Antibody-drug conjugates, their preparation methods, and applications

Abstract

In the field of biopharmaceutical technology, we provide an antibody-drug conjugate obtained by conjugating an antibody with the compound shown in formula (I), a method for preparing the same, and its applications, which have a good inhibitory effect on the proliferation of solid tumors. [Formula 1] JPEG2026529123000061.jpg44170

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 2023110584395, filed on August 21, 2023, and this application refers to all of the aforementioned Chinese Patent Application.

[0002] This invention relates to the field of biopharmaceutical technology, and more specifically to antibody-drug conjugates, methods for preparing them, and their applications. [Background technology]

[0003] Antibody drug conjugates (ADCs) are effective, novel targeted therapies consisting of three parts: an antibody, a linker, and a toxin. The toxin (payload) is the crucial component that exerts the drug's effect, the antibody is the crucial component that exerts the targeting action, while the linker not only connects the two but also influences the ADC's stability, drug release mechanism, and antibody conjugation method. A desirable payload should not only kill target tumor cells but also non-target tumor cells (those without the target antigen), thus exhibiting a bystander effect without causing toxicity to normal cell tissue (those without the target antigen). The desirable linker must be stable in the circulatory system and, upon reaching the tumor microenvironment or tumor cells (endocytosis), release the payload to kill the tumor cells or cells in the tumor microenvironment.

[0004] Exatecan and its derivatives are effective DNA topoisomerase I inhibitors, and have already demonstrated very good efficacy as a payload in the field of HER2-positive tumor treatment. The development of more drug conjugates with antitumor activity using exatecan as a payload is of significant importance. [Overview of the project]

[0005] The present invention provides a low-toxicity antibody-drug conjugate formed by linking a stable linker and a TOP1 isomerase inhibitor, which has a good inhibitory effect on the growth of solid tumors.

[0006] In one aspect of the present invention, an antibody-drug conjugate represented by formula (I) is provided.

Chemical formula

[0007] In one embodiment, the antibody is obtained by conjugating the antibody with a compound of formula (I-1) (payload-linker), [ka] In the formula, L 1 , L 2 , R 2 , L p The definitions of each are the same as those of the compound in formula (I).

[0008] Z' is a linker group that is compatible with Z and can be conjugated to an antibody.

[0009] In one embodiment, Z' is [ka] (maleimide group) [ka] Selected from, where R j The halogen is selected from halogens, preferably bromine or iodine.

[0010] In one embodiment, Z' is selected from maleimide, a bromoacetyl group, or an iodoacetyl group.

[0011] In one embodiment, as shown in formula (I-2), the linking site between the antibody and the payload-linker is the mercapto group of cysteine ​​after the disulfide bond between the antibody chains has been reduced and cleaved.

[0012] [ka] Here, [ka] This represents an antibody.

[0013] In one embodiment, the linkage between the payload-linker and antibody cysteine ​​is achieved by either the substitution of the bromine atom in the bromoacetyl linker group with a mercapto group between antibody chains, or by Michael addition conjugation with the linker group of the maleimide structure.

[0014] In one embodiment, R 2 This is selected from hydrogen, deuterium, C1-C3 alkyl groups, C1-C3 alkoxy groups, -C(=O)C1-C3 alkyl groups, or -S(=O)2C1-C3 alkyl groups. Preferably, R 2 This is selected from hydrogen, deuterium, methyl group, ethyl group, methoxy group, ethoxy group, formyl group, acetyl group, methanesulfonyl group, or ethanesulfonyl group. Preferably, R 2 This is selected from hydrogen, a methyl group, a methoxy group, a formyl group, or a methanesulfonyl group.

[0015] In one embodiment, L 11 , L 12 , L 13 Each of these is independently selected from non-existent ones, -C=O-, -CH2-, -CH2O-, or -OCH2-. Preferably, L 1 This is selected from -C(=O)CH2OCH2-, -C(=O)CH2O-, -C(=O)CH2, or -CH2-. Preferably, L 2 The group is selected from C1-C3 alkylene groups. Preferably, L 2 The group is selected from a methylene group and an ethylene group.

[0016] In one embodiment, L 1If R is -C(=O)CH2O-, 2 The group is selected from C1-C6 alkyl groups, preferably C1-C3 alkyl groups, and more preferably a methyl group.

[0017] In one embodiment, L 1 If R is -C(=O)CH2-, 2 The group is selected from C1 to C6 alkoxy groups, preferably a -C1 to C3 alkoxy group, and more preferably a methoxy group.

[0018] In one embodiment, L p It is selected from peptide residues consisting of 1, 2, 3, or 4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), or leucine (Leu). Preferably, L p -Val-Cit-, -Gly-Lys-, -Gly-Leu-, -Val-Ala-, -Gly-Phe-, -GLy-Gly-Lys-, -Gly-Gly-Phe-, -Gly-Val-Ala-, -Gly-Ala-, -Gly-Gly-Val-, -Gly-Leu-Val-, -Gly-Phe-Gly- or -Gly-Gly-Leu-, Preferably, L p teeth, [ka] Selected from.

[0019] In one embodiment, L j teeth, [ka] Selected from, [ka] The position indicated by the arrow represents the point where the antibody is bound. [ka] The position indicated by is L z It indicates that it is connected to the base, Preferably, L z These are -C(=O)-C1~C8 alkylene groups and -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=O)-(CH2) 1-4 -NR 1 (CH2)2- is selected, Preferably, L z is -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=O)-(CH2) 1-4 -NR 1 (CH2)2- is selected, Preferably, R 1 It is selected from C1-C3 alkyl-carboxyl groups, Preferably, L z is -C(=O)-(CH2CH2O)2-CH2CH2NH- or [ka] Selected from, Preferably, Z is [ka] Selected from.

[0020] In one embodiment, the antibody-drug conjugate is obtained by conjugating a compound of formula (II) or formula (III) to an antibody, [ka] Here, R 2 , L P The definitions of each are the same as those of the compound in formula (I), where Z' is a linker group that can be conjugated to an antibody and is compatible with Z, preferably a maleimide, bromoacetyl, or iodoacetyl group. Preferably, the antibody-drug conjugate is subjected to physiological conditions. [ka] It releases an antitumor compound, Here, R 21 The group is selected from C1-C6 alkyl groups, preferably C1-C3 alkyl groups, and more preferably a methyl group.

[0021] R 22 The group is selected from C1 to C6 alkoxy groups, preferably a -C1 to C3 alkoxy group, and more preferably a methoxy group.

[0022] In one embodiment, the physiological environment is under the action of a protease.

[0023] Preferably, [ka] It releases an antitumor compound.

[0024] In one embodiment, the antibody is an antibody against a tumor-associated antigen, Preferably, the tumor-associated antigen is one or more selected from Her2, Nectin-4, Trop2, 5T4, B7H3, ROR1, or Claudin18.2, and the antibody may be a monospecific or multispecific antibody.

[0025] Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, or liver cancer. Preferably, the antibody is HS627 antibody, IP140B antibody, or Nectin-4 antibody.

[0026] Preferably, the antibody comprises a heavy chain of amino acid sequence SEQ ID NO:1 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO:2 or any variant thereof. Preferably, the antibody comprises a heavy chain of amino acid sequence SEQ ID NO:3 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO:4 or any variant thereof. Preferably, the antibody comprises a heavy chain of amino acid sequence SEQ ID NO:5 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO:6 or any variant thereof.

[0027] In one embodiment, the drug-antibody ratio of the antibody-drug conjugate is 2 to 8, more preferably 3.5 to 4.5 or 7.5 to 8.

[0028] In one embodiment, the antibody may be IgG1, IgG2, IgG3, or IgG4, and preferably IgG1.

[0029] In one embodiment, the antibody-drug conjugate is [ka] This compound is obtained by conjugating an antibody with it.

[0030] The present invention [ka] This provides an antibody-drug conjugate.

[0031] Here, n is the drug-antibody ratio of the antibody-drug conjugate, and is preferably 8 ± 0.5.

[0032] Another aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0033] Another aspect of the present invention provides the use of the antibody-drug conjugate or the pharmaceutical composition in the preparation of antitumor drugs.

[0034] Another aspect of the present invention provides a method for treating a tumor disease, which includes the step of administering a therapeutically effective amount of the antibody-drug conjugate or the pharmaceutical composition to a patient in need thereof.

[0035] Preferably, the tumor is a solid tumor.

[0036] Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, or liver cancer. [Brief explanation of the drawing]

[0037] [Figure 1] The DAR test results for ADC1 in Example 9 are shown. [Figure 2] The DAR test results for ADC2 in Example 9 are shown. [Figure 3] The DAR test results for ADC3 in Example 10 are shown. [Figure 4] The DAR test results for ADC4 in Example 11 are shown. [Figure 5] The DAR test results for ADC5 in Example 12 are shown. [Figure 6] The DAR test results for ADC6 in Example 13 are shown. [Figure 7] The DAR test results for ADC7 in Example 14 are shown. [Figure 8] The tumor growth curve diagram for the BXPC-3 model in Test Example 1 is shown. [Figure 9] The image shows a tumor photograph taken after dissection of the BXPC-3 model in Test Example 1. [Figure 10] The NCI-H1975 model tumor growth curve diagram for Test Example 1 is shown. [Figure 11] The image shows a tumor photograph after dissection of the NCI-H1975 model in Test Example 1. [Figure 12] The tumor growth curve diagram for the MDA-MB-231 model in Test Example 1 is shown. [Figure 13]The image shows a tumor photograph taken after dissection of the MDA-MB-231 model in Test Example 1. [Figure 14] The tumor growth curve diagram for the KYSE-150 model in Test Example 1 is shown. [Figure 15] The image shows a tumor photograph taken after dissection of the KYSE-150 model in Test Example 1. [Figure 16] The drug blood concentration-time curves for ADC5-TAB individuals in cynomolgus monkeys after intravenous injection of 22 and 35 mg / kg of ADC5 at the 1st, 3rd, and 4th doses (Days 1, 38, and 52) in Test Example 2 are shown. [Figure 17] The drug blood concentration-time curves for individual toxin molecules in plasma of cynomolgus monkeys that received intravenous injections of ADC5@35 mg / kg on the third (Day 38, left) and fourth (Day 52) doses in Test Example 2 are shown. [Figure 18] The drug-time curves for ADC5 (ADC drug) and ADC5-TAB (total antibody) in serum after intravenous injection of ADC5 into cynomolgus monkeys in Test Example 2 are shown. [Figure 19] The drug-time curve of serum payload (free toxin molecules) after intravenous injection of ADC5 into cynomolgus monkeys in Test Example 2 is shown. [Modes for carrying out the invention]

[0038] I. Definition In this invention, unless otherwise specified, the scientific and technical terms used herein have meanings that are generally understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are all widely used terms and common procedures in the relevant fields. In addition, to better understand this invention, definitions and interpretations of related terms are provided below.

[0039] As used herein, unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means rounded up or down to the nearest integer within 10% of a given value or range.

[0040] In this specification, any mention of “some examples,” “some embodiments,” or “some embodiments” refers to a subset of all possible examples, but it should be understood that “some examples” may be the same subset or different subsets of all possible examples, and may be combined with each other in a non-contradictory manner.

[0041] As used herein, unless otherwise stated, the terms “include,” “incorporate,” “have,” and “contain” should generally be understood to be open and non-restrictive, including their grammatical equivalents, and not to exclude, for example, other unlisted elements or steps.

[0042] As used herein, the term “alkyl group” means a straight-chain or branched-chain saturated aliphatic hydrocarbon group which contains 1 to 20 carbon atoms. 1~6 "Alkyl alkyl group" means a linear or branched alkyl group consisting of 1 to 6 carbon atoms, more preferably C 1~4 It is an alkyl group, most preferably C 1~3These are alkyl groups. Specific examples of alkyl groups include, but are not limited to, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, and various branched isomers thereof.

[0043] The term "alkoxy group" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where the definition of alkyl is as described above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy groups. Alkoxy groups may be optionally substituted or unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydrogen, nitro, chlorine, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester groups.

[0044] [ka] This refers to the linking site of a chemical bond. Note that the structural fragment described in the present invention (e.g., -L) 11 -L 12 -L 13 - or -L z -L j-) unless otherwise specified, represents a combination of the corresponding bases in left-to-right order, for example, -L 12 If - is a C1-C2 alkylene-O-, then the left side of the C1-C2 alkylene-O- is -L. 11 - is connected to -L at the right end. 13 This indicates that it is linked to -.

[0045] In the antibody-drug conjugate of this application, for example, in formula (I), the "-" between the drug-linker fragment and the antibody is intended to represent the linkage relationship between the antibody and the fragment, and is not intended to limit the conjugate to one antibody linked to one drug-linker fragment. As is well known in the art, since a single antibody has multiple interchain disulfide bonds, a single antibody can be linked to one or more drugs.

[0046] As used herein, the term “substituted” means that any one or more hydrogen atoms on a particular atom are substituted with a substituent, and may include deuterium and hydrogen variants, provided that the specific valence is normal and the substituted compound is stable. When the substituent is an oxo group (i.e., =O), it means that two hydrogen atoms are substituted. Oxo substitution does not occur in aromatic groups. The term “optionally substituted” or “optionally substituted” means that substitution may or may not occur, and unless otherwise specified, the type and number of substituents may be any, provided that they are chemically feasible.

[0047] Although the term "arbitrarily substituted" is not explicitly used, it should be understood that when substitutions occur at any position on a ring (e.g., an antilipid ring or an aromatic ring), the position and number of substitutions can be arbitrary, provided they are chemically feasible.

[0048] As used herein, the term “antibody” is used in its broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments or synthetic polypeptides having one or more CDRs or derived from CDR sequences, insofar as they exhibit the required biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. “Antibody” may also mean immunoglobulins and immunoglobulin fragments, and includes any fragments that retain the binding specificity of full-length immunoglobulins, whether naturally occurring or partially or completely synthetically produced (e.g., by recombination), containing at least a portion of the variable region of an immunoglobulin molecule. Thus, an antibody includes any protein having a binding domain homologous or substantially homologous to the immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments, e.g., antitumor stem cell antibody fragments. As used herein, the term antibody therefore includes, but is not limited to, synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fv(dsFv) linked via disulfide bonds, Fd fragments, Fd' fragments, single-chain Fv(scFv), single-chain Fab(scFab), diabodies, anti-idiotype (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. Antibodies according to this specification include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b) (the terms "type" and "class," and "subtype" and "subclass" are interchangeable in this specification). Natural or wild-type (i.e., obtained from population members that have not been artificially manipulated) antibodies and immunoglobulins are generally heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H).Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end, followed by a constant domain at the other end. "Non-manipulated" means a treatment that is not intended to contain or express a foreign antigen-binding molecule. The wild type may mean the most common allele or type found in a population, or an antibody obtained from an unmanipulated animal compared to the allele or polymorphism, or a variant or derivative obtained from any form of manipulation, such as mutation, recombination, or modification of the amino acids of the antigen-binding molecule.

[0049] As used herein, the term “drug-antibody ratio (DAR)” refers to the average number of payload molecules (antitumor compounds or drugs) attached to a single monoclonal antibody.

[0050] As used herein, the term “pharmaceutically acceptable excipient” means a non-toxic, inert solid, semi-solid substance or liquid filler, diluent, packaging material or auxiliary formulation or any type of excipient that is compatible with patients, most preferably mammals, more preferably humans, and suitable for delivery to target substances without impairing the activity of the active reagent. The drug carriers and excipients of the present invention can better conform to the active ingredient, and the resulting pharmaceutical formulation can have a elution rate, storage stability and impurity content that meets the standards.

[0051] Typically, the antibody-drug conjugate of the present invention can be administered in a suitable dosage form with one or more medicinal carriers. These dosage forms are applicable to oral, rectal, topical, orally, and other parenteral administration (e.g., subcutaneous, intramuscular, intravenous, etc.). For example, other dosage forms suitable for parenteral administration include injectable preparations. The above dosage forms can be prepared from the active ingredient of the present invention and one or more carriers or excipients by general pharmaceutical methods. The above carriers must be compatible with the active ingredient of the present invention or other excipients.

[0052] In embodiments of the present invention, the pharmaceutical composition may include any method of administration, such as oral administration, spray inhalation, rectal administration, intranasal administration, transbuccal administration, topical administration, parenteral administration, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or administration using an implantable reservoir. In the case of oral administration, the compound of the present invention may be in any orally acceptable formulation form, including but not limited to tablets, capsules, aqueous solutions, or aqueous suspensions. The carrier used for tablets generally contains lactose and corn starch, and a lubricant, such as magnesium stearate, may also be added. The diluent used for capsule formulations generally contains lactose and dried corn starch. Aqueous suspension formulations are usually used by mixing the active ingredient with a suitable emulsifier and suspending agent. Sweeteners, flavorings, or colorings may be further added to the above oral formulations if necessary. The compound of the present invention may further be administered in the form of a sterile injection formulation, which includes sterile injection water or oil suspension or sterile injection solution. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Sterilized non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media.

[0053] The pharmaceutical compositions of the present invention are prepared, quantified, and administered in accordance with medical practice standards. The "therapeutic effective dose" of the active ingredient of the present invention is determined by factors such as the specific disease being treated, the individual being treated, the cause of the disease, the target of the drug, and the method of administration.

[0054] As used herein, “therapeutic effective dose” means an amount that can produce function or activity in a patient (e.g., human and / or animal) and is tolerable to humans and / or animals.

[0055] As used herein, “patient” means an animal, most preferably a mammal, and more preferably a human.

[0056] As used herein, “treatment” means reducing, slowing the progression of, attenuating, preventing, or maintaining an existing disease or condition (e.g., cancer). “Treatment” further includes curing, preventing the progression of, or reducing to some extent one or more symptoms of a disease or condition.

[0057] II. Examples To further clarify the object, technical proposal and advantages of the present invention, the present invention is described in more detail below, and the embodiments described therein should not be considered limiting to the present invention, and all other embodiments obtained without the creative effort of a person skilled in the art are all within the scope of the protection of the present invention.

[0058] Before further detailing the embodiments of the present invention, the nouns and terms relating to the embodiments of the present invention will be explained, and these nouns and terms relating to the embodiments of the present invention will be used in the following interpretation.

[0059] All raw materials and equipment used in the specific embodiments of this disclosure are known products and can be obtained by purchasing commercially available products.

[0060] Abbreviation: Fmoc: 9-Fluorenylmethoxycarbonyl, DCM: Dichloromethane, DMF: N,N-Dimethylformamide, THF: Tetrahydrofuran, HOBT: 1-Hydroxybenzotriazole, EDCI: 1-Ethyl-(3-Dimethylaminopropyl)carbodiimide hydrochloride, DIPEA: Diisopropylethylamine, TFA: Trifluoroacetic acid, HATU: 2-(7-Azabenzotriazole)-N,N,N',N'-Tetramethyluronium hexafluorophosphate, DIC: N,N'-Diisopropylcarbodiimide, NHS: N-Hydroxysuccinimide, DBU: 1,8-Disazobisspiro[5.4.0]undeca-7-ene, DMA: N,N-Dimethylaniline. Val: Valine, its structural formula is, [ka] And Ala: Alanine, its structural formula is, [ka] Gly: Glycine, its structural formula is [ka] And Phe: phenylalanine, its structural formula is, [ka] And Leu: Leucine, its structural formula is, [ka] The peptide residue L is the one mentioned above. P This can be obtained by amino acid condensation methods known in the art, but the present invention does not particularly limit this.

[0061] In this invention, an antibody and a linker L for binding to the antibody are provided. j This can be obtained by concatenating in a manner known in this field. For example, L j Structure [ka] (or [ka] ) If this is the case, then this is [ka] (or [ka] This can be obtained by the reaction and linkage of a reactive group such as a mercapto group on an antibody, but the present invention is not particularly limited thereto.

[0062] The IgG1 monoclonal antibody (Human IgG1, kappa isotype Control) used in Test Example 1 was purchased from Sino-Biological, and its catalog number is HG1K.

[0063] In this example, HS627, IP140B antibody, and Nectin-4 antibody are used to prepare the ADC, but are not limited to these. The heavy chain amino acid sequence of the HS627 antibody is as follows (SEQ ID NO:1): EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNHYTQKSLSLSPG The light chain amino acid sequence is as follows (SEQ ID NO:2): DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO:3): QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREMQFGWELLGAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The light chain amino acid sequence is as follows (SEQ ID NO:4): EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFGQGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The heavy chain amino acid sequence of the Nectin-4 antibody is as follows (SEQ ID NO: 5): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The light chain amino acid sequence is as follows (SEQ ID NO:6): DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0064] DAR value testing and calculation: Based on RP-HPLC-MS test results, the DAR value of the ADC was analyzed using a Waters Acquity UPLC I-Class / Xevo G2-XS QTOF instrument.

[0065] RP-HPLC parameter settings: Chromatography column PLRP-S 1000A 5μm, column temperature 70°C. Mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, flow rate 0.2 mL / min. Mobile phase gradients are 20-50%B for 18 minutes, 50-95%B for 5 minutes, 95-20%B for 0.1 minutes, and 20-20%B for 6.9 minutes.

[0066] MS parameter settings: capillary voltage 2.50kV, cone voltage 100V, mass spectrometry range m / z: 200~4000, MSE collision energy 20~45 eV, ion source temperature 120℃, nebulize temperature 500℃, nebulize flow rate 1000 L / Hr, internal standard leucine enkephalin. The sample buffer for the test was diluted to 1 mg / mL, TCEP with a final concentration of 50 mmol / L was added, incubated at 37℃ for 20 min, and 5 μL of sample was added. Light chain peaks were identified, and the peak area percentage was calculated; the sum of peak areas was 100. Similarly, heavy chain peaks were identified, and the peak area percentage was calculated; the sum of peak areas was 100. The weighted peak areas of the heavy and light chains were calculated by multiplying the peak area percentage by the corresponding drug load. The formula for calculating the DAR value is DAR = 2 * (Σ light chain weighted peak area + Σ heavy chain weighted peak area) / 100.

[0067] Example 1: Synthesis of 2-(((tert-butoxycarbonyl)(methyl)amino)oxy)acetic acid (2) [ka] Step 1: In a 300 mL three-necked flask, N-methylhydroxylamine hydrochloride (20 g, 239.5 mmol), water (60 mL), and THF (60 mL) were added sequentially. The mixture was cooled to 0°C under an ice bath, sodium bicarbonate (20.1 g, 239.3 mmol) was added, and di-tert-butyl dicarbonate (47.0 g, 215.3 mmol) was slowly added dropwise. The mixture was stirred at 0°C for 1 hour, and the reaction was allowed to proceed at room temperature for 3 hours. The mixture was extracted with ethyl acetate (2 × 50 mL), the organic phases were combined, washed with saturated saline (50 mL), washed with 0.5 N dilute hydrochloric acid (30 mL), washed again with saturated saline (50 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain pale yellow oily liquid compound 1 (33 g, yield 93.7%, HPLC purity 98%). 1 H NMR (500MHz, DMSO-d6) δ 9.25 (s, 1H), 3.00 (s, 3H), 1.40 (s, 9H).

[0068] Step 2: Under ice bath cooling, add 100 mL of 1.5 N NaOH aqueous solution and compound 1 (10 g, 67.9 mmol) to a 300 mL three-necked flask. While stirring, add 50 mL of bromoacetic acid (9.4 g, 67.7 mmol) aqueous solution, stir at 0°C for 0.5 hours, extract with ethyl acetate (2 × 50 mL), adjust the pH of the aqueous phase to 2 with 1 N HCl, extract with dichloromethane (3 × 50 mL), wash with saturated saline solution, dry with anhydrous MgSO4, filter, concentrate to obtain pale yellow oily liquid compound 2 (12 g, yield 86.1%, HPLC purity 95%). 1 H NMR(500MHz,DMSO-d6)δ 12.42(s,1H),4.37(s,2H),3.09(s,3H),1.43(d,J=5.8Hz,9H), LCMS:[M-1] + 204.12 (Theoretical value: 205.10).

[0069] Example 2: Synthesis of (S)-N-(chloromethyl)-2-(1,3-dioxoisoindorin-2-yl)propionamide (4) [ka] Step 1: Add phthaloylalanine (5 g, 22.8 mmol), DCM (175 mL), and DMF (100 μL) to a 500 mL single-necked flask, cool to 0°C in an ice bath, add oxalyl chloride (5.79 g, 45.6 mmol) dropwise under argon gas protection, stir at 0°C for 1.5 hours, concentrate, dissolve the crude product in DCM (90 mL), cool to 0°C, add 7 M ammonia-methanol solution (9.78 mL, 69.1 mmol) dropwise, stir at room temperature for 2 hours to react, add n-hexane, filter, and dry to obtain compound 3 (4.5 g, yield 92%, HPLC 97%). 1 H NMR(600MHz,DMSO-d6)δ 7.84(d,J=4.9Hz,3H),7.63(s,1H),7.45(s,1H),7.19(d,J=10.6Hz,1H),4.69(q,J=7.2Hz,1H),1.56(d,J=7.3Hz,3H),LCMS:[M+1] + 219.05 (Theoretical value: 218.07).

[0070] Step 2: Compound 3 (1 g, 4.5 mmol) was dissolved in DCM (10 mL), TMS-Cl (10 mL) and paraformaldehyde (550.4 mg, 18 mmol) were added, the tube was sealed, the temperature was raised to 40°C, and the reaction was allowed to proceed for 1.5 hours. After cooling, the mixture was filtered and concentrated to obtain Compound 4 (1.22 g, 100% yield, 96% HPLC purity). 1 H NMR(500MHz,CDCl3)δ 7.85(dd,J=5.4,3.1Hz,2H),7.78-7.72(m,2H),7.19(s,1H),5.23-5.13(m,2H),5.03-4.74(m,2H),1.69(d,J=7.3Hz,3H),LCMS:[M+1] + 266.98 (Theoretical value: 266.05).

[0071] Example 3: Synthesis of Fmoc-PEG2-Gly-Val(8) [ka] Step 1: Add 500 mL of dichloromethane to a 1000 mL single-necked flask and, while stirring, sequentially add Fmoc-NH-PEG2-CH2CH2-COOH (30 g, 75 mmol), HOBT (12.15 g, 90 mmol), EDCI (17.25 g, 90 mmol), glycine tert-butyl (10.32 g, 78.75 mmol), and DIPEA (10.16 g, 90 mmol). React while stirring at room temperature for 2 hours, wash with saturated saline, saturated sodium bicarbonate aqueous solution, and 0.5 M hydrochloric acid, dry over anhydrous magnesium sulfate, filter, concentrate to obtain compound 5 (36.2 g, yield 94%, HPLC 98%). 1 H NMR(600MHz,CDCl3)δ 7.78-7.67(m,2H),7.60(d,J=7.3Hz,2H),7.41-7.34(m,2H),7.34-7.24(m,2H),6.78(s,1H),5.60(s,1H) ),4.45-4.15(m,2H),3.90-4.00(m,2H),3.80-3.33(m,10H),2.90-2.42(m,3H),1.44(s,9H),LCMS:[M+1] + 513.14 (Theoretical value: 512.25).

[0072] Step 2: Add dichloromethane (400 mL) and compound 5 (36.2 g, 70 mmol) to a 1000 mL single-necked flask, stir homogeneously, then add trifluoroacetic acid (100 mL) dropwise, stir overnight at room temperature to react, concentrate to obtain compound 6 (32.2 g, 100% yield, HPLC 97%). 1 H NMR(600MHz,CDCl3)δ 8.88(s,1H),7.72(d,J=7.4Hz,2H),7.60-7.50(m,2H),7.40-7.33(m,2H),7.30-7.20(m,2H),7.19(s,1H),5.68(s,1H),7.45-7.33(m ,2H),4.45-4.35(m,2H),4.25-4.15(m,2H),4.00(d,J=4.8Hz,1H),3.69(t,J=5.7Hz,2H),3.60-3.20(m,8H),2.50(m,2H),LCMS:[M+1] + 457.08 (Theoretical value: 456.19).

[0073] Step 3: Add 300 mL of dichloromethane to a 1000 mL single-necked flask and, while stirring, sequentially add compound 6 (5 g, 10.87 mmol), HOBT (1.76 g, 13.04 mmol), EDCI (2.5 g, 13.04 mmol), L-valine tert-butyl hydrochloride (1.91 g, 11 mmol), and DIPEA (2.84 g, 22 mmol). Stir at room temperature for 2 hours, wash with saturated saline, saturated sodium bicarbonate aqueous solution, and 0.5 M hydrochloric acid, dry over anhydrous magnesium sulfate, filter, concentrate to obtain compound 7 (5.5 g, yield 82%, HPLC 97%). 1 1H NMR (600MHz, CDCl3)δ 7.80-7.70(m,2H),7.61(d,J=7.4Hz,2H),7.41-7.33(m,2H),7.31-7.25(m,2H ),6.97(d,J=8.6Hz,1H),5.98(t,J=5.4Hz,1H),4.50-4.36(m,3H),4.21(t,J=7 .0Hz,1H),4.05-3.95(m,2H),3.75-3.65(m,1H),3.64-3.50(m,6H),3.45-3.30 (m,2H),2.51(s,1H),2.20-2.10(m,1H),1.43(s,9H),0.90(m,6H),LCMS:[M+1] + 612.05 (Theoretical value: 611.32).

[0074] Step 4: Add dichloromethane (50 ml) and compound 7 (5.5 g, 9 mmol) to a 1000 mL single-necked flask. While stirring, add trifluoroacetic acid (10 mL) dropwise, continue stirring overnight, and concentrate to obtain compound 8 (4.99 g, 100% yield, HPLC 96%). 11H NMR (600 MHz, CDCl3) δ 9.41 (s, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.64 - 7.53 (m, 3H), 7.36 (q, J = 7.7 Hz, 2H), 7.28 (q, J = 7.4 Hz, 2H), 5.90 - 5.84 (s, 1H), 5.27 (s, 0H), 4.52 - 4.33 (m, 3H), 4.25 - 4.13 (m, 1H), 4.14 - 4.09 (m, 1H), 4.07 - 4.01 (m, 1H), 3.97 (m, 1H), 3.70 (s, 2H), 3.57 (m, 3H), 3.52 (t, J = 5.3 Hz, 2H), 3.41 - 3.23 (m, 2H), 3.22 (s, 1H), 2.51 (q, J = 10.5, 5.5 Hz, 2H), 2.17 (dt, J = 13.4, 6.6 Hz, 1H), 0.91 (dd, J = 18.1, 6.8 Hz, 5H), LCMS: [M + 1] + 556.14 (theoretical value: 555.26).

[0075] Example 4: Synthesis of N-(bromoacetyl-PEG2-Gly-Val-Ala-ha-N(Me)-aminooxyacetyl) exatecan (15)

Chemical formula

[0076] Step 2: Add DCM (100 mL) and compound 9 (10.5 g, 16.9 mmol) to a 250 mL three-necked flask, stir, cool to 0°C, add TFA (25 mL) dropwise, stir at room temperature for 3 hours to react, concentrate, add ethyl acetate / petroleum ether (1:1), filter, dry to obtain compound 10 (10 g, yield 93%, HPLC 87%). 1 H NMR(600MHz,DMSO-d6)δ 8.87(d,J=8.6Hz,1H),7.78(d,J=10.8Hz,1H),7.30(s,1H),6.42(s,0H),5.75(s,1H),5 .60(dt,J=8.7,4.4Hz,1H),5.42(s,2H),5.28(s,2H),4.68-4.58(m,2H),3.18(p,J=6.3 ,5.4Hz,2H),2.84(s,3H),2.39(d,J=1.8Hz,3H),2.24(m,J=14.0,4.8Hz,1H),2.15(m,J =10.4,7.4,4.4Hz,1H),1.85(m,J=21.4,7.3Hz,2H),0.86(t,J=7.3Hz,3H),LCMS:[M+1] + 523.38 (Theoretical value: 522.19).

[0077] Step 3: Add compound 10 (6 g, 9.4 mmol), DMF (30 mL), and compound 4 (3 g, 11.2 mmol) to a 100 mL three-necked flask, stir at 0-5°C, add DIPEA (3.7 g, 28.6 mmol) dropwise, stir at room temperature for 1.5 hours to react, add methyl tert-butyl ether (300 mL), remove the supernatant, dissolve the black solid in 50 mL of DCM, add 15 g of silica gel and mix, purify by silica gel column chromatography to obtain compound 11 (6.2 g, yield 87.4%, HPLC 92%). 1 H NMR(500MHz,DMSO-d6)δ 8.72-8.66(m,1H),8.49(d,J=8.8Hz,1H),7.79-7.71(m,1H),7.70(s,3H),7.28(s,1H),6.51(s,1H) ),5.39(d,J=17.2Hz,3H),5.23-5.19(s,1H),5.00(s,1H),4.68(q,J=7.3Hz,1H),4.18(s,3H),4.1 2-4.01(m,1H),3.18(d,J=17.1Hz,1H),3.07(d,J=17.6Hz,1H),2.37(s,3H),2.00-1.92(m,2H),1. 87-1.80(m,2H),1.29(m,J=7.2Hz,3H),1.25-1.05(m,1H),0.84(q,J=13.5,7.5Hz,3H),LCMS:[M+1] + 753.12 (Theoretical value: 752.26).

[0078] Step 4: Compound 11 (0.1 g, 0.13 mmol) was dissolved in MeOH (5 mL), hydrazine hydrate (39.9 mg, 0.79 mmol) was added, the tube was sealed, the temperature was raised to 60°C, the reaction was allowed to proceed for 2 hours, the temperature was cooled, the reaction mixture was poured into saturated saline (100 mL), extracted with ethyl acetate, and concentrated to obtain compound 12 (0.07 g, yield 77%, HPLC 95%). 11H NMR (500 MHz, DMSO-d6) δ 8.91 (t, J = 6.0 Hz, 1H), 8.58 (d, J = 8.9 Hz, 1H), 8.17 (s, 3H), 7.66 (d, J = 10.8 Hz, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 5.58 (td, J = 8.3, 7.9, 4.8 Hz, 1H), 5.39 (s, 2H), 5.19 (d, J = 19.0 Hz, 1H), 5.01 (d, J = 18.9 Hz, 1H), 4.35 (dd, J = 13.2, 6.3 Hz, 1H), 4.25 (dd, J = 12.1, 4.7 Hz, 1H), 4.20 (d, J = 2.3 Hz, 2H), 3.97 - 3.88 (m, 1H), 3.18 (dt, J = 17.1, 5.9 Hz, 1H), 3.13 - 3.03 (m, 1H), 2.53 (s, 3H), 2.31 (d, J = 1.9 Hz, 3H), 2.26 - 2.09 (m, 2H), 1.93 - 1.77 (m, J = 7.2 Hz, 2H), 1.37 (d, J = 6.9 Hz, 3H), 0.86 (t, J = 7.3 Hz, 3H). LCMS: [M+1] + 623.15 (theoretical value: 622.26).

[0079] Step 5: Weigh compound 12 (70 mg, 0.112 mmol), dissolve it in DMF (2 mL), add DIPEA (29 mg, 0.224 mmol), stir and dissolve. Then sequentially add compound 8 (62.16 mg, 0.112 mmol) and HATU (42.56 mg, 0.112 mmol). Stir and react at room temperature for 1 h, concentrate, and purify by silica gel column chromatography to obtain compound 13 (81 mg, yield 62%, HPLC 96%). 11H NMR (500MHz, DMSO-d6)δ 8.50(d,J=8.9Hz,1H),8.24(t,J=6.3Hz,1H),8.11(t,J=5.7Hz,1H),7.99(d,J =7.2Hz,1H),7.86(d,J=7.5Hz,2H),7.74(dd,J=9.8,7.4Hz,2H),7.67(d,J=7.5 Hz,2H),7.39(t,J=7.4Hz,2H),7.35-7.26(m,4H),6.52(s,1H),5.75(s,2H),5. 59(dt,J=9.2,6.1Hz,1H),5.41(d,J=2.0Hz,2H),5.25(d,J=18.9Hz,1H),5.09( d,J=18.8Hz,1H),4.33-3.99(m,8H),3.70(qd,J=16.6,5.7Hz,2H),3.57(t,J= 6.5Hz,2H),3.46(s,4H),3.39(d,J=6.0Hz,2H),3.26-3.06(q,J=6.1Hz,5H),2. 48(d,J=2.4Hz,3H),2.41-2.29(m,5H),2.17(q,J=6.5Hz,2H),1.93-1.77(m,3H ),1.14(d,J=7.1Hz,3H),0.86(t,J=7.3Hz,3H),0.82-0.66(m,6H),LCMS:[M+1] + 1160.24 (Theoretical value: 1159.50).

[0080] Step 6: Add compound 13 (81 mg, 0.069 mg) to a 10 mL single-necked flask, dissolve with DMF (1 mL), add piperidine (100 μL), react at room temperature for 1 hour, add methyl tert-butyl ether (20 mL), centrifuge, remove the supernatant, and dry to obtain compound 14 (32 mg, yield 49%, HPLC 94%). 1H NMR(500MHz,DMSO-d6)δ 8.53(d,J=8.9Hz,1H),8.28(t,J=6.3Hz,1H),8.13(t,J=5.7Hz,1H),8.01(d,J=7.2Hz,1H),7.77(t,J=10.2Hz,5H),7.30(s,1H),6.53(s,1H) ,5.60(dt,J=8.9,6.0Hz,1H),5.41(s,2H),5.27(d,J=18.9Hz,1H),5.13(d,J=19.0Hz,1H),4.28(dd,J=13.2,6.7Hz,1H),4.24-4.12(m,3H), 4.12-4.03(m,2H),3.79-3.64(m,2H),3.63-3.48(m,8H),3.27-3.08(m,2H),2.97(h,J=5.7Hz,2H),2.47(s,3H),2.37(dd,J=7.8,4.3Hz,5H) ,2.18(q,J=6.4Hz,2H),1.85(qq,J=14.0,7.2Hz,3H),1.15(d,J=7.1Hz,3H),0.86(t,J=7.3Hz,3H),0.71(dd,J=13.3,6.8Hz,6H),LCMS:[M+1] + 938.02 (Theoretical value: 937.43).

[0081] Step 7: In a 10 mL single-necked flask, DCM (3 mL), compound 14 (32 mg, 0.034 mmol), bromoacetic acid (9.5 mg, 0.068 mmol), and DIC (8.6 mg, 0.068 mmol) were sequentially added, and the mixture was stirred at room temperature for 90 minutes to react. The mixture was then concentrated and purified by silica gel column chromatography to obtain a yellow solid compound 15 (22 mg, yield 61%, HPLC 97%). 1H NMR(500MHz,DMSO-d6)δ 8.49(d,J=8.9Hz,1H),8.33(t,J=5.7Hz,1H),8.24(t,J=6.2Hz,1H),8.11(t,J=5.8Hz,1H),7.98(d,J=7.1Hz,1H),7.75(d,J=8.5Hz,1H),7.70(d, J=10.8Hz,1H),7.28(s,1H),5.64-5.54(m,1H),5.40(s,2H),5.23(d,J= 18.9Hz,1H),5.04(d,J=18.9Hz,1H),4.33-3.97(m,6H),3.85(s,2H),3.7 0(qd,J=16.5,5.7Hz,3H),3.57(t,J=6.5Hz,4H),3.52-3.37(m,4H),3.2 6-3.17(m,4H),3.14-3.06(m,1H),2.47(s,3H),2.41-2.28(m,5H),2.24- 2.11(m,J=6.9,6.0Hz,2H),1.84(dh,J=20.8,7.0Hz,3H),1.14(d,J=7.1Hz,3H),0.85(t,J=7.3Hz,3H),0.70(dd,J=11.4,6.8Hz,6H),LCMS:[M+1] + 1058.24 (Theoretical value: 1057.36).

[0082] Example 5: N-(maleimidoethylamine(N-carboxyethyl)-Gly-Ala-ha-N(Me)-aminooxyacetyl)exatecan (16) [ka] Step 1: Add compound 12 (200 mg, 0.32 mmol) to a single-necked flask containing 5 mL of anhydrous DMF, stir, and dissolve thoroughly. Prepare a clean flask, weigh maleimideethylamine-N,N-diacetic acid (226.1 mg, 0.64 mmol) and DIC (81.0 mg, 0.64 mmol), shake, and dissolve in a mixed solvent of dichloromethane (2 mL) and DMF (0.5 mL). Place the DMF solution of compound 12 in an ice bath and add the mixed solution of DIC and maleimideethylamine-N,N-diacetic acid. Allow the system to rise naturally to room temperature, stir overnight, remove dichloromethane under reduced pressure, load by wet reverse-phase column chromatography, and purify the product (ACN% = 25-40) to obtain a white solid compound 16 (100 mg, yield 36%). 1 H NMR(600MHz,DMSO-d6)δ 12.39(s,1H),8.53(d,J=8.9Hz,1H),8.38(t,J=6.2Hz,1H),7.76(dd,J=13.8,8.9Hz,2H),7.31(s,1H),6.95(s ,2H),6.52(s,1H),5.65-5.57(m,1H),5.43(s,2H),5.27(d,J=19.0Hz,1H),5.17(d,J=18.8Hz,1H),4.27-4.08 (m,6H),3.42(t,J=6.3Hz,3H),3.19-3.11(m,3H),2.68(t,J=6.5Hz,2H),2.48(s,4H),2.39(d,J=1.8Hz,4H),2 .19(q,J=6.3Hz,2H),1.86(dp,J=21.4,7.2Hz,3H),1.13(d,J=7.1Hz,3H),0.87(t,J=7.3Hz,4H),LC / MS:[M+1] + 862.22 (Theoretical value: 860.35).

[0083] Example 6: N-(maleimidoethylamine(N-carboxyethyl)-Gly-PEG2-Gly-Val-Ala-ha-N(Me)-aminooxyacetyl)exatecan (17) [ka] Step 1: Add compound 14 (200 mg, 0.213 mmol) to a single-necked flask containing 5 mL of anhydrous DMF, stir, and dissolve thoroughly. Prepare a clean flask, weigh maleimideethylamine-N,N-diacetic acid (151.89 mg, 0.43 mmol) and DIC (54.44 mg, 0.43 mmol), shake, and dissolve in a mixed solvent of dichloromethane (2 mL) and DMF (0.5 mL). Place the DMF solution of compound 14 in an ice bath and add the mixed solution of DIC and maleimideethylamine-N,N-diacetic acid. Allow the system to rise naturally to room temperature and stir overnight. Remove dichloromethane under reduced pressure, load and purify by wet reverse-phase column chromatography (ACN% = 25-35%) to obtain a white solid compound 1 (84 mg, yield 33.5%). 1 H NMR(600MHz,DMSO-d6)δ 8.55(d,J=9.0Hz,1H),8.33-8.16(m,2H),8.08(s,1H),7.80(dd,J=22.2,10.7Hz,2H),7.3 1(s,1H),6.99(s,1H),6.52(s,1H),5.61(dd,J=9.2,5.5Hz,1H),5.42(s,2H),5.29(d,J=1 9.0Hz,1H),5.18(d,J=18.7Hz,1H),4.28(dd,J=13.1,6.7Hz,1H),4.22-4.16(m,2H),4.14 (dd,J=12.2,5.2Hz,1H),4.10-4.06(m,1H),4.05-3.99(m,1H),3.73(dd,J=16.5,5.7Hz,1 H),3.66(dd,J=16.6,5.8Hz,1H),3.57(t,J=6.5Hz,2H),3.45(d,J=3.0Hz,6H),3.38(t,J= 6.2Hz,5H),3.25-3.12(m,8H),2.70(t,J=6.5Hz,1H),2.47(s,4H),2.39(d,J=5.0Hz,3H), 2.35(t,J=6.5Hz,2H),2.19(p,J=6.9,5.8Hz,2H),1.90-1.80(m,3H),1.13(d,J=7.1Hz,3H ),0.87(t,J=7.3Hz,3H),0.81(t,J=6.5Hz,1H),0.71(dd,J=13.7,6.7Hz,5H),LCMS:[M+1] +1176.45 (Theoretical value: 1175.49).

[0084] Example 7: Synthesis of N-(bromoacetyl-PEG2-Gly-Val-Ala-ha-N(OMe)-aminoacetyl)exatecan (23) [ka] Step 1: Add exatecan methanesulfonate (100 mg, 0.19 mmol) and TEA (25 mg, 0.24 mmol) to 1 mL of DMF, stir, and dissolve. Add bromoacetic acid (40 mg, 0.28 mmol) and HATU (85 mg, 0.22 mmol), stir at room temperature for 1 hour, and react. Remove DMF under reduced pressure, separate and purify by silica gel column chromatography to obtain the intermediate exatecan-N-bromoacetamide (90 mg, yield 86%). LCMS:[M+1] + 557.1 (Theoretical value: 556.39).

[0085] Step 2: Exatecan-N-bromoacetamide was dissolved in DMF (1 mL), methoxyamine hydrochloride (126 mg, 1.5 mmol) and TEA (182 mg, 1.8 mmol) were added, and the mixture was stirred at room temperature for 1 hour to allow it to react. The DMF was removed under reduced pressure, and the mixture was separated and purified by silica gel column chromatography to obtain compound 18 (80 mg, yield 86%). 1 1H NMR (500MHz, DMSO-d6)δ 8.50-8.46(m,1H),7.80(d,J=10.8Hz,1H),7.30(d,J=1.9Hz,1H),6.83(td,J=6. 2,1.8Hz,1H),6.52(d,J=1.9Hz,1H),5.61-5.55(m,1H),5.42(s,2H),5.22(s,2H) ),4.01-3.87(m,1H),3.42(d,J=6.1Hz,3H),3.17(t,J=6.4Hz,2H),2.40(s,3H), 2.22-2.11(m,2H),1.86(dp,J=21.1,7.1Hz,2H),0.90-0.83(m,3H),LCMS:[M+1] + 523.20 (Theoretical value: 522.19).

[0086] Step 3: Add DMF (12 mL) to a 50 mL three-necked flask, and sequentially add compound 18 (1.68 g, 2.2 mmol), DIPEA (1.17 g, 9.1 mmol), and compound 4 (1.75 g, 6.6 mmol) at 0-5°C, stirring and reacting at 5-10°C for 2 hours, add methyl tert-butyl ether (60 mL), discard the supernatant, and compound 19 (LCMS[M+H] + Compound 20 (1.08 g, 50% total yield from 2 steps) was obtained by obtaining a value of 753.27 (theoretical value: 752.26), dissolving it in DCM (25 mL), and adding 80% hydrazine hydrate (0.94 g, 15 mmol) dropwise while stirring, stirring overnight at room temperature, concentrating, dissolving it in DMF and water, then liquid fractionation, and lyophilization to obtain compound 20 (1.08 g, 50% total yield from 2 steps). 1 H NMR(500MHz,DMSO-d6)δ 8.91(t,J=6.0Hz,1H),8.51(d,J=8.7Hz,1H),8.11(s,3H),7.78(d,J=10.9Hz,1H),7.31 (s,1H),6.54(s,1H),5.63-5.54(m,1H),5.42(s,2H),5.19(s,2H),4.35-4.39(m,1H),4. 25-4.30(m,1H),3.88(s,1H),3.50(s,3H),3.25-3.06(m,2H),2.39(s,3H),2.29-2.11( m,2H),1.81-1.93(m,2H),1.37(d,J=7.0Hz,3H),0.87(t,J=7.3Hz,3H),LCMS(ESI)[M+H] + :623.20 (Theoretical value: 622.26).

[0087] Step 4: Add compound 8 (0.68 g, 1.22 mmol) and DCM (9 mL) to a 25 mL single-necked flask, cool to 0°C, and sequentially add EDCI (0.25 g, 1.30 mmol) and NHS (0.15 g, 1.52 mmol). Stir and react at room temperature for 2 hours. At 0°C under argon gas protection, add DIPEA (0.19 g, 1.47 mmol) dropwise to the reaction mixture. Slowly add a DMF solution of compound 20 (0.9 g, 1.25 mmol) (diluted in 5 mL of DMF) dropwise to the reaction system. React overnight at room temperature, remove DMF under vacuum, and purify by silica gel column chromatography to obtain compound 21 (500 mg, yield 32%). 1 H NMR(500MHz,DMSO-d6)δ 8.50(d,J=8.7Hz,1H),8.39(t,J=6.1Hz,1H),8.13(t,J=5.7Hz,1H),8.09(d,J=7.2Hz,1H),7.91(d,J=7.5Hz,2H),7.80(t,J=10.4 Hz,2H),7.72(d,J=7.4Hz,2H),7.44(t,J=7.4Hz,2H),7.38-7.26(m,4H),6.56(s,1H),5.67-5.56(m,1H),5.45(s,2H),5.23(s,2H) ),4.35-4.12(m,5H),3.82-3.69(m,2H),3.62(t,J=6.4Hz,2H),3.55-3.45(m,6H),3.43(t,J=5.8Hz,2H),3.22-3.12(m,4H),2.45 -2.35(m,5H),2.32-2.12(m,2H),1.95-1.83(m,3H),1.34-1.19(m,8H),0.90(t,J=7.3Hz,3H),0.74-0.80(m,6H),LCMS(ESI)[M+H] + :1160.26 (Theoretical value: 1159.50).

[0088] Step 5: Add compound 21 (422 mg, 0.36 mmol) to DMF (2 mL), stir and dissolve, add DBU (28 mg, 1.84 mmol) dropwise at 0°C, stir at room temperature for 2 hours, add methyl tert-butyl ether (21 g, 3.66 mmol) solution in DMF (1.9 mL) dropwise, stir at room temperature for 2 hours, add methyl tert-butyl ether (20 mL), remove the supernatant, dissolve the viscous substance in DMF, purify the liquid phase, freeze-dry to obtain compound 23 (100 mg, 26% total yield from step 2), 1 H NMR(500MHz,DMSO)δ 8.47(d,J=8.0Hz,1H),8.39-8.24(m,2H),8.16-8.01(m,2H),7.77(d,J=10.0Hz, 2H),7.30(s,1H),5.59(s,1H),5.42(s,2H),5.19(s,2H),4.38-4.08(m,4H),3.90 -3.58(m,14H),3.50-3.14(m,10H),2.43-2.33(m,4H),2.19(s,2H),1.92-1.82(m ,3H),1.20(d,J=6.6Hz,3H),0.87(t,J=6.7Hz,3H),0.73(dd,J=13.8,6.3Hz,5H), 13 C NMR(126MHz,DMSO-d6)δ 173.65,172.90,170.98,170.88,169.32,168.70,166.55,163.10,161.12,157.16,152.84,15 0.43,145.69,140.84,136.81,126.10,124.23,124.08,122.11,119.59,110.25,97.14,72.81 ,69.97,69.89,69.19,67.16,65.71,60.62,59.30,58.35,57.74,50.07,48.67,44.92,42.42, 36.33,31.07,30.77,29.92,28.19,19.45,18.54,18.34,11.47,11.43,8.21,LCMS(ESI)[M+H] + :1058.26 (Theoretical value: 1057.36).

[0089] Example 8: N-(m-ethylamine(N-carboxyethyl)-Gly-Ala-ha-N(OMe)-aminoacetyl)exatecan (24) [ka] Step 1: Add compound 20 (80 mg, 0.129 mmol) and DMF (5 mL) to a 50 mL round-bottom flask and stir until completely dissolved (Solution 1). Next, prepare a 10 mL reaction flask and add maleimide ethylamine-N,N-diacetic acid (91 mg, 0.258 mmol), DIC (32.51 mg, 0.258 mmol), dichloromethane (4 mL), and DMF (2 L). Shake thoroughly until completely dissolved (Solution 2). Place the DMF solution of compound 20 (Solution 1) in an ice bath and, while stirring, add Solution 2 dropwise to Solution 1. Remove the ice bath and allow the reaction system to rise naturally to room temperature, stirring overnight. Remove the dichloromethane under reduced pressure and purify by reverse-phase chromatography column (ACN% = 35%) to obtain a yellow powder compound 24 (32 mg, yield 28.8%). 1 H NMR(600MHz,DMSO-d6)δ 8.48(d,J=8.9Hz,1H),8.43(t,J=6.3Hz,1H),7.78(d,J=10.8Hz,2H),7.30(s,1H),6.96(s,1H),6. 52(s,1H),5.61-5.56(m,1H),5.42(s,2H),5.25-5.17(m,2H),4.28-4.21(m,2H),4.19-4.13(m,1H) ,3.49-3.42(m,6H),3.18(t,J=5.5Hz,5H),2.70(t,J=6.8Hz,2H),2.39(s,3H),2.22-2.13(m,2H), 1.85(dq,J=21.6,6.9Hz,2H),1.21(dd,J=32.6,6.2Hz,5H),0.87(t,J=7.4Hz,4H),LCMS(ESI)[M+H] + 862.05 (theoretical value 860.31).

[0090] Example 9: Preparation of IP140B antibody-drug conjugates ADC1 and ADC2 (1) Preparation of ADC1 (DAR 8) [ka] IP140B antibody (10.0 mg / mL, 10 mg, 0.066 mmol) was prepared, the pH was adjusted to 7.2 with 1 M Na2HPO4 solution, then 25 μL of 0.1 M ethylenediaminetetraacetate disodium solution was added, followed by the preparation of the TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and the mixture was reacted on a turntable at room temperature (25°C) for 4 hours.

[0091] Compound 15 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed uniformly, and reacted on a turntable at room temperature for 16 hours. After the reaction was complete, low molecular weight molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, and the pH was adjusted to 6.2 to obtain the antibody-drug conjugate ADC1 (3.2 mg / mL, 2 mL). The results of the RP-HPLC-MS test are shown in Figure 1. Here, A and B are HPLC results, and C and D are MS results. The mean value calculated by RP-MS was n=7.9, and the MS results show that one linker-payload is linked to the antibody light chain (L) and three linker-payloads are linked to the heavy chain (H).

[0092] (2) Preparation of ADC2 (DAR 4) [ka] IP140B antibody (10.0 mg / mL, 10 mg, 0.066 mmol) was prepared, the pH was adjusted to 7.2 with 1 M Na2HPO4 solution, the formulated TCEP·HCl solution (10 mM, 0.02 mL) was added, and the reaction was carried out on a turntable at 10°C for 2 hours.

[0093] Compound 15 (0.59 mg, 0.56 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed uniformly, and reacted on a turntable at room temperature for 16 hours. After the reaction was complete, low molecular weight molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, and the pH was adjusted to 6.2 to obtain the antibody-drug conjugate ADC2 (3.0 mg / ml, 2 mL). The results of the RP-HPLC-MS test are shown in Figure 2. Here, A and B are the HPLC results, and C is the MS result. The mean value calculated by RP-MS was n=3.9, and the MS results show that two linker-payloads are linked to the antibody light-heavy chain (HL).

[0094] Example 10: Preparation of HS627 antibody-drug conjugate ADC3 [ka] HS627 antibodies (10.0 mg / mL, 10 mg, 0.066 mmol) were prepared, the pH was adjusted to 7.2 with 1 M Na2HPO4 solution, then 25 μL of 0.1 M ethylenediaminetetraacetate disodium solution was added, followed by the preparation of the TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and the mixture was reacted on a turntable at room temperature (25°C) for 4 hours.

[0095] Compound 15 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed uniformly, and reacted on a turntable at room temperature for 16 hours. After the reaction was complete, low molecular weight molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, and the pH was adjusted to 6.2 to obtain the antibody-drug conjugate ADC3 (3.0 mg / ml, 2 mL). The results of the RP-HPLC-MS test are shown in Figure 3. Here, A and B are HPLC results, and C and D are MS results. The mean value calculated by RP-MS was n=7.8, and the MS results show that one linker-payload is linked to the light chain (L) and three linker-payloads are linked to the heavy chain (H).

[0096] Example 11: Preparation of Nectin-4 antibody-drug conjugate ADC4 [ka] Nectin-4 antibodies (10.0 mg / mL, 10 mg, 0.066 mmol) were prepared, the pH was adjusted to 6.2 with 1 M Na2HPO4 solution, then 25 μL of 0.1 M ethylenediaminetetraacetate disodium solution was added, followed by the preparation of the TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and the mixture was reacted on a turntable at room temperature (25°C) for 4 hours.

[0097] Compound 15 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed uniformly, and reacted on a turntable at room temperature for 16 hours. After the reaction was complete, low molecular weight molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, and the pH was adjusted to 6.2 to obtain the antibody-drug conjugate ADC4 (3.0 mg / mL, 2 mL). The results of the RP-HPLC-MS test are shown in Figure 4. Here, A and B are HPLC results, and C and D are MS results. The mean value calculated by RP-MS was n=7.9, and the MS results show that one linker-payload is linked to the light chain (L) and three linker-payloads are linked to the heavy chain (H).

[0098] Example 12: Preparation of IP140B antibody-drug conjugate ADC5 [ka] IP140B antibody (10.0 mg / mL, 10 mg, 0.066 mmol) was prepared, the pH was adjusted to 7.2 with 1 M Na2HPO4 solution, then 25 μL of 0.1 M ethylenediaminetetraacetate disodium solution was added, followed by the preparation of the TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and the mixture was reacted on a turntable at room temperature (25°C) for 4 hours.

[0099] Compound 23 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed uniformly, and reacted on a turntable at room temperature for 16 hours. After the reaction was complete, low molecular weight molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, and the pH was adjusted to 6.2 to obtain the antibody-drug conjugate ADC5 (3.2 mg / mL, 2 mL). The results of the RP-HPLC-MS test are shown in Figure 5. Here, A and B are HPLC results, and C and D are MS results. The mean value calculated by RP-MS was n=7.8, and the MS results show that one linker-payload is linked to the light chain (L) and three linker-payloads are linked to the heavy chain (H).

[0100] Example 13: Preparation of HS627 antibody-drug conjugate ADC6 [ka] HS627 antibodies (10.0 mg / mL, 10 mg, 0.066 mmol) were prepared, the pH was adjusted to 7.2 with 1 M Na2HPO4 solution, then 25 μL of 0.1 M ethylenediaminetetraacetate disodium solution was added, followed by the preparation of the TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and the mixture was reacted on a turntable at room temperature (25°C) for 4 hours.

[0101] Compound 23 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed uniformly, and reacted on a turntable at room temperature for 16 hours. After the reaction was complete, low molecular weight molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, and the pH was adjusted to 6.2 to obtain the antibody-drug conjugate ADC6 (3.0 mg / mL, 2 mL). The RP-HPLC-MS test results are shown in Figure 6. Here, A and B are HPLC results, and C and D are MS results. The average value calculated by RP-MS was n=7.8, and the MS results show that one linker-payload is linked to the light chain (L) and three linker-payloads are linked to the heavy chain (H).

[0102] Example 14: Preparation of Nectin-4 antibody drug conjugate ADC7 [ka] Nectin-4 antibodies (10.0 mg / mL, 10 mg, 0.066 mmol) were prepared, the pH was adjusted to 6.2 with 1 M Na2HPO4 solution, then 25 μL of 0.1 M ethylenediaminetetraacetate disodium solution was added, followed by the preparation of the TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and the mixture was reacted on a turntable at room temperature (25°C) for 4 hours.

[0103] Compound 23 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA, added to the above solution system, mixed uniformly, and reacted on a turntable at room temperature for 16 hours. After the reaction was complete, low molecular weight molecules were removed using a NAP-5 gel column (Cytiva), the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, and the pH was adjusted to 6.2 to obtain the antibody-drug conjugate ADC7 (3.0 mg / mL, 2 mL). The RP-HPLC-MS test results are shown in Figure 7. Here, A and B are HPLC results, and C and D are MS results. The average value calculated by RP-MS was n=7.9, and the MS results show that one linker-payload is linked to the light chain (L) and three linker-payloads are linked to the heavy chain (H).

[0104] Test Example 1: In vivo activity of ADC in inhibiting tumor growth Method for testing tumor inhibitory activity against ADC nude mouse transplant tumor models (cell-derived xenografts, CDX): Pancreatic cancer cells BXPC-3, lung adenocarcinoma cells NCI-H1975, triple-negative breast cancer cells MDA-MB-231, and esophageal squamous cell carcinoma cells KYSE-150 were cultured in a monolayer in vitro. When the cell saturation reached 80%-90%, the cells were digested with pancreatin-EDTA, centrifuged, the supernatant was discarded, and the cells were resuspended in PBS. The cell suspension was adjusted to an appropriate concentration, and BXPC-3, NCI-H1975, MDA-MB-231, and KYSE-150 cells (2-10 × 10⁶) were cultured in a monolayer. 6 Cells (0.1 mL) were subcutaneously inoculated into BALB / c nude mice, and the growth status of the animals and transplanted tumors was regularly observed. When the tumor volume reached 100-200 mm², 3 Once the tumors had grown to a certain extent, the animals were randomly divided into groups based on tumor volume and body weight, with 6 animals per group. Intravenous injections were administered regularly. Twice a week, the longest diameter a (mm) and shortest diameter b (mm) of the tumor were measured using calipers, and the mouse body weight was also measured. V = 1 / 2 × a × b 2 (mm 3The tumor volume (V) was calculated using the formula ), where a and b represent the length and width of the tumor, respectively. A growth curve was plotted, and finally, the tumor was dissected and weighed. In statistical analysis, the tumor inhibition results were obtained by analyzing the tumor volume and body weight of the tumor-bearing mice at the end of the experiment using GraphPad Prism software.

[0105] Main evaluation metrics: Tumor growth inhibition rate (TGI): TGI(%)=[1-(avT i-0 / avC i-0 ) × 100%, here, avT i-0 This is calculated by subtracting the average tumor volume on the day administration began for the treatment group from the average tumor volume on a specific day for the treatment group, where avCi-0 is calculated by subtracting the average tumor volume on the day administration began for the vehicle control group from the average tumor volume on a specific day for the vehicle control group.

[0106] [Table 1] BIW was administered twice a week, on days 0, 3, 7, 10, 14, and 17, and patients were observed until day 33.

[0107] Here, IgG1 is a monoclonal antibody tool sequence with no target selectivity, and therefore has no affinity for human cells.

[0108] Compound 10 is the toxic (antitumor compound) portion of compound 15, and its structural formula is: [ka] That is the case.

[0109] Experimental results: ADC1 exhibited stronger tumor growth inhibitory activity at three doses of 0.8 mg / kg, 2 mg / kg, and 5 mg / kg than the positive control gemcitabine, the antibody alone, the toxin alone, a simple mixture of antibody and toxin, and ADC without targeting activity, and also showed dose-dependent inhibitory activity.

[0110] [Table 2] BIW was administered twice a week, on days 0, 4, 7, and 11, and patients were observed until day 21.

[0111] Experimental results: The experimental groups, at three doses of 1 mg / kg, 3 mg / kg, and 6 mg / kg, all showed stronger tumor growth inhibitory activity than simple mixtures of antibody and toxin, or ADCs without targeting activity. The tumor inhibitory effect of the 1 mg / kg ADC group was even superior to that of 10 mg / kg paclitaxel. The two dose groups, 3 mg / kg and 6 mg / kg, almost completely inhibited tumor growth, and in the 6 mg / kg dose group, tumors in 50% of cancer-bearing mice completely disappeared.

[0112] [Table 3] BIW was administered twice a week, on days 0, 3, and 7, and patients were observed until day 25.

[0113] Structural formula of IP140B-Dxd (where n=7.89): [ka] Experimental results: Both doses of ADC1 (3 mg / kg and 6 mg / kg), ADC5 (3 mg / kg), and ADC2 (6 mg / kg and 12 mg / kg) all showed stronger tumor growth inhibitory activity than the positive control paclitaxel, a simple mixture of antibody and toxin, and ADCs without targeted activity.

[0114] [Table 4] BIW was administered twice a week, on days 0, 3, 7, and 10, and patients were observed until day 21.

[0115] Experimental results: ADC1 at three doses of 1 mg / kg, 3 mg / kg, and 6 mg / kg, and ADC2 at two doses of 6 mg / kg and 12 mg / kg, all showed stronger tumor growth inhibitory activity than the positive control paclitaxel and ADCs without targeted activity.

[0116] Test Example 2: Study on the toxicity of ADC in cynomolgus monkeys 1. A single dose of ADC was administered, and dose tolerance, pharmacokinetics, and toxicity were investigated. After a single intravenous injection of ADC into cynomolgus monkeys, the pharmacokinetic properties of the drug in the monkeys were investigated, and the toxic expression in the animals was observed.

[0117] Test method: In this study, two groups were established: a 22 / 35 mg / kg group and a 60 mg / kg group, with two monkeys per group, and an equal number of females and males (the first and second doses in the low-dose group were 22 mg / kg, and the third and fourth doses were 35 mg / kg). Each group of cynomolgus monkeys received intravenous injection of the ADC formulation at the appropriate concentration according to the administration volume of 5 mL / kg. Two consecutive doses were administered every two weeks, followed by a third dose 22 days after dissolution and recovery, and a fourth dose 2 weeks later. The day of the first dose in the 22 / 35 mg / kg group was defined as day 1 of the study, and the day of the first dose in the 60 mg / kg group was defined as day 5 of the study. During the administration period, the general condition of the cynomolgus monkeys was observed daily, and their body weight and food intake were measured once a week. Three days after the initial administration, blood samples were taken intravenously from each group of cynomolgus monkeys, and on days 19, 42, 56, and 66 of the study, blood samples were taken from the 22 / 35 mg / kg group for hematological and biochemical tests. On day 66 of the study, the 22 / 35 mg / kg group of cynomolgus monkeys were anesthetized and euthanized. After necropsy, organ weights were measured, and bone marrow smears were prepared. Histopathological examinations were performed on the heart, liver, spleen, lungs, kidneys, ovaries, cervix, testes, inguinal lymph nodes, and administration site (including blood vessels) of each group of cynomolgus monkeys.

[0118] Pharmacokinetics: After a single intravenous infusion of different doses of ADC drugs in cynomolgus monkeys, blood samples were collected at multiple consecutive time points. Blood samples were collected from the upper extremity veins of cynomolgus monkeys in the 22 / 35 mg / kg group before the 1st and 3rd doses, 0.0167 hours after the 2nd dose, and 0.167, 4, 24, 48, 72, 96, 168, and 336 hours after the 1st, 3rd, and 4th doses. The concentration of ADC-TAB in serum was detected by ELISA, and the concentration of low molecular weight toxins in plasma was detected by LC-MS / MS. Toxicity parameters such as AUClast were calculated.

[0119] Toxicity studies: After single intravenous infusions of different doses of ADC drugs into cynomolgus monkeys, animal tolerance and drug-related toxic expressions were examined using multiple methods, including clinical observation, body weight and food intake, hematology, blood biochemistry, urinalysis, and dissection.

[0120] Test results: During the study period, the general condition of the cynomolgus monkeys was good, their spontaneous activity was normal, and no obvious toxic reactions were observed. During the administration period, there were no obvious abnormal changes in the food intake of male and female cynomolgus monkeys in the 22 / 35 mg / kg groups. There were no abnormalities in the body weight of the cynomolgus monkeys after two consecutive doses at 22 mg / kg. There were no obvious changes in the body weight of the cynomolgus monkeys after two consecutive doses at an increased dose of 35 mg / kg. Three to four days after the first dose, the food intake and body weight of the cynomolgus monkeys in the 60 mg / kg group decreased slightly.

[0121] During the study period, no significant abnormal changes were observed in the blood biochemical indicators of female and male cynomolgus monkeys in each group at each detection point.

[0122] Pharmacokinetics: After a single intravenous infusion of ADC into cynomolgus monkeys, the ADC remained stable in the monkeys' blood (see Figure 16), the concentration of free toxin was extremely low (see Figure 17), and the ADC was slowly released in the monkeys, indicating a stable conjugation mechanism.

[0123] Toxicity Report: After a single intravenous infusion of ADC in cynomolgus monkeys, the animals demonstrated good tolerance, no severe or intolerable drug-related toxicity occurred, indicating that the safety of ADC is controllable and can support further clinical research.

[0124] 2. Consideration of organs and pharmacokinetic parameters that may cause toxicity due to repeated administration. In toxicity studies in which the test substance was intravenously injected once every three weeks for six consecutive weeks (a total of three times) into cynomolgus monkeys, the properties, degree, dose-response, temporal relationship, and reversibility of the test substance that could cause toxic reactions were observed. This allowed for the identification of toxic target organs or tissues, as well as the study of its toxic kinetic characteristics. The study also investigated the effects on the cardiovascular system, central nervous system, respiratory function, and injection site, providing information for the safety of clinical use.

[0125] Test method: This study included four groups: a control group (ADC5 buffer solution) and groups receiving ADC5 at 9, 18, and 36 mg / kg. Each group consisted of 10 cynomolgus monkeys, with an equal number of males and females. Each group received either the control product (ADC5 buffer solution) or the corresponding concentration of ADC5 intravenously at a volume of 5 mL / kg. The monkeys were administered once every three weeks for six consecutive weeks, for a total of three doses, followed by a six-week recovery period. The day of the first dose was day 1 of the administration period, and the day after the last dose was day 1 of the recovery period.

[0126] Blood samples were collected from each group of cynomolgus monkeys at the time of the first dose, before the final dose, immediately after the end of the dose (0-2 minutes after the end of the dose), and at 4, 8, 24, 48, 72, 96, 168, 240, 336, and 504 hours after the end of the dose, and at 672 hours after the end of the final dose. The concentrations of ADC-TAB (total antibody) and ADC (antibody-drug conjugate) in the monkey serum at each time point were detected by ELISA, and the concentration of free toxins in the cynomolgus monkey plasma at each time point was detected by LC-MS / MS, and toxicity parameters such as AUC were calculated.

[0127] Test results: During the administration period, some female and male cynomolgus monkeys in the 18 and 36 mg / kg groups showed a decrease in food intake. In addition, no significant abnormal changes were observed in the food intake of female and male cynomolgus monkeys in the 9 mg / kg group or in the body weight of female and male cynomolgus monkeys in each group of ADC5 during the administration and recovery periods.

[0128] No arrhythmias were observed in the lead II electrocardiograms of female and male cynomolgus monkeys in each group of ADC5 at 4-5, 24, 72, 168, 336, and 504 hours after the first dose, 4 hours after the final dose, and after the recovery period. No obvious abnormalities were found in lead II electrocardiogram indicators such as heart rate, P wave duration, PR interval, QRS wave duration, QT interval, RR interval, and calibration QT interval, as well as in blood pressure and respiratory detection-related indicators. No obvious abnormalities were found in various blood biochemical indicators, urinary indicators, lymphocytes, cytokines, bone marrow smears, organs, etc.

[0129] Pharmacokinetics: Under these study conditions, cynomolgus monkeys were intravenously injected with 9, 18, and 36 mg / kg of ADC5 once every three weeks for six consecutive weeks (a total of three injections), followed by a six-week rest period. ADC5 was stable in the blood of cynomolgus monkeys (see Figure 18), the concentration of free toxin was extremely low (see Figure 19), and ADC was released slowly in cynomolgus monkeys, indicating a stable conjugation pattern that supports a clinical Q3W administration plan.

[0130] Toxicity Report: After repeated intravenous infusions of ADC in cynomolgus monkeys, the animals showed good tolerance, with any mild symptoms such as occasional food depletion resolving, and no severe or intolerant drug-related toxicity occurring. This indicates that the safety of ADC is controllable and supports further clinical research.

Claims

1. An antibody-drug conjugate shown in formula (I), 【Chemistry 1】 In the formula, R ２ is hydrogen, deuterium, C １ ~C ６ Alkyl alkyl group, C １ ~C ６ Selected from alkoxy groups, acyl groups, and sulfonyl groups, L １ is selected from -L １１ -L １２ -L １３ -, where L １１ , L １２ , L １３ are each independently non-existent, -C=O-, C １ ~C ２ alkylene group or C １ ~C ２ alkylene-O-, provided that when L １１ is -C=O-, R ２ is not hydrogen, L ２ C １ ~C ６ Alkylene group or C １ ~C ６ Selected from the acyl group, the C １ ~C ６ Alkylene group or C １ ~C ６ The acyl group can optionally consist of one or more R groups. ３ Replaced by, R ３ C is either phenyl-substituted or unsubstituted. １ ~C ６ Alkyl alkyl group, C １ ~C ６ Selected from alkoxy groups, L ｐ It is a peptide residue composed of 1 to 7 amino acids, Z is -L ｚ -L ｊ - is selected from, and here, L ｚ This is something that does not exist, -C (=O)-C １ ~C ８ Alkylene group, -C(=O)-(CH ２ CH ２ O) ２－６ -CH ２ CH ２ NH- or -C(=O)-(CH ２ ) ０－６ -NR １ (CH ２ ) ０－６ - Selected from, L ｊ It is a linker that can be conjugated to an antibody, R １ is, -C １ ~C ６ Alkyl-carboxyl group or -C １ ~C ６ Selected from alkyl-amino groups, Ab is an antibody, an antibody-drug conjugate as shown in formula (I).

2. R ２ is hydrogen, deuterium, C １ ~C ３ Alkyl alkyl group, C １ ~C ３ Alkoxy group, -C(=O)C １ ~C ３ Alkyl group or -S (=O) ２ C １ ~C ３ Selected from alkyl groups, Preferably, R ２ This is selected from hydrogen, deuterium, methyl group, ethyl group, methoxy group, ethoxy group, formyl group, acetyl group, methanesulfonyl group, or ethanesulfonyl group. Preferably, R ２ The antibody-drug conjugate according to claim 1, wherein is selected from hydrogen, a methyl group, a methoxy group, a formyl group, or a methanesulfonyl group.

3. L １１ , L １２ , L １３ These are independent entities that do not exist: -C=O- and -CH. ２ -ien-CH ２ O- or -OCH ２ - Selected from, Preferably, L １ is -C(=O)CH ２ OCH ２ -, -C(=O)CH ２ O-, -C(=O)CH ２ or -CH ２ - Selected from, Preferably, L ２ C １ ~C ３ Selected from alkylene groups, Preferably, L ２ The antibody-drug conjugate according to claim 1 or 2, wherein is selected from a methylene group and an ethylene group.

4. L ｐ It is selected from peptide residues consisting of 1, 2, 3, or 4 amino acids selected from phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala), or leucine (Leu), Preferably, L ｐ This is selected from -Val-Cit-, -Gly-Lys-, -Gly-Leu-, -Val-Ala-, -Gly-Phe-, -Gly-Gly-Lys-, -Gly-Gly-Phe-, -Gly-Val-Ala-, -Gly-Ala-, -Gly-Gly-Val-, -Gly-Leu-Val-, -Gly-Phe-Gly- or -Gly-Gly-Leu-, Preferably, L ｐ teeth, 【Chemistry 2】 An antibody-drug conjugate according to any one of claims 1 to 3, selected from the above.

5. L ｊ teeth, 【Transformation 3】 Selected from, 【Chemistry 4】 The position indicated by the arrow represents the point where the antibody is bound. 【Transformation 5】 The position indicated by is L ｚ It indicates that it is connected to the base, Preferably, L ｚ is -C(=O)-C １ ~C ８ alkylene group, -C(=O)-(CH ２ CH ２ O) ２－６ -CH ２ CH ２ NH- or -C(=O)-(CH ２ ) １－４ -NR １ (CH ２ ) ２ selected from, Preferably, L ｚ is -C(=O)-(CH ２ CH ２ O) ２－６ -CH ２ CH ２ NH- or -C(=O)-(CH ２ ) １－４ -NR １ (CH ２ ) ２ - Selected from, Preferably, R １ is, -C １ ~C ３ Selected from alkyl-carboxyl groups, Preferably, L ｚ is -C(=O)-(CH ２ CH ２ O) ２ -CH ２ CH ２ NH- or 【Transformation 6】 Selected from, Preferably, Z is 【Transformation 7】 An antibody-drug conjugate according to any one of claims 1 to 4, selected from the above.

6. The antibody-drug conjugate is obtained by conjugating an antibody with a compound of formula (II) or formula (III). 【Transformation 8】 Here, R ２ , L ｐ The definitions of each are the same as those of the compound in formula (I), where Z' is a linker group that can be conjugated to an antibody and is compatible with Z, preferably a maleimide group, a bromoacetyl group, or an iodoacetyl group. Preferably, the antibody-drug conjugate is subjected to physiological conditions. 【Chemistry 9】 It releases an antitumor compound, Here, R ２１ C １ ~C ６ Selected from alkyl groups, preferably -C １ ~C ３ It is an alkyl group, more preferably a methyl group, R ２２ C １ ~C ６ Selected from alkoxy groups, preferably -C １ ~C ３ It is an alkoxy group, more preferably a methoxy group, Preferably, 【Chemistry 10】 An antibody-drug conjugate according to any one of claims 1 to 5, which releases an antitumor compound.

7. The aforementioned antibody is an antibody against a tumor-associated antigen, Preferably, the tumor-associated antigen is one or more selected from Her2, Nectin-4, Trop2, 5T4, B7H3, ROR1, or Claudin18.

2. Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, or liver cancer. Preferably, the antibody comprises a heavy chain of amino acid sequence SEQ ID NO:1 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO:2 or any variant thereof. Preferably, the antibody comprises a heavy chain of amino acid sequence SEQ ID NO:3 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO:4 or any variant thereof. Preferably, the antibody comprises a heavy chain of amino acid sequence SEQ ID NO: 5 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO: 6 or any variant thereof. Preferably, the drug-antibody ratio of the antibody-drug conjugate is 2 to 8, and more preferably 3.5 to 4.5 or 7.5 to 8, according to any one of claims 1 to 6.

8. The aforementioned antibody-drug conjugate is 【Chemistry 11】 The antibody-drug conjugate according to any one of claims 1 to 7, which is obtained by conjugating the compound to an antibody.

9. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. An antibody-drug conjugate according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9, used in the preparation of an antitumor drug, Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, or liver cancer.

11. A method for treating a tumor disease, comprising the step of administering to a patient in need of such treatment an effective amount of an antibody-drug conjugate according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9, Preferably, the tumor is a solid tumor, Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer, or liver cancer.

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