Antibody-conjugated drug, and preparation method and application thereof

Abstract

Relates to the technical field of biological medicines, and provides an antibody-coupled drug obtained by coupling a compound shown in a formula (I) and an antibody, and a preparation method and application thereof, and the antibody-coupled drug has a relatively good inhibition effect on growth of solid tumors.

Description

Antibody-drug conjugates, preparation methods and applications thereof

[0001] This application claims priority to Chinese patent application No. 2023110584395, filed on August 21, 2023, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to an antibody-drug conjugate, a preparation method and application thereof. Technical Background

[0003] Antibody drug conjugates (ADCs) are a new and effective targeted drug composed of three components: an antibody, a linker, and a toxin. The toxin (payload) is the key component for drug efficacy, while the antibody is crucial for targeting. The linker not only connects the two but also influences the stability of the ADC, the drug release mechanism, and the antibody conjugation method. An ideal payload should be able to kill both targeted tumor cells and non-targeted tumor cells (those lacking the target antigen), known as bystander effects, while being non-toxic to normal cells and tissues (those lacking the target antigen). An ideal linker should be stable in the circulation and release its payload upon reaching the tumor microenvironment or tumor cells (endocytosis), thereby killing tumor cells or cells in the tumor microenvironment.

[0004] Ixitectin and its derivatives are potent DNA topoisomerase I inhibitors that have demonstrated promising results as payloads in the treatment of HER2-positive tumors. The development of more drug-drug conjugates with anti-tumor activity using ixitectin as a payload is of great significance.

[0005] Summary of the Invention

[0006] The present invention provides a low-toxic antibody-drug conjugate formed by connecting a stable linker and a TOP1 isomerase inhibitor, which has a good inhibitory effect on the growth of solid tumors.

[0007] In one aspect of the present invention, an antibody-drug conjugate is provided, represented by formula (I):

[0008] Where,

[0009] R 2 is selected from hydrogen, deuterium, C1-C6 alkyl, C1-C6 alkoxy, acyl, and sulfonyl;

[0010] L 1 Selected from -L 11 -L 12 -L13 -; among them, L 11 、L 12 、L 13 are independently selected from absent, -C=O-, C1-C2 alkylene or C1-C2 alkylene-O-, provided that when L 11 When -C=O-, R 2 Not hydrogen;

[0011] L 2 is selected from C1-C6 alkylene or C1-C6 acyl, wherein the C1-C6 alkylene or C1-C6 acyl is optionally substituted by one or more R 3 replace;

[0012] R 3 Selected from phenyl-substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy;

[0013] L P is a peptide residue consisting of 1-7 amino acids, for example, 2-7 amino acids;

[0014] Z is selected from -L z -L j -, where L z Selected from absent, -C(=O)-C1-C8 alkylene, -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=o)-(CH2)0-6-NR 1 (CH2) 0-6 -;L j A linker that can be coupled to an antibody;

[0015] R 1 Selected from -C1-C6 alkyl-carboxyl or -C1-C6 alkyl-amino;

[0016] Ab is an antibody.

[0017] In one embodiment, the antibody is obtained by coupling a compound of formula (I-1) (payload-linker) to an antibody:

[0018] Where, L 1 、L 2 、R 2 、L P Each defines a compound of formula (I).

[0019] Z' is a linker group compatible with Z and capable of coupling to an antibody.

[0020] In one embodiment, Z' is selected from (maleimide), Among them, R j is selected from halogen, preferably bromine or iodine.

[0021] In one embodiment, Z' is selected from maleimide, bromoacetyl, or iodoacetyl.

[0022] In one embodiment, as shown in formula (I-2), the linking site between the antibody and the payload-linker is the thiol group of cysteine ​​after the disulfide bond between the antibody chains is reduced and opened.

[0023] in, Indicates antibodies.

[0024] In one embodiment, the payload-linker is connected to the antibody cysteine ​​by replacing the bromine atom in the bromoacetyl linker group with the sulfhydryl group between the antibody chains or by Michael addition coupling with a maleimide structure linker group.

[0025] In one embodiment, R 2 is selected from hydrogen, deuterium, C1-C3 alkyl, C1-C3 alkoxy, -C(=O)C1-C3 alkyl or -S(=O)2C1-C3 alkyl;

[0026] Preferably, R 2 is selected from hydrogen, deuterium, methyl, ethyl, methoxy, ethoxy, formyl, acetyl, methylsulfonyl or ethylsulfonyl;

[0027] Preferably, R 2 is selected from hydrogen, methyl, methoxy, formyl or methylsulfonyl.

[0028] In one embodiment, L 11 、L 12 、L 13 are each independently selected from absent, -C=O-, -CH2-, -CH2O- or -OCH2-;

[0029] Preferably, L 1 Selected from -C(=O)CH2OCH2-, -C(=O)CH2O-, -C(=O)CH2 or -CH2-;

[0030] Preferably, L 2 Selected from C1-C3 alkylene;

[0031] Preferably, L 2 Selected from methylene and ethylene.

[0032] In one embodiment, when L 1 When it is -C(=O)CH2O-, R 2It is selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl.

[0033] In one embodiment, when L 1 When it is -C(=O)CH2-, R 2 It is selected from C1-C6 alkoxy, preferably -C1-C3 alkoxy, more preferably methoxy.

[0034] In one embodiment, L p selected from the group consisting of 1, 2, 3 or 4 peptide residues consisting of phenylalanine (Phe), glycine (Gly), valine (Val), alanine (Ala) or leucine (Leu);

[0035] Preferably, L p 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-;

[0036] Preferably, L p Selected from

[0037] In one embodiment, L j Selected from The positions shown indicate attachment to antibodies. The position shown indicates the same as L z The groups are connected;

[0038] Preferably, L z Selected from -C(=O)-C1-C8 alkylene, -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=O)-(CH2) 1-4 -NR 1 (CH2)2-;

[0039] Preferably, L z Selected from -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=O)-(CH2) 1-4 -NR 1 (CH2)2-;

[0040] Preferably, R 1Selected from -C1-C3 alkyl-carboxyl;

[0041] Preferably, L z Selected from -C(=O)-(CH2CH2O)2-CH2CH2NH- or

[0042] Preferably, Z is selected from

[0043] In one embodiment, the antibody-drug conjugate is obtained by conjugating a compound of formula (II) or formula (III) to an antibody:

[0044] Among them, R 2 、L P For each compound of formula (I), Z' is a linker group compatible with Z and capable of coupling to an antibody, preferably maleimide, bromoacetyl or iodoacetyl;

[0045] Preferably, the antibody-drug conjugate releases the following anti-tumor compounds under physiological conditions:

[0046] Among them, R 21 It is selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl.

[0047] R 22 It is selected from C1-C6 alkoxy, preferably -C1-C3 alkoxy, more preferably methoxy.

[0048] In one embodiment, the physiological environment is under the action of a protease.

[0049] The following anti-tumor compounds are preferably released:

[0050] In one embodiment, the antibody is an antibody against a tumor-associated antigen;

[0051] Preferably, the tumor-associated antigen is selected from one or more antibodies selected from Her2, Nectin-4, Trop2, 5T4, B7H3, ROR1 or Claudin18.2; the antibody may be a monospecific or multispecific antibody.

[0052] Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer or liver cancer;

[0053] Preferably, the antibody is HS627 antibody, IP140B antibody or Nectin-4 antibody.

[0054] Preferably, the antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 1 or any variant thereof, and a light chain having an amino acid sequence of SEQ ID NO: 2 or any variant thereof;

[0055] Preferably, the antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 3 or any variant thereof, and a light chain having an amino acid sequence of SEQ ID NO: 4 or any variant thereof;

[0056] Preferably, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 5 or any variant thereof, and a light chain having the amino acid sequence of SEQ ID NO: 6 or any variant thereof.

[0057] In one embodiment, the drug-antibody ratio of the antibody-drug conjugate is 2-8, more preferably 3.5-4.5 or 7.5-8.

[0058] In one embodiment, the antibody may be IgG1, IgG2, IgG3, IgG4, preferably IgG1.

[0059] In one embodiment, the antibody-drug conjugate is obtained by conjugating the following compound to an antibody:

[0060] The present invention provides the following antibody-drug conjugates:

[0061] Wherein, n is the drug-antibody ratio of the antibody-drug conjugate, preferably 8±0.5.

[0062] Another aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate and a pharmaceutically acceptable carrier.

[0063] Another aspect of the present invention provides use of the antibody-drug conjugate or the pharmaceutical composition in the preparation of anti-tumor drugs.

[0064] Another aspect of the present invention provides a method for treating tumor diseases, comprising the step of administering a therapeutically effective amount of the antibody-drug conjugate or the pharmaceutical composition to a patient in need thereof.

[0065] Preferably, the tumor is a solid tumor.

[0066] Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer or liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 shows the DAR test results of ADC1 in Example 9.

[0068] FIG2 shows the DAR test results of ADC2 in Example 9.

[0069] FIG3 shows the DAR test results of ADC3 in Example 10.

[0070] FIG4 shows the DAR test results of ADC4 in Example 11.

[0071] FIG5 shows the DAR test results of ADC5 in Example 12.

[0072] FIG6 shows the DAR test results of ADC6 in Example 13.

[0073] FIG7 shows the DAR test results of ADC7 in Example 14.

[0074] FIG8 shows a tumor growth curve of the BXPC-3 model in Test Example 1.

[0075] FIG9 shows a photo of the tumor after dissection of the BXPC-3 model in Test Example 1.

[0076] FIG10 shows a tumor growth curve of the NCI-H1975 model in Test Example 1. FIG.

[0077] FIG11 shows a tumor photograph of the NCI-H1975 model after dissection in Test Example 1.

[0078] FIG12 shows a tumor growth curve of the MDA-MB-231 model in Test Example 1. FIG.

[0079] FIG13 shows a tumor photograph of the MDA-MB-231 model after dissection in Test Example 1.

[0080] FIG14 shows a graph of the tumor growth curve of the KYSE-150 model in Test Example 1.

[0081] FIG15 shows a tumor photograph of the KYSE-150 model after dissection in Test Example 1.

[0082] FIG16 shows the individual drug-time curves of ADC5-TAB in the serum of cynomolgus monkeys after the 1st, 3rd, and 4th intravenous injections of 22 and 35 mg / kg of ADC5 were performed in cynomolgus monkeys in Test Example 2 (Day 1, 38, and 52).

[0083] FIG17 shows the individual drug-dose curves of toxin molecules in the plasma of cynomolgus monkeys after the third (Day 38, left) and fourth (Day 52, right) intravenous injections of ADC5@35 mg / kg in Test Example 2.

[0084] FIG18 shows a drug-time curve of ADC5 (ADC drug) and ADC5-TAB (total anti-) in serum after intravenous injection of ADC5 in cynomolgus monkeys in Test Example 2.

[0085] FIG19 shows a drug-time curve of the serum payload (free toxin molecules) after intravenous injection of ADC5 in cynomolgus monkeys in Test Example 2. DETAILED DESCRIPTION

[0086] I. Definition

[0087] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0088] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0089] In the description herein, references to “some embodiments,” “some implementation schemes,” or “some implementation plans” describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0090] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0091] As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group containing 1 to 20 carbon atoms. 1-6 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, more preferably C 1-4 Alkyl, most preferably C 1-3Alkyl. Specific examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched chain isomers thereof.

[0092] The term "alkoxy" refers to-O-(alkyl) and-O-(unsubstituted cycloalkyl), wherein the definition of alkyl is as described above. The limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydrogen, nitro, chloro, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0093] Refers to the chemical bond connection. It should be noted that the structural fragments described in the present invention (such as -L 11 -L 12 -L 13 -or-L z -L j -), when there is no special explanation, it represents the corresponding groups connected in order from left to right, for example, when -L 12 When - is C1-C2 alkylene-O-, it means that the left side of C1-C2 alkylene-O- is connected to -L 11 -, right end connected to -L 13 -.

[0094] It is understood that in the antibody-drug conjugates of the present application, for example, in Formula (I), the "-" between the drug-linker fragment and the antibody is intended to indicate the connection between the antibody and the fragment, and is not intended to limit the connection between one antibody and one drug-linker fragment. It is well known in the art that an antibody can be linked to one or more drugs due to the presence of multiple interlinking disulfide bonds.

[0095] As used herein, the term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom 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 replaced. Oxo substitution does not occur on aromatic groups. The term "optionally substituted" or "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

[0096] Although the term "optionally substituted" does not appear, if substitution occurs at any position on a ring (eg, an aliphatic ring or an aromatic ring), it should be understood that the position and number of substitution are arbitrary as long as it is chemically feasible.

[0097] As used herein, the term "antibody" is used in the broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments or synthetic polypeptides carrying one or more CDRs or derived from CDR sequences, as long as these polypeptides exhibit the desired biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. "Antibody" may also refer to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetically (e.g., recombinantly), including any fragment that retains the binding specificity of a full-length immunoglobulin comprising at least a portion of the variable region of an immunoglobulin molecule. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-tumor stem cell antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. The antibodies provided herein include members of any immunoglobulin class (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) ("type" and "class," as well as "subtype" and "subclass" are used interchangeably herein). Natural or wild-type (i.e., obtained from members of a population that has not been artificially manipulated) antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). One end of each heavy chain has a variable domain (VH), followed by multiple constant domains. One end of each light chain has a variable domain (VL) and the other end has a constant domain. The so-called "unmanipulated" means without the purpose of containing or expressing foreign antigen-binding molecules. Wild-type can refer to the most common allele or species found in a population or to antibodies obtained from unmanipulated animals, compared to alleles or polymorphisms, or variants or derivatives that have been obtained by some form of manipulation, such as mutagenesis, using recombinant methods, etc., to change the amino acids of the antigen-binding molecule.

[0098] As used herein, the term "drug-to-antibody ratio (DAR)" refers to the average number of payload molecules (anti-tumor compounds or drugs) attached to a single mAb.

[0099] As used herein, the term "pharmaceutically acceptable excipient" refers to a non-toxic, inert, solid, semi-solid substance or liquid filler, diluent, encapsulating material, or auxiliary formulation, or any other type of excipient, that is compatible with the patient, preferably a mammal, more preferably a human, and suitable for delivering the active agent to the target site without interrupting the agent's activity. The pharmaceutical carriers and excipients of the present invention are preferably compatible with the active ingredient, resulting in pharmaceutical formulations that meet standards for dissolution, storage stability, and impurity content.

[0100] Typically, the antibody-drug conjugates of the present invention can be administered in a suitable dosage form with one or more pharmaceutical carriers. These dosage forms are suitable for oral, rectal, topical, oral, and other parenteral administrations (e.g., subcutaneous, intramuscular, intravenous, etc.). For example, dosage forms suitable for other parenteral administrations include injections, etc. The above dosage forms can be prepared from the active ingredient of the present invention and one or more carriers or excipients via a general pharmaceutical method. The above carriers need to be compatible with the active ingredient of the present invention or other excipients.

[0101] In embodiments of the present invention, the pharmaceutical composition may include any of the following modes of administration: oral, spray inhalation, rectal, nasal, buccal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or administration via an explanted reservoir. When administered orally, the compounds of the present invention may be formulated into any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions, or aqueous suspensions. Carriers used in tablets generally include lactose and corn starch, and lubricants such as magnesium stearate may also be added. Diluents used in capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations typically combine the active ingredient with a suitable emulsifier and suspending agent. If desired, sweeteners, flavorings, or coloring agents may be added to the above oral formulations. The compounds of the present invention may also be administered in the form of sterile injectable formulations, including sterile water for injection or oil suspensions or sterile injectable solutions. Useful carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium, such as mono- or diglycerides.

[0102] The pharmaceutical compositions of the present invention are formulated, dosed, and administered in a manner consistent with standard medical practice. A "therapeutically effective amount" of the active ingredients of the present invention is determined by factors such as the specific condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the route of administration.

[0103] As used herein, "therapeutically effective amount" refers to an amount that can produce a function or activity in a patient (eg, a human and / or animal) and can be accepted by a human and / or animal.

[0104] As used herein, "patient" refers to an animal, preferably a mammal, more preferably a human.

[0105] As used herein, "treat" refers to alleviating, slowing the progression, attenuating, preventing, or maintaining an existing disease or condition (e.g., cancer). "Treatment" also includes curing, preventing the development of, or alleviating to some extent, one or more symptoms of a disease or condition.

[0106] II. Examples

[0107] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0108] Before further explaining the embodiments of the present invention in detail, the nouns and terms involved in the embodiments of the present invention are explained. The nouns and terms involved in the embodiments of the present invention are subject to the following interpretations.

[0109] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products and are obtained by purchasing commercially available products.

[0110] abbreviation:

[0111] 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-diazobisspiro[5.4.0]undec-7-ene; DMA: N,N-dimethylaniline. Val: valine, whose structural formula is Ala: alanine, its structural formula is Gly: glycine, its structural formula is Phe: phenylalanine, its structural formula is Leu: Leucine, its structural formula is The peptide residue L P It can be obtained by amino acid condensation methods known in the art, and the present invention is not particularly limited thereto.

[0112] In the present invention, the antibody and the linker L for connecting the antibody j It can be obtained by connecting by methods known in the art. For example, when Lj The structure is (or ), which can be through (or ) is reacted with a thiol or other reactive group on an antibody to form a ligand, and the present invention does not impose any particular limitation on this.

[0113] The IgG1 monoclonal antibody (Human IgG1, kappa isotype Control) in Test Example 1 was purchased from Sino-Biological, catalog number: HG1K.

[0114] In this example, ADCs prepared using, but not limited to, HS627, IP140B antibodies, and Nectin-4 antibodies, have the following amino acid sequence for the heavy chain of HS627 antibody (SEQ ID NO: 1):

[0115] The light chain amino acid sequence is as follows (SEQ ID NO: 2):

[0116] The heavy chain amino acid sequence of the IP140B antibody is as follows (SEQ ID NO: 3):

[0117] The light chain amino acid sequence is as follows (SEQ ID NO: 4):

[0118] The amino acid sequence of the nectin-4 antibody heavy chain is as follows (SEQ ID NO: 5):

[0119] The light chain amino acid sequence is as follows (SEQ ID NO: 6):

[0120] DAR value testing and calculation: Based on the 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.

[0121] RP-HPLC parameters: PLRP-S 1000A 5 μm column, column temperature 70°C. Mobile phase A consisted of 0.1% formic acid in water, mobile phase B consisted of 0.1% formic acid in acetonitrile, flow rate 0.2 mL / min. Mobile phase gradient was 20-50% B over 18 minutes; 50-95% B over 5 minutes; 95-20% B over 0.1 minutes; and 20-20% B over 6.9 minutes.

[0122] MS parameters were set as follows: capillary voltage 2.50 kV, cone voltage 100 V, mass analysis range m / z 200 to 4000, MSE collision energy 20 to 45 eV, ion source temperature 120°C, nebulizer temperature 500°C, nebulizer flow rate 1000 L / Hr, and internal standard leucine enkephalin. The sample was diluted to 1 mg / mL in sample buffer, TCEP was added to a final concentration of 50 mmol / L, and the sample was incubated at 37°C for 20 min. A 5 μL injection was performed. Light chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Similarly, heavy chain peaks were identified and peak area percentages were calculated, with the sum of the peak areas being 100. Weighted peak areas for the heavy and light chains were calculated by multiplying the peak area percentages by the corresponding drug loading. The DAR value was calculated as: DAR = 2*(∑weighted light chain peak area + ∑weighted heavy chain peak area) / 100.

[0123] Example 1: Synthesis of 2-(((tert-Butoxycarbonyl)(methyl)amino)oxy)acetic acid (2)

[0124] 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 in sequence, cooled to 0°C in 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 h, reacted at room temperature for 3 h, extracted with ethyl acetate (2 x 50 mL), and the organic phases were combined, washed with saturated brine (50 mL), washed with 0.5 N dilute hydrochloric acid (30 mL), and then washed with saturated brine (50 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a light yellow oily liquid compound 1 (33 g, yield 93.7%, HPLC purity 98%); 1 H NMR (500MHz, DMSO-d6) δ9.25 (s, 1H), 3.00 (s, 3H), 1.40 (s, 9H).

[0125] Step 2: Under ice cooling, 1.5N NaOH aqueous solution (100 mL) and compound 1 (10 g, 67.9 mmol) were added to a 300 mL three-necked flask, and bromoacetic acid (9.4 g, 67.7 mmol) aqueous solution (50 mL) was added under stirring, and stirred at 0 ° C for 0.5 h, ethyl acetate (2x50 mL), and the aqueous phase was adjusted to pH = 2 with 1N HCl, extracted with dichloromethane (3x50 mL), washed with saturated brine, dried over anhydrous MgSO4, filtered and concentrated to give a light yellow oily liquid compound 2 (12 g, yield 86.1%, HPLC purity 95%); 1H NMR (500MHz, DMSO-d6) δ12.42 (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).

[0126] Example 2: Synthesis of (S)-N-(chloromethyl)-2-(1,3-dioxoisoindolin-2-yl)propionamide (4)

[0127] Step 1: In a 500 mL single-necked bottle, phthalylalanine (5 g, 22.8 mmol), DCM (175 mL), and DMF (100 μL) were added, cooled to 0°C in an ice bath, and oxalyl chloride (5.79 g, 45.6 mmol) was added dropwise under argon protection. The mixture was stirred at 0°C for 1.5 h, concentrated, and the crude product was dissolved in DCM (90 mL), cooled to 0°C, and 7 M ammonia methanol solution (9.78 mL, 69.1 mmol) was added dropwise. The reaction was stirred at room temperature for 2 h, and n-hexane was added. The mixture was filtered and dried to give compound 3 (4.5 g, yield 92%, HPLC 97%). 1 H NMR (600MHz, DMSO-d6) δ7.84 (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).

[0128] Step 2: Compound 3 (1 g, 4.5 minol) 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 for reaction for 1.5 h, the mixture was cooled, filtered, and concentrated to give compound 4 (1.22 g, yield 100%, HPLC purity 96%): 1 H NMR (500MHz, CDCl3) δ7.85 (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).

[0129] Example 3: Synthesis of Fmoc-PEG2-Gly-Val (8)

[0130] Step 1: In a 1000 mL single-necked flask, dichloromethane (500 mL) was added, and Fmoc-NH-PEG2-CH2CH2-COOH (30 g, 75 mmol), HOBT (12.15 g, 90 mmol), EDCI (17.25 g, 90 mmol), glycine tert-butyl ester (10.32 g, 78.75 mmol), and DIPEA (10.16 g, 90 mmol) were added in sequence under stirring. The reaction was stirred at room temperature for 2 h, and the mixture was washed with saturated brine, saturated sodium bicarbonate aqueous solution, and 0.5 M hydrochloric acid, dried over anhydrous magnesium sulfate, filtered, and concentrated to give compound 5 (36.2 g, yield 94%, HPLC 98%). 1 H NMR (600MHz, CDCl3) δ7.78-7.67 (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).

[0131] Step 2: In a 1000 mL single-necked flask, dichloromethane (400 mL) and compound 5 (36.2 g, 70 mmol) were added and stirred uniformly. Trifluoroacetic acid (100 mL) was added dropwise and the mixture was stirred at room temperature overnight. The mixture was concentrated to obtain compound 6 (32.2 g, 100% yield, HPLC 97%). 1 H NMR (600MHz, CDCl3) δ8.88 (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).

[0132] Step 3: In a 1000 mL single-necked bottle, dichloromethane (300 mL) was added, and 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 ester hydrochloride (1.91 g, 11 mmol) and DIPEA (2.84 g, 22 mmol) were added in sequence under stirring, and stirred at room temperature for 2 h. The mixture was washed with saturated brine, saturated sodium bicarbonate aqueous solution, and 0.5 M hydrochloric acid, dried over anhydrous magnesium sulfate, filtered, and concentrated to give compound 7 (5.5 g, yield 82%, HPLC 97%); 1 H NMR (600MHz, CDCl3) δ7.80-7.70 (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).

[0133] Step 4: In a 1000 mL single-necked flask, dichloromethane (50 mL) and 7 (5.5 g, 9 mmol) were added, and trifluoroacetic acid (10 mL) was added dropwise with stirring. Stirring was continued overnight and the mixture was concentrated to obtain compound 8 (4.99 g, 100% yield, HPLC 96%). 1H NMR (600MHz, CDCl3) δ9.41 (s, 1H), 7.72 (d, J=7.4Hz, 2H), 7.64-7.53 (m, 3H), 7.36 (q, J=7.7Hz, 2H), 7.28 (q , J=7.4Hz, 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=5.3Hz, 2H), 3.41-3.23(m, 2H), 3.22(s , 1H), 2.51 (q, J=10.5, 5.5Hz, 2H), 2.17 (dt, J=13.4, 6.6Hz, 1H), 0.91 (dd, J=18.1, 6.8Hz, 5H); LCMS: [M+1] + 556.14 (theoretical value: 555.26).

[0134] Example 4: Synthesis of N-(bromoacetyl-PEG2-Gly-Val-Ala-ha-N(Me)-aminooxyacetyl)exitecan (15)

[0135] Step 1: Under argon protection, compound 2 (5.8 g, 28.3 mmol), DMF (50 mL), HATU (10.7 g, 28.2 mmol), exitecan mesylate (10 g, 18.8 mmol), and DIPEA (6.1 g, 47.2 mmol) were added sequentially to a 100 mL three-necked flask, stirred to dissolve the solution, heated to room temperature and reacted for 6 h, poured into 500 mL of ice water, filtered, and the filter cake was rinsed with water three times. The filter cake was dried in a forced air drying oven (50 ° C, 24 h) to obtain compound 9 (10.5 g, yield 90%, HPLC 89%); 1H NMR (600MHz, DMSO-d6) δ8.81 (d, J=8.6Hz, 1H), 7.72 (d, J=10.7Hz, 1H), 7.28-7.23 (m, 1H), 6.51 (d , J=1.9Hz, 1H), 5.65-5.59 (m, 1H), 5.40 (d, J=3.8Hz, 2H), 5.23-5.14 (m, 1H), 5.11 (d, J=19.0Hz, 1H ), 4.44-4.35 (m, 2H), 3.16 (d, J = 6.6Hz, 2H), 3.07 (s, 3H), 2.36-2.31 (m, 3H), 2.23 (dq, J = 14.6, 5.0 Hz, 2H), 2.19 (s, 2H), 1.85 (m, J=21.4, 7.3Hz, 2H), 1.20 (s, 9H), 0.86 (t, J=7.3Hz, 3H); LCMS: [M+1] + 623.45 (theoretical value: 622.24).

[0136] Step 2: DCM (100 mL) and compound 9 (10.5 g, 16.9 mmol) were added to a 250 mL three-necked flask, stirred, cooled to 0°C, and TFA (25 mL) was added dropwise. The mixture was stirred at room temperature for 3 h, concentrated, and ethyl acetate / petroleum ether (1:1) was added. The mixture was filtered and dried to obtain compound 10 (10 g, yield 93%, HPLC 87%). 1 H NMR (600MHz, DMSO-d6) δ8.87 (d, J=8.6Hz, 1H), 7.78 (d, J=10.8Hz, 1H), 7.30 (s, 1H), 6.42 (s, 0 H), 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), 2.1 5 (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).

[0137] Step 3: Compound 10 (6 g, 9.4 mmol), DMF (30 mL) and compound 4 (3 g, 11.2 mmol) were added to a 100 mL three-necked flask and stirred at 0-5°C. DIPEA (3.7 g, 28.6 mmol) was added dropwise and stirred at room temperature for 1.5 h. Methyl tert-butyl ether (300 mL) was added and the supernatant was removed. The black solid was dissolved in 50 mL of DCM and 15 g of silica gel was added and the sample was mixed. The product was purified by silica gel column chromatography to give compound 11 (6.2 g, yield 87.4%, HPLC 92%). 1 H NMR (500MHz, DMSO-d6) δ8.72-8.66 (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), 4.12-4.01 (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, 2 H), 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).

[0138] 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 for reaction for 2 h, the temperature was lowered, the reaction solution was poured into saturated brine (100 mL), extracted with ethyl acetate, and concentrated to give compound 12 (0.07 g, yield 77%, HPLC 95%); 1H NMR (500MHz, DMSO-d6) δ8.91 (t, J=6.0Hz, 1H), 8.58 (d, J=8.9Hz, 1H), 8.17 (s, 3H ), 7.66 (d, J=10.8Hz, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 5.58 (td, J=8.3, 7.9, 4.8Hz, 1H), 5.39 (s, 2H), 5.19 (d, J=19.0Hz, 1H), 5.01 (d, J=18.9Hz, 1H), 4.35 (dd, J=13. 2, 6.3Hz, 1H), 4.25 (dd, J=12.1, 4.7Hz, 1H), 4.20 (d, J=2.3Hz, 2H), 3.97-3.88 (m, 1H), 3.18 (dt, J=17.1, 5.9Hz, 1H), 3.13-3.03 (m, 1H), 2.53 (s, 3H), 2.31 (d, J=1.9Hz, 3H), 2.26-2 .09 (m, 2H), 1.93-1.77 (m, J=7.2Hz, 2H), 1.37 (d, J=6.9Hz, 3H), 0.86 (t, J=7.3Hz, 3H).LCMS: [M+1] + 623.15 (theoretical value: 622.26).

[0139] Step 5: Compound 12 (70 mg, 0.112 mmol) was weighed and dissolved in DMF (2 mL). DIPEA (29 mg, 0.224 mmol) was added and stirred to dissolve. Compound 8 (62.16 mg, 0.112 mmol) and HATU (42.56 mg, 0.112 mmol) were added sequentially. The mixture was stirred at room temperature for 1 h, concentrated, and purified by silica gel column chromatography to obtain compound 13 (81 mg, yield 62%, HPLC 96%). 1H NMR (500MHz, DMSO-d6) δ8.50 (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, 2 H), 7.67 (d, J=7.5Hz, 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, 5 H), 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).

[0140] Step 6: In a 10 mL single-necked vial, compound 13 (81 mg, 0.069 mg) was added, dissolved in DMF (1 mL), piperidine (100 μL) was added, and the mixture was reacted at room temperature for 1 h. Methyl tert-butyl ether (20 mL) was added, the mixture was centrifuged, the supernatant was removed, and the mixture was dried to obtain compound 14 (32 mg, yield 49%, HPLC 94%): 1H NMR (500MHz, DMSO-d6) δ8.53 (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=1 8.9Hz, 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), 3.2 7-3.08 (m, 2H), 2.97 (h, J=5.7Hz, 2H), 2.47 (s, 3H), 2.37 (dd, J=7.8, 4.3H z, 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).

[0141] Step 7: In a 10 mL single-necked vial, 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 added sequentially. The reaction was stirred at room temperature for 90 min, concentrated, and purified by silica gel column chromatography to obtain compound 15 as a yellow solid (22 mg, yield 61%, HPLC 97%). 1H NMR (500MHz, DMSO-d6) δ8.49 (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, 2 H), 5.23 (d, J=18.9Hz, 1H), 5.04 (d, J=18.9Hz, 1H), 4.33-3.97 (m, 6H), 3.85 (s, 2H), 3.70 (qd, J=16.5, 5.7Hz, 3H), 3.57 (t, J=6.5Hz, 4H), 3.52-3.37 (m, 4H), 3.26-3.17(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).

[0142] Example 5: N-(Maleimidoethylamine (N-carboxyethyl)-Gly-Ala-ha-N(Me)-aminooxyacetyl)exitecan (16)

[0143] Step 1: Add compound 12 (200 mg, 0.32 mmol) to a single-necked flask containing 5 mL of anhydrous DMF and stir to fully dissolve. In a separate clean flask, weigh maleimidoethylamine-N,N-diacetic acid (226.1 mg, 0.64 mmol) and DIC (81.0 mg, 0.64 mmol) and shake to dissolve them 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 maleimidoethylamine-N,N-diacetic acid. Allow the system to warm to room temperature and stir overnight. Remove the dichloromethane under reduced pressure and purify the product by reverse-phase column chromatography (ACN% = 25-40) to obtain compound 16 (100 mg, 36% yield) as a white solid. 1H NMR (600MHz, DMSO-d6) δ12.39 (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=18.8 Hz, 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).

[0144] Example 6: N-(Maleimidoethylamine (N-carboxyethyl)-Gly-PEG2-Gly-Val-Ala-ha-N(Me)-aminooxyacetyl)exitecan (17)

[0145] Step 1: Add compound 14 (200 mg, 0.213 mmol) to a single-necked flask containing 5 mL of anhydrous DMF and stir to dissolve thoroughly. In a separate clean flask, weigh maleimidoethylamine-N,N-diacetic acid (151.89 mg, 0.43 mmol) and DIC (54.44 mg, 0.43 mmol) and shake to dissolve them 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 maleimidoethylamine-N,N-diacetic acid. Allow the system to warm to room temperature and stir overnight. Remove the dichloromethane under reduced pressure and purify by reverse-phase column chromatography (ACN% = 25-35) to obtain compound 1 (84 mg, 33.5% yield) as a white solid. 1H NMR (600MHz, DMSO-d6) δ8.55 (d, J=9.0Hz, 1H), 8.33-8.16 (m, 2H), 8.08 (s, 1H), 7.80 (dd, J=2 2.2, 10.7Hz, 2H), 7.31 (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=19.0Hz, 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, 1H), 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).

[0146] Example 7: Synthesis of N-(bromoacetyl-PEG2-Gly-Val-Ala-ha-N(OMe)-aminoacetyl)exitecan (23)

[0147] Step 1: In 1 mL of DMF, add isotecan mesylate (100 mg, 0.19 mmol) and TEA (25 mg, 0.24 mmol), stir to dissolve, add bromoacetic acid (40 mg, 0.28 mmol) and HATU (85 mg, 0.22 mmol), stir and react at room temperature for 1 h, remove DMF under reduced pressure, and separate and purify by silica gel column chromatography to obtain the intermediate isotecan-N-bromoacetamide (90 mg, yield 86%); LCMS: [M+1] + 557.1 (theoretical value: 556.39).

[0148] Step 2: Dissolve exitecan-N-bromoacetamide in DMF (1 mL), add methoxyamine hydrochloride (126 mg, 1.5 mmol) and TEA (182 mg, 1.8 mmol), and stir at room temperature for 1 h. Remove DMF under reduced pressure, and separate and purify by silica gel column chromatography to obtain compound 18 (80 mg, yield 86%). 1 H NMR (500MHz, DMSO-d6) δ8.50-8.46 (m, 1H), 7.80 (d, J = 10.8Hz, 1H), 7.30 (d, J = 1.9Hz, 1 H), 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: [N+1] + 523.20 (theoretical value: 522.19).

[0149] Step 3: DMF (12 mL) was added to a 50 mL three-necked flask, and compound 18 (1.68 g, 2.2 mmol), DIPEA (1.17 g, 9.1 mmol), and compound 4 (1.75 g, 6.6 mmol) were added in sequence at 0-5 ° C. The mixture was stirred at 5-10 ° C for 2 h, and methyl tert-butyl ether (60 mL) was added. The supernatant was discarded to obtain compound 19 (LCMS [M+H] + : 753.27; theoretical value: 752.26), dissolved in DCM (25 mL), 80% hydrazine hydrate (0.94 g, 15 mmol) was added dropwise with stirring, stirred at room temperature overnight, concentrated, dissolved in DMF and water, and then liquid phase preparation was performed. After lyophilization, compound 20 (1.08 g, two-step combined yield 50%) was obtained: 1H NMR (500MHz, DMSO-d6) δ8.91 (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), 4.35-4.3 9(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).

[0150] Step 4: Compound 8 (0.68 g, 1.22 mmol) and DCM (9 mL) were added to a 25 mL single-necked bottle, cooled to 0°C, and EDCI (0.25 g, 1.30 mmol) and NHS (0.15 g, 1.52 mmol) were added in sequence. The mixture was stirred at room temperature for 2 h. DIPEA (0.19 g, 1.47 mmol) was added dropwise to the reaction solution at 0°C under argon protection. A DMF solution of compound 20 (0.9 g, 1.25 mmol) (diluted with 5 mL DMF) was slowly added dropwise to the reaction system. The reaction was allowed to proceed at room temperature overnight. DMF was removed under vacuum and purified by silica gel column chromatography to give compound 21 (500 mg, 32% yield); 1H NMR (500MHz, DMSO-d6) δ8.50 (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=10.4Hz, 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).

[0151] Step 5: Compound 21 (422 mg, 0.36 mmol) was added to DMF (2 mL), stirred and dissolved, DBU (28 mg, 1.84 mmol) was added dropwise at 0°C, stirred at room temperature for 2 h, methyl tert-butyl ether (21 mL) was added, and the supernatant was removed to obtain compound 22 (LCMS [M+H] + : 938.23: theoretical value: 937.43), dissolved in DMF (1.1 mL), stirred, and a solution of bromoacetic anhydride (0.95 g, 3.66 mmol) in DMF (1.9 mL) was added dropwise at 0°C, stirred at room temperature for 2 h, methyl tert-butyl ether (20 mL) was added, the supernatant was removed, and the viscous material was dissolved in DMF, purified by liquid phase, and lyophilized to obtain compound 23 (100 mg, two-step combined yield 26%); 1H NMR (500MHz, DMSO) δ8.47 (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) δ173.65, 172.90, 170.98, 170.88, 169.32, 168.70, 166.55, 163.10, 161.12, 1 57.16, 152.84, 150.43, 145.69, 140.84, 136.81, 126.10, 124.23, 124.08, 122.11, 119.59, 110.25, 9 7.14, 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, 4 2.42, 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).

[0152] Example 8: N-(m-ethylamine (N-carboxyethyl)-Gly-Ala-ha-N(OMe)-aminoacetyl)exitecan (24)

[0153] Step 1: Add compound 20 (80 mg, 0.129 mmol) and DMF (5 mL) to a 50 mL round-bottom flask and stir to fully dissolve it (solution 1). Take another 10 mL reaction bottle, 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 mL), and shake it thoroughly to dissolve it (solution 2). Place the DMF solution of compound 20 (solution 1) in an ice bath, add solution 2 dropwise to solution 1 with stirring, remove the ice bath, allow the reaction system to naturally warm to room temperature, and stir overnight. Remove dichloromethane under reduced pressure, and purify by reverse phase chromatography (ACN% = 35%) to obtain compound 24 as a yellow powder (32 mg, yield 28.8%); 1 H NMR (600MHz, DMSO-d6) δ8.48 (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).

[0154] Example 9: Preparation of IP140B Antibody-Drug Conjugates ADC1 and ADC2

[0155] (1) Preparation of ADC1 (DAR8)

[0156] Take IP140B antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 4 h.

[0157] Compound 15 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. Mix thoroughly and allow to react at room temperature for 16 hours on a rotating disk. After completion, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH 6.2, to obtain the antibody-drug conjugate ADC1 (3.2 mg / mL, 2 mL). The RP-HPLC-MS results are shown in Figure 1, where A and B are HPLC results, and C and D are MS results. The RP-MS calculated average: n = 7.9; the MS results showed that the antibody light chain (L) was linked to one linker-payload, and the heavy chain (H) was linked to three linker-payloads.

[0158] (2) Preparation of ADC2 (DAR4)

[0159] IP140B antibody (10.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 with 1 M Na2HPO4 solution. The prepared TCEP·HCl solution (10 mM, 0.02 mL) was added and reacted at 10°C on a rotating turntable for 2 h.

[0160] Compound 15 (0.59 mg, 0.56 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. Mix thoroughly and allow to react at room temperature for 16 hours on a rotating turntable. After completion, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH 6.2, to obtain the antibody-drug conjugate ADC2 (3.0 mg / mL, 2 mL). The RP-HPLC-MS results are shown in Figure 2, where A and B are HPLC results and C is the MS result. The RP-MS calculated average: n = 3.9; the MS results showed that the antibody light and heavy chains (HL) were connected to two linker-payloads.

[0161] Example 10: Preparation of HS627 Antibody-Drug Conjugate ADC3

[0162] HS627 antibody (10.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 with 1 M Na2HPO4 solution. Then, 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL) was added. The reaction was carried out at room temperature at 25°C with a rotating turntable for 4 h.

[0163] Compound 15 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. Mix thoroughly and allow to react at room temperature for 16 h on a rotating disk. After completion, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH 6.2, to obtain the antibody-drug conjugate ADC3 (3.0 mg / mL, 2 mL). The RP-HPLC-MS results are shown in Figure 3, where A and B are HPLC results, and C and D are MS results. The RP-MS calculated average: n = 7.8; the MS results showed that the light chain (L) was linked to one linker-payload and the heavy chain (H) was linked to three linker-payloads.

[0164] Example 11: Preparation of Nectin-4 Antibody-Drug Conjugate ADC4

[0165] Take nectin-4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 6.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 4 h.

[0166] Compound 15 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. Mix thoroughly and allow to react at room temperature for 16 h on a rotating disk. After completion, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH 6.2, to obtain antibody-drug conjugate ADC4 (3.0 mg / mL, 2 mL). The RP-HPLC-MS results are shown in Figure 4, where A and B are HPLC results, and C and D are MS results. The RP-MS calculated average: n = 7.9; MS results showed that the light chain (L) was linked to one linker-payload and the heavy chain (H) was linked to three linker-payloads.

[0167] Example 12: Preparation of IP140B Antibody-Drug Conjugate ADC5

[0168] Take IP140B antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 7.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 4 h.

[0169] Compound 23 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. Mix thoroughly and allow to react at room temperature for 16 hours on a rotating disk. After completion, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH 6.2, to obtain antibody-drug conjugate ADC5 (3.2 mg / mL, 2 mL). The RP-HPLC-MS results are shown in Figure 5, where A and B are HPLC results, and C and D are MS results. The RP-MS calculated average: n = 7.8; the MS results showed that the light chain (L) was linked to one linker-payload and the heavy chain (H) was linked to three linker-payloads.

[0170] Example 13: Preparation of HS627 Antibody-Drug Conjugate ADC6

[0171] HS627 antibody (10.0 mg / mL, 10 mg, 0.066 mmol) was taken and the pH was adjusted to 7.2 with 1 M Na2HPO4 solution. Then, 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL) was added, and the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL) was added. The reaction was carried out at room temperature at 25°C with a rotating turntable for 4 h.

[0172] Compound 23 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. Mix thoroughly and allow to react at room temperature for 16 h on a rotating disk. After completion, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH 6.2, to obtain antibody-drug conjugate ADC6 (3.0 mg / mL, 2 mL). The RP-HPLC-MS results are shown in Figure 6, where A and B are HPLC results, and C and D are MS results. The RP-MS calculated average: n = 7.8; MS showed that the light chain (L) was attached to one linker-payload, and the heavy chain (H) was attached to three linker-payloads.

[0173] Example 14: Preparation of Nectin-4 Antibody-Drug Conjugate ADC7

[0174] Take nectin-4 antibody (10.0 mg / mL, 10 mg, 0.066 mmol), adjust the pH to 6.2 with 1 M Na2HPO4 solution, then add 0.1 M disodium ethylenediaminetetraacetic acid solution (25 μL), add the prepared TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) solution (10 mM, 0.04 mL), and react at room temperature 25°C on a rotating turntable for 4 h.

[0175] Compound 23 (0.99 mg, 0.93 mmol) was dissolved in 0.1 mL of DMA and added to the above solution. Mix thoroughly and allow to react at room temperature for 16 h on a rotating disk. After completion, small molecules were removed using a NAP-5 gel column (Cytiva), and the buffer was replaced with a 20 mM histidine-histidine hydrochloride solution, pH 6.2, to obtain antibody-drug conjugate ADC7 (3.0 mg / mL, 2 mL). The RP-HPLC-MS results are shown in Figure 7, where A and B are HPLC results, and C and D are MS results. The RP-MS calculated average: n = 7.9; the MS results showed that the light chain (L) was linked to one linker-payload and the heavy chain (H) was linked to three linker-payloads.

[0176] Test Example 1: In vivo tumor growth inhibition activity of ADC

[0177] ADC nude mouse xenograft (cell-derived xenograft, CDX) tumor inhibition activity test method: 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 monolayer in vitro. When the cell saturation reached 80%-90%, they were digested with trypsin-EDTA, centrifuged and the supernatant was discarded. The cells were resuspended in PBS and the cell suspension was adjusted to an appropriate concentration. BXPC-3, NCI-H1975, MDA-MB-231, and KYSE-150 cells (2-10×10 6 cells / 0.1 mL) were subcutaneously inoculated into BALB / c nude mice, and the animals and transplanted tumor growth were observed regularly. 3 Animals were randomly divided into groups of 6 per group based on tumor volume and body weight. The drugs were administered intravenously at regular intervals. Tumor long diameter (a) (mm), short diameter (b) (mm ... 2 (mm 3), where a and b represent tumor length and width, respectively. A growth curve was plotted, and the tumors were removed and weighed. Statistical analysis was performed using GraphPad Prism software based on the data of tumor volume and weight of tumor-bearing mice at the end of the experiment to obtain the tumor inhibition results.

[0178] Main evaluation indicators:

[0179] Tumor growth inhibition (TGI):

[0180] TGI (%) = [1-(avT i-0 / avC i-0 )]×100%; where avT i-0 is the average tumor volume of the dosing group on a specific day, minus the average tumor volume of the dosing group on the day dosing was started; where avCi-0 is the average tumor volume of the vehicle control group on a specific day, minus the average tumor volume of the vehicle control group on the day dosing was started.

[0181] 1. BXPC-3 CDX model

[0182] BIW was administered twice a week on days 0, 3, 7, 10, 14, and 17, and the drug was observed on day 33.

[0183] Among them, IgG1 is a monoclonal antibody tool sequence with no target selectivity and has no affinity for human cells.

[0184] Compound 10 is the toxin (antitumor compound) portion of compound 15, and its structural formula is

[0185] Experimental results: The three doses of ADC1, 0.8 mg / kg, 2 mg / kg and 5 mg / kg, all showed stronger tumor growth inhibition activity than gemcitabine, single antibody, single toxin, simple mixture of antibody and toxin, and ADC without targeting effect, and had dose-dependent inhibitory activity.

[0186] 2. NCI-H1975 model

[0187] BIW was administered twice a week on days 0, 4, 7, and 11, and the drug was observed until the 21st day.

[0188] Experimental results: All three experimental doses (1 mg / kg, 3 mg / kg, and 6 mg / kg) demonstrated stronger tumor growth inhibition activity than simple mixtures of antibodies and toxins, as well as non-targeted ADCs. The 1 mg / kg ADC1 group exhibited even greater tumor suppression than 10 mg / kg of paclitaxel. The 3 mg / kg and 6 mg / kg doses almost completely inhibited tumor growth, with 50% of tumor-bearing mice in the 6 mg / kg group experiencing complete tumor disappearance.

[0189] 3. MDA-MB-231 CDX model

[0190] BIW was administered twice a week on days 0, 3, and 7, and the drug was observed until the 25th day.

[0191] The structural formula of IP140B-Dxd is (where n=7.89):

[0192] Experimental results: Two doses of ADC1 (3 mg / kg and 6 mg / kg), ADC5 (3 mg / kg), and two doses of ADC2 (6 mg / kg and 12 mg / kg) all showed stronger tumor growth inhibition activity than Paditaxel, a simple mixture of antibodies and toxins, and ADCs without targeting.

[0193] KYSE-150 CDX model

[0194] BIW was administered twice a week on days 0, 3, 7, and 10, and the drug was observed until the 21st day.

[0195] Experimental results: The three doses of ADC1 (1 mg / kg, 3 mg / kg and 6 mg / kg) and the two doses of ADC2 (6 mg / kg and 12 mg / kg) all showed stronger tumor growth inhibition activity than paclitaxel and ADCs without targeting effects.

[0196] Test Example 2: ADC cynomolgus monkey toxicology study

[0197] 1. Single-dose ADC to investigate dose tolerance, pharmacokinetics, and toxicity

[0198] After a single intravenous infusion of ADC in crab-eating monkeys, the pharmacokinetic properties of the drug in monkeys were investigated and the toxicity of the animals was observed.

[0199] Test method:

[0200] This study set up two groups, namely 22 / 35mg / kg and 60mg / kg groups, with 2 monkeys in each group, half male and half female (the first and second doses of the low-dose group were 22mg / kg, and the third and fourth doses were 35mg / kg). Each group of crab-eating macaques were intravenously injected with the corresponding concentration of ADC preparation at a dosage volume of 5mL / kg. The ADC preparation was administered once every 2 weeks for 2 consecutive times. The third dose was administered 22 days after elution recovery, and the fourth dose was administered 2 weeks later. The day of the first dose of the 22 / 35mg / kg group was defined as the first day of the study, and the day of the first dose of the 60mg / kg group was defined as the fifth day of the study. The general condition of the cynomolgus monkeys was observed every day during the dosing period, and their body weight and food intake were measured once a week. Three days after the first dose, venous blood was collected from each group of cynomolgus monkeys and on the 19th, 42nd, 56th, and 66th days of the experiment, as well as from the 22 / 35 mg / kg group of cynomolgus monkeys for hematological and blood biochemical examinations. On the 66th day of the final experiment, the 22 / 35 mg / kg group of cynomolgus monkeys were anesthetized and euthanized, and gross anatomical observations, organ weight measurements, and bone marrow smears were performed. Histopathological examinations were performed on the heart, liver, spleen, lungs, kidneys, ovaries, cervix, epididymis, inguinal lymph nodes, and administration sites (including blood vessels) of each group of cynomolgus monkeys.

[0201] Pharmacokinetics: Following single intravenous infusions of different doses of the ADC in cynomolgus monkeys, blood samples were collected at multiple time points. Blood samples were collected from the upper limb vein in the 22 / 35 mg / kg groups before the first and third doses, 0.0167 hours after the second dose, and 0.167, 4, 24, 48, 72, 96, 168, and 336 hours after the first, third, and fourth doses. Serum ADC-TAB concentrations were determined by ELISA, and plasma small molecule toxin concentrations were determined by LC-MS / MS. Toxicokinetic parameters such as AUClast were calculated.

[0202] Toxicity study: After a single intravenous infusion of different doses of ADC drugs in crab-eating macaques, the animals' tolerance and drug-related toxicity were evaluated through clinical observation, body weight and food intake, hematology, blood biochemistry, urine, gross anatomy, and other aspects.

[0203] Test results:

[0204] During the trial, the cynomolgus monkeys were in good general condition, with normal autonomous activities and no obvious toxic reactions. During the dosing period, no significant abnormal changes in food intake were observed in male and female cynomolgus monkeys in the 22 / 35 mg / kg groups. The weight of the cynomolgus monkeys remained normal after two consecutive doses of 22 mg / kg. No significant changes in weight were observed after two consecutive doses of 35 mg / kg. Three to four days after the first dose, the food intake and weight of the cynomolgus monkeys in the 60 mg / kg group decreased slightly.

[0205] At any time point during the experiment, no obvious abnormal changes were observed in the blood biochemical parameters of male and female crab-eating macaques in each group.

[0206] Pharmacokinetics: After a single intravenous infusion of ADC in cynomolgus monkeys, ADC was stable in the blood of cynomolgus monkeys (see Figure 16) and the concentration of free toxin was very low (see Figure 17), indicating that ADC was slowly released in cynomolgus monkeys and the coupling mode was stable.

[0207] Toxicity manifestations: After a single intravenous infusion of ADC in crab-eating macaques, the animals tolerated it well and showed no severe or intolerable drug-related toxicity, indicating that the safety of ADC is controllable and can support its further clinical research.

[0208] 2. Repeated administration to investigate possible toxic organs and pharmacokinetic parameters

[0209] The toxicity study was conducted in crab-eating monkeys with intravenous injection once every 3 weeks for 6 consecutive weeks (3 times in total). The nature, extent, dose-effect, time-effect relationship and reversibility of the toxic reactions that may be caused by the test article were observed, the toxic target organs or target tissues were determined, and its toxicokinetic characteristics were studied. The effects on the cardiovascular system, central nervous system, respiratory function and administration site were also examined to provide a reference for the safety of clinical use.

[0210] Test method:

[0211] This study consisted of four groups: a control group (ADC5 formulation buffer) and ADC5@9, 18, and 36 mg / kg groups, each consisting of 10 cynomolgus macaques, half male and half female. Each group received an intravenous injection of the control substance (ADC5 formulation buffer) or the corresponding concentration of ADC5 at a volume of 5 mL / kg. Dosing was repeated once every three weeks for six consecutive weeks, for a total of three doses. The drug was then discontinued and observed for six weeks. The first day of the dosing period was designated as day 1, and the day after the last dose was designated as day 1 of the recovery period.

[0212] Blood samples were collected from each group of crab-eating macaques before the first and last doses, immediately after the end of the dose (0-2 minutes after the end of the dose), and 4, 8, 24, 48, 72, 96, 168, 240, 336, and 504 hours after the dose; 672 hours after the last dose, and the concentrations of ADC-TAB (total antibody) and ADC (antibody-drug conjugate) in the monkey serum at each time point were detected by ELISA. The concentration of free toxin in the crab-eating macaque plasma at each time point was detected by LC-MS / MS, and toxicological parameters such as AUC were calculated.

[0213] Test results:

[0214] During dosing, some male and female cynomolgus monkeys in the 18 and 36 mg / kg groups showed decreased food intake. Otherwise, no significant changes in food intake were observed in the 9 mg / kg group or in body weight in the ADC5 groups during the dosing and recovery periods.

[0215] No arrhythmias were observed in lead II electrocardiograms of male and female cynomolgus monkeys in all ADC5 groups 4-5, 24, 72, 168, 336, and 504 hours after the first dose, 4 hours after the last dose, and at the end of the recovery period. No significant abnormalities were observed in lead II electrocardiograms (ECGs) of heart rate, P wave duration, PR interval, QRS duration, QT interval, RR interval, and corrected QT interval, as well as in blood pressure and respiratory tests. No significant abnormalities were observed in various blood biochemical indices, urine indices, lymphocytes, cytokines, bone marrow smears, or organ function tests.

[0216] Pharmacokinetics:

[0217] Under the conditions of this study, cynomolgus monkeys received intravenous injections of ADC5 at doses of 9, 18, and 36 mg / kg every three weeks for six consecutive weeks (three doses total), followed by a six-week recovery period. ADC5 was stable in the monkeys' blood (see Figure 18), and free toxin concentrations were very low (see Figure 19), demonstrating slow release of the ADC and a stable conjugation mechanism in the monkeys, supporting a Q3W dosing schedule for clinical use.

[0218] Toxicity manifestations: After repeated intravenous infusion of ADC in crab-eating macaques, the animals tolerated it well. Mild manifestations such as occasional reduction in food intake were recovered, and no severe or intolerable drug-related toxicity was shown, indicating that the safety of ADC is controllable and can support its further clinical research.

Claims

1. Antibody-drug conjugate represented by formula (I): In the formula, R 2 is selected from hydrogen, deuterium, C1-C6 alkyl, C1-C6 alkoxy, acyl, sulfonyl; L 1 Selected from -L 11 -L 12 -L 13 -;in, L 11 , L 12 , L 13 are each independently selected from absent, -C=O-, C1-C2 alkylene or C1-C2 alkylene-O-, provided that when L 11 When -C=O-, R 2 Not hydrogen; L 2 is selected from C1-C6 alkylene or C1-C6 acyl, wherein the C1-C6 alkylene or C1-C6 acyl is optionally substituted by one or more R 3 replace; R 3 Selected from phenyl-substituted or unsubstituted C1-C6 alkyl, C1-C6 alkoxy; L P is a peptide residue consisting of 1-7 amino acids; Z is selected from -L z -L j -, where L z Selected from absent, -C(=O)-C1-C8 alkylene, -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=O)-(CH2) 0- 6-NR 1 (CH2) 0-6 -;L j A linker that can be coupled to an antibody; R 1 Selected from -C1-C6 alkyl-carboxyl or -C1-C6 alkyl-amino; Ab is an antibody.

2. The antibody-drug conjugate according to claim 1, wherein R 2 is selected from hydrogen, deuterium, C1-C3 alkyl, C1-C3 alkoxy, -C(=O)C1-C3 alkyl or -S(=O)2C1-C3 alkyl; Preferably, R 2 is selected from hydrogen, deuterium, methyl, ethyl, methoxy, ethoxy, formyl, acetyl, methylsulfonyl or ethylsulfonyl; Preferably, R 2 is selected from hydrogen, methyl, methoxy, formyl or methylsulfonyl.

3. The antibody-drug conjugate according to claim 1 or 2, wherein: L 11 , L 12 , L 13 Each is independently selected from absent, -C=O-, -CH2-, -CH2O- or -OCH2-; Preferably, L1 is selected from -C(=O)CH2OCH2-, -C(=O)CH2O-, -C(=O)CH2 or -CH2-; Preferably, L 2 Selected from C1-C3 alkylene; Preferably, L 2 Selected from methylene and ethylene.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein: Lp is selected from a peptide residue 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 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 Selected from 5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein: L j Selected from The positions shown indicate attachment to antibodies. The position shown indicates that the z The groups are connected; Preferably, L z Selected from -C(=O)-C1-C8 alkylene, -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=O)-(CH2) 1-4 -NR 1 (CH2)2-; Preferably, L z Selected from -C(=O)-(CH2CH2O) 2-6 -CH2CH2NH- or -C(=O)-(CH2) 1-4 -NR 1 (CH2)2-; Preferably, R 1 Selected from -C1-C3 alkyl-carboxyl; Preferably, L z Selected from -C(=O)-(CH2CH2O)2-CH2CH2NH- or Preferably, Z is selected from 6. The antibody-drug conjugate according to any one of claims 1 to 5, wherein: The antibody-drug conjugate is obtained by coupling the compound of formula (II) or formula (III) with an antibody: Among them, R 2 , L P In each of the compounds of formula (I), Z' is a linker group that is compatible with Z and can be coupled to an antibody, preferably a maleimide group, a bromoacetyl group or an iodoacetyl group; Preferably, the antibody-drug conjugate releases the following anti-tumor compounds under physiological conditions: Among them, R 21 Selected from C1-C6 alkyl, preferably -C1-C3 alkyl, more preferably methyl; R 22 Selected from C1-C6 alkoxy, preferably -C1-C3 alkoxy, more preferably methoxy; The following anti-tumor compounds are preferably released:

7. The antibody-drug conjugate according to any one of claims 1 to 6, wherein: The antibody is a tumor-associated antigen antibody; Preferably, the tumor-associated antigen is selected from one or more antibodies of 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, lung 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 having an amino acid sequence of SEQ ID NO: 1 or any variant thereof, and a light chain having an amino acid sequence of SEQ ID NO: 2 or any variant thereof; Preferably, the antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 3 or any variant thereof, and a light chain having an amino acid sequence of SEQ ID NO: 4 or any variant thereof; Preferably, the antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 5 or any variant thereof, and a light chain having an amino acid sequence of SEQ ID NO: 6 or any variant thereof; Preferably, the drug-antibody ratio of the antibody-drug conjugate is 2-8, more preferably 3.5-4.5 or 7.5-8.

8. The antibody-drug conjugate according to any one of claims 1 to 7, wherein: The antibody-drug conjugate is obtained by conjugating the following compound to an antibody:

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

10. Use of the antibody-drug conjugate according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of an anti-tumor drug; Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, lung 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 a therapeutically effective amount of the antibody-drug conjugate according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 to a patient in need thereof; Preferably, the tumor is a solid tumor; Preferably, the tumor is selected from breast cancer, lung cancer, colorectal cancer, esophageal cancer, gastric cancer, lung cancer, kidney cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, pancreatic cancer or liver cancer.

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