Recognition antibody of antibody drug conjugate and application thereof

Abstract

The invention relates to a recognition antibody of an antibody drug conjugate and application thereof. The recognition antibody is specifically combined with a compound with a structure as shown in a formula I or pharmaceutically acceptable salt thereof. The provided recognition antibody can specifically recognize drug molecules in the antibody drug conjugate, so that the content of the antibody drug conjugate is effectively measured.

Description

Recognition antibodies of antibody-drug conjugates and their applications

[0001] This application claims the benefit of Chinese Patent Application No. 2023109828098, filed August 4, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention belongs to the field of biotechnology, and in particular relates to an antibody-drug conjugate recognition antibody and its application. Background Art

[0003] Antibody-drug conjugates (ADCs) with cytotoxic drugs conjugated to antibodies (which bind to antigens expressed on the surface of cancer cells and are also capable of cellular internalization) can selectively deliver drugs to cancer cells, and are therefore expected to cause the drug to accumulate in cancer cells and kill cancer cells. Similar to the development of small molecule compounds and antibodies, when antibody-drug conjugates are developed as drug products, pharmacokinetic studies (PK studies) are essential. This is because, by implementing PK studies in people based on the understanding of the correlation between the PK study results and pharmacological study results and safety test results in animals, information useful for planning clinical trial design and considering effectiveness and safety in people can be obtained.

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

[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 a protein capable of recognizing the antibody-drug conjugate and labeled with a marker with the complex to form a further complex; and then, (3) detecting the marker based on color / light generated by an enzymatic reaction.

[0006] Therefore, in order to accurately measure 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.

[0007] Summary of the Invention

[0008] The present invention addresses the technical problem of the lack of antibodies for recognizing antibody-drug conjugates in the prior art. The present invention provides an antibody-drug conjugate recognition antibody and its use. The antibody provided by the present invention can specifically bind to an antibody-drug conjugate conjugated with exitecan or its derivatives, effectively determining the content of the antibody-drug conjugate.

[0009] The first aspect of the present invention provides a recognition antibody D, which specifically binds to a compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof.

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

[0011] In the present invention, the recognition antibody D can recognize the compound having the structure shown in Formula I. In a preferred embodiment, the recognition antibody D can recognize the structure shown in Formula I in an antibody-drug conjugate.

[0012] In a preferred embodiment, R 1 In 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.

[0013] In a preferred embodiment, R 1 In the C 1-6 Alkylene is Preferably, for More preferably, for The "1" position is connected to O, and the "2" position is connected to the carbonyl group.

[0014] In a preferred embodiment, R 1 In the C 3-6 Cycloalkylene is cyclopropylene, cyclobutylene, cyclopentylene or cyclohexylene, preferably cyclobutylene, more preferably

[0015] In a preferred embodiment, R 1 In the C 3-6 Cycloalkylene is Preferably, for More preferably, for The "1" position is connected to O, and the "2" position is connected to the carbonyl group.

[0016] In a preferred embodiment, R 1 for Preferably, R 1 for wherein the "1" position is connected to O and the "2" position 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 shown in Formula I-1 or a pharmaceutically acceptable salt thereof, a compound shown in Formula I-2 or a pharmaceutically acceptable salt thereof, or a compound shown in Formula I-3 or a pharmaceutically acceptable salt thereof,

[0018] R 1 is as defined in the preceding item;

[0019] G and G 1 is a linking group (the linking group can connect the compound represented by Formula I-1 or a pharmaceutically acceptable salt thereof to T);

[0020] T is a ligand, and the T is connected to G through the sulfhydryl or amino group on it. 1 forming a covalent bond;

[0021] n is a natural number or decimal from 1 to 16; (n is the drug-ligand coupling rate).

[0022] In a preferred embodiment, G is

[0023] In a preferred embodiment, G 1 for The "1" position is connected to T, and the "2" position is connected to O.

[0024] In a preferred embodiment, the T is a ligand targeting the following targets: HER2, HER3, B7H3, B7H4, TROP2, GPC3, DLL3, Claudin 18.2, CD30, CD33, CD70 or EGFR.

[0025] In a preferred embodiment, the T is an anti-HER2 antibody or an anti-TROP2 antibody.

[0026] In a preferred embodiment, the anti-HER2 antibody is trastuzumab or a variant thereof.

[0027] In a preferred embodiment, the anti-TROP2 antibody is Sacituzumab, M1, M2 or M3 or variants thereof.

[0028] In a preferred embodiment, the T is trastuzumab or sacituzumab.

[0029] In a preferred embodiment, n is a natural number or a decimal number from 1 to 9.

[0030] In a preferred embodiment, n is a natural number or a decimal number from 3 to 9, such as 3.67 or 8.05.

[0031] In a preferred embodiment, the compound represented by formula I-1 is any of the following compounds:

[0032] In a preferred embodiment, for

[0033] In a preferred embodiment, for

[0034] In a preferred embodiment, the compound shown in formula I-2 is any one of the following compounds:

[0035] In a preferred embodiment, the compound represented by formula I-3 is any one of the following compounds:

[0036] Wherein, T is a ligand, and n is a natural number or decimal from 1 to 16;

[0037] Preferably, the compound represented by formula I-3 is any one of the following compounds:

[0038] n is a natural number or decimal from 1 to 16, such as 8.05;

[0039] n is a natural number or decimal from 1 to 16, for example, 3.67,

[0040] n is a natural number or decimal from 1 to 16, for example, 8.05.

[0041] 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.

[0042] In a preferred embodiment, in the recognition antibody D, the heavy chain variable region includes HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, and HCDR3 as shown in SEQ ID NO:9; and the light chain variable region includes LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11, and LCDR3 as shown in SEQ ID NO:12.

[0043] In a preferred embodiment, in the identification antibody D, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 5, or a sequence identity thereof of 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%, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 6, or a sequence identity thereof of 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%.

[0044] In a preferred embodiment, in the recognition antibody D, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 6.

[0045] In a preferred embodiment, the recognition antibody D is selected from murine antibodies, humanized antibodies and chimeric antibodies.

[0046] In a preferred embodiment, the recognition antibody D is a full-length antibody, Fab, Fab', F(ab')2 or Fv, such as scFv.

[0047] 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 murine or human antibody or a variant thereof, and the light chain constant region is the κ chain or λ chain of a murine or human antibody or a variant thereof.

[0048] In a preferred embodiment, the recognition antibody D includes 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.

[0049] In a preferred embodiment, the recognition antibody D comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13; the amino acid sequence of the light chain is shown in SEQ ID NO: 14.

[0050] In a preferred embodiment, the recognition antibody D includes 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.

[0051] In a preferred embodiment, the recognition antibody D comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 15; the amino acid sequence of the light chain is shown in SEQ ID NO: 16.

[0052] In a preferred embodiment, the recognition antibody D specifically binds to the compound shown in formula I-3, T or G 1 .

[0053] A second aspect of the present invention provides an identification antibody X, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 7, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 8, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 9; and the light chain variable region comprises a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 10, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 12.

[0054] In the present invention, the recognition antibody X can recognize an antibody-drug conjugate.

[0055] In a preferred embodiment, in the identification antibody X, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 7, HCDR2 as shown in SEQ ID NO: 8, and HCDR3 as shown in SEQ ID NO: 9; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO: 12.

[0056] In a preferred embodiment, in the identification antibody X, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 5, or a sequence identity thereof of 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%, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 6, or a sequence identity thereof of 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%.

[0057] In a preferred embodiment, in the recognition antibody X, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 6.

[0058] In a preferred embodiment, the recognition antibody X is selected from a mouse antibody, a humanized antibody and a chimeric antibody.

[0059] In a preferred embodiment, the recognition antibody X is a full-length antibody, Fab, Fab', F(ab')2 or Fv, such as scFv.

[0060] In a preferred embodiment, when the recognition antibody X is a full-length antibody, the antibody 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 lambda chain of a murine or human antibody or a variant thereof.

[0061] In a preferred embodiment, the recognition antibody X comprises a heavy chain and a light chain, wherein the heavy chain has 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 to SEQ ID NO: 13; and the light chain has 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 to SEQ ID NO: 14.

[0062] In a preferred embodiment, the recognition antibody X comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13; the amino acid sequence of the light chain is shown in SEQ ID NO: 14.

[0063] In a preferred embodiment, the recognition antibody X comprises a heavy chain and a light chain, wherein the heavy chain has 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 to SEQ ID NO: 15; and the light chain has 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 to SEQ ID NO: 16.

[0064] In a preferred embodiment, the antibody X comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 15; the amino acid sequence of the light chain is shown in SEQ ID NO: 16.

[0065] The third aspect of the present invention provides a nucleic acid (polynucleotide), which encodes the recognition antibody as described in the first aspect or the second aspect.

[0066] The fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the third aspect.

[0067] The fifth aspect of the present invention provides a transformant, which comprises the nucleic acid as described in the third aspect or the recombinant expression vector as described in the fourth aspect.

[0068] The sixth aspect of the present invention provides a method for in vitro detection of an antibody-drug conjugate, comprising the step of contacting the recognition antibody according to the first aspect or the second aspect with a sample to be tested containing the antibody-drug conjugate.

[0069] In a preferred embodiment, the antibody-drug conjugate is as described in the first aspect as the compound represented by formula I-3 or a pharmaceutically acceptable salt thereof.

[0070] In a preferred embodiment, the sample to be tested is serum, such as mammalian serum.

[0071] The seventh aspect of the present invention provides a detection reagent, which comprises the recognition antibody as described in the first aspect or the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, and / or the transformant as described in the fifth aspect.

[0072] The eighth aspect of the present invention provides a kit, which comprises the recognition antibody as described in the first aspect or the second aspect, the nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the transformant as described in the fifth aspect, and / or the detection reagent as described in the seventh aspect.

[0073] The ninth aspect of the present invention provides the use of the recognition antibody described in the first or second aspect, the nucleic acid described in the third aspect, the recombinant expression vector described in the fourth aspect, the transformant described in the fifth aspect, or the detection reagent described in the seventh aspect in the preparation of a detection reagent. In a preferred embodiment, the detection reagent is used to detect an antibody-drug conjugate in a sample to be tested.

[0074] The tenth aspect of the present invention provides a method for preparing a recognition antibody, the method comprising culturing the transformant as described in the fifth aspect, and obtaining the recognition antibody from the culture.

[0075] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0076] The reagents and raw materials used in the present invention are commercially available.

[0077] The present invention provides a drug-recognizing antibody for use in an antibody-drug conjugate. The antibody can specifically bind to the drug molecule exotecan or its derivatives in the antibody-drug conjugate, effectively determining the content of the antibody-drug conjugate.

[0078] the term

[0079] Unless otherwise specified, the terms used in this invention have the following meanings:

[0080] It will be understood by those skilled in the art that the structural formulas used in the present invention to describe groups are based on the conventions used in the art. It means that the corresponding group is connected to other fragments and groups in the compound through this site.

[0081] The "-" at the end of a group means that the group is connected to other fragments in the molecule through this site. For example, -CH2-CH(CH3)- means that the CH2 and CH therein are connected to other fragments in the molecule.

[0082] In the claims of this application, the term "multiple" in the phrase "satisfies one or more of the following conditions" refers to 2, 3, 4, or more conditions. The maximum value of "more" is the maximum number of conditions recited in each claim. For example, if a claim recites four conditions, the term "one or more" in the phrase "satisfies one or more of the following conditions" in that claim can be any integer between 1 and 4, such as 1, 2, 3, or 4.

[0083] The term "pharmaceutically acceptable salt" refers to salts prepared from compounds of the present invention with relatively nontoxic, pharmaceutically acceptable acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a neat solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a neat 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.

[0084] The term "alkylene" refers to a substituent formed by eliminating two hydrogen atoms from a saturated straight-chain or branched alkane. The two hydrogen atoms eliminated can be on the same carbon atom or on different carbon atoms (for example, the two hydrogen atoms eliminated are on carbon atoms at the two 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)-; C3 alkylene (i.e., butylene) refers to -CH2-CH2-CH2-CH2-, -CH2-CH2-CH(CH2)-, -CH2-CH(CH2)-, or -CH(CH2-CH2-CH2)-.

[0085] The term "cycloalkylene" refers to a substituent formed by removing two hydrogen atoms from a cycloalkane. The two hydrogen atoms removed can be on the same ring carbon atom or on different ring carbon atoms. Here, "cycloalkane" refers to a substituent with a specified number of ring carbon atoms (e.g., C3 to C 10 , C3~C6), saturated cyclic alkanes whose ring atoms consist only of carbon atoms. For example, C4 cycloalkylene (i.e. cyclopropylene) can be

[0086] The term "antibody-drug conjugate (ADC)" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker. The ADC may be a monoclonal antibody or antibody fragment linked to a biologically active cytotoxic drug via a stable linker.

[0087] The term "antibody" generally refers to immunological binding agents and extends to all antibodies from all species, including dimers, trimers and multimers; bispecific antibodies; chimeric antibodies; fully human antibodies; humanized antibodies; recombinant and remodeled antibodies and their fragments. The term "antibody" can refer to any antibody-like molecule with an antigen-binding region, and the term includes small molecule fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), linear antibodies, diabodies, etc.

[0088] The antibody in the "antibody-drug conjugate" of the present invention can be anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-TNF-α antibody, anti-TROP2 antibody, 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 or more, for example, trastuzumab, certolizumab, pertuzumab or adalimumab.

[0089] The term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, a hybridoma secreting a mouse-specific monoclonal antibody is generated. The variable region gene is then cloned from the mouse hybridoma cell. The human constant region gene can then be cloned as needed. The mouse variable region gene and the human constant region gene are then linked to form a chimeric gene, which is then inserted into an expression vector. The chimeric antibody molecule can then be expressed in either eukaryotic or prokaryotic systems.

[0090] The term "humanized antibody", also known as CDR-grafted antibody, generally refers to an antibody produced by transplanting mouse CDR sequences into the variable region framework of a human antibody, i.e., different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database.

[0091] The terms "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody can be both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: 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 can be: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phagedisplay), transgenic mouse antibody preparation technology (transgenicmouse) and single B cell antibody preparation technology, etc.

[0092] The term "CDR" generally refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to 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 immunological 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 CDRs can be applied to CDR1, CDR2 and CDR3 of the light chain variable domain (CDRL1, CDRL2, CDRL3 or L1, L2, L3), and CDR1, CDR2 and CDR3 of the heavy chain variable domain (CDR H1, CDRH2, CDRH3 or H1, H2, H3).

[0093] 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, humans, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] FIG1 is a schematic diagram of the IC50 standard curve fitting of P-II-3.

[0095] FIG2 is a schematic diagram of the IC50 standard curve fitting of P-III-20.

[0096] FIG3 is a schematic diagram of IC50 standard curve fitting of DXd. DETAILED DESCRIPTION

[0097] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0098] Example 1 Preparation of toxin (payload)

[0099] Preparation Example 1

[0100] first step:

[0101] Under nitrogen, DIEA (500 mg, 3.87 mmol) was added to a solution of KI4 (900 mg, 1.69 mmol), HATU (691 mg, 1.88 mmol), and 3a (320 mg, 2.00 mmol) in DMF (18 mL) at 0°C. The mixture was stirred at 25°C for 3 hours. TLC (EA) indicated complete reaction of the starting materials. The reaction solution was added dropwise to 320 mL of deionized water and filtered to afford 850 mg of a gray solid (yield: 87%).

[0102] Step 2:

[0103] To a solution of 3b (100 mg, 0.174 mmol) in MeOH / DCM (1 / 1, 3 mL) was added solid NaHCO (42 mg, 0.50 mmol) and stirred at 25°C for 3 hours. TLC (EA) indicated the reaction was complete. The reaction solution was filtered, dried at low temperature, slurried with aq. HCl (0.5 M, 10 mL), filtered, and purified by prep-HPLC (0.1% TFA) and lyophilized to afford 15 mg of P-II-3 as a gray solid. Yield: 16%.

[0104] MS m / z (ESI): 534 [M+1].

[0105] 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).

[0106] Preparation Example 2

[0107] first step:

[0108] Under nitrogen, DIEA (486 mg, 3.76 mmol) was added to a 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 (18 mL) at 0°C. The mixture was stirred at 25°C for 3 hours. TLC (EA) indicated complete reaction of the starting materials. The reaction solution was added dropwise to 320 mL of deionized water and filtered to afford 910 mg of a gray solid (yield: 84%).

[0109] Step 2:

[0110] To a solution of 4b (100 mg, 1.58 mmol) in MeOH / DCM (1 / 1, 3 mL) was added NaHCO3 (42 mg, 0.50 mmol) as a solid and stirred at 25°C for 3 h. TLC (EA) showed that the reaction was complete. The reaction solution was filtered, dried at low temperature, slurried with aq.HCl (0.5 M, 10 mL) and filtered. The product was lyophilized by prep-HPLC (0.1% TFA) to give 13 mg of P-II-4 as a yellow solid, yield: 14%.

[0111] MS m / z (ESI): 534 [M+1].

[0112] 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).

[0113] Preparation Example 3

[0114] Under nitrogen, DIEA (61 mg, 0.47 mmol) was added dropwise to a solution of KI4 (100 mg, 0.188 mmol), HATU (86 mg, 0.23 mmol), and 7a (22 mg, 0.21 mmol) in DMF (2 mL). The mixture was allowed to react at 25°C for 2.5 hours. LCMS indicated complete reaction. The reaction solution was added to 20 mL of water, whereupon P-III-20 solid precipitated. Filtration afforded 13 mg of solid, yield: 11%.

[0115] MS m / z (ESI): 522 [M+1].

[0116] 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).

[0117] Preparation Example 4

[0118] first step

[0119] Under nitrogen, DIEA (60.6 mg, 0.47 mmol) was added dropwise to a solution of KI4 (100 mg, 0.19 mmol), HATU (85.7 mg, 0.23 mmol), and 23a (21.5 mg, 0.21 mmol) in DMF (2 mL). The mixture was reacted at 0°C for 2 hours. LCMS showed complete reaction. The reaction solution was added dropwise to 20 mL of water with stirring. The precipitated solid was filtered to afford 60.2 mg of a gray solid, P-III-30, in a 61% yield.

[0120] MS-ESI: m / z 522.2[M+H]+.

[0121] 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).

[0122] Example 2 Preparation of linker-payload

[0123] Connector-Toxin X1

[0124] first step

[0125] To a solution of 27a (5.00 g, 43.0 mmol) and NaHCO₃ (10.9 g, 129 mmol) in DMF (50 mL) was added benzyl bromide (11.0 g, 64.6 mmol) dropwise under nitrogen. The mixture was allowed to react at 25°C for 17 hours. TLC (PE / EA = 2 / 1) indicated completion of the reaction. The reaction solution was added to 500 mL of water and extracted twice with EA (250 mL). The mixture was separated, washed with saturated aqueous sodium chloride (500 mL), dried over anhydrous Na₂SO₄, and concentrated through a column chromatography (PE:EA = 3:2) to afford 5.1 g of a colorless liquid (yield: 57.1%).

[0126] Step 2

[0127] Under nitrogen, a solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) at 0°C. The mixture was allowed to react at 25°C for 2 hours. TLC (PE / EA = 1 / 2) indicated the reaction was complete. The reaction solution was added to 200 mL of water and extracted twice with EA (200 mL). The mixture was dried over anhydrous Na2SO4 and concentrated through a column chromatography (PE / EA = 3 / 2) to afford 1.56 g of a white solid (yield: 26%).

[0128] Step 3

[0129] To a solution of 27c (800 mg, 1.55 mmol) in EtOH (8 mL) and EA (8 mL) was added Pd / C (80 mg) under hydrogen at 0°C, and the mixture was stirred at 0°C for 2.5 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered through celite, washed with EA (200 mL), and the filter cake was concentrated and dissolved in THF (20 mL). The product was then dried to afford 600 mg of a white solid (91% yield).

[0130] Step 4

[0131] Under nitrogen, DIEA (152 mg, 1.18 mmol) was added to a solution of 27d (220 mg, 0.515 mmol), KI4 (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol) in DMF (6 mL) at 0°C. The mixture was allowed to react for 2 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to aqueous citric acid (pH = 4) (150 mL), filtered, and the filter cake was washed with 175 mL of water, filtered, and pulled dry using an oil pump to afford 260 mg of a brown solid (yield: 66%).

[0132] Step 5

[0133] Under nitrogen, diethylamine (8 mL) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in DCM (30 mL) at 0°C. The mixture was allowed to react for 3 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to 600 mL of petroleum ether at 0°C, resulting in the precipitation of a solid. After the solid was allowed to adsorb to the bottom of the flask, the solution was decanted and pumped dry with an oil pump to afford 90 mg of a brown solid (yield: 47.1%).

[0134] Step 6

[0135] Under nitrogen, HATU (74 mg, 0.19 mmol) was added to a solution of 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol), and DIEA (50 mg, 0.39 mmol) in DMF (2.5 mL) at 0°C and allowed to react for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added to a pH 4 aqueous citric acid solution (30 mL) at 0°C. A flocculent solid precipitated, which was filtered and purified by preparative chromatography (DCM / MecOH = 10 / 1) to afford 9.2 mg of a light yellow solid, X1, in a 6% yield.

[0136] MS m / z (ESI): 1074 [M+1].

[0137] 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).

[0138] Connector-Toxin X2

[0139] first step

[0140] 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 mixture was allowed to react at 25°C for 17 hours. TLC (PE / EA = 3 / 1) confirmed the complete reaction. The reaction mixture was added to water (200 mL) and extracted with EA (250 mL). The mixture was washed with saturated NaCl, dried over anhydrous Na2SO4, and concentrated to obtain 8.7 g of 34b as a colorless liquid (93% yield). MS-ESI: m / z 195.1 [M+H]+.

[0141] Step 2

[0142] 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) were dissolved in THF (20 mL) under nitrogen atmosphere and cooled to 0°C. A solution of 43c (7.7 g, 39.6 mmol) in THF (10 mL) was added dropwise and allowed to react at 0°C for 2 hours. TLC (PE / EA = 2 / 1) showed that most of the starting material had reacted. The reaction solution was poured into 100 mL of water and extracted with DCM (100 mL). The mixture was separated and washed with saturated NaCl, dried over anhydrous Na2SO4, and filtered through a column (PE / EA = 1 / 1) to give 3.9 g of a colorless viscous product 34d (yield: 39%).

[0143] MS-ESI: m / z 503.3[M+H]+.

[0144] Step 3

[0145] To a solution of 34d (1.9 g, 3.78 mmol) in EtOH (100 mL) and EA (100 mL) was added Pd / C (1 g, 10 wt.%) under hydrogen at 0°C for 3 hours. TLC (PE / EA = 2 / 1) indicated the reaction was complete. The reaction mixture was filtered through celite, and the filter cake was washed with EA / EtOH (1:1, 100 mL × 3). The filtrate was concentrated and dissolved in THF (50 mL × 3) and the mixture was dried by spin drying. This was repeated three times to afford 1 g of 34e as a gray solid in a 64% yield.

[0146] MS-ESI: m / z 435.2[M+Na]+.

[0147] Step 4

[0148] Under nitrogen, DIEA (303 mg, 2.35 mmol) was added dropwise to a solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) in DMF (20 mL) at 0°C. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, allowed to stand for 5 minutes, and filtered. The filter cake was dissolved in DCM / MeOH (10:1, 100 mL), dried, and then spin-dried. The mixture was purified by column chromatography (EA:MeOH = 30:1) to afford 600 mg of 34f as a yellow solid, in a yield of 77%.

[0149] MS-ESI: m / z 830.3[M+H]+.

[0150] Step 5

[0151] Under nitrogen, diethylamine (5 mL) was added dropwise to a solution of 34f (150 mg, 0.18 mmol) in DCM (5 mL) at 0°C. The mixture was allowed to react for 2 hours at 0°C. LCMS indicated the reaction was complete. Petroleum ether (100 mL x 6) was added to the reaction mixture, causing solid precipitation. After allowing the solid to settle, the solution was decanted and pumped dry with an oil pump to yield 120 mg of a white powder (34 g). LCMS indicated a 70% product content, for a yield of 76%.

[0152] MS-ESI: m / z 608.3[M+H]+.

[0153] Step 6

[0154] Under nitrogen, a solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to a solution of 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) at 0°C. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated complete reaction of the starting materials. The reaction solution was directly purified by a reverse phase column using an eluent (MeCN / MeOH = 1 / 1): HO = 60%:40%) to afford 14.8 mg of a yellow solid, X2, in a 14% yield.

[0155] MS-ESI: m / z 1062.4[M+H]+.

[0156] 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).

[0157] Connector-Toxin X3

[0158] Referring to the experimental method of linker-toxin X2, linker-toxin X3 was synthesized.

[0159] Preparation of Deruxtecan

[0160] Derutecan was synthesized according to the method provided in Example 58 on page 163 of the specification of patent application CN104755494A.

[0161] Example 3 Preparation of Antibody Drug Conjugates

[0162] The following antibodies can be prepared according to conventional antibody methods, for example, after constructing a vector, transfecting eukaryotic cells such as HEK293 cells (Life Technologies Cat No. 11625019), and then purified and expressed.

[0163] Sequence of Trastuzumab:

[0164] Light chain (SEQ ID NO: 1)

[0165] Heavy chain (SEQ ID NO: 2)

[0166] Sequence of sacituzumab

[0167] Light chain (SEQ ID NO: 3)

[0168] Heavy chain (SEQ ID NO: 4)

[0169] Preparation of anti-HER2 antibody drug conjugate ADC-Ⅱ-9:

[0170] At 37°C, a solution of the antibody trastuzumab in PB buffer (0.04 M PB buffer at pH 7.0; 35 mg, 15 mg / mL, 0.236 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.355 mL, 1.77 μmol). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg / mL, and 2.0 mL of the solution was removed for further reaction.

[0171] The linker-toxin X1 (4.51 mg, 2.84 μmol) was dissolved in 0.10 mL of DMSO, added to the above 2.0 mL solution, placed in a water bath shaker, and oscillated at 25°0 for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.04 M PB buffer 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.

[0172] LC-MS calculated average value n=8.05.

[0173] Preparation of anti-TROP2 antibody drug conjugate ADC-III-28:

[0174] At 25°C, a solution of sacituzumab in PB buffer (0.04 M PB buffer at pH 7.0; 56.3 mg, 11 mg / mL, 0.381 μmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (5 mM, 0.251 mL, 1.255 μmol). The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction.

[0175] The linker-toxin X2 (4.05 mg, 3.81 μmol) was dissolved in 0.10 mL of DMSO, added to the above solution, placed in a water bath shaker, and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.02 M histidine buffer solution at pH = 5.5) to obtain the exemplary product ADC-III-28 in a histidine solution (4.83 mg / mL, 25.9 mg), which was stored refrigerated at 4°C.

[0176] LC-MS calculated average value n=3.67.

[0177] Preparation of ADC-III-9:

[0178] The preparation of ADC-III-9 was specifically synthesized by referring to the method provided in Preparation Example 1.44 on page 275 of WO2022068878A1.

[0179] LC-MS calculated average value n=8.05.

[0180] Preparation of Trastuzumab deruxtecan (T-DXd):

[0181] Trastuzumab deruxtecan (T-DXd) was prepared by referring to the method provided in Example 58 on page 163 of the specification of CN104755494A or a commonly used method disclosed in the art.

[0182] Example 4 Preparation of Antibodies Recognizing Antibody Drug Conjugates

[0183] 4.1 Preparation of Antigens

[0184] KLH-coupled Deruxtecan (Deruxtecan-KLH) was used for immunization, and BSA-coupled Deruxtecan (Deruxtecan-BSA) was used for ELISA detection.

[0185] The ADC antigens used for screening were T-DXd, ADC-II-9 and ADC-III-9, and the corresponding small molecules DXd, P-II-3 and P-III-20.

[0186] 4.2 Antibody Preparation

[0187] Nine Balb / c mice (purchased from WuXi AppTec) were immunized with Deruxtecan-KLH. Antibody production in sera was assessed by ELISA. Mice with high titers exceeding 1:32,000 were selected, and their splenocytes were electrofused with myeloma cells. Positive cell lines were screened by ELISA. Two rounds of subcloning revealed hybridoma cell lines specific for small molecules DXd, P-II-9, and P-III-20, and large molecules T-DXd, ADC-II-9, and ADC-III-9. One cell line, 2335CT81.1.1, was selected for antibody production, and the antibody SG201221AA was obtained by protein G affinity purification.

[0188] Experimental methods and results:

[0189] Immunization / Blood Collection: Nine Balb / c mice were immunized with Deruxtecan-KLH. The first immunization used Deruxtecan-KLH with Freund's complete adjuvant (Sigma), the second and third immunizations used Deruxtecan-KLH with Freund's incomplete adjuvant (Sigma), and the fourth and fifth booster immunizations used Deruxtecan-KLH with PBS. Animals were injected every two weeks. Blood was collected from mice before immunization (first bleed), after the third injection (second bleed), after the first booster immunization (third bleed), and after the second booster immunization (fourth bleed).

[0190] ELISA test process:

[0191] The antigen (Deruxtecan-BSA) was diluted with coating solution (10 mM PBS, pH 7.4) to a concentration of 1.25 μg / mL, 100 μL per well, and incubated at 4°C overnight. Empty the liquid and pat dry the residual liquid, rinse three times with washing solution (PBST); add 200 μL of blocking solution (2% BSA) to each well and incubate at 37°C for 1-1.5 hours; empty the liquid and pat dry the residual liquid, rinse three times with washing solution (PBST); add 100 μL of primary antibody (i.e., immunized mouse serum to be tested) to each well and incubate at 37°C for 1-1.5 hours; empty the liquid and pat dry the residual liquid, rinse three times with washing solution (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 pat dry the residual liquid, rinse five times with washing solution (PBST); pat dry the residual liquid in the well, add 100 μL of TMB color development solution to each well, develop at 37°C for 5-10 minutes; add 50 μL of 2M H2SO4 to each well to stop color development, and immediately read the OD value at 450 nm.

[0192] The test results showed that the mouse serum titer reached above 1:32000. Mice with high titer were selected, and their spleen cells were taken for electrofusion with myeloma cells.

[0193] Electrofusion process:

[0194] Isolate mouse spleen cells and prepare a splenocyte suspension. Wash once with IMDM medium. Discard the supernatant and add 5 mL of red blood cell lysis buffer. Lyse at room temperature for 5 minutes. Add IMDM medium to 30 mL. Centrifuge at 1500 rpm for 5 minutes. Resuspend the splenocytes in fresh IMDM medium and count the cells.

[0195] Myeloma cells (FO) were collected, washed twice with IMDM medium, and the cells were counted.

[0196] Splenocytes and FO cells were mixed at a ratio of 2:1 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 and electrofusion buffer was added to adjust the concentration of splenocytes to 1-2×10 7 / mL, add the cell suspension to the cell electrofusion chamber, and start the electrofusion program on the electrofusion instrument. After the electrofusion is completed, let the fused cells stand for 5 minutes. Transfer the fused cells into the screening culture medium containing HAT, and transfer the cells to 20 culture plates covered with feeder cells (peritoneal macrophages), 200μL / well. Place the cell plate in a carbon dioxide incubator at 37℃ and 5% CO2 for culture. On the fifth day of culture, replace the HAT culture medium with HT culture medium. Perform antibody ELISA detection of the fused cells on the 8th day.

[0197] Many small molecule-specific clones were screened through fusion, and only 8 clones fused to 2335CT were selected for subcloning.

[0198] Subcloning process:

[0199] The first subcloning was performed using the gradient dilution method. Cell counting was performed using the red blood cell counting method. 300 cells were added to 10 mL of complete culture medium (IMDM culture medium containing 15% fetal bovine serum and 1% HT). 200 μL of cell suspension was added to each well of the 4 columns of a 96-well cell culture plate, with approximately 6 cells per well. 5 mL of complete culture medium was added to the remaining cell solution and mixed. 200 μL of cell suspension was added to each well of the other 4 columns of the 96-well cell culture plate, with approximately 2-3 cells per well. 5 mL of complete culture medium was added to the remaining cell solution and mixed. 200 μL of cell suspension was added to each well of the remaining 4 columns of the 96-well cell culture plate, with approximately 1 cell per well. The culture plate was placed in a carbon dioxide incubator at 37°C and 5% CO2 for culture.

[0200] The second subcloning was performed using the limiting dilution method. 100 cells were added to 20 mL of complete culture medium (IMDM medium containing 10% fetal bovine serum). 200 μL of the cell suspension was added to each well of a 96-well cell culture plate, resulting in approximately one cell per well. The plate was incubated at 37°C in a 5% CO2 incubator for 7 days. Specific positive clones were then screened by ELISA.

[0201] After two rounds of subcloning and ELISA screening, three clones specific for 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.

[0202] Subtype identification:

[0203] Using IsoStrip TM Mouse monoclonal antibody typing kit was prepared according to the instructions, and clone 2335CT81.1.1, IgG1, κ was obtained.

[0204] Antibody production: Cell culture: Cells (clone 2335CT81.1.1) were added to the cell culture flask at a cell density of 1-2×10 5 After 5 days, the culture medium was added to the maximum volume, and when the cell survival rate dropped to 20%, the cell supernatant was collected and purified to obtain the antibody SG201221AA.

[0205] Antibody testing:

[0206] 1) Specificity detection:

[0207] The specificity of the antibody SG201221AA was tested using ELISA, and three antigens were used for simultaneous testing: T-DXd, ADC-II-9, or ADC-III-9. The results are shown in Table 1 below.

[0208] Table 1

[0209] Conclusion: Antibody SG201221AA specifically binds to T-DXd, ADC-Ⅱ-9, or ADC-III-9. The titer of antibody SG201221AA is above 1:500,000.

[0210] 2) Competitive ELISA:

[0211] IC50 determination

[0212] Dilute the antigen ADC (T-DXd, ADC-II-9, or ADC-III-9) in coating buffer (10 mM PBS, pH 7.4) to a concentration of 1.25 μg / mL. 100 μL was added to each well and incubated overnight at 4°C. Pour the solution and tap off any remaining liquid. Rinse three times with wash buffer. Add 200 μL of blocking buffer (2% BSA-PBS) to each well and incubate at 37°C for 1 hour. Pour the solution and tap off any remaining liquid. Rinse three times with wash buffer. Add 100 μL of sample (a 1:1 volume ratio of antibody SG201221AA and a small molecule (DXd, P-II-3, or P-III-20)) to each well and incubate at 37°C for 1-1.5 hours. Pour the solution and tap off any remaining liquid. Rinse three times with wash 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 wells and pat dry. Rinse five times with washing solution. Pat dry any remaining liquid in the wells. Add 100 μL of color development solution to each well and develop at 37°C for 5-10 minutes. Stop development by adding 50 μL of 2M H₂SO₄ to each well and immediately read the OD value at 450 nm.

[0213] Experimental results:

[0214] A: P-II-3(P2C)

[0215] A1: ELISA experiment selects the optimal reaction concentration of antigen and antibody. The experimental results are shown in Table 2:

[0216] Table 2

[0217] Conclusion: The coating concentration of antigen ADC-Ⅱ-9 was 0.25 μg / mL and the detection concentration of antibody was 0.031 μg / mL for competition experiment with small molecule P-II-3.

[0218] A2: IC50 determination. The experimental results are shown in Table 3:

[0219] Table 3

[0220] According to the ELISA test results, the average of 6 replicate OD values ​​was taken to calculate IC50, and the results are shown in Figure 1. Experimental conclusion: The IC50 of P-II-3 (P2C) is 0.396 ng / mL.

[0221] B:P-III-20

[0222] B1: ELISA experiment to select the optimal reaction concentration of antigen and antibody. The experimental results are shown in Table 4:

[0223] Table 4

[0224] Conclusion: The coating concentration of antigen ADC-III-9 was 0.25 μg / mL and the detection concentration of antibody was 0.031 μg / mL for competition experiment with small molecule P-III-20.

[0225] B2: IC50 determination. The experimental results are shown in Table 5:

[0226] Table 5

[0227] Based on the ELISA test results, the average of 6 replicate OD values ​​was taken to calculate IC50, and the results are shown in Figure 2. Experimental conclusion: The IC50 of P-III-20 is 0.282 ng / mL.

[0228] C.DXd

[0229] C1: ELISA experiment to select the optimal reaction concentration of antigen and antibody. The experimental results are shown in Table 6:

[0230] Table 6

[0231] Conclusion: The coating concentration of antigen T-DXd was 0.25 μg / mL and the detection concentration of antibody was 0.063 μg / mL for competition experiment with small molecule DXd.

[0232] C2: IC50 determination, the experimental results are shown in Table 7

[0233] Table 7

[0234] According to the ELISA test results, the average of 6 repeated OD values ​​was taken to calculate IC50. The results are shown in Figure 3. The conclusion is: the IC50 of DXd is 2.693 ng / mL.

[0235] 4.3 Purification and hybridoma sequencing

[0236] Purity detection: After protein G affinity purification, reducing SDS-PAGE, and gel imaging analysis system analysis, the antibody purity of SG201221AA was 95.1%.

[0237] According to the technical manual of RNA-easy Isolation Reagent (Vazyme), total RNA was isolated from hybridoma cell 2335CT81.1.1. Then, according to the technical manual of SMARTScribe Reverse Transcriptase (TaKaRa), total RNA was reverse transcribed into cDNA using isotype-specific antisense primers or universal primers. VH and VL antibody fragments were amplified according to the standard operating procedure (SOP) of GenScript's rapid amplification of cDNA ends (RACE). The amplified antibody fragments were cloned into standard cloning vectors respectively. Colony PCR was performed to screen for clones with the correct size insert. For each fragment, no less than five colonies with the correct size insert were sequenced. The sequences of different clones were compared, and the consensus sequences of these clones were provided.

[0238] Table 8. VH and VL amino acid sequences of SG201221AA antibody

[0239] 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.

[0240] Table 9. Identified heavy and light chain CDR sequences of SG201221AA antibody (Kabat)

[0241] 4.4 Construction of mouse Fc monoclonal antibody

[0242] The heavy chain variable region of the resulting SG201221AA antibody was combined with the Fc region of murine IgG2A, and the light chain variable region was combined with the kappa light chain constant region of murine IgG2A. The recombinant antibody was expressed and purified. The heavy and light chain sequences of the recombinant antibody are as follows:

[0243] C544YHI220-1_2335CT-mIg2A-kappa

[0244] Heavy chain (SEQ ID NO: 13)

[0245] Light chain (SEQ ID NO: 14)

[0246] 4.5 Construction of human Fc monoclonal antibodies

[0247] The heavy chain variable region of the obtained SG201221AA antibody was combined with the Fc of human IgG1, and the light chain variable region was combined with the kappa light chain constant region of human IgG1, and the chimeric antibody was expressed and purified. The heavy and light chain sequences of the chimeric antibody are as follows:

[0248] Monoclonal Antibody-Human Fc

[0249] C8709HB100-1_C4773GL030 antibody

[0250] Heavy chain (SEQ ID NO: 15)

[0251] Light chain (SEQ ID NO: 16)

[0252] Example 5: Determination of serum concentrations in non-clinical studies

[0253] Using the mouse antibody SG201221AA obtained in Example 4, methods for measuring the concentration of antibody-drug conjugates in mouse and cynomolgus monkey serum, and methods for measuring the concentration of antibody-drug conjugates in mouse tumor tissue were developed.

[0254] Analysis method for HER2-ADC (ADC-II-9) concentration in serum:

[0255] 1)Payload (P-Ⅱ-3): LC-MS / MS

[0256] After the sample was thawed at room temperature, the internal standard was added, followed by 0.2% formic acid-50% methanol-acetonitrile. After vortex mixing, the sample was centrifuged at 18000g for 10 min at 4°C. The supernatant was transferred to an injection vial and placed in an injector for injection testing.

[0257] The standard curve concentrations were 0.1, 0.2, 0.5, 2, 5, 10, 40, and 50 ng / mL.

[0258] P-Ⅲ-20 and P-Ⅲ-30 can be determined by similar methods.

[0259] 2) Detection of HER2-ADC concentration in serum:

[0260] Serum: ELISA was used to detect the HER2-ADC concentration in mouse / cynomolgus monkey serum.

[0261] (1) Coating: Dilute the biotinylated SG201221AA antibody 1:500 with coating solution (0.05 M CBS) and add it to the SA enzyme-labeled plate (Streptavidin High Binding Capacity Coated 96-Well Plates) at 100 μL / well. Cover with the membrane and leave it at 37°C for overnight coating.

[0262] (2) Washing: Discard the coating solution, wash three times with washing solution, 300 μL / well, and pat dry.

[0263] (3) Blocking: Add blocking solution, 300 μL / well, cover with film, and incubate on a shaker at 15-25°C for 2 h ± 20 min.

[0264] (4) Washing: Wash three times with washing solution, 300 μL / well, and pat dry.

[0265] (5) Sample addition: According to the sample arrangement diagram in the Watson system, add NSB (1% mouse / cynomolgus macaque blank serum), standard curve sample solution, quality control sample solution, and test sample solution to the ELISA plate at 100 μL / well. Cover with film 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.

[0266] (6) Washing: Wash three times with washing solution, 300 μL / well, and pat dry.

[0267] (7) Add detection antibody: dilute goat anti-human IgG and monkey ads-HRP in sample diluent at a ratio of 1:10,000, add 100 μL / well, cover with film, and incubate on a shaker at 15-25°C for 1 h±5 min.

[0268] (8) Washing the plate: Wash three times with washing solution, 300 μL / well, and pat dry.

[0269] (9) Color development: Add TMB color development solution, 100 μL / well, cover with film, and incubate at 15-25°C in the dark for 3-20 min to develop color.

[0270] (10) Termination: Add stop solution, 100 μL / well, to terminate the reaction.

[0271] (11) Detection: Use an enzyme-labeled instrument to read the OD value of each well at a detection wavelength of 450 nm (reference wavelength of 630 nm).

[0272] Other ADCs, such as HER3-ADC, TROP2-ADC, B7H3-ADC, etc., can be determined using similar methods.

[0273] 3) ELISA test of total antibody concentration in serum:

[0274] (1) Coating: Dilute human HER2 protein (His tag) with coating solution (0.05 M CBS) to a final concentration of 1 μg / mL, add to the ELISA plate, 100 μL / well, cover with film, and incubate at 2-8°C for overnight coating.

[0275] (2) Washing: Discard the coating solution, wash three times with washing solution, 300 μL / well, and pat dry.

[0276] (3) Blocking: Add blocking solution, 300 μL / well, cover with film, and incubate on a shaker at 15-25°C for 2 h ± 20 min.

[0277] (4) Washing: Wash three times with washing solution, 300 μL / well, and pat dry.

[0278] (5) Sample addition: According to the sample arrangement diagram in the Watson system, add NSB (1% mouse / cynomolgus macaque blank serum), standard curve sample solution, quality control sample solution, and test sample solution to the ELISA plate at 100 μL / well. Cover with film 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.

[0279] (6) Washing: Wash three times with washing solution, 300 μL / well, and pat dry.

[0280] (7) Add detection antibodies: dilute Goat Anti-Human IgG and Monkey ads-HRP with sample diluent at a ratio of 1:10000, add 100 μL / well, cover with film, and incubate on a shaker at 15-25°C for 1 h±5 min.

[0281] (8) Washing the plate: Wash three times with washing solution, 300 μL / well, and pat dry.

[0282] (9) Color development: Add TMB color development solution, 100 μL / well, cover with film, and incubate at 15-25°C in the dark for 3-20 min to develop color.

[0283] (10) Termination: Add stop solution, 100 μL / well, to terminate the reaction.

[0284] (11) Detection: Use an enzyme-labeled instrument to read the OD value of each well at a detection wavelength of 450 nm (reference wavelength of 630 nm).

[0285] Other total antibodies, such as anti-HER3 antibodies, anti-TROP2 antibodies, anti-B7H3 antibodies, etc. can be measured using similar methods.

[0286] Example 6: PK study of human gastric cancer NCI-N87 subcutaneous transplant tumor model in nude mice

[0287] The effects of a single intravenous injection of ADC-II-9 on subcutaneous xenografts in NCI-N87 nude mice were investigated. Serum drug concentrations in tumor-bearing nude mice were measured to investigate drug PK. Human gastric cancer NCI-N87 cells were purchased from the American Type Culture Collection. Female BALB / c nude mice, 5-7 weeks old, were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0288] Nude mice were subcutaneously inoculated with NCI-N87 cells and the tumors were grown to 200-300 mm. 3 Mice were given a single intravenous injection of ADC-Ⅱ-9 1.5 and 5 mg / kg. Four mice were killed at 5 minutes, 4 hours, 24 hours, 48 ​​hours, 72 hours, and 168 hours after administration (four mice at each time point). Before killing, blood was collected from the retroorbital venous plexus of the mice, and the serum was separated. The serum drug concentration was detected using the mouse Fc monoclonal antibody SG201221AA.

[0289] The results are shown in Tables 10-12 below.

[0290] Table 10 Pharmacokinetic parameters of ADC-II-9 in serum of mice after intravenous injection of ADC-II-9

[0291] Table 11 Pharmacokinetic parameters of total antibody in serum of mice after intravenous injection of ADC-Ⅱ-9

[0292] Table 12 Pharmacokinetic parameters of P-II-3 in serum of mice after intravenous injection of ADC-II-9

[0293] Note: If there are less than 3 data points, no pharmacokinetic parameter calculation will be performed.

[0294] Conclusion: After a single dose of ADC-Ⅱ-9, the serum ADC and total anti-dose ratio increased, and the exposure of the two was similar, and significantly higher than that of serum P-Ⅱ-3.

[0295] Example 7: PK study in cynomolgus monkeys

[0296] Cynomolgus monkeys (3 monkeys / sex / group) received a single 30-minute intravenous infusion of ADC-II-9 at 1, 3, and 10 mg / kg. Serum and plasma samples were collected before dosing (before the start of the IV infusion) and at 0.0167 hours (immediately after the IV 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) after dosing. Serum samples were analyzed for ADC-II-9 and total antibody, and plasma samples were analyzed for free P-II-3.

[0297] The results are shown in Tables 13-15 below.

[0298] Table 13 Average pharmacokinetic parameters of P-II-3 after intravenous administration of ADC-II-9 in male and female cynomolgus monkeys (n=6, 3 females and 3 males)

[0299] ND: Not sure.

[0300] --:not applicable.

[0301] Table 14 Average pharmacokinetic parameters of ADC-II-9 after intravenous administration of ADC-II-9 to male and female cynomolgus monkeys (n=6, 3 females and 3 males)

[0302] Table 15 Average pharmacokinetic parameters of total antibody after intravenous administration of ADC-II-9 in male and female cynomolgus monkeys (n=6, 3 females and 3 males)

[0303] Note: The actual PK parameters are calculated based on the start of intravenous infusion.

[0304] Conclusion: After a single dose of ADC-Ⅱ-9, the serum ADC and total anti-ADC levels in cynomolgus monkeys increased at a similar dose ratio, with exposure levels similar to those in serum and significantly higher than those in serum P-Ⅱ-3. No significant gender differences were observed.

Claims

1. A recognition antibody D, wherein the recognition antibody D specifically binds to a compound having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof, R 1 C 3-6 Cycloalkylene or C 1-6 Alkylene, It means that the structure shown in Formula I is connected to other fragments in the compound through this site.

2. The recognition antibody D according to claim 1, characterized in that It meets one or both of the following conditions: (1)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; preferably, the C 1-6 The alkylene group is More preferably, for Best, for wherein the "1" position is connected to O and the "2" position is connected to a carbonyl group; and (2)R 1 In the C 3-6 The cycloalkylene group is cyclopropylene, cyclobutylene, cyclopentylene or cyclohexylene, preferably cyclobutylene, more preferably Best, for Further optimally, for Among them, the "1" position is connected to O, and the "2" position is connected to the carbonyl group.

3. The recognition antibody D according to claim 1 or 2, characterized in that: R 1 for Preferably, R 1 for wherein the "1" position is connected to O, and the "2" position 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.

4. The recognition antibody D according to any one of claims 1 to 3, characterized in that The compound comprising the structure shown in Formula I or a pharmaceutically acceptable salt thereof is a compound shown in Formula I-1 or a pharmaceutically acceptable salt thereof, a compound shown in Formula I-2 or a pharmaceutically acceptable salt thereof, or a compound shown in Formula I-3 or a pharmaceutically acceptable salt thereof, R 1 The definition as in any one of claims 1 to 3; G and G 1 is a connecting group; T is the ligand; n is a natural number or decimal number from 1 to 16.

5. The recognition antibody D according to claim 4, characterized in that It meets one or both of the following conditions: (1) The T is a ligand targeting the following targets: HER2, HER3, B7H3, B7H4, TROP2, GPC3, DLL3, Claudin 18.2, CD30, CD33, CD70 or EGFR; Preferably, the T is an anti-HER2 antibody or an anti-TROP2 antibody; More preferably, the anti-HER2 antibody is trastuzumab or a variant thereof; the anti-TROP2 antibody may be sacituzumab, M1, M2 or M3 or a variant thereof; and (2)n is a natural number or decimal from 1 to 9.

6. The recognition antibody D according to claim 4 or 5, characterized in that It meets one or more of the following conditions: (1) G is (2) G 1 for The "1" position is connected to T, and the "2" position is connected to O; (3) T is trastuzumab or certolizumab; and (4) n is a natural number or decimal from 3 to 9, such as 3.67 or 8.

05.

7. The recognition antibody D according to any one of claims 1 to 6, characterized in that The compound comprising the structure shown in Formula I is any of the following compounds: Wherein, T is a ligand, and n is a natural number or decimal from 1 to 16; Preferably, for for for n is a natural number or decimal from 1 to 16, such as 8.05; for n is a natural number or decimal from 1 to 16, for example, 3.67, for n is a natural number or a decimal number from 1 to 16, for example, 8.

05.

8. The recognition antibody D according to any one of claims 1 to 7, characterized in that: The recognition antibody D comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 comprises 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; Preferably, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8 and HCDR3 as shown in SEQ ID NO:9; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11 and LCDR3 as shown in SEQ ID NO:12; More preferably, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:5, 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 to SEQ ID NO:5, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:6, 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 to SEQ ID NO:6; Further more preferably, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

6.

9. The recognition antibody D according to any one of claims 1 to 8, characterized in that It meets one or both of the following conditions: (1) the recognition antibody D is selected from a mouse antibody, a humanized antibody and a chimeric antibody; and (2) The recognition antibody D is a full-length antibody, Fab, Fab', F(ab')2 or Fv. When the recognition antibody D is a full-length antibody, the recognition antibody D also includes 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; preferably, the recognition antibody D is a scFv.

10. The recognition antibody D according to claim 8 or 9, characterized in that It is the following option 1 or option 2: Scheme 1: The identification antibody D comprises a heavy chain and a light chain, wherein the heavy chain has 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; and the light chain has 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; Preferably, the recognition antibody D comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13; the amino acid sequence of the light chain is shown in SEQ ID NO: 14; Scheme 2: The identification 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 of the antibody 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; Preferably, the recognition antibody D comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO:15; the amino acid sequence of the light chain is shown in SEQ ID NO:

16.

11. An antibody that recognizes X, characterized in that The recognition antibody X comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 comprises 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; Preferably, the heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:7, HCDR2 with an amino acid sequence as shown in SEQ ID NO:8 and HCDR3 with an amino acid sequence as shown in SEQ ID NO:9; and the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:10, LCDR2 with an amino acid sequence as shown in SEQ ID NO:11 and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

12.

12. The recognition antibody X according to claim 11, characterized in that: the heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:5, 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 to SEQ ID NO:5, and the light chain variable region having an amino acid sequence as set forth in SEQ ID NO:6, 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 to SEQ ID NO:6; Preferably, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:

6.

13. The recognition antibody X according to claim 11 or 12, characterized in that It meets one or both of the following conditions: (1) the recognition antibody X is selected from a mouse antibody, a humanized antibody and a chimeric antibody; and (2) The recognition antibody X is a full-length antibody, Fab, Fab', F(ab')2 or Fv. When the recognition antibody X is a full-length antibody, the recognition antibody 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 mouse or human antibody or a variant thereof, and the light chain constant region is a κ chain or λ chain of a mouse or human antibody or a variant thereof; Preferably, the recognition antibody X is scFv.

14. The recognition antibody X according to claim 11, characterized in that It is either Scheme I or Scheme II below: Scheme I: The identification antibody X comprises a heavy chain and a light chain, wherein the heavy chain has 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; and the light chain has 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; Preferably, the recognition antibody X comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13; the amino acid sequence of the light chain is shown in SEQ ID NO: 14; Scheme II: The identification antibody X comprises a heavy chain and a light chain, wherein the heavy chain has 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; and the light chain has 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; Preferably, the recognition antibody X comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 15; the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:

16.

15. A nucleic acid, characterized in that The nucleic acid encodes the recognition antibody D according to any one of claims 1 to 10 or the recognition antibody X according to any one of claims 11 to 14.

16. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid of claim 15.

17. A transformant, characterized in that: The transformant comprises the nucleic acid according to claim 15 or the recombinant expression vector according to claim 16.

18. A method for in vitro detection of antibody-drug conjugates, characterized in that: The method comprises the step of contacting the recognition antibody D according to any one of claims 1 to 10 or the recognition antibody X according to any one of claims 11 to 14 with a sample to be tested containing an antibody-drug conjugate; Preferably, the antibody-drug conjugate is the compound shown in formula I-3 or a pharmaceutically acceptable salt thereof in the recognition antibody D according to any one of claims 4 to 7; Preferably, the sample to be tested is serum, such as mammalian serum.

19. A detection reagent, characterized in that: The detection reagent comprises the recognition antibody D according to any one of claims 1 to 10 or the recognition antibody X according to any one of claims 11 to 14, the nucleic acid according to claim 15, the recombinant expression vector according to claim 16, and / or the transformant according to claim 17.

20. A kit, characterized in that The kit comprises the recognition antibody D according to any one of claims 1 to 10 or the recognition antibody X according to any one of claims 11 to 14, the nucleic acid according to claim 15, the recombinant expression vector according to claim 16, the transformant according to claim 17, and / or the detection reagent according to claim 19.

21. A method for preparing a recognition antibody for an antibody-drug conjugate, characterized in that: The method comprises culturing the transformant according to claim 17, and obtaining the recognition antibody from the culture.

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