; pat the ELISA plate dry, add 3% BSA / PBST, and incubate at room temperature for 2 hours; pat the blocked ELISA plate dry and rinse the ELISA plate 3 times with washing buffer.

[0282] 1.2 Sample addition: Dilute the four anti-idiotypic antibodies (S-412-8, S-322-4, S-401-2, S-255-11) with 1% BSA / PBST to 10 μg / mL as the starting concentration, and dilute them 3-fold to a total of 11 points, with 1% BSA / PBST as the 0 point; add the above samples to the ELISA plate, set up duplicate wells, and incubate at room temperature for 2 hours.

[0283] 1.3 Add detection antibody: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer; dilute goat anti-mouse IgG HRP-labeled secondary antibody (Jackson ImmunoResearch, catalog number 115-035-003) 20,000 times with 1% BSA / PBST and add to the ELISA plate, and incubate at room temperature for 1 hour.

[0284] 1.4 Color development: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer; add TMB substrate (KPL, catalog number 5120-0047) that has been equilibrated to room temperature to the ELISA plate and incubate at room temperature in the dark for about 10 minutes; add 1M H3PO4 to terminate the reaction.

[0285] 1.5 Plate reading: After the color development reaction is terminated, use 650nm as the reference wavelength and read the OD value at 450nm.

[0286] 2. Test results

[0287] The results are as follows Figure 1 As shown in Table 3 , the four anti-idiotypic antibodies were able to bind to the anti-B7-H3 humanized mAb, and there was no significant difference in the binding ability.

[0288] Table 3 Binding data of anti-idiotypic antibody and anti-B7-H3 humanized monoclonal antibody

[0289]

[0290] Example 2: Application of anti-idiotypic antibodies in drug immunogenicity ADA detection

[0291] 1. Experimental steps

[0292] 1.1 Coating and blocking: dilute the anti-B7-H3 humanized monoclonal antibody to 2 μg / mL with coating solution and incubate at 2-8°C overnight; pat the ELISA plate dry and rinse the ELISA plate 3 times with washing buffer; pat the ELISA plate dry, add 3% BSA / PBST, and incubate at room temperature for 2 hours; pat the blocked ELISA plate dry and rinse the ELISA plate 3 times with washing buffer.

[0293] 1.2 Prepare the samples to be tested: dilute the four anti-idiotypic antibodies (S-412-8, S-322-4, S-401-2, S-255-11) with 1% BSA / PBST to 10 μg / mL as the starting concentration, and perform 3-fold gradient dilution for a total of 11 points, with 1% BSA / PBST as the zero point.

[0294] 1.3 Sample addition: Label the anti-B7-H3 humanized monoclonal antibody with EZ-LINK NHS-LC-LC-Biotin (Thermo, Cat#21343) according to the instructions to obtain anti-B7-H3 antibody-Biotin, which was diluted to 0.2 μg / mL with 1% BSA / PBST. Add 50 μl / well of each diluted anti-idiotypic antibody sample and anti-B7-H3 antibody-Biotin to the ELISA plate, set up duplicate wells, and incubate at room temperature for 2 hours.

[0295] 1.4 Add SA-HRP: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer. Dilute SA-HRP (Southern Biotech, Cat. No. 7105-05) with 1% BSA / PBST at 1:20000 and add to the ELISA plate, incubate at room temperature for 1 hour.

[0296] 1.5 Color development: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer. Add TMB substrate (KPL, catalog number 5120-0047) that has been equilibrated to room temperature to the ELISA plate and incubate at room temperature in the dark for about 10 minutes. Add 1M H3PO4 to terminate the reaction.

[0297] 1.6 Plate reading: After the color development reaction is terminated, use 650nm as the reference wavelength and read the OD value at 450nm.

[0298] 2. Test results

[0299] The results are as follows Figure 2 As shown in Table 4, the two anti-idiotypic antibodies S-401-2 and S-412-8 performed well and can be used as positive controls for ADA analysis.

[0300] Table 4 Anti-idiotypic antibody ADA analysis data

[0301]

[0302] Example 3: Application of anti-idiotypic antibodies in drug neutralizing antibody detection

[0303] 1. Experimental procedures

[0304] 1.1 Coating and blocking: Dilute B7-H3 extracellular protein (produced by Maiwei (Shanghai) Biotechnology Co., Ltd.) to 2 μg / mL with coating solution and incubate at 2-8°C overnight; Pat the ELISA plate dry and rinse the ELISA plate 3 times with washing buffer; Pat the ELISA plate dry, add 3% BSA / PBST, and incubate at room temperature for 2 hours; Pat the blocked ELISA plate dry and rinse the ELISA plate 3 times with washing buffer.

[0305] 1.2 Prepare the samples to be tested: dilute the four anti-idiotypic antibodies (S-412-8, S-322-4, S-401-2, S-255-11) with 1% BSA / PBST to 10 μg / mL as the starting concentration, and perform 3-fold gradient dilution for a total of 11 points, with 1% BSA / PBST as the zero point.

[0306] 1.3 Sample addition: Use EZ-LINK NHS-LC-LC-Biotin (Thermo, Cat#21343) to label the anti-B7-H3 humanized monoclonal antibody according to the instructions to obtain anti-B7-H3 antibody-Biotin, which is diluted to 20 ng / mL with 1% BSA / PBST. Add 50 μl / well of each diluted anti-idiotypic antibody sample and anti-B7-H3 antibody-Biotin to the ELISA plate, set up duplicate wells, and incubate at room temperature for 2 hours.

[0307] 1.4 Add SA-HRP: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer. Dilute SA-HRP (Southern Biotech, Cat. No. 7105-05) with 1% BSA / PBST at 1:20000 and add to the ELISA plate, incubate at room temperature for 1 hour.

[0308] 1.5 Color development: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer. Add TMB substrate (KPL, catalog number 5120-0047) that has been equilibrated to room temperature to the ELISA plate and incubate at room temperature in the dark for about 10 minutes. Add 1M H3PO4 to terminate the reaction.

[0309] 1.6 Plate reading: After the color development reaction is terminated, use 650nm as the reference wavelength and read the OD value at 450nm.

[0310] 2. Test results

[0311] The results are as follows Figure 3 As shown in Table 5, S-322-4 and S-255-11 are target non-blocking anti-idiotypic antibodies, and S-401-2 and S-412-8 are target blocking anti-idiotypic antibodies. The two target blocking antibodies can be used as positive controls for neutralizing antibody analysis.

[0312] Table 5 Anti-idiotypic antibody and neutralizing antibody analysis data

[0313]

[0314] Example 4: Application of anti-idiotypic antibodies in non-clinical pharmacokinetic analysis

[0315] The anti-B7-H3 humanized monoclonal antibody drug was intravenously injected into nude mice at a dose of 3 mg / kg, and a total of 6 mice were administered in parallel. 200 μL of blood was collected from the vein at 3h, 1 day, 3 days, 7 days, 10 days, 14 days and 21 days after administration. After the blood was allowed to stand for 30 minutes, the serum was separated by centrifugation at 1000 rpm for 30 minutes, and then the drug concentration in the serum was determined by ELISA. The specific determination method is based on the anti-idiotypic antibody in the present invention, and the method is as follows:

[0316] 1. Experimental procedures

[0317] 1.1 Coating and blocking: dilute the anti-idiotypic antibody S-412-8 antibody to 1 μg / mL with coating solution and coat overnight at 2-8°C. Pat the ELISA plate dry, rinse the ELISA plate 3 times with washing buffer and then pat dry, add 3% BSA / PBST, and incubate at room temperature for 2 hours. Pat the blocked ELISA plate dry, and rinse the ELISA plate 3 times with washing buffer.

[0318] 1.2 Dilution of test samples, standard curve samples and quality control products:

[0319] The anti-B7-H3 humanized monoclonal antibody was diluted to 100 ng / mL (ULOQ), 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.12 ng / mL, 1.56 ng / mL (LLOQ) and 0.78 ng / mL (anchor point) with 5% mixed blank mouse serum (purchased by Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.). The blank control (0 ng / mL) was used as part of the standard curve, but was not involved in the standard curve fitting.

[0320] The anti-B7-H3 humanized monoclonal antibody was used to prepare three concentration quality controls with 5% mixed blank mouse serum, the concentrations were: 80 ng / mL (HQC, high concentration quality control), 20 ng / mL (MQC, medium concentration quality control), and 4.0 ng / mL (LQC, low concentration quality control).

[0321] Blood samples were taken at different time points after mice were given anti-B7-H3 humanized monoclonal antibody (produced by Maiwei (Shanghai) Biotechnology Co., Ltd.) and first diluted 20 times with 1% BSA / PBST, and then diluted to appropriate multiples with 5% mixed blank mouse serum.

[0322] 1.3 Sample addition: Add the above-mentioned test samples, standard curve samples and quality control samples to the ELISA plate and incubate at room temperature for 2 hours.

[0323] 1.4 Add detection antibody: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer. Use EZ-LINK NHS-LC-LC-Biotin (Thermo, Cat#21343) to label the anti-idiotypic antibody S-322-4 according to the instructions, and dilute the obtained S-322-4-Biotin to 2 μg / mL with 1% BSA / PBST, add it to the ELISA plate, and incubate at room temperature for 1 hour.

[0324] 1.5 Add SA-HRP: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer. Dilute SA-HRP with 1% BSA / PBST at 1:40000 and add to the ELISA plate, incubate at room temperature for 1 hour.

[0325] 1.6 Color development: After incubation, pat the ELISA plate dry and rinse the ELISA plate 6 times with washing buffer. Add TMB substrate and incubate at room temperature in the dark for about 10 minutes. Add 1M H3PO4 to terminate the reaction.

[0326] 1.7 Plate reading: After the color development reaction is terminated, use 650nm as the reference wavelength and read the OD value at 450nm.

[0327] 1.8 Concentration calculation: Perform four-parameter fitting of the standard curve concentration and OD value, and use the fitting equation to calculate the drug concentration of the sample to be tested.

[0328] 2. Test results

[0329] The results are shown in Tables 6 to 7 and Figure 4-5 As shown, in the analysis of anti-B7-H3 humanized monoclonal antibody drugs, good concentration-absorbance linearity is shown, and the accuracy of all quality control samples in the test is within the range of 80.0-120.0%, meeting the methodological requirements. The concentration-time curve of the anti-B7-H3 humanized monoclonal antibody in rats after drug administration shows a normal drug pharmacokinetic curve, indicating that the anti-idiotypic antibodies S-412-8 and S-322-4 based on the present invention can well detect the concentration of anti-B7-H3 humanized monoclonal antibody drugs in the blood.

[0330] Table 6 Anti-B7-H3 humanized monoclonal antibody standard curve measurement results

[0331]

[0332]

[0333] Table 7 Anti-B7-H3 humanized monoclonal antibody drug measurement results

[0334]

[0335] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An anti-idiotypic antibody, characterized in that comprising a first anti-idiotypic antibody, a second anti-idiotypic antibody, a third anti-idiotypic antibody and / or a fourth anti-idiotypic antibody; (I), CDR1, CDR2 and CDR3 of the heavy chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 1, 2 and 3, respectively; and The CDR1, CDR2 and CDR3 of the light chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 11, 12 and 13, respectively; or (II), CDR1, CDR2 and CDR3 of the heavy chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 21, 22 and 23, respectively; and The CDR1, CDR2 and CDR3 of the light chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 31, 32 and 33, respectively; or (III), CDR1, CDR2 and CDR3 of the heavy chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 41, 42 and 43, respectively; and The CDR1, CDR2 and CDR3 of the light chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No.51, 52 and 53, respectively; or (IV), CDR1, CDR2 and CDR3 of the heavy chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 61, 62 and 63, respectively; and The CDR1, CDR2 and CDR3 of the light chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No.71, 72 and 73, respectively; or (V) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (I) to (IV); or (VI) A sequence having an identity of 80% or more to the amino acid sequence shown in any one of (I) to (V).

2. The anti-idiotypic antibody according to claim 1, characterized in that (i) FR1, FR2, FR3 and FR4 of the heavy chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 4, 5, 6 and 7, respectively; and FR1, FR2, FR3 and FR4 of the light chain of the first anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 14, 15, 16 and 17, respectively; or (ii) FR1, FR2, FR3 and FR4 of the heavy chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 24, 25, 26 and 27, respectively; and FR1, FR2, FR3 and FR4 of the light chain of the second anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 34, 35, 36 and 37, respectively; or (iii) FR1, FR2, FR3 and FR4 of the heavy chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 44, 45, 46 and 47, respectively; and FR1, FR2, FR3 and FR4 of the light chain of the third anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No. 54, 55, 56 and 57, respectively; or (iv) FR1, FR2, FR3 and FR4 of the heavy chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID Nos. 64, 65, 66 and 67, respectively; and FR1, FR2, FR3 and FR4 of the light chain of the fourth anti-idiotypic antibody have the amino acid sequences shown in SEQ ID No.74, 75, 76 and 77, respectively; or (v) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (i) to (iv); or (vi) A sequence having 80% or more homology with the amino acid sequence shown in any one of (i) to (v).

3. The anti-idiotypic antibody according to claim 1 or 2, characterized in that (A), the variable region of the heavy chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 9; and The variable region of the light chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 19; or (B), the variable region of the heavy chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 29; and The variable region of the light chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.39; or (C) the variable region of the heavy chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 49; and The variable region of the light chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.59; or (D), the variable region of the heavy chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.69; and The variable region of the light chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.79; or (E) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (A) to (D); or (F) A sequence having 80% or more homology with the amino acid sequence shown in any one of (A) to (E).

4. The anti-idiotypic antibody according to any one of claims 1 to 3, characterized in that (a) the signal peptide of the heavy chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 8; and The signal peptide of the light chain of the first anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.18; or (b) the signal peptide of the heavy chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No. 28; and The signal peptide of the light chain of the second anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.38; or (c) the signal peptide of the heavy chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.48; and The signal peptide of the light chain of the third anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.58; or (d) the signal peptide of the heavy chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.68; and The signal peptide of the light chain of the fourth anti-idiotypic antibody has the amino acid sequence shown in SEQ ID No.78; or (e) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (a) to (d); or (f) A sequence having 80% or more homology with the amino acid sequence shown in any one of (a) to (e).

5. The anti-idiotypic antibody according to any one of claims 1 to 4, characterized in that The anti-idiotypic antibodies include target blocking antibodies and target non-blocking antibodies; The target blocking antibody comprises a first anti-idiotypic antibody and a third anti-idiotypic antibody; The target non-blocking antibodies include a second anti-idiotypic antibody and a fourth anti-idiotypic antibody.

6. Biomaterial, characterized in that Include any of the following: (I), a nucleic acid encoding the anti-idiotypic antibody according to any one of claims 1 to 5; and / or (II), a nucleic acid encoding the anti-idiotypic antibody according to any one of claims 1 to 5 and an acceptable carrier; and / or (III) a host that secretes the anti-idiotypic antibody according to any one of claims 1 to 5; and / or (IV), the anti-idiotypic antibody according to any one of claims 1 to 5 which is chemically labeled or biologically labeled; and / or (V) The anti-idiotypic antibody according to any one of claims 1 to 5 coupled to a carrier.

7. Use of any of the following in blocking the binding between antibodies and antigens: (I), the anti-idiotypic antibody according to any one of claims 1 to 5; and / or (II) The biomaterial as described in claim 6.

8. The use according to claim 7, characterized in that The antibody includes anti-B7-H3 humanized monoclonal antibody; the antigen includes B7-H3 extracellular protein.

9. Application of any of the following in detecting the immunogenicity of drugs: (I), the anti-idiotypic antibody according to any one of claims 1 to 5; and / or (II) The biomaterial as described in claim 6.

10. The use according to claim 9, characterized in that The immunogenicity of the test drug is based on ADA in the test sample; The drug includes an anti-B7-H3 humanized monoclonal antibody drug.

11. Application of any of the following in nonclinical pharmacokinetic analysis: (I), the anti-idiotypic antibody according to any one of claims 1 to 5; and / or (II) The biomaterial as described in claim 6.

12. Use of the anti-idiotypic antibody according to any one of claims 1 to 5 or the biomaterial according to claim 6 in the preparation of any of the following neutralizing antibodies, reagents and / or kits: (I), anti-B7-H3 humanized monoclonal antibody; and / or (II) Detection of ADA; and / or (III) Detect drug concentration.

13. The use according to claim 12, characterized in that The drug includes an anti-B7-H3 humanized monoclonal antibody drug.

14. A reagent and / or a kit, characterized in that: Include any of the following: (I), the anti-idiotypic antibody according to any one of claims 1 to 5; and / or (II) The biomaterial as described in claim 6.

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