Antibodies for antibody drug conjugates and uses thereof

Abstract

Disclosed are an antibody drug conjugate recognition antibody and use thereof. The recognition antibody specifically binds to a compound comprising a structure as shown in Formula I or a pharmaceutically acceptable salt thereof. The provided recognition antibody can specifically recognize a drug molecule in an antibody drug conjugate, thereby effectively determining the content of the antibody drug conjugate.

Description

[0001] This application claims priority to Chinese patent application 2023109828098, filed on August 4, 2023. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a recognition antibody for an antibody-drug conjugate and its application. Background Technology

[0003] Antibody-drug conjugates (ADCs), which conjugate with cytotoxic drugs that bind to antigens expressed on the surface of cancer cells and can also be internalized within the cells, can selectively deliver drugs to cancer cells, and are therefore expected to cause drug accumulation within the cancer cells and kill them. Similar to the development of small molecule compounds and antibodies, pharmacokinetic studies (PK studies) are essential when developing antibody-drug conjugates into drug products. This is because conducting PK studies in humans, based on an understanding of the correlation between PK study results in animals and pharmacological and safety trial results, provides information useful for planning clinical trial design and considering efficacy and safety in humans.

[0004] PK studies of antibody-drug conjugates are generally conducted by quantifying the plasma concentration of the administered antibody-drug conjugate. An example of a method for quantifying the plasma concentration of antibody-drug conjugates is the ELISA method.

[0005] For example, the concentration of an antibody-drug conjugate in plasma can be quantified by the following steps: (1) contacting the antibody-drug conjugate with a plate having an antigen immobilized thereon to form a complex; (2) contacting the complex with a protein capable of recognizing the antibody-drug conjugate and labeled with a marker to form a further complex; and then, (3) detecting the marker based on the color / light generated by an enzymatic reaction.

[0006] Therefore, in order to accurately determine the concentration of antibody-drug conjugates with specific structures in the blood, it is necessary to develop an antibody that can specifically recognize the drug in the antibody-drug conjugate with a specific structure. Summary of the Invention

[0007] The technical problem this invention aims to solve is the lack of recognition antibodies for antibody-drug conjugates in existing technologies. This invention provides a recognition antibody for antibody-drug conjugates and its applications. Using the antibody provided by this invention, antibody-drug conjugates conjugated with eczemab or its derivatives can be specifically bound, effectively determining the content of the antibody-drug conjugate.

[0008] A first aspect of the present invention provides a recognition antibody D that specifically binds to a compound comprising a structure as shown in Formula I or a pharmaceutically acceptable salt thereof.

[0009]

[0010] R 1 C 3-6 Cycloalkyl or C 1-6 Alkylene This indicates that the structure shown in Formula I is connected to other segments in the compound through this site.

[0011] In this invention, the recognition antibody D can recognize compounds with structures as shown in Formula I. In a preferred embodiment, the recognition antibody D can recognize antibody-drug conjugates with structures as shown in Formula I.

[0012] In a certain preferred solution, R 1 In, the C 1-6 The alkylene group is methylene, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH2-CH3)-, -C(CH3)2-, -CH2-CH(CH3)- or butylene.

[0013] In a certain preferred solution, R 1 In, the C 1-6 alkylene Better place, for Better, for The "1" position is connected to O, and the "2" position is connected to the carbonyl group.

[0014] In a certain preferred solution, R 1 In, the C 3-6 The cycloalkylene group is cyclopropylene, cyclobutylene, cyclopentylene, or cyclohexylene, preferably cyclobutylene, and more preferably cyclohexylene.

[0015] In a certain preferred solution, R 1 In, the C 3-6 Cycloalkylene is Better place, for Better, for The "1" position is connected to O, and the "2" position is connected to the carbonyl group.

[0016] In a certain preferred solution, R 1 for Ideally, R1 for Where position "1" is connected to O, and position "2" is connected to the carbonyl group; more preferably, R 1 for The "1" position is connected to O, and the "2" position is connected to the carbonyl group.

[0017] In a preferred embodiment, the compound comprising the structure shown in Formula I or a pharmaceutically acceptable salt thereof is a compound or a pharmaceutically acceptable salt thereof shown in Formula I-1, a compound or a pharmaceutically acceptable salt thereof shown in Formula I-2, or a compound or a pharmaceutically acceptable salt thereof shown in Formula I-3.

[0018]

[0019] R 1 The definition is as stated in the previous item;

[0020] G and G 1 The linking group (the linking group can connect a compound as shown in Formula I-1 or a pharmaceutically acceptable salt thereof to T);

[0021] T is a ligand, wherein T interacts with G via its thiol or amino group. 1 Forming covalent connections;

[0022] n is a natural number or decimal from 1 to 16; (n is the drug ligand coupling rate).

[0023] In a certain preferred solution, G is

[0024] In a certain preferred solution, G 1 for The "1" bit is connected to T, and the "2" bit is connected to O.

[0025] In a preferred embodiment, T is a ligand targeting the following targets: HER2, HER3, B7H3, B7H4, TROP2, GPC3, DLL3, Claudin 18.2, CD30, CD33, CD70, or EGFR.

[0026] In a preferred embodiment, T is an anti-HER2 antibody or an anti-TROP2 antibody.

[0027] In a preferred embodiment, the anti-HER2 antibody is trastuzumab or a variant thereof.

[0028] In a preferred embodiment, the anti-TROP2 antibody is Sacituzumab, M1, M2, or M3 or a variant thereof.

[0029] In a preferred embodiment, T is either trastuzumab or sacituzumab.

[0030] In a preferred scheme, n is a natural number or decimal from 1 to 9.

[0031] In a preferred embodiment, n is a natural number or decimal between 3 and 9, such as 3.67 or 8.05.

[0032] In a preferred embodiment, the compound represented by Formula I-1 is any of the following compounds:

[0033]

[0034] In a certain preferred solution, for

[0035] In a certain preferred solution, for

[0036] In a preferred embodiment, the compound represented by Formula I-2 is any of the following compounds:

[0037]

[0038]

[0039] In a preferred embodiment, the compound represented by formula I-3 is any of the following compounds:

[0040]

[0041] Where T is the ligand and n is a natural number or decimal from 1 to 16;

[0042] Preferably, the compound represented by formula I-3 is any of the following compounds:

[0043]

[0044] n is a natural number or decimal from 1 to 16, for example, 8.05;

[0045]

[0046] n is a natural number or decimal from 1 to 16, for example, 3.67.

[0047]

[0048] n is a natural number or decimal from 1 to 16, for example, 8.05.

[0049] In a preferred embodiment, the recognition antibody D includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1 having the amino acid sequence shown in SEQ ID NO:7, HCDR2 having the amino acid sequence shown in SEQ ID NO:8, and HCDR3 having the amino acid sequence shown in SEQ ID NO:9. The light chain variable region includes LCDR1 having the amino acid sequence shown in SEQ ID NO:10, LCDR2 having the amino acid sequence shown in SEQ ID NO:11, and LCDR3 having the amino acid sequence shown in SEQ ID NO:12.

[0050] In a preferred embodiment, the recognition antibody D comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, amino acid sequences as shown in SEQ ID NO:7; and the light chain variable region comprising LCDR1, SEQ ID NO:10, LCDR2, and SEQ ID NO:12, amino acid sequences as shown in SEQ ID NO:11.

[0051] In a preferred embodiment, the recognition antibody D comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:5, and a light chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:6.

[0052] In a preferred embodiment, the amino acid sequence of the heavy chain variable region in the recognition antibody D is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6.

[0053] In a preferred embodiment, the recognition antibody D is selected from murine antibodies, humanized antibodies, and chimeric antibodies.

[0054] In a preferred embodiment, the recognition antibody D is a full-length antibody, Fab, Fab', F(ab')2, or Fv, such as scFv.

[0055] In a preferred embodiment, when the recognition antibody D is a full-length antibody, the recognition antibody D further includes a heavy chain constant region and a light chain constant region; the heavy chain constant region is the heavy chain constant region of a mouse or human antibody or a variant thereof, and the light chain constant region is the κ chain or λ chain of a mouse or human antibody or a variant thereof.

[0056] In a preferred embodiment, the recognition antibody D comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence as shown in SEQ ID NO:13 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:13; the light chain having an amino acid sequence as shown in SEQ ID NO:14 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:14.

[0057] In a preferred embodiment, the recognition antibody D comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:13, and the amino acid sequence of the light chain is shown in SEQ ID NO:14.

[0058] In a preferred embodiment, the recognition antibody D comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence as shown in SEQ ID NO:15 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:15; the light chain having an amino acid sequence as shown in SEQ ID NO:16 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:16.

[0059] In a preferred embodiment, the recognition antibody D comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:15, and the amino acid sequence of the light chain is shown in SEQ ID NO:16.

[0060] In a preferred embodiment, the recognition antibody D specifically binds to the structure shown in Formula I in the compound shown in Formula I-3. T or G 1 .

[0061] A second aspect of the present invention provides a recognition antibody X, the recognition antibody X comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 having an amino acid sequence as shown in SEQ ID NO:7, HCDR2 having an amino acid sequence as shown in SEQ ID NO:8, and HCDR3 having an amino acid sequence as shown in SEQ ID NO:9; the light chain variable region comprising LCDR1 having an amino acid sequence as shown in SEQ ID NO:10, LCDR2 having an amino acid sequence as shown in SEQ ID NO:11, and LCDR3 having an amino acid sequence as shown in SEQ ID NO:12.

[0062] In this invention, the recognition antibody X can recognize antibody-drug conjugates.

[0063] In a preferred embodiment, the recognition antibody X comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, respectively; and the light chain variable region comprising LCDR1, SEQ ID NO:10, LCDR2, and SEQ ID NO:12, respectively.

[0064] In a preferred embodiment, the recognition antibody X comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity as shown in SEQ ID NO:5, and a light chain variable region having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity as shown in SEQ ID NO:6.

[0065] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the recognition antibody X is as shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:6.

[0066] In a preferred embodiment, the recognition antibody X is selected from murine antibodies, humanized antibodies, and chimeric antibodies.

[0067] In a preferred embodiment, the recognition antibody X is a full-length antibody, Fab, Fab', F(ab')2, or Fv, such as scFv.

[0068] In a preferred embodiment, when the recognition antibody X is a full-length antibody, the antibody further includes a heavy chain constant region and a light chain constant region; the heavy chain constant region is the heavy chain constant region of a mouse or human antibody or a variant thereof, and the light chain constant region is the κ chain or λ chain of a mouse or human antibody or a variant thereof.

[0069] In a preferred embodiment, the recognition antibody X comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence as shown in SEQ ID NO:13 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:13; the light chain having an amino acid sequence as shown in SEQ ID NO:14 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:14.

[0070] In a preferred embodiment, the recognition antibody X comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:13, and the amino acid sequence of the light chain is shown in SEQ ID NO:14.

[0071] In a preferred embodiment, the recognition antibody X comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence as shown in SEQ ID NO:15 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:15; the light chain having an amino acid sequence as shown in SEQ ID NO:16 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with SEQ ID NO:16.

[0072] In a preferred embodiment, the antibody X comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO:15; and the amino acid sequence of the light chain is shown in SEQ ID NO:16.

[0073] A third aspect of the present invention provides a nucleic acid (polynucleotide) that encodes a recognition antibody as described in the first or second aspect.

[0074] A fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the third aspect.

[0075] A fifth aspect of the present invention provides a transformant comprising a nucleic acid as described in the third aspect or a recombinant expression vector as described in the fourth aspect.

[0076] A sixth aspect of the present invention provides a method for in vitro detection of antibody-drug conjugates, the method comprising the step of contacting a recognition antibody as described in the first or second aspect with a test sample containing an antibody-drug conjugate.

[0077] In a preferred embodiment, the antibody-drug conjugate is as described in the first aspect as a compound of formula I-3 or a pharmaceutically acceptable salt thereof.

[0078] In a preferred embodiment, the sample to be tested is serum, such as mammalian serum.

[0079] A seventh aspect of the present invention provides a detection reagent comprising a recognition antibody as described in the first or second aspect, a nucleic acid as described in the third aspect, a recombinant expression vector as described in the fourth aspect, and / or a transformant as described in the fifth aspect.

[0080] An eighth aspect of the present invention provides a kit comprising a recognition antibody as described in the first or second aspect, a nucleic acid as described in the third aspect, a recombinant expression vector as described in the fourth aspect, a transformant as described in the fifth aspect, and / or a detection reagent as described in the seventh aspect.

[0081] A ninth aspect of the present invention provides the use of recognition antibodies as described in the first or second aspect, nucleic acids as described in the third aspect, recombinant expression vectors as described in the fourth aspect, transformants as described in the fifth aspect, or detection reagents as described in the seventh aspect in the preparation of detection reagents. In a preferred embodiment, the detection reagent is used to detect antibody-drug conjugates in a sample to be tested.

[0082] A tenth aspect of the present invention provides a method for preparing a recognition antibody, the method comprising culturing a transformant as described in the fifth aspect and obtaining the recognition antibody from the culture.

[0083] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0084] The reagents and raw materials used in this invention are all commercially available.

[0085] The significant advantages of this invention are: it provides a drug recognition antibody for antibody-drug conjugates. Using the antibody provided by this invention, the drug molecule eczema or its derivatives in the antibody-drug conjugate can be specifically bound, effectively determining the content of the antibody-drug conjugate.

[0086] the term

[0087] Unless otherwise specified, the terms used in this invention have the following meanings:

[0088] Those skilled in the art will understand that, according to conventions used in the art, the structural formulas of the groups described in this invention are... This refers to the fact that the corresponding group is connected to other fragments or groups in the compound through this site.

[0089] The "-" at the end of a group indicates that the group is connected to other segments in the molecule through that site. For example, -CH2-CH(CH3)- means that CH2 and CH are connected to other segments in the molecule.

[0090] In the claims of this application, "one or more" in "satisfying one or more of the following conditions" means 2, 3, 4 or more, and the maximum value of "more" is the maximum number of conditions recited in each claim. For example, if a claim recites 4 conditions, then "one or more" in "satisfying one or more of the following conditions" in that claim is any integer from 1 to 4, such as 1, 2, 3 or 4.

[0091] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. When the compounds of the present invention contain both relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts.

[0092] The term "alkylene" refers to a substituent formed by eliminating two hydrogen atoms from a saturated straight-chain or branched alkane. The two eliminated hydrogen atoms can be on the same carbon atom or on different carbon atoms (e.g., the two eliminated hydrogen atoms are on the carbon atoms at both ends). Thus, C1 alkylene (i.e., methylene) refers to -CH2-; C2 alkylene (i.e., ethylene) refers to -CH2-CH2- or -CH(CH3)-; C3 alkylene (i.e., propylene) refers to -CH2-CH2-CH2-, -CH(CH2-CH3)-, -C(CH3)2- or -CH2-CH(CH3)-; and C3 alkylene (i.e., butylene) refers to -CH2-CH2-CH2-CH2-, -CH2-CH2-CH(CH2)-, -CH2-CH(CH2-CH2)- or -CH(CH2-CH2-CH2)-.

[0093] The term "cycloalkylene" refers to a substituent formed by eliminating two hydrogen atoms from a cycloalkane. The two eliminated hydrogen atoms can be on the same carbon atom or on different carbon atoms in the ring. "Cycloalkane" refers to a cycloalkane with a specified number of carbon atoms in the ring (e.g., C3-C4). 10 C3-C6 cyclic alkanes are saturated cyclic alkanes whose ring atoms consist only of carbon atoms. For example, C4 cycloalkylene (i.e., cyclopropylene) can be...

[0094] The term "antibody-drug conjugate (ADC)" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker. An ADC can refer to a monoclonal antibody or antibody fragment linked to a biologically active cytotoxic drug via a stable linker.

[0095] The term "antibody" generally refers to the immunological binding agent extended to all antibodies from all species, including dimer, trimer, and multimer antibodies; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and modified antibodies, and their fragments. The term "antibody" can also refer to any antibody-like molecule with an antigen-binding region, including small molecule fragments such as Fab′, Fab, F(ab′)2, single-domain antibodies (DABs), Fv, scFv (single-chain Fv), linear antibodies, biantibodies, etc.

[0096] In this invention, the antibody in the "antibody-drug conjugate" can be an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-TNF-α antibody, an anti-TROP2 antibody, or an anti-Claudin antibody. 18.2 Antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-ADAM9 antibody or anti-Mesothelin antibody, for example, trastuzumab, sacituzumab, pertuzumab, adalimumab.

[0097] The term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies can be constructed. The variable region gene can then be cloned from the murine hybridoma cells. The constant region gene of the human antibody can be cloned as needed. The murine variable region gene and the human constant region gene can be linked to form a chimeric gene, which is then inserted into an expression vector. Chimeric antibody molecules can then be expressed in eukaryotic or prokaryotic systems.

[0098] The term "humanized antibody," also known as a CDR-grafted antibody, generally refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database.

[0099] The term "fully human antibody," also known as "fully human monoclonal antibody," refers to an antibody whose variable and constant regions can both be human-derived, thus eliminating immunogenicity and toxicity. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. The antibody or ligand described in this application can be a fully human monoclonal antibody. Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology, etc.

[0100] The term “CDR” generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242; Chothia et al., “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography,” J. Mol. Biol. 262:732 (1996). As used in this application, the Kabat definition of CDR can be applied to CDR1, CDR2, and CDR3 (CDRL1, CDRL2, CDRL3 or L1, L2, L3) of light chain variable structural domains, and CDR1, CDR2, and CDR3 (CDR H1, CDRH2, CDRH3 or H1, H2, H3) of heavy chain variable structural domains.

[0101] The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans. Attached Figure Description

[0102] Figure 1 This is a schematic diagram of the IC50 standard curve fitting for P-II-3.

[0103] Figure 2 This is a schematic diagram of the IC50 standard curve fitting for P-III-20.

[0104] Figure 3 This is a schematic diagram of the IC50 standard curve fitting for DXd. Detailed Implementation

[0105] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0106] Example 1: Preparation of the toxin (payload)

[0107] Preparation Example 1

[0108]

[0109] first step:

[0110] Under nitrogen protection, DIEA (500 mg, 3.87 mmol) was added to an 18 mL solution of DMF containing KI4 (900 mg, 1.69 mmol), HATU (691 mg, 1.88 mmol), and 3a (320 mg, 2.00 mmol) at 0 °C. The mixture was stirred at 25 °C for 3 hours. TLC (EA) showed that the starting materials reacted completely. The reaction solution was added dropwise to 320 mL of deionized water and filtered to give 850 mg of a gray solid, yield: 87%.

[0111] Step Two:

[0112] To 3b (100 mg, 0.174 mmol), NaHCO3 (42 mg, 0.50 mmol) solid was added to MeOH / DCM (1 / 1, 3 mL) and stirred at 25 °C for 3 hours. TLC (EA) showed that the reaction was complete. The reaction solution was filtered, evaporated to dryness at low temperature, slurried with aq. HCl (0.5 M, 10 mL), filtered again, and prepared by prep-HPLC (0.1% TFA). The solution was then lyophilized to obtain 15 mg of P-II-3 as a gray solid. Yield: 16%.

[0113] MS m / z (ESI): 534 [M+1].

[0114] 1H-NMR (400MHz, DMSO-d6): 8.45(d,1H),7.81(d,1H),7.32(s,1H),6.52(m,1H),5.58-5.56(m,1H),5.44(s,2H),5.14(dd,2H) ,3.96(m,1H),3.48(m,1H),3.19(m,2H),2.53-2.28(m,3H),2.48(s,3H),2.20-2.00(m,4H),1.95-1.80(m,2H),0.89(t,3H).

[0115] Preparation Example 2

[0116]

[0117] first step:

[0118] Under nitrogen protection, DIEA (486 mg, 3.76 mmol) was added to an 18 mL solution of KI 4 (1.00 g, 1.88 mmol), HATU (691 mg, 1.88 mmol), and 4a (310 mg, 1.88 mmol) in DMF at 0 °C. The mixture was stirred at 25 °C for 3 hours. TLC (EA) showed that the starting materials reacted completely. The reaction solution was added dropwise to 320 mL of deionized water and filtered to give 910 mg of a gray solid, yield: 84%.

[0119] Step Two:

[0120] To 4b (100 mg, 1.58 mmol) of MeOH / DCM (1 / 1, 3 mL), NaHCO3 (42 mg, 0.50 mmol) solid was added and stirred at 25 °C for 3 hours. TLC (EA) showed that the reaction was complete. The reaction solution was filtered, evaporated to dryness at low temperature, slurried with aq.HCl (0.5 M, 10 mL), filtered, and lyophilized by prep-HPLC (0.1% TFA) to obtain 13 mg of P-II-4 as a yellow solid, yield: 14%.

[0121] MS m / z (ESI): 534 [M+1].

[0122] 1H-NMR (400MHz, DMSO-d6): 8.39(d,1H),7.77(d,1H),7.29(s,1H),6.52(m,1H),5.58-5.54(m,1H),5.41(s,2H),5.18-5.06(m,3H),4.39 -4.33(m,1H),3.19-3.07(m,2H),2.97-2.82(m,1H),2.49-2.36(m,2H),2.38(s,3H),2.20-1.96(m,4H),1.93-1.79(m,2H),0.87(t,3H).

[0123] Preparation Example 3

[0124]

[0125] Under nitrogen protection, DIEA (61 mg, 0.47 mmol) was added dropwise to a DMF (2 mL) solution containing KI4 (100 mg, 0.188 mmol), HATU (86 mg, 0.23 mmol), and 7a (22 mg, 0.21 mmol). After the addition was complete, the reaction was carried out at 25 °C for 2.5 hours. LC-MS showed that the starting material reacted completely. The reaction solution was added to 20 mL of water, and P-III-20 solid precipitated. Filtering yielded 13 mg, yield: 11%.

[0126] MS m / z (ESI): 522 [M+1].

[0127] 1 H-NMR (400MHz, DMSO-d6): 8.44(d,1H),7.80(d,1H),7.32(s,1H),6.54(m,1H),5.60-5.50(m,1H),5.44(s,2H),5.22(dd, 2H),4.10-4.00(m,1H),3.30-3.17(m,2H),2.41(s,3H),2.38-2.10(m,4H),1.96-1.80(m,2H),1.11(d,3H),0.89(t,3H).

[0128] Preparation Example 4

[0129]

[0130]

[0131] first step

[0132] Under nitrogen protection, DIEA (60.6 mg, 0.47 mmol) was added dropwise to a DMF (2 mL) solution containing KI4 (100 mg, 0.19 mmol), HATU (85.7 mg, 0.23 mmol), and 23a (21.5 mg, 0.21 mmol). After the addition was complete, the reaction was carried out at 0 °C for 2 hours. LC-MS showed that the starting material reacted completely. The reaction solution was added dropwise to 20 mL of water and stirred. After the solid precipitated, it was filtered to give 60.2 mg of gray solid P-III-30, yield: 61%.

[0133] MS-ESI:m / z 522.2[M+H]+.

[0134] 1 H NMR(400MHz, DMSO-d6)δ8.42(d,J=8.7Hz,1H),7.79(d,J=11.0Hz,1H),7.30(s,1H), 6.53(s,1H),5.62–5.53(m,1H),5.42(s,2H),5.30–5.16(m,2H),4.63(d,J=4.6Hz,1 H),4.09–3.99(m,1H),3.22–3.11(m,2H),2.40(s,3H),2.28(dd,J=13.7,7.2Hz,1H) ,2.22–2.08(m,3H),1.94–1.78(m,2H),1.08(d,J=6.1Hz,3H),0.87(t,J=7.3Hz,3H).

[0135] Example 2: Preparation of the linker-payload

[0136] Connector - Toxin X1

[0137]

[0138]

[0139] first step

[0140] Under nitrogen protection, benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a DMF (50 mL) solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol), and the reaction was carried out at 25 °C for 17 hours. TLC (PE / EA = 2 / 1) showed that the reaction was complete. The reaction solution was added to 500 mL of water and extracted twice with EA (250 mL). After separation, the solution was washed with saturated sodium chloride aqueous solution (500 mL), dried over anhydrous Na2SO4, concentrated, and column filtered (PE:EA = 3:2) to give 5.1 g of colorless liquid, yield: 57.1%.

[0141] Step 2

[0142] Under nitrogen protection, a solution of 27b (4.50 g, 21.8 mmol) in THF (30 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol), TsOH (800 mg, 4.65 mmol), and reacted at 0 °C for 2 hours at 25 °C. TLC (PE / EA = 1 / 2) showed the reaction was complete. The reaction solution was added to 200 mL of water, extracted twice with EA (200 mL), dried over anhydrous Na2SO4, and concentrated by column chromatography (PE / EA = 3 / 2) to give 1.56 g of a white solid, yield: 26%.

[0143] Step 3

[0144] Under hydrogen atmosphere and at 0°C, Pd / C (80 mg) was added to a mixed solution of EtOH (8 mL) and EA (8 mL) at 27°C (800 mg, 1.55 mmol), and the mixture was stirred at 0°C for 2.5 hours. LC-MS showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with EA (200 mL), concentrated, dissolved in THF (20 mL), and evaporated to dryness to obtain 600 mg of white solid, yield: 91%.

[0145] Step 4

[0146] Under nitrogen protection, at 0°C, DIEA (152 mg, 1.18 mmol) was added to a DMF (6 mL) solution of 27 d (220 mg, 0.515 mmol), KI4 (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol), and the reaction was carried out at 0°C for 2 hours. LC-MS showed that the reaction was complete. The reaction solution was added to an aqueous citric acid solution (pH = 4) (150 mL), filtered, and the filter cake was washed with 175 mL of water. After drying, the solution was pulled dry with an oil pump to obtain 260 mg of brown solid, yield: 66%.

[0147] Step 5

[0148] Under nitrogen protection, diethylamine (8 mL) was added dropwise to a 30 mL solution of DCM (27°C, 260 mg, 0.309 mmol) at 0 °C, and the reaction was carried out at 0 °C for 3 hours. LC-MS showed that the reaction was complete. The reaction solution was added to a 600 mL solution of petroleum ether at 0 °C, and a solid precipitated out. After standing for the solid to be adsorbed to the bottom of the flask, the solution was poured out and dried using an oil pump to obtain 90 mg of brown solid, yield: 47.1%.

[0149] Step 6

[0150] Under nitrogen protection, HATU (74 mg, 0.19 mmol) was added to a DMF (2.5 mL) solution containing 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol), and DIEA (50 mg, 0.39 mmol) at 0 °C, and the reaction was carried out at 0 °C for 2 hours. LC-MS showed that the basic reaction was complete. At 0 °C, the reaction solution was added to an aqueous solution of citric acid (pH = 4) (30 mL), and flocculent solid precipitated. After filtration, the solid was analyzed by preparative agar (DCM / MecOH = 10 / 1) to give 9.2 mg of a pale yellow solid (X1), yield: 6%.

[0151] MS m / z (ESI): 1074 [M+1].

[0152] 1 H-NMR (400MHz, MeOD): 7.65(d,1H),7.62(s,1H),7.30-7.21(m,5H),6.79(s,2H),5.69-5.65(m, 1H),5.57(d,1H),5.43-5.10(m,3H),4.70(d,2H),4.48-4.39(m,2H),4.10-4.05(m,1H),4.01-3 .75(m,5H),3.46(t,2H),3.22-3.15(m,2H),3.07-3.00(m,1H),2.75(m,1H),2.62(m,1H),2.45( s,3H),2.37-2.20(m,6H),2.10-2.02(m,2H),2.00-1.92(m,2H)1.68-1.57(m,6H),1.01(t,3H).

[0153] Connector - Toxin X2

[0154]

[0155]

[0156] first step

[0157] 34a (5 g, 48.0 mmol) and K₂CO₃ (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), and benzyl bromide (12.3 g, 72.0 mmol) was added dropwise. The reaction was carried out at 25 °C for 17 hours. TLC (PE / EA = 3 / 1) was used to determine if the reaction was complete. The reaction solution was added to water (200 mL), extracted with EA (250 mL), washed with saturated NaCl, dried over anhydrous Na₂SO₄, and concentrated by column chromatography (PE:EA = 2:1) to give 8.7 g of colorless liquid 34b, yield 93%. MS-ESI: m / z 195.1 [M+H]⁺.

[0158] Step 2

[0159] Dissolve 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) in THF (20 mL), protect under nitrogen atmosphere and cool to 0 °C. Add 43c (7.7 g, 39.6 mmol) of THF (10 mL) dropwise. After addition, react at 0 °C for 2 hours. TLC (PE / EA = 2 / 1) shows that most of the starting material has reacted. Pour the reaction solution into 100 mL of water, extract with DCM (100 mL), separate the layers, wash with saturated NaCl, dry with anhydrous Na2SO4, and pass through a column (PE / EA = 1 / 1) to give 3.9 g of colorless viscous substance 34d, yield: 39%.

[0160] MS-ESI: m / z 503.3[M+H]+.

[0161] Step 3

[0162] Under hydrogen atmosphere and at 0°C, Pd / C (1 g, 10 wt.%) was added to a mixed solution of EtOH (100 mL) and EA (100 mL) containing 34d (1.9 g, 3.78 mmol), and the reaction was carried out at 0°C for 3 hours. TLC (PE / EA = 2 / 1) showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with EA / EtOH (1:1, 100 mL × 3). The filtrate was concentrated, dissolved in THF (50 mL × 3), and evaporated to dryness. This process was repeated three times to obtain 1 g of gray solid 34e, yield: 64%.

[0163] MS-ESI: m / z 435.2 [M+Na]+.

[0164] Step 4

[0165] Under nitrogen protection, DIEA (303 mg, 2.35 mmol) was added dropwise to a DMF (20 mL) solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) at 0 °C. After the addition was complete, the reaction was allowed to proceed at 0 °C for 2 hours. LC-MS showed the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, and allowed to stand for 5 minutes. After filtration, the filter cake was dissolved in a DCM / MeOH (10:1, 100 mL) solution, dried, and stirred by rotary evaporation. Column chromatography (EA:MeOH = 30:1) yielded 600 mg of a yellow solid 34f, with a yield of 77%.

[0166] MS-ESI: m / z 830.3[M+H]+.

[0167] Step 5

[0168] Under nitrogen protection, diethylamine (5 mL) was added dropwise to a 34f (150 mg, 0.18 mmol) DCM (5 mL) solution at 0 °C, and the reaction was carried out at 0 °C for 2 hours. LCMS showed that the reaction was complete. Petroleum ether solution (100 mL × 6) was added to the reaction solution, and a solid precipitated. After standing to allow the solid to settle, the solution was poured off and then dried using an oil pump, yielding 34 g of 120 mg white powder. LCMS showed that the product content was 70%, and the yield was 76%.

[0169] MS-ESI:m / z 608.3[M+H]+.

[0170] Step 6

[0171] Under nitrogen protection, HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) solutions at 0 °C, and the reaction was carried out at 0 °C for 2 h. LC-MS showed that the starting material reacted completely. The reaction solution was directly passed through a reversed-phase column with eluent ((MeCN / MeOH = 1 / 1):H2O = 60%:40%) to purify 14.8 mg of yellow solid x 2, yield 14%.

[0172] MS-ESI: m / z 1062.4[M+H]+.

[0173] 1 H NMR(400MHz, Methanol-d4)δ7.69–7.61(m,2H),7.22–7.16(m,2H),7.16–7.09(m,3H),6.76(s,2H),5.70–5.64(m,1H),5.6 0(d,J=16.4Hz,1H),5.40–5.31(m,2H),5.26(d,J=19.0Hz,1H),4.65–4.50(m,7H),4.25–4.16(m,1H),3.87(d,J=16.7Hz,1 H),3.83–3.76(m,3H),3.72(d,J=17.0Hz,2H),3.44(t,J=7.1Hz,2H),3.25–3.17(m,2H),3.10–3.02(m,1H),2.92–2.83(m, 1H),2.45–2.39(m,5H),2.32–2.20(m,5H),1.97–1.89(m,2H),1.63–1.50(m,4H),1.34–1.20(m,6H),0.99(t,J=7.3Hz,3H).

[0174] Connector - Toxin X3

[0175] Following the experimental method used for connector-toxin X2, connector-toxin X3 was synthesized.

[0176]

[0177] Preparation of Deruxtecan

[0178]

[0179] Delutecan was synthesized according to the method provided in Example 58 on page 163 of the specification in patent application CN104755494A.

[0180] Example 3: Preparation of Antibody-Drug Conjugates

[0181] The following antibodies can be prepared using conventional antibody methods. For example, after constructing a vector, they can be transfected into eukaryotic cells such as HEK293 cells (Life Technologies Cat No. 11625019) and purified for expression.

[0182] The sequence of Trastuzumab:

[0183] Light chain (SEQ ID NO:1)

[0184]

[0185] Heavy chain (SEQ ID NO:2)

[0186]

[0187] Sacituzumab sequence

[0188] Light chain (SEQ ID NO:3)

[0189]

[0190] Heavy chain (SEQ ID NO:4)

[0191]

[0192]

[0193] Preparation of anti-HER2 antibody-drug conjugate ADC-II-9:

[0194]

[0195] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5mM, 0.355mL, 1.77μmol) was added to a PB buffer solution of the antibody Trastuzumab (0.04M PB buffer solution with pH=7.0; 35mg, 15mg / mL, 0.236μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours. The reaction was then stopped. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0mg / mL, and 2.0mL of the solution was taken out and used for further reaction.

[0196] The adapter-toxin X1 (4.51 mg, 2.84 μmol) was dissolved in 0.10 mL of DMSO and added to the above 2.0 mL solution. The mixture was placed in a water bath and shaken at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.04 M PB buffer aqueous solution at pH 7.0 containing 0.002 M EDTA) to obtain a PB solution of the exemplary product ADC-II-9 (7.00 mg / mL, 15 mg), which was stored frozen at 4°C.

[0197] The average value n = 8.05 was calculated using LC-MS.

[0198] Preparation of anti-TROP2 antibody-drug conjugate ADC-III-28:

[0199]

[0200] At 25°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5mM, 0.251mL, 1.255μmol) was added to a PB buffer solution of antibody Sacituzumab (0.04M PB buffer solution of pH=7.0; 56.3mg, 11mg / mL, 0.381μmol). The solution was placed in a water bath shaker and shaken at 25°C for 3 hours, after which the reaction was stopped.

[0201] The linker-toxin X2 (4.05 mg, 3.81 μmol) was dissolved in 0.10 mL of DMSO and added to the above solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.02 M histidine buffer aqueous solution at pH = 5.5) to obtain a histidine solution of the exemplary product ADC-III-28 (4.83 mg / mL, 25.9 mg), which was stored at 4 °C.

[0202] The average value calculated by LC-MS is n = 3.67.

[0203] Preparation of ADC-III-9:

[0204] The preparation of ADC-III-9 was specifically carried out by referring to the method provided in Preparation Example 1.44 on page 275 of WO2022068878A1.

[0205]

[0206] The average value n = 8.05 was calculated using LC-MS.

[0207] Preparation of Trastuzumab deruxtecan (T-DXd):

[0208] Trastuzumab deruxtecan (T-DXd) was prepared by referring to the method provided in Example 58 on page 163 of the specification in CN104755494A or by other commonly used methods in the art.

[0209] Example 4: Preparation of antibodies for recognizing antibody-drug conjugates

[0210] 4.1 Preparation of antigen

[0211] KLH conjugated with Deruxtecan (Deruxtecan-KLH) is used for immunization, and BSA conjugated with Deruxtecan (Deruxtecan-BSA) is used for ELISA detection.

[0212] The ADC antigens T-DXd, ADC-II-9, and ADC-III-9, and the corresponding small molecules DXd, P-II-3, and P-III-20 were used for screening.

[0213] 4.2 Antibody Preparation

[0214] Nine Balb / c mice (purchased from WuXi AppTec) were immunized with Deruxtecan-KLH immunoglobulin. Antibody production in serum was detected by ELISA. Mice with high titers were selected, and their spleen cells and myeloma cells were electrofused. Positive cell lines were screened by ELISA. After two rounds of subclonal screening, specific hybridoma cell lines against small molecules DXd, P-II-9 and P-III-20, and large molecules T-DXd, ADC-II-9 and ADC-III-9 were identified. One cell line, 2335CT81.1.1, was selected for antibody production, and the antibody SG201221AA was obtained through protein G affinity purification.

[0215] Experimental methods and results:

[0216] Immunization / Blood Collection: Nine Balb / c mice were immunized with Deruxtecan-KLH. The first immunization used Deruxtecan-KLH and Freund's complete adjuvant (Sigma), the second and third immunizations used Deruxtecan-KLH and Freund's incomplete adjuvant (Sigma), and the fourth and fifth immunizations used Deruxtecan-KLH and PBS. Animals were injected every two weeks. Blood was collected from the mice before immunization (first blood collection), after the third injection (second blood collection), after the first booster immunization (third blood collection), and after the second booster immunization (fourth blood collection).

[0217] ELISA testing process:

[0218] The antigen (Deruxtecan-BSA) was diluted with coating buffer (10mM PBS, pH 7.4) to a concentration of 1.25 μg / mL, 100 μL per well, and incubated overnight at 4°C. Empty the liquid and blot dry any remaining liquid. Rinse three times with PBST. Add 200 μL of blocking buffer (2% BSA) to each well and incubate at 37°C for 1-1.5 hours. Empty the liquid and blot dry any remaining liquid. Rinse three times with PBST. Add 100 μL of primary antibody (i.e., the mouse serum obtained from immunization) to each well and incubate at 37°C for 1-1.5 hours. Empty the liquid and blot dry any remaining liquid. Rinse three times with PBST. Add 100 μL of secondary antibody (goat anti-mouse IgG-HRP labeled, Sigma (#A0168)) to each well and incubate at 37°C for 1 hour. Empty the liquid and blot dry any remaining liquid. Rinse five times with PBST. Blot dry any remaining liquid in the wells. Add 100 μL of TMB chromogenic buffer to each well and incubate at 37°C for 5-10 minutes. Add 50 μL of 2M H2SO4 to each well to stop the chromogenic process and immediately read the 450 nm OD value.

[0219] The test results showed that the mouse serum titer reached 1:32000 or higher. Mice with high titers were selected, and their spleen cells and myeloma cells were electrofusion.

[0220] Electrical Fusion Process:

[0221] Mouse spleen cells were isolated and suspended in IMDM medium. The supernatant was discarded, and 5 mL of erythrocyte lysis buffer was added. The cells were lysed at room temperature for 5 minutes. IMDM medium was added to a final volume of 30 mL. The cells were centrifuged at 1500 rpm for 5 minutes. The spleen cells were resuspended in fresh IMDM medium and cell counting was performed.

[0222] Collect myeloma cells (FO), wash twice with IMDM medium, and count the cells.

[0223] Spleen cells and FO cells were mixed at a 2:1 ratio and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the mixed cells were washed twice with electrofusion buffer (Qiwenbio (#CEB005), 20 mL). The supernatant was discarded again, and electrofusion buffer was added to adjust the spleen cell concentration to 1-2 × 10⁻⁶ cells / mL. 7 Add the cell suspension to the electrofusion chamber at a concentration of 200 μL / well and start the electrofusion program on the electrofusion instrument. After electrofusion is complete, let the fused cells stand for 5 minutes. Transfer the fused cells to selection medium containing HAT, and then transfer the cells to a culture plate containing 20 feeder cells (peritoneal macrophages) at a concentration of 200 μL / well. Incubate the cell plates at 37°C in a 5% CO2 incubator. On the fifth day of culture, replace the HAT medium with HT medium. Perform antibody ELISA detection of the fused cells on the eighth day.

[0224] Many small molecule-specific clones were obtained through fusion screening, and only 8 clones fused with 2335CT were selected for subcloning.

[0225] Subcloning process:

[0226] The first subcloning was performed using a serial dilution method. Cell counting was performed using a red blood cell count method. 300 cells were added to 10 mL of complete culture medium (IMDM medium containing 15% fetal bovine serum and 1% HT). 200 μL of cell suspension was added to each well of a 96-well cell culture plate, resulting in approximately 6 cells per well. 5 mL of complete culture medium was added to the remaining cell suspension, and the mixture was repeated. 200 μL of cell suspension was added to each well of another 4 wells of the 96-well cell culture plate, resulting in approximately 2-3 cells per well. Another 5 mL of complete culture medium was added to the remaining cell suspension, and the mixture was repeated. 200 μL of cell suspension was added to each well of the remaining 4 wells of the 96-well cell culture plate, resulting in approximately 1 cell per well. The culture plates were then incubated at 37°C in a 5% CO2 incubator.

[0227] The second subcloning was performed using a limiting dilution method. 100 cells were added to 20 mL of complete culture medium (IMDM medium containing 10% fetal bovine serum), and 200 μL of cell suspension was added to each well of a 96-well cell culture plate, with approximately one cell per well. The culture plates were incubated at 37°C in a 5% CO2 incubator for 7 days, and specific positive clones were screened using an ELISA assay.

[0228] Through two rounds of subcloning and ELISA screening, three clones specific to small molecules DXd, P-II-3 and P-III-20 were obtained: 2335CT81.1.1, 2335CT115.2.3 and 2335CT117.1.3. Clone 2335CT81.1.1 was selected for antibody production.

[0229] Subtype identification:

[0230] Using IsoStrip TM The mouse monoclonal antibody typing kit was prepared according to the instructions to obtain clone 2335CT81.1.1, IgG1, κ.

[0231] Antibody production: Cell culture: Add cells (clone 2335CT81.1.1) to cell culture flasks, with a cell density of 1-2 × 10⁻⁶ cells / year. 5 / mL. After 5 days, the culture medium was increased to the maximum amount. When the cell viability dropped to 20%, the cell supernatant was collected and purified to obtain antibody SG201221AA.

[0232] Antibody testing:

[0233] 1) Specific detection:

[0234] The antibody SG201221AA was specifically detected using ELISA, and three antigens were used for detection simultaneously: T-DXd, ADC-II-9, or ADC-III-9. The results are shown in Table 1 below.

[0235] Table 1

[0236]

[0237] Conclusion: Antibody SG201221AA specifically binds to T-DXd, ADC-II-9, or ADC-III-9. The titer of antibody SG201221AA reaches 1:500,000 or higher.

[0238] 2) Competitive ELISA:

[0239] IC50 measurement

[0240] Dilute the antigen ADC (T-DXd, ADC-II-9, or ADC-III-9) with coating buffer (10mM PBS, pH 7.4) to a concentration of 1.25 μg / mL, 100 μL per well, and incubate overnight at 4°C. Empty the container and blot dry any remaining liquid, then wash three times with washing buffer. Add 200 μL of blocking buffer (2% BSA-PBS) to each well and incubate at 37°C for 1 hour. Empty the container and blot dry any remaining liquid, then wash three times with washing buffer. Add 100 μL of sample (antibody SG201221AA and small molecules (DXd, P-II-3, or P-III-20) at a 1:1 volume ratio) to each well and incubate at 37°C for 1–1.5 hours. Empty the container and blot dry any remaining liquid, then wash three times with washing buffer. Add 100 μL of secondary antibody (HRP-goat anti-mouse IgG) to each well and incubate at 37°C for 1–1.5 hours. Empty the liquid and blot dry any remaining liquid. Rinse 5 times with washing solution. Blot dry any remaining liquid in the wells, add 100 μL of colorimetric reagent to each well, and develop at 37°C for 5-10 min. Add 50 μL of 2M H2SO4 to each well to stop the color development and immediately read the 450 nm OD value.

[0241] Experimental results:

[0242] A: P-II-3 (P2C)

[0243] A1: The optimal antigen-antibody reaction concentration was selected for the ELISA experiment, and the experimental results are shown in Table 2:

[0244] Table 2

[0245]

[0246]

[0247] Conclusion: The antigen ADC-II-9 coating concentration of 0.25 μg / mL and the antibody detection concentration of 0.031 μg / mL were used in the competition experiment with the small molecule P-II-3.

[0248] A2: IC50 determination, the experimental results are shown in Table 3:

[0249] Table 3

[0250]

[0251] Based on the ELISA test results, the IC50 was calculated by averaging the OD values ​​of six replicates. The results are as follows: Figure 1 As shown. Experimental conclusion: The IC50 of P-II-3 (P2C) is 0.396 ng / mL.

[0252] B: P-III-20

[0253] B1: The optimal antigen-antibody reaction concentration was selected for the ELISA experiment, and the experimental results are shown in Table 4:

[0254] Table 4

[0255]

[0256] Conclusion: The antigen ADC-III-9 coating concentration of 0.25 μg / mL and the antibody detection concentration of 0.031 μg / mL were used in the competition experiment with the small molecule P-III-20.

[0257] B2: IC50 determination, the experimental results are shown in Table 5:

[0258] Table 5

[0259]

[0260] Based on the ELISA test results, the IC50 was calculated by averaging the OD values ​​of six replicates. The results are as follows: Figure 2 As shown. Experimental conclusion: The IC50 of P-III-20 is 0.282 ng / mL.

[0261] C.DXd

[0262] C1: The optimal antigen-antibody reaction concentration was selected for the ELISA experiment, and the experimental results are shown in Table 6.

[0263] Table 6

[0264]

[0265] Conclusion: The antigen T-DXd coating concentration of 0.25 μg / mL and the antibody detection concentration of 0.063 μg / mL were used to compete with the small molecule DXd in the experiment.

[0266] C2: IC50 determination, the experimental results are shown in Table 7.

[0267] Table 7

[0268]

[0269]

[0270] Based on the ELISA test results, the IC50 was calculated by averaging the OD values ​​of six replicates. The results are as follows: Figure 3 As shown, the conclusion is that the IC50 of DXd is 2.693 ng / mL.

[0271] 4.3 Purification and Hybridoma Sequencing

[0272] Purity test: After protein G affinity purification, SDS-PAGE and gel imaging analysis showed that the antibody purity of SG201221AA was 95.1%.

[0273] Total RNA was isolated from hybridoma cells 2335CT81.1.1 according to the RNA-easy Isolation Reagent (Vazyme) manual. Then, the total RNA was reverse transcribed into cDNA using isotype-specific antisense primers or universal primers according to the SMARTScribe Reverse Transcriptase (TaKaRa) manual. Antibody fragments from VH and VL were amplified according to the GenScript cDNA end rapid amplification (RACE) standard operating procedure (SOP). The amplified antibody fragments were cloned into standard cloning vectors. Colony PCR was performed to screen clones with inserts of the correct size. For each fragment, at least five colonies with inserts of the correct size were sequenced. Sequences of different clones were compared, and the common sequences of these clones were identified.

[0274] Table 8. VH and VL amino acid sequences of SG201221AA antibody

[0275]

[0276] The amino acid sequence of the heavy chain variable region signal peptide used is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region signal peptide used is shown in SEQ ID NO:18.

[0277] Table 9. Identified heavy and light chain CDR sequences (Kabat) of SG201221AA antibody

[0278]

[0279] 4.4 Construction of mouse Fc monoclonal antibodies

[0280] The heavy chain variable region of the obtained SG201221AA antibody was combined with the Fc region of mouse IgG2A, and the light chain variable region was combined with the kappa light chain constant region of mouse IgG2A. The recombinant antibody was then expressed and purified. The heavy and light chain sequences of the recombinant antibody are as follows:

[0281] C544YHI220-1_2335CT-mIg2A-kappa

[0282] Heavy chain (SEQ ID NO:13)

[0283]

[0284] Light chain (SEQ ID NO:14)

[0285]

[0286] 4.5 Construction of human Fc monoclonal antibodies

[0287] The heavy chain variable region of the obtained SG201221AA antibody was combined with the Fc region of human IgG1, and the light chain variable region was combined with the kappa light chain constant region of human IgG1. The chimeric antibody was then expressed and purified. The heavy and light chain sequences of the chimeric antibody are as follows:

[0288] Monoclonal antibody - human Fc

[0289] C8709HB100-1_C4773GL030 antibody

[0290] Heavy chain (SEQ ID NO:15)

[0291]

[0292]

[0293] Light chain (SEQ ID NO:16)

[0294]

[0295] Example 5: Determination of serum concentration in non-clinical studies

[0296] Using the mouse antibody SG201221AA obtained in Example 4, methods were developed for measuring the concentration of antibody-drug conjugates in the serum of mice and cynomolgus monkeys, as well as for measuring the concentration of antibody-drug conjugates in mouse tumor tissues.

[0297] Analytical methods for serum HER2-ADC (ADC-II-9) concentration:

[0298] 1) Payload (P-Ⅱ-3): LC-MS / MS

[0299] After the sample is thawed at room temperature, an internal standard is added, followed by 0.2% formic acid-50% methanol-acetonitrile. After vortexing and mixing, the sample is centrifuged at 18000g for 10 minutes at 4℃. The supernatant is then transferred to a vial and injected into the syringe for analysis.

[0300] The standard curve concentrations were 0.1, 0.2, 0.5, 2, 5, 10, 40, and 50 ng / mL.

[0301] P-Ⅲ-20 and P-Ⅲ-30 can be determined using similar methods.

[0302] 2) Detection of HER2-ADC concentration in serum:

[0303] Serum: The concentration of HER2-ADC in mouse / cynomolgus monkey serum was detected by ELISA.

[0304] (1) Coating: Dilute the biotinylated SG201221AA antibody 1:500 with coating buffer (0.05M CBS) and add it to SA microplate (Streptavidin High Binding Capacity Coated 96-Well Plates), 100 μL / well, cover with membrane, and incubate overnight at 37°C.

[0305] (2) Washing: Discard the coating solution, wash 3 times with washing solution, 300 μL / well, and pat dry.

[0306] (3) Blocking: Add blocking solution, 300 μL / well, cover with membrane, and incubate on a shaker at 15-25℃ for 2h±20min.

[0307] (4) Washing: Wash 3 times with washing solution, 300μL / well, and pat dry.

[0308] (5) Sample addition: According to the sample arrangement diagram in the Watson system, add NSB (1% mouse / cynomolgus monkey blank serum), standard curve sample solution, quality control sample solution, and test sample solution to the microplate at 100 μL / well. Cover with membrane and incubate on a shaker at 15–25°C for 2 h ± 20 min. The standard curve concentrations are 5000, 2500, 1250, 625, 313, 156, and 78.1 ng / mL.

[0309] (6) Washing: Wash 3 times with washing solution, 300μL / well, and pat dry.

[0310] (7) Add detection antibodies: Dilute goat anti-human IgG and monkey ads-HRP with sample diluent at a ratio of 1:10000, 100 μL / well, cover with membrane, and incubate on a shaker at 15-25℃ for 1 h ± 5 min.

[0311] (8) Washing: Wash 3 times with washing solution, 300μL / well, and pat dry.

[0312] (9) Color development: Add TMB color development solution, 100 μL / well, cover with film, and let stand at 15-25℃ in the dark for 3-20 min to develop color.

[0313] (10) Termination: Add 100 μL of termination solution to terminate the reaction.

[0314] (11) Detection: Use an ELISA reader to read the OD value of each well at a detection wavelength of 450 nm (reference wavelength of 630 nm).

[0315] Other ADCs, such as HER3-ADC, TROP2-ADC, and B7H3-ADC, can be measured using similar methods.

[0316] 3) ELISA detection of total antibody concentration in serum:

[0317] (1) Coating: Dilute human HER2 protein (His tag) with coating buffer (0.05M CBS) to a final concentration of 1μg / mL, add 100μL / well to the microplate, cover with membrane, and incubate overnight at 2-8℃.

[0318] (2) Washing: Discard the coating solution, wash 3 times with washing solution, 300 μL / well, and pat dry.

[0319] (3) Blocking: Add blocking solution, 300 μL / well, cover with membrane, and incubate on a shaker at 15-25℃ for 2h±20min.

[0320] (4) Washing: Wash 3 times with washing solution, 300μL / well, and pat dry.

[0321] (5) Sample addition: According to the sample arrangement diagram in the Watson system, add NSB (1% mouse / cynomolgus monkey blank serum), standard curve sample solution, quality control sample solution, and test sample solution to the microplate at 100 μL / well. Cover with membrane and incubate on a shaker at 15–25°C for 2 h ± 20 min. The standard curve concentrations are 5000, 2500, 1250, 625, 313, 156, and 78.1 ng / mL.

[0322] (6) Washing: Wash 3 times with washing solution, 300μL / well, and pat dry.

[0323] (7) Add detection antibodies: Dilute Goat Anti-Human IgG and Monkey ads-HRP with sample diluent at a ratio of 1:10000, 100 μL / well, cover with membrane, and incubate on a shaker at 15-25℃ for 1 h ± 5 min.

[0324] (8) Washing: Wash 3 times with washing solution, 300μL / well, and pat dry.

[0325] (9) Color development: Add TMB color development solution, 100 μL / well, cover with film, and let stand at 15-25℃ in the dark for 3-20 min to develop color.

[0326] (10) Termination: Add 100 μL of termination solution to terminate the reaction.

[0327] (11) Detection: Use an ELISA reader to read the OD value of each well at a detection wavelength of 450 nm (reference wavelength of 630 nm).

[0328] Other total antibodies, such as anti-HER3 antibody, anti-TROP2 antibody, and anti-B7H3 antibody, can be detected using similar methods.

[0329] Example 6: PK Study of Human Gastric Cancer NCI-N87 Nude Mouse Subcutaneous Xenograft Tumor Model

[0330] This study investigated the effect of a single intravenous injection of ADC-II-9 on subcutaneous xenograft tumors in NCI-N87 nude mice. Serum drug concentrations in tumor-bearing nude mice were measured to assess pharmacokinetic (PK) profiles. Human gastric cancer NCI-N87 cells were purchased from American Type Culture Collection. BALB / c Nude mice, 5-7 weeks old, female, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0331] Nude mice were subcutaneously inoculated with NCI-N87 cells, and tumors were allowed to grow to 200-300 mm. 3 Mice were given a single intravenous injection of ADC-II-9 at doses of 1.5 mg / kg or 5 mg / kg. Four mice were sacrificed at 5 min, 4 h, 24 h, 48 h, 72 h, and 168 h after administration (four mice at each time point). Blood was collected from the retroocular venous plexus of the mice before sacrifice, and serum was separated. The serum drug concentration was detected using a murine Fc monoclonal antibody of SG201221AA.

[0332] The results are shown in Table 10-12 below.

[0333] Table 10. Pharmacokinetic parameters of ADC-II-9 in mouse serum after intravenous injection of ADC-II-9

[0334]

[0335] Table 11 Pharmacokinetic parameters of total antibodies in mouse serum after intravenous injection of ADC-II-9

[0336]

[0337] Table 12 Pharmacokinetic parameters of P-II-3 in mouse serum after intravenous injection of ADC-II-9

[0338]

[0339] Note: If there are fewer than 3 "-" data points, pharmacokinetic parameter calculations will not be performed.

[0340] Conclusion: After a single dose of ADC-II-9, the serum ADC and total anti-approximate dose ratio increased, and the exposure levels of the two were similar and significantly higher than those of serum P-II-3.

[0341] Example 7: A Comparison Study of Crab-Eating Macaques

[0342] Cynomolgus monkeys were administered ADC-II-9 at single intravenous infusions of 1, 3, and 10 mg / kg (3 monkeys / sex / group) over 30 minutes. Serum and plasma samples were collected before administration (before the start of the intravenous infusion) and at 0.0167 hours (just after the end of the infusion), 0.167 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours (day 2), 48 hours (day 3), 72 hours (day 4), 96 hours (day 5), 168 hours (day 8), 240 hours (day 11), 336 hours (day 15), 504 hours (day 22), 672 hours (day 29), and 1008 hours (day 43). Serum samples were used to analyze ADC-II-9 and total antibodies, while plasma samples were used to analyze free P-II-3.

[0343] The results are shown in Table 13-15 below.

[0344] Table 13 Mean pharmacokinetic parameters of P-II-3 after intravenous administration of ADC-II-9 to male and female cynomolgus monkeys (n=6, 3 females and 3 males)

[0345]

[0346]

[0347] ND: Uncertain.

[0348] --:not applicable.

[0349] Table 14 Mean pharmacokinetic parameters of ADC-II-9 after intravenous infusion in male and female cynomolgus monkeys (n=6, 3 females and 3 males)

[0350]

[0351] Table 15 Mean pharmacokinetic parameters of total antibody after intravenous administration of ADC-II-9 to male and female cynomolgus monkeys (n=6, 3 females and 3 males)

[0352]

[0353] Note: Actual PK parameters are calculated at the start of intravenous infusion.

[0354] Conclusion: After a single dose of ADC-II-9, the serum ADC and total anti-approximate dose ratio of cynomolgus monkeys increased, and the exposure levels of the two were similar and significantly higher than those of serum P-II-3; no significant sex differences were observed.

Claims

1. An identifying antibody D, wherein the identifying antibody D specifically binds to a compound comprising a structure as shown in Formula I, or a pharmaceutically acceptable salt thereof, ， R 1 is C 3-6 cycloalkylene or C 1-6 alkylene, " indicates the point of attachment of the moiety to the rest of the compound as shown in Formula I; the identifying antibody D comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 7, HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 8, and HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 9; the light chain variable region comprises LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 10, LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 11, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO:

12.

2. The identifying antibody D according to claim 1, characterized in that, which satisfies one or both of the following conditions: (1) R 1 In particular, the C 1-6 alkylene is methylene, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(CH2-CH3)-, -C(CH3)2-, -CH2-CH(CH3)- or butylene; and (2) R 1 In particular, the C 3-6 Cycloalkylene is cyclopropylene, cyclobutylene, cyclopentylene or cyclohexylene.

3. The identifying antibody D according to claim 2, characterized in that, which satisfies one or both of the following conditions: (1) R 1 In particular, the C 1-6 alkylene is or ; and (2) R 1 In particular, the C 3-6 Cycloalkylene is cyclobutylene.

4. The identifying antibody D according to claim 3, characterized in that, which satisfies one or both of the following conditions: (1) R 1 In particular, the C 1-6 In the alkylene group, is or ; and (2) R 1 In particular, the C 3-6 Cycloalkylene is .

5. The identifying antibody D according to claim 4, characterized in that, which satisfies one or both of the following conditions: (1) R 1 In particular, the C 1-6 In the alkylene group, is or wherein the "1" position is attached to O and the "2" position is attached to the carbonyl group; and (2) R 1 In particular, the C 3-6 In the cycloalkylene group, is or .

6. The identifying antibody D according to claim 5, characterized in that, R 1 In particular, the C 3-6 In the cycloalkylene group, is or wherein, wherein the "1 " position is attached to O and the "2" position is attached to the carbonyl group.

7. The identifying antibody D of claim 1, wherein: R 1 For , or .

8. The identifying antibody D of claim 7, wherein: R 1 For , , or where the "1" position is attached to O and the "2" position is attached to the carbonyl group.

9. The identifying antibody D of claim 8, wherein: R 1 For , Or wherein the "1" position is attached to O and the "2" position is attached to the carbonyl group.

10. The identifying antibody D of claim 1, wherein, the compound comprising a structure as shown in Formula I, or a pharmaceutically acceptable salt thereof is a compound as shown in Formula I-1, or a pharmaceutically acceptable salt thereof, a compound as shown in Formula I-2, or a pharmaceutically acceptable salt thereof, or a compound as shown in Formula I-3, or a pharmaceutically acceptable salt thereof, 、 、 ， R 1 of claim 1 ; G and G 1 is a linking group; T is a ligand; n is a natural number or a decimal number from 1 to 16.

11. The identifying antibody D according to claim 10, characterized in that, which satisfies one or both of the following conditions: (1) the T is a ligand targeting HER2, HER3, B7H3, B7H4, TROP2, GPC3, DLL3, Claudin 18.2, CD30, CD33, CD70, or EGFR; and (2) n is a natural number or a decimal number from 1 to 9.

12. The identifying antibody D according to claim 11, characterized in that, the T is an anti-HER2 antibody or an anti-TROP2 antibody.

13. The identifying antibody D according to claim 12, characterized in that, the anti-HER2 antibody is trastuzumab; the anti-TROP2 antibody is Sacituzumab.

14. The identifying antibody D of claim 10, wherein, which satisfies one or more of the following conditions: (1) G is ; (2) G 1 To wherein the "1 " position is attached to T and the "2" position is attached to O; (3) the T is trastuzumab or Sacituzumab; and (4) n is a natural number or a decimal number from 3 to 9.

15. The identifying antibody D according to claim 14, characterized in that, n is 3.67 or 8.

05.

16. The identifying antibody D of claim 1, wherein the compound comprising a structure as shown in Formula I is any one of the following compounds: 、 、 、 、 、 、 、 、 、 ， wherein T is a ligand, and n is a natural number or a decimal number from 1 to 16.

17. The identifying antibody D of claim 16, wherein For Or ; or, for or ； or, to n is a natural number or a decimal number from 1 to 16; or, to n is a natural number or a decimal number from 1 to 16, or, to n is a natural number or a decimal number from 1 to 16.

18. The identifying antibody D of claim 17, wherein To n is 8.05; or, to n is 3.67, or, to n is 8.

05.

19. The identifying antibody D of any one of claims 1-18, wherein, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 5 or has at least 80% sequence identity to SEQ ID NO: 5, and the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 6 or has at least 80% sequence identity to SEQ ID NO:

6.

20. The identifying antibody D of claim 19, wherein, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO:

6.

21. The identifying antibody D of any one of claims 1-18, wherein, which satisfies one or both of the following conditions: (1) the identifying antibody D is selected from a murine antibody, a humanized antibody, and a chimeric antibody; and (2) the recognizing antibody D is a full-length antibody, Fab, Fab', F(ab')2, or Fv, and when the recognizing antibody D is a full-length antibody, the recognizing antibody D further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is a heavy chain constant region of a murine or human antibody or a variant thereof, and the light chain constant region is a kappa chain or a lambda chain of a murine or human antibody or a variant thereof.

22. The identifying antibody D of claim 21, wherein, The recognizing antibody D is an scFv.

23. The identifying antibody D of any one of claims 1-18, wherein, It is the following scheme 1 or scheme 2: Scheme 1: the recognizing antibody D comprises a heavy chain and a light chain, the heavy chain has an amino acid sequence as shown in SEQ ID NO: 13 or has at least 80% sequence identity with SEQ ID NO: 13; the light chain has an amino acid sequence as shown in SEQ ID NO: 14 or has at least 80% sequence identity with SEQ ID NO: 14; Scheme 2: the recognizing antibody D comprises a heavy chain and a light chain, the heavy chain has an amino acid sequence as shown in SEQ ID NO: 15 or has at least 80% sequence identity with SEQ ID NO: 15; the light chain of the antibody has an amino acid sequence as shown in SEQ ID NO: 16 or has at least 80% sequence identity with SEQ ID NO:

16.

24. The identifying antibody D of claim 23, wherein, It is the following scheme 1 or scheme 2: Scheme 1: the recognizing antibody D comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 13; the amino acid sequence of the light chain is as shown in SEQ ID NO: 14; Scheme 2: the recognizing antibody D comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 15; the amino acid sequence of the light chain is as shown in SEQ ID NO:

16.

25. A nucleic acid, comprising: The nucleic acid encodes the recognizing antibody D according to any one of claims 1-24.

26. A recombinant expression vector comprising, The recombinant expression vector comprises the nucleic acid according to claim 25.

27. A transformant characterized in that, The transformant comprises the nucleic acid according to claim 25 or the recombinant expression vector according to claim 26.

28. A method of detecting an antibody drug conjugate in vitro, characterized in that, The method comprises the step of contacting the recognizing antibody D according to any one of claims 1-24 with a sample to be tested comprising an antibody drug conjugate.

29. The method of claim 28, wherein, The antibody drug conjugate is the compound as shown in formula I-3 or a pharmaceutically acceptable salt thereof in the recognizing antibody D according to any one of claims 10-18.

30. The method of claim 28, wherein, The sample to be tested is serum.

31. The method of claim 30, wherein, The sample to be tested is serum of a mammal.

32. A detection reagent, characterized by, The detection reagent comprises the recognizing antibody D according to any one of claims 1-24, the nucleic acid according to claim 25, the recombinant expression vector according to claim 26, and / or the transformant according to claim 27.

33. A kit comprising, The kit comprises the recognizing antibody D according to any one of claims 1-24, the nucleic acid according to claim 25, the recombinant expression vector according to claim 26, the transformant according to claim 27, and / or the detection reagent according to claim 32.

34. A method of preparing an antibody drug conjugate recognizing an antibody, characterized in that, The method comprises culturing the transformant according to claim 27, and obtaining the recognizing antibody from the culture.

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