MMAE-based compound, and antibody-drug conjugates thereof

Abstract

Provided in the present invention are a monomethyl auristatin E (MMAE)-based compound or a pharmaceutically acceptable salt or ester thereof, a solvate or isotopically labeled substance thereof, and antibody-drug conjugates (ADCs) of the compound. Further provided in the present invention are a method for preparing the MMAE-based compound and antibody-drug conjugates thereof, a pharmaceutical composition containing same, and the use thereof.

Description

MMAE-based compounds and their antibody-drug conjugates

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 2024116203813, filed on November 13, 2024, the entire contents of which are incorporated herein by reference. Invention Field

[0003] This invention relates to compounds based on monomethylaurestatin E (MMAE) or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and antibody-drug conjugates (ADCs) of said compounds. The invention also relates to methods for preparing said MMAE-based compounds and their antibody-drug conjugates, pharmaceutical compositions comprising them, and their uses. Background Technology

[0004] With its increasing incidence, cancer has become a major medical challenge for society today. Cytotoxic agents are an important therapy for cancer, but their low selectivity often kills normal cells, causing severe toxic side effects. Biological macromolecular drugs, such as antibodies or antibody fragments, while possessing high targeting specificity, have limited efficacy in treating solid tumors.

[0005] Antibody-drug conjugates (ADCs) are a new class of targeted drugs for treating tumors or cancers. They mainly consist of three parts: an antibody or its antigen-binding fragment (Ab) responsible for selectively recognizing tumor cell surface antigens, a small molecule toxin (Payload) responsible for killing tumor cells, and a linker that connects the Payload to the Ab. ADCs aim to directly deliver the small molecule toxin to tumor target cells by recognizing tumor cell surface antigens with antibodies, thereby exerting a tumor-killing effect. Therefore, ADCs combine the powerful killing effect of traditional small molecule toxin drugs with the tumor-targeting specificity of antibodies.

[0006] While traditional ADC drugs have produced some therapeutic effects, they may release toxins before reaching the target tissue (premature detachment), leading to off-target toxicity and potential dose-limiting toxicity, resulting in poor safety. Furthermore, traditional ADC drugs have low response rates and are prone to drug resistance after a period of treatment, resulting in less than ideal clinical efficacy.

[0007] Monomethylaurestatin E (MMAE) possesses strong microtubule-inhibiting activity and is a highly cytotoxic compound capable of killing cancer cells. However, when used as a single drug to kill tumors, MMAE often produces intolerable side effects. Therefore, MMAE has not been approved as a single drug for cancer treatment, but rather as a small molecule toxin in the development of antibody-drug conjugates. How to modify MMAE to reduce its off-target toxicity and dose-limiting toxicity, while simultaneously achieving effective tumor cell killing, is a pressing problem to be solved in the development of antibody-drug conjugates.

[0008] Therefore, there is a need to develop novel MMAE-based compounds that, when conjugated to antibodies or antigen fragments, possess sufficient stability in the bloodstream and can be released upon reaching tumor target cells to exert tumor-killing effects, while also exhibiting good safety and tolerability. Furthermore, there is a need for highly effective and safe antibody-drug conjugates (ADCs) that possess good blood circulation stability, good tumor targeting, and good tumor-killing effects, while overcoming the drawbacks of traditional ADCs such as drug resistance. Summary of the Invention

[0009] In one aspect, the present invention provides compounds of formula (I) or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof: T a1 --L a2 ——L a3 --L a4 ——D1 (I)

[0010] in,

[0011] D1 has the structure of formula (Ia):

[0012] in,

[0013] R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose, hexuronic acid, of which R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring;

[0014] Rd2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkyl groups, pentoses, penturonic acid, hexoses, hexuronic acid;

[0015] The wavy line represents the rest of the molecule (i.e., T). a1 -L a2 -L a3 -L a24 - Partial) connection;

[0016] T a1 It is a connector unit or H;

[0017] L a2 There is no or a bridging spacer;

[0018] L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C14, C24, C34, C4 ... 1-6 Substitution of alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3, or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection, C end and L a4 Connection or when L a2 If it does not exist, connect it to D1;

[0019] L a4 It either does not exist or is a cuttable connector or a self-destructing connector.

[0020] The inventors have discovered that, compared to conventional MMAEs, the compounds of formula (I) of this invention exhibit significantly improved hydrophilicity. Antibody-drug conjugates (ADCs) using these compounds as linker-payloads demonstrate sufficient stability in the bloodstream. This is because the dual-cleavage mechanism of the compounds of formula (I) reduces the shedding of toxin molecules in the bloodstream, mitigating off-target toxicity and dose-limiting toxicity, resulting in good safety and tolerability. Specifically, some compounds of this invention contain glycoside units, allowing the ADC to release toxin molecules only after reaching the target antigen, further reducing off-target toxicity and dose-limiting toxicity.

[0021] The inventors have also discovered that a dual-toxin ADC drug comprising the compound of the present invention and another toxin exhibits excellent tumor-killing activity and good tolerability, even showing significantly superior tumor-killing activity compared to single-toxin ADC drugs. For ADC drugs, linking two toxin molecules to the same antibody makes the actual tumor-killing effect, safety, and drug resistance unpredictable. For example, it cannot be guaranteed that both toxin molecules will be successfully delivered to tumor target cells and released to exert a tumor-killing effect; it is unclear whether their tumor-killing effects are synergistic, additive, or antagonistic; nor is it clear how to improve their therapeutic index, safety, and tolerability. Moreover, the effective dose and toxic dose of the two toxin molecules are usually different, making it difficult to ensure that both toxin molecules reach the effective dose without reaching the toxic dose. The inventors have discovered that the dual-toxin ADC drug of the present invention has good blood circulation stability, low off-target toxicity, can effectively target tumor cells, release an effective dose of toxin molecules, exert excellent tumor-killing activity, and exhibits good tolerability and low drug resistance. In particular, the dual-toxin ADC drug of the present invention has shown significantly superior tumor-killing effects compared to single-toxin ADCs in a variety of cell and tumor models (e.g., CDX or PDX).

[0022] The substances of the present invention, including linker toxin compounds of formula (I) or antibody-drug conjugates of formula (II) or (III) or their pharmaceutically acceptable salts or esters, solvates or isotope-labeled forms, have high cytotoxicity and good safety and tolerability, and can be used to treat or prevent tumors or cancers, including but not limited to solid tumors or hematologic malignancies or metastatic lesions.

[0023] Therefore, the present invention provides linker toxin compounds of formula (I) or antibody-drug conjugates of formula (II) or (III), or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, methods for their preparation, pharmaceutical compositions comprising them, and their applications. Various aspects of the invention are described in detail below.

[0024] definition

[0025] The terminology and scientific and technical terms used in this invention generally have the meanings commonly understood by those skilled in the art, unless otherwise stated.

[0026] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0027] The term “about” when used in conjunction with a numeric value means a range of numeric values ​​that have a lower limit of 5%, 4%, 3%, 2%, or 1% smaller than the specified numeric value and an upper limit of 5%, 4%, 3%, 2%, or 1% larger than the specified numeric value.

[0028] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0029] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, they also cover situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0030] A hyphen ("-") not between two letters or symbols indicates the linking site of a substituent. For example, -OR4 indicates that the group is linked to the rest of the molecule via an oxygen atom. The "-" may be omitted when the linking site of the substituent is obvious to those skilled in the art (e.g., for halogens, CN, OH, etc.).

[0031] The term "halogen" or "halogenated" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I), preferably fluorine, chlorine or bromine.

[0032] The term "alkyl" alone or as part of other groups refers to a fully saturated straight-chain or branched hydrocarbon group consisting of carbon and hydrogen atoms. Preferably, the alkyl group has 1-6 carbon atoms (C6H ... 1-6 Alkyl groups, 1-4 carbon atoms (C 1-4 Alkyl group or 1-3 carbon atoms (C 1-3 Alkyl groups. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (Pr) (including n-propyl and isopropyl), butyl (Bu) (including n-butyl, isobutyl, sec-butyl and tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl, etc.), hexyl, heptyl, octyl, etc.

[0033] The term "alkylene" refers to an alkyl group as defined above, but which is divalent, i.e. has two single bonds attached to two other groups. Non-limiting examples of alkylene include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0034] The term "alkenyl" alone or as part of other groups refers to a straight-chain or branched hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one double bond. Preferably, the alkenyl group has 2-6 carbon atoms (C6H ... 2-6 alkenyl), 2-4 carbon atoms (C 2-4 Alkenyl group or 2-3 carbon atoms (C 2-3 Alkenyl). Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, allyl, butenyl, isobutenyl, pentenyl, isopentenyl, and hexenyl.

[0035] The term "alkynyl" alone or as part of other groups refers to a straight-chain or branched hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one triple bond. Preferably, the alkynyl group has 2-6 carbon atoms (C6H ... 2-6 acetylsyl group), 2-4 carbon atoms (C 2-4 (alkynyl group) or 2-3 carbon atoms (C 2-3 (Alynyl group). Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, propynyl, butynyl, isobutynyl, penynyl, isopentenynyl, and hexynyl.

[0036] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above, linked by an oxygen atom. Preferably, the alkoxy group has 1-6 carbon atoms (C... 1-6 alkoxy group), 1-4 carbon atoms (C 1-4 alkoxy group or 1-3 carbon atoms (C 1-3 Alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0037] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) halogens. It will be understood that when there is more than one halogen substituent, the halogen substituents may be the same or different, and may be located on the same or different carbon atoms. Preferably, the haloalkyl group is C10. 1-6 Haloalkyl, C 1-4 Halogenated alkyl or C 1-3 Haloalkyl groups. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, chloromethyl, difluoromethyl, dichloromethyl, fluorochloromethyl, trifluoromethyl, trichloromethyl, dichlorofluoromethyl, difluoroethyl, trifluoroethyl, trichloroethyl, difluorochloroethyl, difluoropropyl, and trifluoropropyl.

[0038] The term “haloalkenyl” refers to an alkenyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The term “haloynyl” refers to an ynyl group as defined herein, which is substituted with one or more halogen groups as defined herein. The meaning of “halogenated” as defined for “haloalkyl” may apply to both “haloalkenyl” and “haloynyl”.

[0039] The term "heteroaryl" refers to a monocyclic or bicyclic group having one or more, preferably 1, 2, 3, 4, 5, or 6 heteroatoms independently selected from N, O, or S, with the remaining ring members being carbon, wherein at least one ring is aromatic, and the other rings (if present) may be aromatic or non-aromatic. Any N and S heteroatoms of the heteroaryl group may optionally be oxidized (e.g., in NO, SO, SO2) and said any N heteroatomium may optionally be quaternized (e.g., in [NR]). + Cl - [NR] + OH - (In Chinese). The heteroaryl group is preferably a 5-8 membered heteroaryl group (e.g., monocyclic, nitrogen-containing). Representative examples of heteroaryl groups include, but are not limited to: pyrrole, furanyl, thiophene, pyrazolyl, imidazolyl, oxazolyl, isothiazolyl, thiazolyl, isothiazolyl, triazolyl, pyridinyl, pyrazinyl, pyranyl, thiaranyl, oxazinyl, oxadiazinyl, indole, isoyindole, azaindole (e.g., 7-azaindole, 6-azaindole, 5-azaindole, 4-azaindole), etc. The heteroaryl group can be attached to the rest of the compound via a carbon atom or a heteroatom, provided it is chemically feasible.

[0040] The term "subunit" or "sub...unit" refers to a divalent group derived by removing two hydrogen atoms from a molecule.

[0041] The term "OH" represents a hydroxyl group.

[0042] The term "SH" stands for thiol group.

[0043] The term "NH2" represents amino.

[0044] The term "-CO-" or "-C(=O)-" represents a carbonyl group.

[0045] The term "-SO-" represents a sulfinyl group.

[0046] The term "-SO2-" represents a sulfonyl group.

[0047] The term "-COOH" represents a carboxyl group.

[0048] The term "NO2" stands for nitro.

[0049] The term "leaving group" refers to an atom or functional group that readily detaches from a molecule during a chemical reaction. Examples include, but are not limited to: halogens; such as F, Cl, or Br; sulfonyl groups, such as methanesulfonyl or p-toluenesulfonyl; sulfonyloxy groups, such as alkylsulfonyloxy (e.g., methanesulfonyloxy), trifluoromethylsulfonyloxy, or arylsulfonyloxy (e.g., p-toluenesulfonyloxy); tertiary amine groups (e.g., Me3N). + or Et3N + ); or diazonium salts.

[0050] The term "polyol group" refers to an alkyl group as defined above containing a plurality of (e.g., 2-10, e.g., 3, 4, 5, 6, 7, or 8) hydroxyl groups, optionally containing one or more (e.g., 2, 3, or 4) other groups (e.g., amino, carbonyl). Non-limiting examples of "polyol group" include, for example... Preferred

[0051] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. The common amino acids discussed in this article are written according to their usual usage. See, for example, Immunology-A Synthesis (2 nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations well known in the art. For example, amino acids may be selected from phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), and glutamine (Gln).

[0052] The term "pentose," also known as a five-carbon sugar, refers to a monosaccharide containing five carbon atoms. Pentoses include D- and / or L-pentoses. Examples of pentoses include, but are not limited to, xylose, arabinose, ribose, and deoxyribose.

[0053] The term "hexose," also known as a six-carbon sugar, refers to a monosaccharide containing six carbon atoms. Hexoses include D- and / or L-pentoses. Examples of hexoses include, but are not limited to, glucose, galactose, mannose, and fructose.

[0054] The term "penturonic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a pentose sugar as defined above to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyuronic acid and arabinuronic acid.

[0055] The term "hexuronic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a hexose as defined above to a carboxyl group. Examples of hexuronic acids include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0056] The term "disaccharide" refers to a disaccharide formed by two molecules of monosaccharides (such as pentoses and / or hexoses as defined above) through a glycosidic bond. Examples of disaccharides include, but are not limited to, sucrose, lactose, and maltose.

[0057] The term "trisaccharide" refers to a sugar formed by three monosaccharide molecules (such as pentoses and / or hexoses as defined above) through glycosidic bonds.

[0058] The term "glycosidic bond" refers to the chemical bond that connects the sugar and the other hydroxyl-containing compound (such as an alcohol, phenol, or another sugar) to form an acetal derivative through dehydration condensation of the hydroxyl group on the hemiacetal structure of a sugar.

[0059] It should be understood that when referring to pentose, hexose, penturonic acid, hexuronic acid, disaccharide, and trisaccharide as a group in a structure, it refers to a group formed by the dehydration condensation reaction between the hydroxyl group (e.g., at position 1, 2, 3, 4, 5, or 6, preferably position 1) of the corresponding sugar or uronic acid and the hydroxyl group or other group of another molecule. It can also be referred to as pentose, hexose, etc. Preferably, the sugar or uronic acid is covalently linked to other structures or partially via a glycosidic bond.

[0060] The expressions “optional,” “optional,” or “optionally” mean that the event described below may or may not occur, and the expression includes both the occurrence and non-occurrence of the event. For example, “optionally substituted by…” includes both the case where the event is not substituted and the case where it is substituted. “Optional substituent” indicates that the substituent may or may not be present.

[0061] When any variable appears more than once in a structural formula, it is defined independently each time it appears. For example, the expression "optionally substituted with one or more substituents independently selected from..." means substitution by one or more independently selected substituents, which can be the same or different. Combinations of substituents and / or variables are permitted, as long as a stable compound is produced.

[0062] The term "substance of the invention" includes linker toxin compounds of formula (I) or its sub-forms, antibody-drug conjugates of formula (II) or (III) or their sub-forms, or their salts or esters, solvates (e.g., hydrates), tautomers, stereoisomers, prodrugs, metabolites, or isotope-labeled substances (e.g., deuterated or tritated). In some embodiments, "substance of the invention" specifically refers to the compounds or conjugates of the embodiments, or their salts or esters, solvates (e.g., hydrates), tautomers, stereoisomers, prodrugs, metabolites, or isotope-labeled substances (e.g., deuterated or tritated).

[0063] In this document, the designation of a general formula includes the general formula itself, any subformulation thereof, and any embodiment falling within the scope of the general formula. For example, the designation of formula (II) includes formulas (IIa), (II-1) to (II-11), and any embodiment falling within the scope of formula (II).

[0064] The term "medicinal" refers to a substance or composition that, when administered to animals such as humans, does not produce adverse reactions, allergic reactions, or other unwanted reactions.

[0065] The term "medicinal salt" refers to those salts that retain the biological effectiveness of the substances of the present invention and are not biologically or otherwise undesirable. The substances of the present invention can be in the form of salts, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts. Acid addition salts can be formed from inorganic or organic acids. Inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, and perchloric acid. Organic acids include formic acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, glutaric acid, adipic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, dihydroxynaphthaleneic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, xylenesulfonic acid, hydroxyethanesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, salicylic acid, oleic acid, nicotinic acid, palmitic acid, stearic acid, furoic acid, hippuric acid, orotic acid, and pyruvic acid. Base addition salts can be formed from organic or inorganic bases, including but not limited to alkali metal salts such as lithium, sodium, or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N-group. Salts can be synthesized from parent compounds using conventional methods.

[0066] Pharmaceutical salts are preferred. However, other salts may also be useful, for example, in separation or purification steps, and can be used during preparation, and are therefore included within the scope of this invention.

[0067] As used herein, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans).

[0068] The term "tautomer" refers to structural isomers with different energies that can interconvert through low energy barriers. For example, proton tautomers (also known as proton shift tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons.

[0069] In this invention, solid lines, solid wedges, or imaginary wedges can be used to depict the valence bonds in the substances of this invention. Solid lines connecting to asymmetric carbon atoms are intended to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or imaginary wedges connecting to asymmetric carbon atoms are intended to indicate the presence of the indicated stereoisomers. In racemic mixtures, solid and imaginary wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the substances of this invention can exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof. The substances of this invention can exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0070] This invention also includes prodrugs of the substances of the invention. The term "prodrug" refers to a chemically modified active or inactive compound that, upon administration to an individual, undergoes physiological processes in vivo (e.g., hydrolysis, metabolism, etc.) to be converted into the substances of the invention. Therefore, in this invention, the term "administration" includes administering a prodrug of the substances of the invention to treat various diseases or conditions, wherein the prodrug is converted into the substances of the invention in vivo. Techniques for manufacturing and using prodrugs are well known to those skilled in the art.

[0071] This invention also includes all pharmaceutically acceptable isotopic labels that are identical to the substances of this invention, but in which one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number than the dominant atomic mass or mass number found in nature. Examples of isotopes suitable for incorporation into the substances of this invention include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H); carbon isotopes (e.g., H); 11 C 13 C and14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 35 S). Designations of compounds of the present invention include their isotopic labels. Similarly, designations of elements or groups also include their isotopes or isotopic labels. For example, the designation "H" or "hydrogen" includes H. 1 H 2 and H 3 .

[0072] This invention also includes metabolites of the substances of this invention, i.e., substances formed in the body after administration of the substances of this invention. These can be produced by, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, etc. Therefore, this invention includes metabolites of the substances of this invention, including compounds obtained by methods that expose the substances of this invention to mammals for a time sufficient to produce their metabolites.

[0073] Some of the substances of this invention can exist in non-solventized and solvated forms, including hydrates. The term "solvent" refers to a complex of solvent molecules with a substance of this invention. Examples of solvents that form solvates include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecules are water. Methods of solvation are well known in the art.

[0074] As used herein, the term "linker" refers to a linker that can be used to covalently link a small molecule toxin or payload to an antibody or its antigen-binding fragment (Ab), and in this invention may be represented as T. a1 -L a2 -L a3 -L a4 -Partial, L a1 -L a2 -L a3 -L a4 -Partial, L b Parts, etc.

[0075] The term "joint unit" refers to a component of a connector (L). a1 or T a1It has reactive groups that can form covalent bonds with functional groups of antibodies or antibody fragments. The role of the linker unit is to attach the linker, or the linker as part of a drug linker conjugate, to the antibody or its antigen-binding fragment.

[0076] As used herein, the term "bridging spacer" refers to one or more linker components covalently attached together to form a divalent moiety, wherein the divalent moiety comprises a divalent amino acid or peptide spacer, etc. (e.g., L...). a3 It is attached to a reactive group or a coupling group.

[0077] The term "cleavable linker" refers to a linker that can utilize the differences in conditions between the bloodstream and tumor cells (e.g., pH, protease hydrolysis, or glutathione concentration) or the specific action of antibodies within lysosomes (e.g., lysosomal proteolytic enzymes) to cleave and release the payload, thereby exerting drug activity. Cleavable linkers include pH-sensitive linkers (e.g., hydrazone linkers), glutathione-sensitive linkers (e.g., disulfide linkers), and enzyme-cleavable linkers (e.g., short peptide linkers, β-glucuronic acid linkers, β-galacturonic acid linkers), etc.

[0078] The term "suicide spacer" refers to a spacer that is embedded between a cleavable linker and a small molecule drug, or is itself part of a cleavable linker. It is capable of spontaneous structural rearrangement, releasing the small molecule drug. Examples of suicide spacers include, for example, the p-aminobenzyloxycarbonyl (PABC) or m-aminobenzyloxycarbonyl classes.

[0079] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a substance obtained by conjugating a small molecule drug payload to an antibody or its antigen-binding fragment (Ab, responsible for targeting function and sometimes also having biological activity) via a linker, including its pharmaceutically usable salts or esters, solvates, isotope labels, stereoisomers, tautomers, etc., unless otherwise indicated or the context is clearly contradictory.

[0080] The term “drug:antibody ratio” or “DAR” refers to the ratio of the drug portion (D1 or D2) to the Ab portion conjugated herein.

[0081] In some embodiments described herein, the DAR value of the ADC molecule is determined by m or n in formula (II) or formula (III). For example, DAR can be an integer from 1 to 20, such as 2-20, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0082] In other embodiments, the DAR value is calculated as the average DAR value of a molecular population in the product, i.e., the overall proportion of the drug portion (D1 or D2) coupled to the Ab portion described herein to the Ab portion in the product, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC). This DAR is referred to herein as the average DAR or the measured DAR. For example, the average DAR can be any value between 0.5 and 20.0. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules containing ADC molecules having the same and / or different DARs. In some embodiments, the ADC of the present invention is in the form of an antibody-drug conjugate comprising one or more of formulas (II) and / or (III) or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled composition thereof.

[0083] The term "individual" or "patient" refers to an animal, preferably a mammal. Examples of individuals include, but are not limited to, primates (e.g., humans and non-human primates such as monkeys), horses, cattle, sheep, cats, dogs, rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual is a person, including children, adolescents, or adults.

[0084] The term "treatment" means (i) treating or preventing a particular disease, symptom, or disorder; (ii) reducing, improving, or eliminating one or more symptoms of a particular disease, symptom, or disorder; and optionally (iii) preventing or delaying the onset of one or more symptoms of a particular disease, symptom, or disorder described herein. In some embodiments, "treatment" means improving at least one bodily parameter, which may not be perceptible to the patient. In other embodiments, "treatment" means regulating a disease or symptom from a physical (e.g., stabilizing perceptible symptoms) or physiological (e.g., stabilizing bodily parameters) or both.

[0085] The term "prevention" refers to the administration of one or more pharmaceutical substances, particularly the compounds of the present invention and / or their pharmaceutically acceptable salts, to an individual with a predisposition to the disease or condition, in order to prevent the individual from contracting the disease.

[0086] The terms “inhibition” and “reduction” refer to the reduction or inhibition of a specific patient, symptom, condition, or disease, or a significant reduction in the baseline activity of a biological activity or process.

[0087] The term "effective dose" refers to the amount of medication required to achieve a desired therapeutic or preventative effect at the necessary dosage and for the required duration. It can be determined by the physician or veterinary practitioner involved and will vary depending on factors such as the compound, the state of the disease being treated, the severity of the disease, the individual's age and relevant health conditions, the route and form of administration, and the judgment of the attending physician or veterinary practitioner. Generally, the "preventive effective dose" will be less than the "therapeutic effective dose."

[0088] The term "preventive effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because the prophylactic dose is administered to the subject before or at an early stage of the disease, the preventive effective dose will be less than the therapeutic effective dose.

[0089] The term "therapeutic effective amount" refers to the amount that, at the required dose and sustained for the required period of time, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or antibody fragment or ADC or composition or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, the "therapeutic effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, and more preferably at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0090] The terms "formulation" or "pharmaceutical composition" refer to a composition suitable for administration to animals, preferably mammals (including humans), comprising at least one active ingredient and at least one inactive ingredient, such as a pharmaceutically acceptable excipient. The formulations of this invention can be any formulation applicable in the art, such as tablets, capsules, liquid formulations, etc.

[0091] The term "pharmaceutical excipient" refers to components in a pharmaceutical preparation other than the active ingredient, including physiologically compatible fillers, solvents, dispersion media, isotonic agents, and absorption delay agents. Examples of pharmaceutically acceptable excipients include, but are not limited to, binders, disintegrants, lubricants, solvents, dispersion media, buffers, excipients, antioxidants, preservatives, or flavoring agents. For more information on pharmaceutical excipients, please refer to "Handbook of Pharmaceutical Excipients," 8th edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago.

[0092] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) antibodies of the present invention or their immunoconjugates such as ADC molecules, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. In some embodiments, the antibodies of the present invention or their immunoconjugates such as ADC molecules and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in separate formulations, and their formulations may be the same or different.

[0093] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0094] When referring to chemical reactions, “processing,” “contacting,” and “reaction” mean the addition or mixing of two or more reagents under appropriate conditions to produce the shown and / or desired product. It should be understood that the reaction producing the shown and / or desired product may not necessarily originate directly from the combination of the two initially added reagents; that is, one or more intermediates may be present in the mixture that ultimately lead to the formation of the shown and / or desired product.

[0095] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants). In some embodiments, the therapeutic agent is an antitumor compound.

[0096] The term "cytotoxic agent" in this invention refers to a substance that inhibits or prevents cell function and / or causes cell death or damage.

[0097] The terms “toxin,” “drug component,” “payload,” and “antibody-drug conjugate” used herein are used interchangeably to refer to the component of an antibody-drug conjugate or drug-linker conjugate responsible for killing tumor cells. After administration, the ADC undergoes lysis between or within target cells, releasing the toxin or its derivatives, thereby exerting its biological activity. To avoid ambiguity, it should be noted that “drug” does not only refer to “medicines” approved by pharmaceutical regulatory authorities, but should also include any compound with potential biological activity in clinical practice or in research and development and academic studies.

[0098] In this document, the term "antibody" encompasses any molecule that specifically recognizes and binds to an antigen. This term covers a wide range of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), chimeric or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0099] The terms "complete antibody," "full antibody," or "full-length antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the full-length antibody heavy chain typically consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The full-length antibody light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in the binding of antibody to antigen, but exhibits a variety of effector functions. In some embodiments, the antibody heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of IgG1.

[0100] The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0101] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It contains a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. Fv fragments consist of the VL and VH domains of the antibody's single arm. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain by using a recombinant approach, connecting the two domains with a synthetic linker peptide, and pairing the VL and VH regions in the single protein chain to form a single-chain Fv (scFv).

[0102] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing an immunoglobulin heavy chain variable domain (VH), a heavy chain constant domain (CH1), a light chain variable domain (VL), and a light chain constant domain (CL). One polypeptide chain contains VH and a constant region selected from CH1 and CL from its N-terminus to its C-terminus, while the other polypeptide chain contains VL and another constant region selected from CL and CH1 from its N-terminus to its C-terminus. The VH and VL domains pair to form an antigen-binding site. In this document, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain," and correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain."

[0103] The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide that extends from EU position 118 to EU position 220 (EU numbering system).

[0104] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimer Fc, for example, two different Fc regions can heterodimerize to form a heterodimeric Fc. In this document, the terms “Fc region,” “Fc portion,” and “dimeric Fc (e.g., heterodimeric Fc)” do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the Fc region of the human IgG heavy chain extends from Asp221 or from Cys226 or from Asp231 to the carboxyl terminus of the heavy chain.

[0105] As used herein, when referring to amino acid positions in domains other than the variable region of an antibody (e.g., constant regions, such as the Fc region), the numbering follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. When a position number and / or amino acid residue is assigned to a particular antibody isotype, it is intended to be applicable to the corresponding position and / or amino acid residue in any other antibody isotype, as is known to those skilled in the art.

[0106] General information about the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0107] The term "amino acid substitution" or "amino acid mutation" refers to replacing at least one amino acid residue in a predetermined parental amino acid sequence with a different "substituted" amino acid residue. This substituted residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).

[0108] The position of the amino acid to be mutated to cysteine ​​is generally indicated by "chain type, mutation position". In this article, unless otherwise specified, LLC represents the lambda light chain, LC represents the kappa light chain, and HC represents the heavy chain. Therefore, "LLC160C" means that the amino acid at position 160 in the EU of the lambda light chain is replaced by cysteine ​​(C). "HC239C" means that the amino acid at position 239 in the EU of the heavy chain is replaced by cysteine ​​(C).

[0109] When referring to the position of an amino acid in the heavy chain in this invention, unless otherwise specified, it refers to the amino acid position based on the IgG1 heavy chain number. That is, it encompasses the amino acid position based on the IgG1 heavy chain number, as well as the corresponding amino acid positions on other heavy chains. For example, when referring to HC239, it encompasses the 239th amino acid of the IgG1 heavy chain under the EU number, and also the position of the 239th amino acid of that IgG1 in other IgG isoforms.

[0110] "Sequence identity" is defined as the percentage of identical residues in an amino acid sequence variant after aligning sequences and, where necessary, introducing gaps to achieve maximum percentage sequence identity.

[0111] "Parental protein (e.g., parental antibody, parental constant region, or parental Fc region)" refers to a protein containing an amino acid sequence in which one or more amino acid residues are to be replaced by one or more other amino acid residues, such as cysteine ​​residues. Parental proteins can contain natural or wild-type sequences. Parental proteins may have existing amino acid sequence modifications (such as additions, deletions, and / or substitutions) relative to other natural, wild-type, or modified forms of proteins. Parental antibodies can target antigens of interest, such as biologically important peptides. In some embodiments, the parental antibody is an antibody against HER2, such as trastuzumab.

[0112] The term “antitumor” or “antitumor effect” refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0113] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Cancer can be in its early, middle, or late stages, or it can be metastatic.

[0114] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. “Tumor” encompasses solid tumors and hematologic malignancies as well as metastatic lesions. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive when used in this article.

[0115] Linker-payload compounds

[0116] In one aspect, the present invention provides compounds of formula (I) or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof: T a1 ——L a2 ——L a3 ——L a4 ——D1 (I)

[0117] in,

[0118] D1 has the structure of formula (Ia):

[0119] in,

[0120] R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose, hexuronic acid, of which R dm Rdn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring;

[0121] R d2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkyl groups, pentoses, penturonic acid, hexoses, hexuronic acid;

[0122] The wavy line represents the rest of the molecule (i.e., T). a1 -L a2 -L a3 -L a4 - Partial) connection;

[0123] T a1 It is a connector unit or H;

[0124] L a2 There is no or a bridging spacer;

[0125] L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C14, C24, C34, C4 ... 1-6 Substitution of alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3, or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection, C end and L a4 Connection or when L a2 If it does not exist, connect it to D1;

[0126] L a4 It either does not exist or is a cuttable connector or a self-destructing connector.

[0127] In some implementations, D1 has the structure of formula (Ib):

[0128] The variables are as defined in this paper.

[0129] In some implementation schemes, R d1 It is H, OH, Where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl group, preferably H.

[0130] In some implementation schemes, R d1 It is H, OH, (For example )or (For example ), where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl group, preferably H.

[0131] In some implementation schemes, R d1 It is H, OH, (For example ), where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl group, preferably H.

[0132] In some implementation schemes, R d1 It is H, OH,

[0133] In some implementation schemes, R d1 It is H, OH,

[0134] In some implementation schemes, R dm R dn and R dp Each is independently H or C 1-4 Alkyl group, preferably H.

[0135] In some implementation schemes, R d2 and R d3 Each independently is: H, C 1-6 Alkyl, C 1-6 Alkoxy,

[0136] In some implementation schemes, R d2 and R d3 Each is C independently1-6 Alkoxy, (For example )or (For example ).

[0137] In some implementation schemes, R d2 and R d3 Each is C independently 1-6 Alkoxy groups, such as C 1-4 Alkyl groups, preferably methoxy, ethoxy, propoxy, or butoxy. In some embodiments, R... d2 and R d3 Each is an methoxy group independently.

[0138] In some implementations, T a1 yes Where Lg is the leaving group, and the wavy line represents the interaction with the rest of the molecule (-L). a2 -L a3 -L a4 -D1 part) connection.

[0139] In some implementations, T a1 yes Where Lg is the leaving group, and the wavy line represents the interaction with the rest of the molecule (-L). a2 -L a3 -L a4 -D1 part) connection.

[0140] In some implementations, T a1 yes For example The wavy line represents the part of the molecule other than (-L). a2 -L a3 -L a4 -D1 part) connection.

[0141] In some implementations, Lg is a halogen, C 1-6 alkylsulfonyl, C 1-6 Halogenated alkyl sulfonyl, optionally C 1-6 Alkyl-substituted C 6-10 Aryl-sulfonyl, C 1-6 Alkylsulfonyloxy, C 1-6 Halogenated alkyl sulfonyloxy, optionally C 1-6 Alkyl-substituted C 6-10 aryl-sulfonyloxy, (C 1-6 Alkyl)3N + Or a diazonium base. For example, Lg is a halogen or C. 1-6Alkyl sulfonyl group. In some embodiments, Lg is F, Cl, Br, or methanesulfonyl. In some embodiments, Lg is methanesulfonyl.

[0142] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CR p R q ) s1 -(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -L M -(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR) p R q ) s1 -(OCH2CH2) t -(CR m R n ) s2 -CO-**;

[0143] Among them, * end and T a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl, L M It is -NH-CO-, -CO-NH-, -CO-, -NH- or -O, and s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0144] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CR p R q ) s1 -(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -NH-CO-(CH2CH2O)t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -CO-NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -CO-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -O-(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR) p R q ) s1 -(OCH2CH2) t -CO-**;

[0145] Among them, * end and T a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl groups, s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0146] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CH2) s1 -(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2)s1 -NH-CO-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -O-(CH2CH2O) t -(CH2) s2 -CO-**; and *-(CH2) s1 -(OCH2CH2) t -CO-**;

[0147] Among them, * end and T a1 Connect, and the **end is connected to L a3 Connect , where s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0148] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CR p R q ) s1 -CO-**;*-(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR) p R q ) s1 -(OCH2CH2) t -CO-**; where the * terminator is related to T. a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl groups, s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0149] In some implementations, L a2 The bridging spacer either does not exist or is selected from the following: *-(CH2) s1 -CO-**;*-(CH2CH2O) t -(CH2) s2 -CO-**; and *-(CH2) s1 -(OCH2CH2) t -CO-**; where the * terminator is related to T. a1 Connect, and the **end is connected to L a3 Connect , where s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0150] In some implementations, L a2 It does not exist or is the formula *-(CR) p R q ) s1 -CO-** bridging spacer, where the * end is connected to T a1 Connect, and the **end is connected to L a3 Connection, R p and R q Each is independently H or C 1-6 Alkyl, preferably H or C 1-4 Alkyl groups, and s1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0151] In some implementations, L a2 It does not exist or is expressed as *-(CH2). s1 -CO-** bridging spacer, where the * end is connected to T a1 Connect, and the **end is connected to L a3 The connection, and s1 are 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6 or 7, more preferably 5 or 6.

[0152] In some implementations, L a2 The following are possible values: -CO-, *-(CH2)-CO-**, *-(CH2)2-CO-**, *-(CH2)3-CO-**, *-(CH2)4-CO-**, *-(CH2)5-CO-**, *-(CH2)6-CO-**, *-(CH2)7-CO-**, or *-(CH2)8-CO-**, preferably *-(CH2)5-CO-**, wherein the * terminus is related to the T terminus. a1 Connect, and the **end is connected to L a3 connect.

[0153] In some implementations, T a1 -L a2 -yes Where s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and the wavy line represents L. a3 connect.

[0154] In some implementations, T a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the wavy line represents L. a3 connect.

[0155] It should be understood that, in this document, the expression "connected to B" includes the case of connecting to B itself, the case of connecting to B's adjacent variables when B does not exist, and the case of connecting to the next adjacent variable when neither B nor the adjacent variable exists, and so on. For example, for L... a2 T a1 -L a2 -or the following -L a1 -L a2 - The expression "with L" a3 "Connectivity" covers L a3 The case of self-connection, when L a3 When it does not exist and L a4 Connection scenarios, and when L a3 and L a4 The case where neither exists when connected to D1. Other similar statements should be understood accordingly.

[0156] In some implementations, L a3 Yes: (i) a short chain containing 1-8 amino acid residues, such as a single amino acid residue or a peptide containing 2, 3, 4, 5, 6, 7 or 8 amino acids, preferably 2, 3 or 4 amino acids, wherein said amino acid is optionally bounded by one or more (e.g. 1, 2, 3 or 4, preferably 1 or 2) C 1-6 Alkyl substitution; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3 or 4, preferably 1 or 2). 1-6 Alkyl substituents; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection (i.e., with T) a -L a2 -Partial connection), C-end and L a4 Connection or when L a4If it does not exist, connect it to D1 (i.e., connect it to -L). a4 -D1 partial connection).

[0157] In some embodiments, each of the amino acids is independently selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His), and threonine (Thr), wherein the amino acid is optionally converted by one or more C 1-6 Alkyl substitution.

[0158] In some embodiments, the amino acid is selected from valine, alanine, glycine, lysine, citrulline, glutamine, glutamic acid, phenylalanine, and leucine, wherein the amino acid is optionally converted by one or more C... 1-6 Alkyl substitution.

[0159] In some embodiments, the amino acid is selected from valine, alanine, glycine, lysine, citrulline, and glutamine, wherein the amino acid (e.g., lysine) is optionally oxidized by one or more C14 groups. 1-6 Alkyl substitution.

[0160] In some embodiments, the amino acid is selected from valine, alanine, glycine, and lysine, wherein the lysine is optionally oxidized by one or more C2O groups. 1-6 Alkyl substitution, preferably N-substitution.

[0161] In some implementations, L a3Selected from: Val, Ala, Gly, Lys, Cit, Gln, Glu, Phe, Leu; Val-Val, Val-Ala, Val-Gly, Val-Lys, Val-Cit, Val-Gln, Val-Glu, Val-Phe, Val-Leu; Ala-Ala, Ala-Gly, Ala-Lys, Ala-Cit, Ala-Gln, Ala-Glu, Ala-Phe, Ala-Leu; Gly-Gly, Gly-Lys, Gly-Cit, Gly-Gln, Gly-Glu, Gly-Phe, Gly-Leu; Lys-Lys, Lys-Cit, Lys-Gln, Lys-Glu, Lys-Phe, Lys-Leu; Cit-Cit, Cit-Gln, Cit-Glu , Cit-Phe, Cit-Leu; Gln-Gln, Gln-Glu, Gln-Phe, Gln-Leu; Glu-Glu, Glu-Phe, Glu-Leu; Ph e-Phe, Phe-Leu, Leu-Leu; Ala-Ala-Ala, Ala-Ala-Asn, Val-Lys-Gly, Gln-Val-Ala, Phe-Ph -NH-(CH2) 2-6 -CO- or absent, wherein the amino acid (preferably lysine) is optionally surrounded by one or more C-. 1-6 Alkyl substitution, preferably N-substitution, and wherein L a3 When L exists a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection (i.e., with T) a1 -L a2 -Connection), and the C end is connected to the L a4 Connection or when L a4 If it does not exist, connect it to D1 (i.e., connect it to -L). a4 -D1- connection). For example, for Val-Ala, it includes the N end of Val connected to L. a2 Connected and Ala's C terminal is connected to L a4 The connection details, and the relationship between Ala's N-terminal and L-terminal. a2 Connected and Val's C end is connected to L a4Connection status.

[0162] In some implementations, L a3 Is Gly, Val-Ala, Val-Cit, Phe-Lys, Val-Lys, Leu-Cit, Val-Lys-Gly, Val-(N6,N6-C 1-6 Alkyl group (-Lys)-Gly, -NH-(CH2) 2-6 -CO- or absent, L is preferred. a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly, or -NH-(CH2). 2-6 -CO-, and L is preferred a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly or -NH-(CH2)2-CO-; where L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connect, and C end is connected to L a4 Connection or when L a4 If it does not exist, connect it to D1.

[0163] In some implementations, L a3 It is -NH-(CH2) 2-4 -CO-, such as -NH-(CH2)2-CO-, -NH-(CH2)3-CO- and -NH-(CH2)4-CO-, preferably -NH-(CH2)2-CO-, wherein L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connect, and C end is connected to L a4 Connection or when L a4 If it does not exist, connect it to D1.

[0164] In some implementations, L a3 yes (Gly) Or -NH-(CH2)2-CO-, where L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1.

[0165] In some implementations, La4 It does not exist or is -NH-CH2-. The right side of the group is connected to D1, and the left side is connected to L. a3 connect.

[0166] In some implementations, L a4 Does not exist or The right side of the group is connected to D1, and the left side is connected to L. a3 connect.

[0167] In some implementation schemes, R L Selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose, hexuronic acid, disaccharide, trisaccharide, and (optionally selected by one or more of C) 1-6 5-8 membered heteroaryl groups substituted with alkyl and nitro groups)-(C 1-4 (alkylene)-O-.

[0168] In some implementation schemes, R L It is H.

[0169] In some implementation schemes, R L It is H. (like ), (like ),or

[0170] In some implementation schemes, R L It is H. (like ),or

[0171] In some implementations, L a4 Does not exist or Terminal C is connected to D1, and terminal N is connected to L. a3 connect.

[0172] In some implementations, L a4 Does not exist or Terminal C is connected to D1, and terminal N is connected to L. a3 connect.

[0173] In some implementations, L a4 yes For example Terminal C is connected to D1, and terminal N is connected to L. a3 connect.

[0174] In some implementation schemes, R d1 Indicates valence bond, L a4 yes R L yes Where R L 1st bit and R d1 Connect and 2 bits with L a4 Connect, and where L a4 The C terminal is connected to D1, and the N terminal is connected to L. a3 connect.

[0175] In some implementation schemes, R d1 Indicates valence bond, L a4 express For example Where L a4 Bit 1 is connected to D1, and bit 2 is connected to R. d1 Connect in a ring, and 3 bits with L a3 connect.

[0176] In some implementations, T a1 ——L a2 ——L a3 ——L a4 —Some of these may be selected from:

[0177] The wavy line indicates a connection to D1, and * indicates a connection to R. d1 connect.

[0178] In some implementations, T a1 ——L a2 ——L a3 ——L a4 —Some of these may be selected from:

[0179] The wavy line indicates a connection to D1, and * indicates a connection to R. d1 connect.

[0180] Understandable, in T a1 -L a2 -L a3 -L a4 -D1 and -L below a1 -L a2 -L a3 -L a4 In -D1, when L a2 L a3and L a4 If any of the variables in the set is missing, the relevant variable is connected to the next adjacent variable. For example, for T... a1 The expression "wavy line represents -L" is used to describe the relationship between the two lines. a2 -L a3 -L a4 "-D1 partial connection" includes via L a2 Connection scenarios, when L a2 When it does not exist, via L a3 Connection scenarios, when L a2 and L a3 When neither exists, via L a4 Connection scenarios, and when L a2 L a3 and L a4 The case where neither of these exists is connected to D1. Other similar statements should be understood in the same way.

[0181] In some embodiments, a compound of formula (I) or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof is provided:

[0182] T a1 -- a2 ——L a3 ——L a4 ——D1 (l)

[0183] in,

[0184] D1 has the structure of formula (Ib):

[0185] in,

[0186] R d1 It is H, OH, (For example );

[0187] R d2 and R d3 Each is C independently 1-4 Alkyl groups, preferably methoxy groups;

[0188] The wavy line represents the rest of the molecule (i.e., T). a1 -L a2 -L a3 -L a4 - Partial) connection;

[0189] T a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the wavy line represents -L. a3 -La4 -D1 connection;

[0190] L a3 yes (Gly) Or -NH-(CH2)2-CO-, where the N-terminus is associated with the L-terminus. a2 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1;

[0191] L a4 Does not exist or (For example ), (For example ),or Preferred is For example Where L a4 When present, the C-terminus connects to D1, and the N-terminus connects to the T-terminus of the molecule. a1 -L a2 -L a3 - Partial connection;

[0192] Or, R d1 Indicates valence bond, L a4 express For example Where L a4 Bit 1 is connected to D1, and bit 2 is connected to R. d1 Linked into rings, and the 3-position with the T of the molecule a1 -L a2 -L a3 - Partial connection.

[0193] In some embodiments, the compounds of formula (I) of the present invention are selected from:

[0194] In some embodiments, the compounds of formula (I) of the present invention are selected from:

[0195] Or its pharmaceutically usable salts or esters, solvates or isotopic labels.

[0196] Antibody-drug conjugates

[0197] In another aspect, antibody-drug conjugates are provided having the compound of formula (I) of the present invention as a linker toxin moiety.

[0198] In one embodiment, an antibody-drug conjugate of formula (II) or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof is provided:

[0199] in,

[0200] Ab is an antibody or its antigen-binding fragment;

[0201] D1 has the structure of formula (Ia):

[0202] in,

[0203] R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose, hexuronic acid, of which R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring;

[0204] R d2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkyl groups, pentoses, penturonic acid, hexoses, hexuronic acid;

[0205] The wavy line represents L. a4 connect;

[0206] L a1 It is a connector unit;

[0207] L a2 There is no or a bridging spacer;

[0208] L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C14, C24, C34, C4 ... 1-6 Substitution of alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3, or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1;

[0209] L a4 There are no non-existent, or the connectors are either cuttable or self-destructive; and

[0210] m can be 1, 2, 3, 4, 5, or 6.

[0211] It should be understood that the expression "wavy line represents L" for D1 is related to... a4 "Connection" includes L a4 The case of self-connection, when L a4 When it does not exist, it is related to the adjacent variable L. a3 Connection scenarios, when L a4 and L a3 When neither exists, the next adjacent variable L a2 Connection scenarios, and when L a4 L a3 and L a2 When neither exists, the next adjacent variable L a1 The connection situation.

[0212] It should also be understood that m refers to the -L affixes of the antibody-drug conjugate molecules of formula (II) that are linked to the Ab. a1 -L a2 -L a3 -L a4 The number of -D1 molecules can also be referred to as the DAR value of ADC molecules.

[0213] In some embodiments, the antibody-drug conjugate of formula (II) of the present invention has the structure of formula (IIa):

[0214] The variables are defined as described in this paper, for example, as defined in equation (I) or (II).

[0215] In some implementations, L a1 yes:

[0216] One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0217] In some implementations, L a1 yes: One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0218] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0219] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0220] It is understandable that the expression L a1 The two "with L" a2 "Connectivity" covers L a2 The case of self-connection, when L a2 When it does not exist and L a3 Connection scenarios, when L a2 and L a3 When neither exists and L a4 Connection scenarios, and when L a2 L a3 and L a4 The case where neither exists when connected to D1. Other similar statements should be understood accordingly.

[0221] In some implementations, -L a1 -L a2 -yes Wherein s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, and t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and the left side of the group is connected to Ab, and the right side is connected to L. a3 connect.

[0222] In some implementations, -L a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the left side of the group is connected to Ab, and the right side is connected to L. a3 connect.

[0223] It is understood that the "right side of the group described" is related to L. a3 "Connectivity" covers L a3 The case of self-connection, when L a3 When it does not exist and L a4 Connection scenarios, and when L a3 and L a4 The case where neither exists when connected to D1. Other similar statements should be understood accordingly.

[0224] In some implementations, -L a1-L a2 -L a3 -L a4 -D1 is selected from:

[0225] The wavy line indicates that the valence key is connected to Ab.

[0226] In some implementations, -L a1 -L a2 -L a3 -L a4 -D1 is selected from:

[0227] The wavy line indicates that the valence key is connected to Ab.

[0228] In some implementations, the antibody-drug conjugate of formula (II) has the following subform:

[0229] Where Ab and m are as defined in this paper.

[0230] In some implementations, the antibody-drug conjugate of formula (II) has the following subform:

[0231] Where Ab and m are as defined in this paper.

[0232] In some implementations, m is 1, 2, 3, or 4. In some implementations, m is 1. In some implementations, m is 2.

[0233] In some embodiments, the antibody-drug conjugates of formula (II) or its sub-forms (including formulas IIa and II-1 to II-11) of the present invention are in the form of a composition comprising one or more of the antibody-drug conjugates or their pharmaceutically acceptable salts or esters, solvates or isotopic labels, and the composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2. In some embodiments, the antibody-drug conjugates of formula (II) or its sub-forms of the present invention, or the composition comprising one or more of the antibody-drug conjugates, have an average DAR selected from any value within the range of 1 ± 0.4, 2 ± 0.4, 3 ± 0.4 or 4 ± 0.4. Preferably, the antibody-drug conjugates of formula (II) or its sub-forms of the present invention, or the composition comprising one or more of the antibody-drug conjugates, have an average DAR of about 1.0. Further preferably, the antibody-drug conjugates of formula (II) of the present invention, or the composition comprising one or more of the antibody-drug conjugates, have an average DAR of about 2.0.

[0234] In some embodiments, the antibody-drug conjugate of formula (II) of the present invention or a composition comprising one or more of the antibody-drug conjugates are those prepared in the examples.

[0235] In another aspect, antibody-drug conjugates of formula (III) are provided:

[0236] in:

[0237] Ab represents an antibody or its antigen-binding fragment;

[0238] D1 has the structure of formula (Ia):

[0239] in,

[0240] R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose or hexuronic acid, wherein R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring;

[0241] R d2 and R d3Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose or hexuronic acid;

[0242] The wavy line represents L. a4 connect;

[0243] L a1 It is a connector unit;

[0244] L a2 There is no or a bridging spacer;

[0245] L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C14, C24, C34, C4 ... 1-6 Substitution of alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3, or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1;

[0246] L a4 The connectors either do not exist or are cuttable or self-destructive connectors.

[0247] L b It is a connector;

[0248] D2 is the drug component, preferably the anti-tumor drug component;

[0249] m is 1, 2, 3, 4, 5, or 6; and

[0250] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0251] It should be understood that m refers to the -L atom linked to Ab in antibody-drug conjugate molecules of formula (III). a1 -L a2 -L a3 -L a4 The number of -D1s can also be referred to as the DAR value of the ADC molecule with respect to D1. n refers to the number of -Ls attached to Abs in antibody-drug conjugates of formula (III). bThe number of -D2 can also be called the DAR value of the ADC molecule relative to D2.

[0252] In some implementations, the antibody-drug conjugate of formula (III) may have the structure of (IIIa):

[0253] The variables are defined as described herein, for example, for equations (I) and / or (II).

[0254] In some implementations, L a1 L a2 L a3 L a4 D1 and L are each as defined herein in the various embodiments and examples and / or aspects of equation (II). In some embodiments, L a1 yes: One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0255] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0256] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0257] In some implementations, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

[0258] In some implementations, -L a1 -L a2 -yes Where s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and one bit is connected to Ab, and two bits are connected to L. a3 connect.

[0259] In some implementations, -L a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and one bit is connected to Ab, and two bits are connected to L. a3 connect.

[0260] The drug moiety D2 can be any antitumor drug, as long as it can be coupled to Ab via a linker. Inside tumor cells, part or all of the linker can be cleaved, releasing the antitumor drug moiety and thus exhibiting an antitumor effect.

[0261] In some embodiments, drug fraction D2 is an antitumor drug fraction having the same mechanism of action as D1. In other embodiments, drug fraction D2 is an antitumor drug fraction having a different mechanism of action than D1.

[0262] In some embodiments, drug fraction D2 can be a cytotoxic agent, a cell growth inhibitor, or an immunosuppressive agent. For example, drug fraction D2 can be a tubulin inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, an antifolate agent, an antimetabolite, a chemotherapy sensitizer, vinca alkaloids, etc. Preferably, drug fraction D2 is a tubulin inhibitor (including tubulin polymerization enhancers and tubulin polymerization inhibitors), a DNA synthesis inhibitor, or a topoisomerase inhibitor (including topoisomerase I inhibitors and topoisomerase II inhibitors). More preferably, drug fraction D2 is a topoisomerase inhibitor, preferably a topoisomerase I inhibitor.

[0263] In some implementations, drug fraction D2 is a camptothecin compound (e.g., ethiotacone, Dxd, SN38), anorectatine compound such as MMAE or MMAF, maytansine compound such as DM1 and DM4, cazithromycin antitumor antibiotic, or atrazomycin antitumor antibiotic.

[0264] In some implementations, the drug portion D2 has the following structure: -QL 2 -L 1 -D 2a

[0265] in:

[0266] Q is -O-, -S-, or -NR 7 -;

[0267] L 2 It does not exist, *-(C 1-10 alkylene)-C(O)N(R 5 )- or *-(C 1-10 alkylene)-N(R 5 )C(O)-; where * indicates that the end is covalently connected to Q, and R 5 Is it H or C? 1-6 alkyl;

[0268] L 1 Is it non-existent or -(C) 1-10(alkylene)-;

[0269] D 2a It has the structure shown in equation (D-1):

[0270] Among them, R 1 and R 6 Selected from H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 2-6 Haloalkenyl and C 2-6 Halogenated alkynyl group; or R 1 and R 6 Together with the carbon atoms they are attached to, they form 5-9 membered rings, such as 5-8 membered rings;

[0271] R 2 It is H, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OR 4 or -SR 4 ;

[0272] R 3 It is H, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl or -OR 4 ;

[0273] Or R 2 and R 3 Together they form -O(CH2) p O- or -O(CF2) p O-, where p is 1 or 2;

[0274] R 4 Is it H or C? 1-4 Alkyl; and

[0275] R 7 Is it H or C? 1-6 alkyl.

[0276] It should be understood that the wavy bond that intersects the benzene ring in formula (D-1) means that formula (D-1) is connected to -QL in any feasible position. 2 -L 1 -connect.

[0277] In some implementation schemes, D 2a It has the structure shown in formula (D-1a) or (D-1b):

[0278] The remaining symbols (e.g., R) 1 R2 R 3 R 6 As defined in this document. It should be understood that the wavy line represents -QL. 2 -L 1 - The location of the connection.

[0279] In some implementation schemes, D 2a It has the structure shown in equation (D-2):

[0280] Among them, each symbol (e.g., R) 1 R 2 R 3 and R 6 As defined above.

[0281] In some implementation schemes, D 2a It has the structure shown in equation (D-2a) or (D-2b):

[0282] Among them, each symbol (e.g., R) 1 R 2 R 3 R 6 (n1 and n2) are as defined above.

[0283] In some implementation schemes, R 1 and R 6 For H, R 2 C 1-6 Alkyl or C 1-6 Alkoxy, R 3 Halogen, preferably -F.

[0284] In some implementations, -L 2 -L 1 -is-(C 1-6 alkylene)-, *-(C 1-6 alkylene)-C(O)N(R 5 )-(C 1-6 alkylene)- or *-(C 1-6 alkylene)-N(R 5 )C(O)-(C 1-6 (alkylene)-, where * indicates that the terminus is covalently connected to Q; and R 5 Is it H or C? 1-6 alkyl.

[0285] In some implementations, -L 2 -L 1 -is-(C 1-6 Alkylene)-.

[0286] In some implementations, -QL 2 -L 1 - can be -OCH2-CH2-CH2-CH2-, -OCH2-CH2-CH2-, or -NH-. It should be understood that the left side of this group is related to L... b Connect, right side with D 2a connect.

[0287] In some implementations, the drug portion D2 or -QL 2 -L 1 -D 2a Selected from:

[0288] In some implementations, connector L b It has the following structure: -ZEX-

[0289] in:

[0290] Connect Z to Ab, and connect X to D;

[0291] Z is selected from

[0292] Where m a1 and m a2 Independently selected integers from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, or 8;

[0293] The carbonyl group on the right end of Z is covalently connected to E;

[0294] E is a short chain containing 1-10 amino acids, such as a single amino acid residue or a peptide residue containing 2-10 amino acids, wherein the short chain is optionally selected by one or more (e.g., 2, 3, or 4) from C. 1-6 Group substitution of alkyl and polyol groups, wherein the N-terminus of the short chain is covalently linked to Z; and

[0295] X is selected from non-existent, -NH-CH2-, or... Where R a1 Selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose, hexuronic acid, disaccharide and trisaccharide; the left end of the group is connected to E and the right end is connected to D2.

[0296] In some implementation schemes, Z is Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5.

[0297] In some implementation schemes, Z is Where, m a2 It is an integer selected from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, preferably 4, and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 2.

[0298] In some implementations, E is a single amino acid residue or a peptide residue containing 2, 3, or 4 amino acids.

[0299] In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, leucine, tyrosine, lysine, citrulline, serine, aspartic acid, asparagine, isoleucine, arginine, and proline, wherein the amino acid (preferably glutamine or glutamic acid) is optionally substituted with one polyol group and optionally substituted with one carbon atom. 1-6 Alkyl substitution.

[0300] In some embodiments, the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally with one carbon atom. 1-6 Alkyl substitution.

[0301] In some embodiments, the polyol group is

[0302] In some embodiments, the substituted glutamine or glutamic acid has the following structure:

[0303] Preferred Where R 8 Is it H or C? 1-6 alkyl.

[0304] In some implementations, E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, or Where R 8 Is it H or C? 1-6 alkyl.

[0305] It should be understood that the above-mentioned E group is covalently connected to Z through the left-side N-terminus.

[0306] In some implementations, X is -NH-CH2- or

[0307] In some implementation schemes, R a1 Selected from: H,

[0308] In some implementation schemes, R a1 yes

[0309] In some implementations, X is -NH-CH2-,

[0310] In some implementations, connector L b -ZEX- has the following structure:

[0311] In some implementations, connector L b -ZEX- has the following structure:

[0312] It is understood that the left end of the structure is connected to the Ab portion.

[0313] In some implementations, -L b The -D2 part is:

[0314] The wavy line indicates a connection to Ab.

[0315] In some implementations, -L b The -D2 part is:

[0316] The wavy line indicates a connection to Ab.

[0317] In some implementations, -L b -D2 can be derived from:

[0318] In some implementations, -L b -D2 can be derived from:

[0319] NT3 as the L-linker toxin of NT3' b The -D2 portion can be obtained as described in WO2021 / 173773. NT2 is the L-type linker toxin of NT2'. b The -D2 portion can be obtained as described in PCT / CN2023 / 086909.

[0320] In some embodiments, the antibody-drug conjugate of formula (III) of the present invention has the following subform:

[0321] Wherein, Ab is an antibody or its antigen-binding fragment;

[0322] m is 1, 2, 3, 4, 5, or 6; and

[0323] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0324] In some embodiments, the antibody-drug conjugate of formula (III) of the present invention has the following subform:

[0325] Wherein, Ab is an antibody or its antigen-binding fragment;

[0326] m is 1, 2, 3, 4, 5, or 6; and

[0327] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0328] In some embodiments, m is 1, 2, 3, or 4. Preferably, m is 1 or 2. In some embodiments, m is 1. In other embodiments, m is 2.

[0329] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, n is 4. In other embodiments, n is 8.

[0330] In some implementations, m is 1 and n is 4. In some implementations, m is 2 and n is 4. In some implementations, m is 1 and n is 8. In some implementations, m is 2 and n is 8.

[0331] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotopic label, is in the form of a composition comprising one or more of the antibody-drug conjugate or its pharmaceutically acceptable salt or ester, solvate, or isotopic label, and the composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2 for drug portion D1 and an average DAR of 2.0-14.0, preferably 4.0-12.0, more preferably 6.0-10.0 for drug portion D2.

[0332] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR selected from any of the following ranges with respect to D1: 1 ± 0.4, 2 ± 0.4, 3 ± 0.4, or 4 ± 0.4. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of about 1.0 or 2.0 with respect to D1.

[0333] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-formulas of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of about 2.0-14.0, preferably 4.0-12.0, more preferably 6.0-10.0 with respect to the drug moiety D2, for example, about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, and the range with any two of these values ​​as endpoints.

[0334] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 1.0 ± 0.4 or 2.0 ± 0.4 for D1 and an average DAR of 4.0 ± 0.4 or 8.0 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 1 ± 0.4 for D1 and an average DAR of 4 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 2 ± 0.4 for D1 and an average DAR of 4 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of said antibody-drug conjugates, has an average DAR of 1 ± 0.4 for D1 and an average DAR of 8 ± 0.4 for D2. In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of the antibody-drug conjugates, has an average DAR of 2 ± 0.4 for D1 and an average DAR of 8 ± 0.4 for D2.

[0335] In some embodiments, the antibody-drug conjugate of formula (III) or its sub-forms of the present invention, or a composition comprising one or more of the antibody-drug conjugates, are those in the examples.

[0336] The various embodiments and examples described herein, and any combination thereof, are equivalent to the linker toxin compounds and antibody-drug conjugates of the present invention, or their pharmaceutically acceptable salts or esters, solvates or isotope labels, etc. For example, the various embodiments and examples described in formula (I), and any combination thereof, are equivalent to formulas (II) and (III) and their sub-formulas, including formulas (IIa), (II-a1) to (II-a11), (II-1) to (II-11), (IIIa), (III-a1) to (III-a24), (III-1) to (III-24).

[0337] In some embodiments, the Ab in the ADC of the present invention (including those of formula (II) or (III)) comprises a cysteine ​​residue. In some embodiments, in the ADC of the present invention, the Ab is linked to a linker via its cysteine ​​residue (e.g., naturally occurring and / or introduced by a cysteine ​​mutation), for example, the linker is coupled to the Ab via a thiol group of the cysteine ​​residue.

[0338] In some embodiments, the Ab is coupled to a linker via a natural cysteine ​​residue, for example, the linker is coupled to the Ab via the thiol group of a cysteine ​​residue with a disulfide bond open (e.g., random coupling).

[0339] In some embodiments, the Ab is coupled to the linker via a cysteine ​​introduced by a cysteine ​​mutation, for example, the Ab is coupled to the linker via a cysteine ​​residue that is not paired or otherwise part of an intramolecular or intermolecular disulfide bond (e.g., cysteine ​​obtained after a cysteine ​​mutation) (e.g., site-directed coupling), or the linker is coupled to the Ab via the thiol group of a cysteine ​​with a disulfide bond open (e.g., random coupling or site-directed coupling).

[0340] In some embodiments, in the ADC molecule of the present invention, the Ab is coupled to a toxin via a linker (e.g., site-directed coupling) by a cysteine ​​introduced by a mutation in its cysteine, and coupled to another toxin via its native cysteine ​​(e.g., random coupling).

[0341] In some embodiments, the Ab in the ADC molecule of the present invention comprises a cysteine ​​introduced by a cysteine ​​mutation, such that the linker is coupled to the antibody via the thiol group of the mutated cysteine. In some embodiments, the Ab comprises, on its heavy or light chain, one or more non-cysteine ​​residues mutated to cysteine, such that the linker is coupled to the cysteine ​​residue, for example, site-directed coupling. Antibodies or antigen-binding fragments thereof comprising the cysteine ​​mutation suitable for use in the ADC of the present invention are described in detail herein, wherein the mutated cysteine ​​residue is coupled to the linker, for example, site-directed coupling.

[0342] In some embodiments, in the ADC molecule of the present invention, the cysteine ​​residue obtained by the Ab through the following mutation is coupled to the linker:

[0343] (i) Cysteine ​​obtained after a mutation of cysteine ​​at position 160 in one or two Lamda light chain constant regions;

[0344] (ii) Cysteine ​​obtained after a mutation of cysteine ​​at position 239 in one or two heavy chain constant regions;

[0345] (iii) Cysteine ​​obtained by one or two Lamda light chain constant regions after a cysteine ​​mutation at position 160 and cysteine ​​obtained by one heavy chain constant region after a cysteine ​​mutation at position 239.

[0346] (iv) Cysteine ​​obtained by mutation of cysteine ​​at position 160 in one or two Lamda light chain constant regions and cysteine ​​obtained by mutation of cysteine ​​at position 239 in two heavy chain constant regions.

[0347] (v) One or two light chain constant regions, such as the cysteine ​​obtained by the cysteine ​​mutation at position 205 in the Kappa light chain constant region.

[0348] (vi) One or two light chain constant regions, such as the Kappa light chain constant region, where cysteine ​​is obtained after a cysteine ​​mutation at positions 164 and 205, and one or two heavy chain constant regions, where cysteine ​​is obtained after a cysteine ​​mutation at position 170; or

[0349] (viii) One or two light chain constant regions, such as the Kappa light chain constant region, with cysteine ​​obtained after a cysteine ​​mutation at position 164, and one or two heavy chain constant regions with cysteine ​​obtained after a cysteine ​​mutation at position 170.

[0350] In some specific embodiments, the ADC molecule of the present invention contains an Ab with a cysteine ​​mutation comprising two heavy chains and two light chains, and the cysteine ​​obtained through the following mutation is coupled to the linker:

[0351] (i) Cysteines obtained after mutation of cysteine ​​at position 239 in the constant regions of the two heavy chains;

[0352] (ii) A cysteine ​​obtained by a heavy chain constant region after a cysteine ​​mutation at position 239;

[0353] (iii) A cysteine ​​obtained by a Lambda light chain constant region after a cysteine ​​mutation at position 160;

[0354] (iv) Cysteines obtained after the cysteine ​​mutation at position 160 in the constant regions of the two Lambda light chains;

[0355] (v) Cysteine ​​obtained from the cysteine ​​mutation at position 160 in the two Lambda light chain constant regions and cysteine ​​obtained from the cysteine ​​mutation at position 239 in the one heavy chain constant region.

[0356] (vi) Cysteines obtained after mutation of cysteine ​​at position 160 in the constant regions of the two Lambda light chains, and cysteines obtained after mutation of cysteine ​​at position 239 in the constant regions of the two heavy chains.

[0357] (vii) Cysteines obtained after mutation of cysteine ​​at position 205 in the constant regions of the two Kappa light chains;

[0358] (viii) Cysteines obtained after mutations at cysteine ​​positions 164 and 205 in the constant regions of the two Kappa light chains, respectively; and cysteines obtained after mutations at cysteine ​​position 170 in the constant regions of the two heavy chains; or

[0359] (ix) The two Kappa light chain constant regions contain cysteine ​​obtained by cysteine ​​mutation at position 164; and the two heavy chain constant regions contain cysteine ​​obtained by cysteine ​​mutation at position 170.

[0360] Any antibody or antigen-binding fragment mentioned herein, or any cysteine ​​obtained by any cysteine ​​mutation obtained herein, can be used for conjugation with a linker, such as site-directed conjugation, to obtain the ADC molecule of the present invention.

[0361] In some embodiments, the linker toxin-L of the ADCs of the present invention a1 -L a2 -L a3 -L a4 -D1 is covalently coupled to the sulfhydryl group of cysteine ​​in the Ab (natural or modified, e.g., introduced via cysteine ​​mutation). Specifically, the linker toxin -L a1 -L a2 -L a3 -L a4 -D1 is coupled to the sulfhydryl group of cysteine ​​introduced by the cysteine ​​mutation in Ab, preferably by site-directed coupling.

[0362] In some embodiments, the linker toxin-L of the ADCs of the present invention b -D2 covalently couples with the thiol group formed by the interchain disulfide bond of the native or modified cysteine ​​residues of Ab (e.g., the thiol group formed after the interchain disulfide bond of the native cysteine ​​residue in Ab is opened). For example, linker toxin -L b -D2 is coupled to the thiol group generated by the opening of the interchain disulfide bond of the natural cysteine ​​in the Ab, preferably by random coupling.

[0363] Antibodies or antigen-binding fragments thereof suitable for the ADCs of this invention

[0364] In some embodiments, the Abs applicable to the ADCs of the present invention (including those of formula (II) or (III)) are, for example, antibodies or antigen-binding fragments thereof in the formulas listed in the present invention. In some embodiments, the Abs applicable to the ADCs of the present invention specifically bind to antigens. In some embodiments, the antigen is a tumor-associated antigen (TAA), a tumor-specific antigen, or an immune checkpoint molecule. In some embodiments, the antigen is selected from HER1, HER2, CD19, CD20, CD22, CD33, CD38, Trop-2, PSMA, CD3, B7H3, PD-1, PD-L1, CD47, immune-activating molecules such as 4-1BB, CD40, OX40, CEACAM5, etc. In some embodiments, the antigen is HER2. In some embodiments, the antigen is CEACAM5. In some embodiments, the antigen is TROP2. In some embodiments, the antigen is B7H3.

[0365] In some embodiments, the Abs suitable for the ADC of the present invention can be internalized by tumor cells.

[0366] In some embodiments, the Ab suitable for the ADC of the present invention is a cysteine-modified antibody or its antigen-binding fragment. The cysteine ​​modification involves mutating one or more amino acids to cysteine ​​on the heavy or light chain of the antibody or its antigen-binding fragment. The thiol group on the cysteine ​​can undergo a nucleophilic reaction with a small toxin molecule having a maleimide linker, thereby conjugating the toxin molecule to the cysteine ​​to prepare a site-specifically conjugated ADC molecule.

[0367] In some embodiments, the antibody or antigen-binding fragment suitable for the ADC of the present invention is an antibody or antigen-binding fragment having a cysteine ​​mutation, for example, the antibody or antigen-binding fragment having a cysteine ​​mutation in the light chain constant region and / or the heavy chain constant region, such as having an LLC160 cysteine ​​mutation in one or two light chain constant regions, and / or having an HC239 cysteine ​​mutation in one or two heavy chain constant regions. In some embodiments, the antibody or antigen-binding fragment contains a 205C mutation in the light chain constant region, for example as described in CN101065151B, which is incorporated herein by reference in its entirety. In some embodiments, the antibody or antigen-binding fragment contains a 170C mutation and a 220S mutation in the heavy chain constant region, and a 164C mutation and a 214S mutation in the light chain constant region.

[0368] In one aspect, the Ab in the formula applicable to this invention has a cysteine ​​mutation at a relatively hidden site in the constant region, thereby providing better stability and / or hydrophilicity to the ADC containing it. As defined herein, a "cysteine ​​mutation" refers to the substitution of a non-cysteine ​​amino acid for a cysteine ​​in a protein.

[0369] In some embodiments, the Ab is an antibody or antigen-binding fragment thereof having a cysteine ​​mutation in a light chain constant region, wherein the antibody or antigen-binding fragment thereof comprises one or two Lambda light chain constant regions and has a cysteine ​​substitution (LLC160C) at position 160 (EU number) of the Lambda light chain constant region.

[0370] In some embodiments, the Ab is an antibody or antigen-binding fragment thereof having a cysteine ​​mutation in the heavy chain constant region, wherein the antibody or antigen-binding fragment thereof comprises one or two heavy chain constant regions and has a cysteine ​​substitution (HC239C) at position 239 (EU number) of the heavy chain constant region.

[0371] In some embodiments, the Ab suitable for use in this invention comprises a heavy chain constant region and / or a light chain constant region, such as a heavy chain constant region and a light chain constant region. The Ab suitable for use in this invention comprises two heavy chain constant regions and two light chain constant regions. In some embodiments, the Ab suitable for use in this invention is a full-length antibody.

[0372] In some embodiments, the light chain constant region is the (human) Lambda light chain constant region. In some embodiments, the Lambda light chain constant region comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:6 and not containing a cysteine ​​mutation. In some embodiments, relative to the wild-type Lambda light chain, such as the amino acid sequence shown in SEQ ID NO:6, the light chain constant region suitable for said Ab has a cysteine ​​mutation at position 160 (Eu number).

[0373] In some embodiments, the constant region of the Lamda light chain with a cysteine ​​mutation at position 160 contains the amino acid sequence shown in SEQ ID NO:7 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7 and contains the amino acid sequence VKAGVCTTTPS (SEQ ID NO:32).

[0374] In some embodiments, the light chain constant region is the (human) Kappa light chain constant region. In some embodiments, the Kappa light chain constant region comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0375] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG1 heavy chain constant region, for example, containing the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:38, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:38, and not containing an amino acid sequence with a cysteine ​​mutation. In some embodiments, relative to the wild-type human IgG1 heavy chain constant region, such as the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:38, the heavy chain constant region suitable for said Ab has a cysteine ​​mutation at position 239 (Eu number).

[0376] In some embodiments, the heavy chain constant region with a cysteine ​​mutation at position 239 contains the amino acid sequence shown in SEQ ID NO:9 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:9 and contains the amino acid sequence GGPCVFLFP (SEQ ID NO:33).

[0377] In some implementations, the Ab includes

[0378] (i) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160;

[0379] (ii) One or two heavy chain constant regions with a cysteine ​​mutation at position 239;

[0380] (iii) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160 and one heavy chain constant region with a cysteine ​​mutation at position 239.

[0381] (iv) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160 and two heavy chain constant regions with a cysteine ​​mutation at position 239.

[0382] (v) One or two light chain constant regions with a cysteine ​​mutation at position 205, such as the Kappa light chain constant region;

[0383] (vi) One or two light chain constant regions having cysteine ​​mutations at positions 164 and 205 and a 214S light chain constant region at position 214, such as the Kappa light chain constant region, and one or two heavy chain constant regions having cysteine ​​mutations at position 170 and a 220S heavy chain constant region at position 220; or

[0384] (viii) One or two light chain constant regions with a cysteine ​​mutation at position 164 and a 214S light chain constant region at position 214, such as the Kappa light chain constant region, and one or two heavy chain constant regions with a cysteine ​​mutation at position 170 and a 220S heavy chain constant region at position 220.

[0385] In some specific implementations, the Ab comprises two heavy chain constant regions and two light chain constant regions, wherein

[0386] (i) The two heavy chain constant regions each contain a cysteine ​​mutation at position 239, and the two light chain constant regions do not contain a cysteine ​​mutation (e.g., the light chain constant region is a Kappa light chain constant region).

[0387] (ii) One heavy chain constant region contains a cysteine ​​mutation at position 239, and another heavy chain constant region does not contain a cysteine ​​mutation at position 239 (or another heavy chain constant region does not contain a cysteine ​​mutation), and two light chain constant regions do not contain a cysteine ​​mutation (e.g., the light chain constant region is a Kappa light chain constant region).

[0388] (iii) The two light chain constant regions are Lambda light chains, and one of the light chain constant regions contains a cysteine ​​mutation at position 160, while the other light chain constant region does not contain a cysteine ​​mutation at position 160.

[0389] (iv) One light chain constant region is the Lambda light chain constant region containing a cysteine ​​mutation at position 160, and the other light chain constant region is the Kappa light chain constant region not containing a cysteine ​​mutation at position 160.

[0390] (v) The two light chain constant regions are Lambda light chains, and both light chain constant regions contain a cysteine ​​mutation at position 160.

[0391] (vi) The two light chain constant regions are Lambda light chains, and both light chain constant regions contain a cysteine ​​mutation at position 160, and one heavy chain constant region contains a cysteine ​​mutation at position 239, and the other heavy chain constant region does not contain a cysteine ​​mutation at position 239 (or the other heavy chain constant region does not contain a cysteine ​​mutation).

[0392] (vii) The two light chain constant regions are Lambda light chains, and the two light chain constant regions each contain a cysteine ​​mutation at position 160, and the two heavy chain constant regions each contain a cysteine ​​mutation at position 239.

[0393] (viii) The two light chain constant regions are Kappa light chains, and the two light chain constant regions each contain a cysteine ​​mutation at position 205.

[0394] (ix) The two light chain constant regions are Kappa light chains, and each light chain constant region contains a cysteine ​​mutation at position 164 and 205, respectively, and a 214S mutation at position 214; the two heavy chain constant regions each contain a cysteine ​​mutation at position 170 and a 220S mutation at position 220; or

[0395] (x) The two light chain constant regions are Kappa light chains, and the two light chain constant regions contain a cysteine ​​mutation at position 164 and a 214S mutation at position 214, respectively; the two heavy chain constant regions contain a cysteine ​​mutation at position 170 and a 220S mutation at position 220, respectively.

[0396] In some implementations, the cysteine ​​mutation contained in the Ab described herein enables the Ab to be coupled to a linker via the mutated cysteine, for example, to achieve site-directed coupling of a toxin.

[0397] In some embodiments, the heavy chain constant region suitable for the Ab of the present invention comprises or is composed of CH1 and Fc regions.

[0398] In some embodiments, CH1 is CH1 of IgG1, for example, comprising the amino acid sequence shown in SEQ ID NO:2 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and not containing an amino acid sequence with a cysteine ​​mutation.

[0399] In some embodiments, the Fc region is an IgG Fc, such as IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, the Fc region is the Fc region of IgG1, for example, containing the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:37, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:37, and not containing an amino acid sequence with a cysteine ​​mutation. In some embodiments, the Fc region is an Fc region with a cysteine ​​mutation at position 239. In some embodiments, the Fc region with a cysteine ​​mutation at position 239 contains the amino acid sequence shown in SEQ ID NO:4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4 and contains the amino acid sequence GGPCVFLFP (SEQ ID NO:33).

[0400] In some embodiments, the Ab of the ADC suitable for use in this invention may also contain modifications in the Fc region that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region contains mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in this invention has an L234A / L235A mutation that reduces binding to the Fcγ receptor. In yet another preferred embodiment, the Fc fragment may have mutations that result in an increased serum half-life, such as mutations that improve the binding of the Fc fragment to FcRn. In some embodiments, the Fc region having a cysteine ​​mutation at position 239 and a mutation that reduces binding to the Fcγ receptor comprises the amino acid sequence shown in SEQ ID NO:5 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5 and comprises the amino acid sequence GGPCVFLFP (SEQ ID NO:33) and the L234A / L235A mutation.

[0401] In some embodiments, when Ab contains two different light chains, or when Ab contains two different heavy chains, the Fc region may contain mutations that facilitate heterodimerization between the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions. Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 regions of the first and second Fc regions are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementary engineered CH3 regions (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). Preferably, based on Knob-in-Hole technology, corresponding Knob mutations and Hole mutations are introduced into the first and second Fc regions. This technique is referenced in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001).

[0402] The Ab suitable for the ADC of this invention can be any form of antibody or antigen-binding fragment thereof known in the art, such as monoclonal, chimeric, humanized, fully human, bispecific, or multispecific antibodies or antibody fragments thereof.

[0403] In some embodiments, the Ab suitable for the ADC of the present invention is derived from antibodies that specifically bind to HER2 or CEACAM, such as trastuzumab. In some embodiments, the Ab suitable for the ADC of the present invention comprises 1, 2, 3, 4, 5, or 6 CDRs of antibodies that specifically bind to HER2 or CEACAM, such as trastuzumab, or mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb12, mAb13, mAb14, or mAb15 as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises one, two, or three heavy chain variable regions (CDRs) of antibodies that specifically bind to HER2 or CEACAM, such as trastuzumab, or mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb12, mAb13, mAb14, or mAb15 as described in the examples, namely HCDR1, HCDR2, and HCDR3. In some embodiments, the Ab suitable for the ADC of the present invention comprises one, two, or three light chain variable regions (CDRs) of antibodies that specifically bind to HER2 or CEACAM, such as trastuzumab, or mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb12, mAb13, mAb14, or mAb15 as described in the examples, namely LCDR1, LCDR2, and LCDR3. In some embodiments, the Ab suitable for the ADC of the present invention comprises three heavy chain variable regions (CDRs) and three light chain variable regions (CDRs) of an antibody specifically binding to HER2 or CEACAM, such as trastuzumab, or mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb12, mAb13, mAb14, or mAb15 as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises the heavy chain variable region and / or light chain variable region of an antibody specifically binding to HER2 or CEACAM, such as trastuzumab, or mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb12, mAb13, mAb14, or mAb15 as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises the cysteine ​​mutation of the present invention.

[0404] In some implementations, the Ab is mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb12, mAb13, mAb14 or mAb15 as described in the embodiments.

[0405] In some embodiments, the Ab is a trastuzumab, such as a trastuzumab having the cysteine ​​mutation of the present invention.

[0406] In some embodiments, the antibody (Ab) suitable for the ADC of the present invention is derived from an antibody that specifically binds to B7H3, such as Ifinatamab. In some embodiments, the Ab suitable for the ADC of the present invention comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody that specifically binds to B7H3, such as Ifinatamab or Ifinatamab_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises 1, 2, or 3 heavy chain variable region CDRs, namely HCDR1, HCDR2, and HCDR3, of an antibody that specifically binds to B7H3, such as Ifinatamab or Ifinatamab_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises 1, 2, or 3 light chain variable region CDRs, namely LCDR1, LCDR2, and LCDR3, of an antibody that specifically binds to B7H3, such as Ifinatamab or Ifinatamab_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an antibody that specifically binds to B7H3, such as Ifinatamab or Ifinatamab_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises the heavy chain variable region and / or light chain variable region of an antibody that specifically binds to B7H3, such as Ifinatamab or Ifinatamab_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises the cysteine ​​mutation of the present invention.

[0407] In some implementations, the Ab is Ifinatamab_LLC160KIH as described in the examples.

[0408] In some embodiments, the Ab is the anti-B7H3 antibody or Ifinatamab described in WO2022102695 (entirely incorporated herein by reference), such as the anti-B7H3 antibody or Ifinatamab described in WO2022102695 (entirely incorporated herein by reference) having the cysteine ​​mutation of the present invention.

[0409] In some embodiments, the antibody (Ab) suitable for the ADC of the present invention is derived from an antibody that specifically binds to TROP2, such as hRS7. In some embodiments, the Ab suitable for the ADC of the present invention comprises 1, 2, 3, 4, 5, or 6 CDRs of an antibody that specifically binds to TROP2, such as Sacituzumab or hRS7_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises 1, 2, or 3 heavy chain variable region CDRs of an antibody that specifically binds to TROP2, such as hRS7 or hRS7_LLC160_KIH as described in the examples, namely HCDR1, HCDR2, and HCDR3. In some embodiments, the Ab suitable for the ADC of the present invention comprises 1, 2, or 3 light chain variable region CDRs of an antibody that specifically binds to TROP2, such as hRS7 or hRS7_LLC160_KIH as described in the examples, namely LCDR1, LCDR2, and LCDR3. In some embodiments, the Ab suitable for the ADC of the present invention comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an antibody that specifically binds to TROP2, such as hRS7 or hRS7_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises the heavy chain variable region and / or light chain variable region of an antibody that specifically binds to TROP2, such as hRS7 or hRS7_LLC160_KIH as described in the examples. In some embodiments, the Ab suitable for the ADC of the present invention comprises the cysteine ​​mutation of the present invention.

[0410] In some implementations, the Ab is hRS7_LLC160KIH as described in the examples.

[0411] In some embodiments, the Ab is the anti-TROP2 antibody or hRS7 described in US 7,238,785 B2 (entirely incorporated herein by reference), such as the anti-TROP2 antibody or hRS7 described in US 7,238,785 B2 (entirely incorporated herein by reference) having the cysteine ​​mutation of the present invention.

[0412] Preparation method

[0413] In one aspect, the antibody-drug conjugate of formula (II) of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotope label, can be prepared by a method comprising the steps of:

[0414] (a) The linker toxin compound of formula (I) is coupled to the thiol group of cysteine ​​of the antibody or its antigen-binding fragment (Ab).

[0415] In another aspect, the antibody-drug conjugate of formula (III) of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotope-labeled form, can be prepared by sequential coupling, for example, by sequential site-directed coupling and conventional coupling. The dual-toxin ADCs of formula (III) prepared by the present invention exhibit good blood circulation stability and contain appropriate proportions of two toxin molecules, demonstrating good safety and tolerability. In particular, the antibody-drug conjugate of formula (III) of the present invention, or its pharmaceutically acceptable salt or ester, solvate, or isotope-labeled form, can be prepared by a method comprising the following steps:

[0416] (a) coupling the linker toxin compound of formula (I) to the thiol group of the cysteine ​​residue of the antibody or its antigen-binding fragment (Ab); and

[0417] (b) Forming a thiol group from the interchain disulfide bond of the antibody or its antigen-binding fragment (Ab), and coupling a linker toxin containing D2 to the thiol group.

[0418] In some implementations, step (a) can be performed via site-specific conjugation, such as thiomab conjugation. Through site-specific conjugation, the linker toxin compound is targeted to the sulfhydryl group of a cysteine ​​residue at a specific site on the antibody or its antigen-binding fragment, forming a site-specific antibody-drug conjugate. This significantly increases the homogeneity and stability of the ADC molecule, reduces off-target toxicity and dose-limiting toxicity, and improves clinical response and safety.

[0419] In some implementations, step (a) may include the following steps:

[0420] (a1) Reduce the antibody or its antigen-binding fragment (Ab) with a reducing agent;

[0421] (a2) Add an oxidizing agent to the reactants in step (a1) to carry out oxidation;

[0422] (a3) Add the linker toxin compound of formula (I) to the product of step (a2) to conjugate and obtain the antibody-drug conjugate.

[0423] In some embodiments, step (a1) is performed in a buffer solution. In some embodiments, the buffer solution has a pH of 5.0-9.0, preferably 6.0-8.0. In some embodiments, the buffer solution is a histidine buffer or a phosphate buffer.

[0424] In some embodiments, the reaction in step (a1) is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-25°C, such as ambient temperature.

[0425] In some embodiments, the antibody concentration in step (a1) is 3-30 mg / mL, preferably 5-15 mg / mL. In some embodiments, the reducing agent in step (a1) is TCEP, such as an aqueous TCEP solution. In some embodiments, the molar ratio of reducing agent to antibody in step (a1) is 5 to 40, preferably 10 to 30, more preferably 10 to 20.

[0426] In some embodiments, the reaction in step (a2) is carried out at pH 5.0-9.0, preferably pH 6.0-8.0.

[0427] In some embodiments, the reaction in step (a2) is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature.

[0428] In some embodiments, the antibody concentration in step (a2) is 3-30 mg / mL, preferably 5-15 mg / mL. In some embodiments, the oxidant in step (a2) is dehydroascorbic acid (dhAA), preferably dissolved in DMSO. In some embodiments, the molar ratio of oxidant to antibody in step (a2) is 5 to 60, preferably 20 to 40.

[0429] In some embodiments, the reaction in step (a3) ​​is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature.

[0430] In some embodiments, in step (a3), the linker toxin compound of formula (I) is added to the reaction system in excess. In some embodiments, the molar ratio of linker toxin compound to antibody in step (a3) ​​is 3 to 20, for example 3 to 8, 2 to 6, or 3 to 6.

[0431] In some embodiments, step (b) can be performed using conventional coupling methods. For example, conventional coupling methods can be used to open the interchain disulfide bonds of the antibody or its antigen-binding fragment and connect the toxin molecule D2. In some embodiments, step (b) may include the following steps:

[0432] (b1) Add a reducing agent to the antibody-drug conjugate obtained in step (a) to reduce it;

[0433] (b2) Add a linker toxin containing D2 to the reactants of step (b1) for conjugation to obtain a bitoxin antibody-drug conjugate.

[0434] In some implementations, step (b1) is performed at pH 5.0-9.0, preferably pH 6.0-8.0.

[0435] In some embodiments, the reaction in step (b1) is carried out at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature or 25°C.

[0436] In some embodiments, the antibody concentration (of the antibody-drug conjugate) in step (b1) is 3-30 mg / mL, preferably 5-15 mg / mL. In some embodiments, the reducing agent in step (b1) is TCEP, such as an aqueous TCEP solution. In some embodiments, the reducing agent:antibody molar ratio in step (b1) is 5 to 40, for example 10 to 20, or 8 to 20.

[0437] In some implementations, step (b2) is performed at a suitable temperature, preferably 15-45°C, more preferably 20-37°C, such as ambient temperature or room temperature.

[0438] In some embodiments, in step (b2), the linker toxin containing D2 is added to the reaction system in excess. In some embodiments, the toxin:antibody molar ratio in step (b2) is 5 to 40, for example 10 to 20 or 10 to 16.

[0439] In some embodiments, the above steps are performed under the reaction conditions disclosed in the examples. It should be noted that embodiments obtained by varying the range or specific values ​​of the reaction conditions disclosed in the examples by 100%, 80%, 60%, 40%, 20%, or 10% are also considered in this invention.

[0440] In some implementations, antibody-drug conjugates can be purified using conventional methods, such as spin desalting, ultrafiltration, or dialysis.

[0441] Pharmaceutical composition and administration

[0442] In another aspect, the present invention provides pharmaceutical compositions comprising substances of the present invention, including compounds of formula (I) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and / or antibody-drug conjugates of formula (II) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0443] The substances of the present invention can be administered to patients in the form of pharmaceutical compositions. The pharmaceutical compositions are in forms suitable for the intended manner of administration (including route and dosage) and therapeutic use, such as liquid, semi-solid, and solid dosage forms. The pharmaceutical compositions can be administered by known methods, such as by injection, oral, rectal, gastric, intracranial, and parenteral administration, including intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intralesional (e.g., intratumoral), intracerebral (parenchymal), intravenous, intraocular, intraarterial, and intraportal injection or infusion.

[0444] In some embodiments, the pharmaceutical composition is a formulation. In some embodiments, the pharmaceutical composition is in the form of an injectable formulation. More preferably, the pharmaceutical composition is in a form suitable for intravenous injection or infusion, such as an aqueous solution for injection or a lyophilized powder for injection. Most preferably, the pharmaceutical composition is in the form of a lyophilized powder formulation, which is prepared for injection with a sterile injection solvent (e.g., water for injection or sterile saline) just before use.

[0445] The pharmaceutical compositions of the present invention can be sustained-release formulations. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being in the form of a shaped article, such as a film or microcapsule. The composition can also be administered topically via an implantable membrane, a sponge, or another suitable material on which the desired molecule is absorbed or encapsulated. In some embodiments, when using an implantable device, said device can be implanted into any suitable tissue or organ and can deliver the desired molecule via diffusion, timed release of a bolus, or continuous administration.

[0446] The pharmaceutical compositions or formulations of the present invention may further comprise additional active ingredients required for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, the additional active ingredients are selected from chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). The active ingredients are suitably combined in amounts effective for the intended use.

[0447] The typical dosage range of the substance of this invention is 0.001-1000 mg of active ingredient / kg body weight / day. This dosage can be administered once daily or in multiple divided doses. The appropriate dosage is determined by the attending physician based on the type and severity of the disease to be treated, the individual's health status and medical history, concomitant medications, the specific compound being administered, and the route of administration. The dosage of the substance of this invention may exceed this range as needed.

[0448] Drug combinations and pillboxes

[0449] In another aspect, the present invention provides pharmaceutical combinations or combination products for treating or preventing tumors or cancer, comprising substances of the present invention, including compounds of formula (I) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and / or antibody-drug conjugates of formula (II) or (III) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and one or more common pharmaceuticals.

[0450] In another aspect, the present invention provides a medicament for treating or preventing tumors or cancer, the medicament containing substances of the present invention, including compounds of formula (I) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and / or antibody-drug conjugates of formula (II) or (III) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and one or more common drugs.

[0451] In some embodiments, the shared drug may have the same or different effects as the substance of the present invention.

[0452] In some implementations, the shared medicine can be any substance that can provide health benefits to the patient, such as relieving or reducing any discomfort the patient experiences due to a disease (e.g., tumor or cancer) or drug treatment.

[0453] In some embodiments, the shared drug may be an antitumor drug. Preferably, the shared drug may be selected from chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). In some embodiments, the shared drug may be a cytotoxic agent, a cell growth inhibitor, or an immunosuppressive drug. For example, the shared drug may be a microtubule inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, an antifolate agent, an antimetabolite, a chemotherapy sensitizer, vinca alkaloids, etc. In some embodiments, the shared drug is a camptothecin compound (e.g., eczema, Dxd, SN38), anorexin compounds such as MMAE or MMAF, maytansine compounds such as DM1 and DM4, cazithromycin-type antitumor antibiotics, or aprotinin-type antitumor antibiotics.

[0454] application

[0455] In another aspect, the present invention provides the use of the substances of the present invention (including compounds of formula (I) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and / or antibody-drug conjugates of formula (II) or (III) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof) in the preparation of medicaments for treating or preventing tumors or cancers. Preferably, the tumor or cancer is a solid tumor or a hematologic malignancy or a metastatic lesion. More preferably, the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer or ovarian cancer.

[0456] On the other hand, the present invention provides a method for treating or preventing tumors or cancer, the method comprising administering to a patient in need of the substances of the present invention (including compounds of formula (I) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof, and / or antibody-drug conjugates of formula (II) or (III) of the present invention or pharmaceutically acceptable salts or esters, solvates or isotopic labels thereof). Preferably, the tumor or cancer is a solid tumor or a hematologic malignancy or a metastatic lesion. More preferably, the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer or ovarian cancer.

[0457] In another aspect, the present invention provides substances of the present invention for treating or preventing tumors or cancer (including compounds of formula (I) of the present invention or their pharmaceutically acceptable salts or esters, solvates or isotopic labels and / or antibody-drug conjugates of formula (II) or (III) of the present invention or their pharmaceutically acceptable salts or esters, solvates or isotopic labels). Preferably, the tumor or cancer is a solid tumor or a hematologic malignancy or a metastatic lesion. More preferably, the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer or ovarian cancer.

[0458] In another aspect, the present invention provides the use of the compound of formula (I) of the present invention or its pharmaceutically acceptable salt or ester, solvate or isotopic label in the preparation of antibody-drug conjugates of formula (II) or formula (III) or its sub-forms.

[0459] The substances of the present invention, including compounds of formula (I) of the present invention or their pharmaceutically acceptable salts or esters, solvates or isotopic labels and / or antibody-drug conjugates of formula (II) or (III) of the present invention or their pharmaceutically acceptable salts or esters, solvates or isotopic labels, have good tumor-killing activity, while also having good tolerability and safety, and do not cause significant behavioral abnormalities or weight fluctuations (such as weight loss) while effectively killing tumor cells.

[0460] The ADCs of this invention have a suitable drug-to-antibody ratio and a uniform distribution of this ratio. Furthermore, the ADCs of this invention are stable in the bloodstream, reducing drug molecule shedding from non-target tissues and mitigating off-target toxicity. The ADCs of this invention also exhibit good tumor tissue targeting, accumulating in the tumor microenvironment, increasing the concentration ratio of active drug molecules within the tumor and in the blood, reducing mechanism-related toxicity of the conjugates, and possessing a good therapeutic index. The small molecule drugs released from the ADCs of this invention can also exert a bystander effect, further killing tumor cells surrounding the target tumor cells that do not express or express low levels of antigens.

[0461] The substances of this invention also have good solubility and physicochemical stability, making them suitable for formulation into pharmaceutical preparations.

[0462] In some embodiments, the tumor or cancer is a solid tumor or a hematologic malignancy, as well as a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors such as cancer. In some embodiments, the cancer is stomach cancer, colon cancer, pancreatic cancer, breast cancer, or ovarian cancer. In some embodiments, the cancer may be in an early, intermediate, or late stage, or metastatic stage. In some embodiments, the tumor is a tumor immune evader.

[0463] In some embodiments, the tumor is a tumor or cancer that is positive for the ADC-targeted antigen. In some embodiments, the tumor is associated with aberrant expression or aberrant activity of the ADC-targeted antigen. In some embodiments, the tumor is associated with aberrant activation of ADC-targeted antigen signaling and its downstream signaling pathways.

[0464] In some embodiments, the ADC targets a tumor-specific antigen, such as CEACAM5, or a tumor-associated antigen, such as HER2. In some embodiments, the tumor or cancer is a HER2-positive tumor or cancer. In some embodiments, the tumor or cancer is a CEACAM5-positive tumor or cancer.

[0465] In some embodiments, an ADC-targeted antigen-positive tumor or cancer refers to the abnormal expression or activity of the ADC-targeted antigen in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has abnormal expression of the ADC-targeted antigen. In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of the ADC-targeted antigen (e.g., compared to a healthy subject). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of the ADC-targeted antigen (e.g., compared to a biological sample from a healthy subject (e.g., corresponding tissue or cells in a healthy subject), or compared to the ADC-targeted antigen in adjacent healthy tissue or cells of the subject.

[0466] In some embodiments, an ADC-targeted antigen-positive tumor or cancer refers to tumor cells in an individual suffering from any type of tumor or cancer that express the ADC-targeted antigen. In some embodiments, the tumor cells of said individual express the ADC-targeted antigen, for example, at a moderate or high level. In some embodiments, an ADC-targeted antigen-positive tumor refers to tumor cells that abnormally express the ADC-targeted antigen. In some embodiments, abnormal expression of the ADC-targeted antigen refers to the expression of the ADC-targeted antigen on the cell membrane of said tumor cells. In some embodiments, abnormal expression of the ADC-targeted antigen refers to a higher expression of the ADC-targeted antigen on tumor cells compared to control cells (e.g., healthy cells from the corresponding tissue of a healthy individual, or healthy cells adjacent to the tumor cells).

[0467] In a preferred embodiment, the tumor cells of the antigen-positive tumor targeted by the ADC have intermediate or high expression of the ADC-targeted antigen.

[0468] In one specific embodiment, the molecules of the present invention, such as any antibody or its antigen-binding fragment or immune conjugate, such as ADC molecules, are capable of killing tumor cells and / or inhibiting tumor cell proliferation, for example, tumor cells expressing the antigen targeted by the ADC.

[0469] In some embodiments, the tumor is a tumor that has already been treated with other treatments such as chemotherapy and / or radiotherapy.

[0470] Depending on its therapeutic use, the ADC molecules or pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as other treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0471] In some embodiments, when the molecules of the present invention (e.g., anti-ADC) are used to treat tumors, the treatment methods include surgery; radiotherapy, local irradiation or focused irradiation, etc.

[0472] In other respects, the present invention provides the use of the molecules of the present invention (e.g., anti-ADC) or compositions or combinations thereof in the production or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0473] In other respects, the present invention also provides molecules of the invention (e.g., anti-ADC), or compositions or pharmaceutical or combination products comprising the invention, for use in therapies, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0474] In other respects, the present invention also provides the use of the molecules of the present invention (e.g., ADCs), or compositions or pharmaceutical or combination products comprising them, for therapeutic purposes, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0475] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein. In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0476] The combination therapy of the present invention covers combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which case the administration of the molecules of the present invention (e.g., antibodies against anti-ADC-targeted antigens or their antigen-binding fragments or their immunoconjugates such as ADC molecules, etc.) or compositions or pharmaceutical preparations containing them may occur before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceutical agents.

[0477] It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Attached Figure Description

[0478] Figures 1-6 show the RP-HPLC and SEC-HPLC chromatograms of the ADCs of the present invention, respectively.

[0479] Figure 7 shows the cell survival % curves of the ADCs tested in Example D1.

[0480] Figure 8 shows the cell survival % curves of the ADCs tested in Example D2.

[0481] Figure 9 shows the cell survival % curves of the ADCs tested in Example D3.

[0482] Figure 10 shows the cell survival % curves of the ADCs tested in Example D4.

[0483] Figure 11 shows the cell survival % curves of the ADCs tested in Example D5.

[0484] Figure 12 shows the cell survival % curves of the ADCs tested in Example D6.

[0485] Figure 13 shows the cell survival % curves of the ADCs tested in Example D7.

[0486] Figure 14 shows the cell survival % curves of the ADCs tested in Example D8.

[0487] Figures 15 and 16 show the tumor inhibition curves and body weight change curves of ADCs in the HT55 tumor-bearing mouse model in Examples D9 and D10.

[0488] Figure 17 shows the cell survival of ADCs in NCI-N87(A), FADU(B), and A375(C) cells in Example D11.

[0489] Figure 18 shows the cell survival of ADCs in SNU601 and RT112 cells in Example D12. Example

[0490] The following embodiments are provided to further illustrate the present invention. It should be understood that they are merely for the purpose of better understanding the present invention and are not intended to limit the scope of the present invention in any way.

[0491] In this invention, when the chemical name and structural formula are inconsistent, the structural formula shall prevail, unless the chemical name, rather than the structural formula, can be inferred from the context as correct. For simplicity, not all hydrogen atoms are explicitly shown in the structural formulas of some compounds given in this invention. When a compound has a vacant valence, it indicates the presence of unshown hydrogen atoms. It is understood that the element can be replaced by its isotope. For example, hydrogen (H) in a compound or ADC can be H₂. 1 H 2 or H 3 .

[0492] Unless otherwise specified, all experimental materials and reagents used in the examples are commercially available or can be readily prepared using methods known to those skilled in the art. Specifically, monomethyl auristatin E (MMAE) was obtained from Jinan Chengquan Chemical Co., Ltd., batch number CQ20230301. DMSO was obtained from Sigma, catalog number 276855. TCEP was obtained from Aldrich, catalog number 646547. Dehydroascorbic acid (dhAA) was obtained from Aldrich, catalog number 261556.

[0493] The RP-HPLC conditions used to determine the DAR value of ADC are as follows:

[0494] The SEC-HPLC conditions used to determine ADC purity are as follows:

[0495] Unless otherwise stated, solvent ratios in this document are by volume. Abbreviations used herein (e.g., for chemical groups and compounds) generally have meanings well-known in the art, unless otherwise indicated.

[0496] Example A. Preparation of linker toxin compounds

[0497] Example A1

[0498] (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8- Diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A1)

[0499] Step 1: Triacetic acid (2S,3R,4S,5S,6S)-2-(5-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A1-2

[0500] Compound A1-1 (17.8 g, 44.8 mmol, supplier: Adamas-beta) and 3-hydroxy-4-nitrobenzaldehyde (5 g, 29.9 mmol) were dissolved in anhydrous acetonitrile (200 mL). Ag₂O (27.8 g, 119.8 mmol) was added at 0 °C. The mixture was heated to 25 °C and stirred in the dark for 24 hours. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A1-2 (9.0 g, yield: 62%). LC-MS: ESI-MS (m / z): [M+H] + =484.1.

[0501] Step 2: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-amino-5-hydroxymethylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A1-3

[0502] Compound A1-2 (9.0 g, 18.6 mmol) was dissolved in ethyl acetate (400 mL), and palladium on carbon (10 wt%, 2.5 g) and triethylamine (376 mg, 3.7 mmol) were added. The reaction system was purged three times with hydrogen and stirred at room temperature for 24 hours under a hydrogen atmosphere. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure to give compound A1-3 (8.0 g, yield: 94%). LC-MS: ESI-MS (m / z): [M+H] + =456.1.

[0503] Step 3: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-5-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester Al-4

[0504] Compound A1-3 (419 mg, 1.0 mmol) and (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionic acid (425 mg, 0.93 mmol) were dissolved in dichloromethane (5 mL) and methanol (1 mL). N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (390 mg, 1.7 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Dichloromethane (50 mL) was added to the reaction mixture, and the solution was washed successively with dilute hydrochloric acid (1 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 9) to give compound A1-4 (500 mg, yield: 63%). LC-MS: ESI-MS (m / z): [M+H] + =848.3.

[0505] Step 4: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-5-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester Al-5

[0506] Compound A1-4 (9.6 g, 11.3 mmol) and bis(4-nitrophenyl) carbonate (5.2 g, 17.1 mmol) were dissolved in dry N,N-dimethylformamide (70 mL), and N,N-diisopropylethylamine (4.4 g, 34.1 mmol) was added dropwise. The mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A1-5 (10.0 g, yield: 87%). LC-MS: ESI-MS (m / z): [M+H] + =1013.3.

[0507] Step 5: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2) ... R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester A1-6

[0508] Compound A1-5 (6.8 g, 6.7 mmol), monomethylaurestatin E (MMAE, 4.0 g, 5.6 mmol), and 1-hydroxybenzotriazole (1.5 g, 11.1 mmol) were dissolved in dry N,N-dimethylformamide (50 mL), and N,N-diisopropylethylamine (2.2 g, 16.7 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give compound A1-6 (8.0 g, yield: 89%). LC-MS: ESI-MS (m / z): [M+H] + =1591.8.

[0509] Step 6: (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)-5-((5S,8S,11S,12R)-11-((S)-sec-Butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A1-7

[0510] Dissolve compound A1-6 (10.0 g, 6.5 mmol) in a mixed solution of tetrahydrofuran and methanol (volume ratio 1 / 1, 100 mL). Under an ice bath condition, slowly dropwise add lithium hydroxide solution (800 mg, 32.5 mmol, 100 mL of water) within 3 hours. Raise the reaction to room temperature and stir for 3 hours. Add diethylamine (20 mL) and stir at room temperature for 6 hours. Concentrate the reaction solution under reduced pressure to 160 mL. Add glacial acetic acid (20 mL) and stir at room temperature for 0.5 hour until the turbid solution becomes a yellow transparent solution. Concentrate the reaction solution under reduced pressure, dissolve the obtained residue in 160 mL of methanol, and purify the crude product by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as an additive) to obtain compound A1-7 (5.6 g, yield: 75%). LC-MS: ESI-MS (m / z): [M+H] + = 1229.7.

[0511] Step 7: (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-But

[0512] yl)-12-(2-((S)-2-((1R,2R)-3-((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A1

[0513] Compound A1-7 (4.1 g, 3.3 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (1.23 g, 4.0 mmol) were dissolved in dry dimethyl sulfoxide (15 mL), and N,N-diisopropylethylamine (645 mg, 5.0 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A1 (3.0 g, yield: 64%). LC-MS: ESI-MS (m / z): [M+H] + =1422.8.

[0514] Example A2

[0515] N-((S)-1-(((S)-1-(((3 2 R,3 3 S,3 4 R,3 5 R,3 6 R,12 2 S,6S,7R,10R,11R,15R,16S,19S,22S)-16-((S)-sec-butyl)-3 3 ,3 4 ,3 5 -Trihydroxy-19,22-diisopropyl-11,15-dimethoxy-7,10,17,23-tetramethyl-4,9,13,18,21,24-hexaoxo-6-phenyl-3 3 ,3 4 ,3 5 ,3 6 -Tetrahydro-3 2 H-2,5,25-trioxa-8,17,20,23-tetraaza-12(2,1)-pyrrolidine-3(2,6)-pyranaza-1(1,3)-benzene-hexaban-1 6 -yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoamide (A2)

[0516] Compound A1 (30 mg, 0.02 mmol) was dissolved in anhydrous dimethyl sulfoxide (1 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6 mg, 0.0317 mmol) and 1-hydroxybenzotriazole (4 mg, 0.03 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 2 / 3, 0.1% formic acid as additive) to give compound A2 (20 mg, yield: 68%). LC-MS: ESI-MS (m / z): [M+H] + =1404.7

[0517] Example A3

[0518] ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propane-2-yl)amino)propyl) )pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester (A3)

[0519] Step 1: ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (9H-fluorene-9-yl)methyl ester A3-2

[0520] Compound A3-1 (MMAE, 500 mg, 0.7 mmol) was dissolved in dry dichloromethane (10 mL), and 9-fluorenyl chloroformate (216 mg, 0.8 mmol) was added. N,N-diisopropylethylamine (135 mg, 1.0 mmol) was added under ice bath conditions, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then washed with dichloromethane (50 mL) and saline solution (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give compound A3-2 (620 mg, yield: 95%). LC-MS: ESI-MS (m / z): [M+H] + =940.2.

[0521] Step 2: Triacetic acid (2R,3S,4S,5R,6R)-2-(acetoxymethyl)

[0522] )-6-((1S,2R)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-11-((S)-sec-butyl)-1-(9H-fluorene-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-acyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamide)-1-phenylpropoxy)tetrahydro-2H-pyran-3,4,5-triyl ester A3-3

[0523] Compound A3-2 (200 mg, 0.2 mmol) and triacetic acid (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl ester (524 mg, 1.1 mmol) were dissolved in a dry mixture of dichloromethane and acetonitrile (2 mL, 1 / 1 v / v). The reaction mixture was cooled to -20 °C, and trifluoromethanesulfonic acid (32 mg, 0.2 mmol) was slowly added dropwise at this temperature. The reaction mixture was slowly heated to 0 °C over 2 hours and quenched with triethylamine (0.1 mL). The mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give compound A3-3 (200 mg, yield: 75%). LC-MS: ESI-MS (m / z): [M+H] + =1270.7.

[0524] Step 3: Triacetic acid (2R,3S,4S,5R,6R)-2-(acetoxymethyl)

[0525] (1S,2R)-2-(2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-1-phenylpropoxy)tetrahydro-2H-pyran-3,4,5-triyl ester A3-4

[0526] Compound A3-3 (200 mg, 0.16 mmol) was dissolved in dichloromethane (5 mL), and ethylenediamine (1 mL) was added. The reaction mixture was stirred at room temperature for 1 hour, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give compound A3-4 (132 mg, yield: 80%). LC-MS: ESI-MS (m / z): [M+H] + =1048.6.

[0527] Step 4: Triacetic acid

[0528] (2R,3R,4S,5S,6R)-2-((1S,2R)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)phenyl)-11-((S (-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-acyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionamide)-1-phenylpropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester A3-5

[0529] Compound A3-4 (132 mg, 0.13 mmol) and (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino))-1-oxopropyl-2-yl)amino)-1-oxobut-2-yl)carbamate (128 mg, 0.19 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (25 mg, 0.19 mmol) and N,N-diisopropylethylamine (66 mg, 0.5 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give compound A3-5 (120 mg, yield: 50%). LC-MS: ESI-MS (m / z): [M+H] +=1588.8.

[0530] Step 5: ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran- 2-yl)oxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl ester A3-6

[0531] Compound A3-5 (100 mg, 0.06 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (4 mL, 1 / 1 v / v). Lithium hydroxide aqueous solution (9 mg, 0.04 mmol, 1 mL) was slowly added dropwise under ice bath conditions. The reaction mixture was slowly brought to room temperature and stirred for 1 hour. Glacial acetic acid (0.3 mL) was added dropwise, and stirring continued for 20 minutes. The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A3-6 (37 mg, yield: 50%). LC-MS: ESI-MS (m / z): [M+H] + =1199.7.

[0532] Step 6: ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propane-2-yl)amino )propyl)pyrrolidone-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolidone-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester A3

[0533] Compound A3-6 (37 mg, 0.03 mmol) was dissolved in dry dimethyl sulfoxide (1 mL), and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (12 mg, 0.04 mmol) and N,N-diisopropylethylamine (6 mg, 0.05 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was quenched dropwise with glacial acetic acid (0.1 mL). The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A3 (25 mg, yield: 60%). LC-MS: ESI-MS (m / z): [M+H] + =1392.8.

[0534] Example A4

[0535] (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy) Propan-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A4)

[0536] Step 1: Triacetic acid

[0537] (2R,3R,4S,5S,6R)-2-((1S,2R)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-3-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxy) (carbonyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-11-((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-acyl)pyrrololin-2-yl)-3-methoxy-2-methylpropionamide)-1-phenylpropoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl ester A4-1

[0538] Compounds A3-4 (100 mg, 0.095 mmol) and A1-5 (145 mg, 0.14 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and 1-hydroxybenzotriazole (19 mg, 0.14 mmol) and N,N-diisopropylethylamine (49 mg, 0.4 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give compound A4-1 (88 mg, yield: 48%). LC-MS: ESI-MS (m / z): [M+H] + =1920.9.

[0539] Step 2: (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1 -((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A4-2

[0540] Compound A4-1 (88 mg, 0.05 mmol) was dissolved in a mixed solution of tetrahydrofuran and methanol (4 mL, 1 / 1 v / v). Lithium hydroxide aqueous solution (9 mg, 0.04 mmol, 1 mL) was slowly added dropwise under ice bath conditions. The reaction mixture was slowly brought to room temperature and stirred for 1 hour. Glacial acetic acid (0.3 mL) was added dropwise, and stirring continued for 20 minutes. The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A4-2 (25 mg, yield: 40%). LC-MS: ESI-MS (m / z): [M+H] + =1391.7.

[0541] Step 3: (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1-((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl) (Oxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A4

[0542] Compound A4-2 (25 mg, 0.02 mmol) was dissolved in dry dimethyl sulfoxide (1 mL), and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid ester (7 mg, 0.02 mmol) and N,N-diisopropylethylamine (3.5 mg, 0.03 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was quenched dropwise with glacial acetic acid (0.1 mL). The crude product was purified by silica gel column chromatography (C18 column, acetonitrile / water = 3 / 10, 0.05% formic acid as additive) to give compound A4 (10 mg, yield: 35%). LC-MS: ESI-MS (m / z): [M+H] + =1584.8.

[0543] Example A5

[0544] (S)-N-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-5,8,11-triazatridecane-13-yl)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-6-(dipropylamino)hexanoamide (A5)

[0545] Step 1: N 2 -(((9H-fluorene-9-yl)methoxy)carbonyl)-N 6 N 6 -Dipropyl-L-lysine tert-butyl ester A5-2

[0546] Compound A5-1 (((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine tert-butyl ester (2 g, 4.72 mmol)) was dissolved in methanol (25 mL), and catalytic amounts of glacial acetic acid (0.1 mL), propionaldehyde (680 mg, 11.8 mmol), and sodium cyanoborohydride (890 mg, 14.2 mmol) were added and stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and diluted with dichloromethane (100 mL), washed with saturated sodium bicarbonate solution (100 mL) and saturated brine (100 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A5-2 (1.96 g, yield: 85%). LC-MS: ESI-MS (m / z): [M+H] + =509.7.

[0547] Step 2: N 6 N 6 -Dipropyl-L-lysine tert-butyl ester A5-3

[0548] Compound A5-2 (1.96 g, 3.86 mmol) was dissolved in N,N-dimethylformamide (10 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by slurrying with methyl tert-butyl ether to give compound A5-3 (1.1 g, yield: 99%). LC-MS: ESI-MS (m / z): [M+H] + =287.5.

[0549] Step 3: N 2-((((9H-fluorene-9-yl)methoxy)carbonyl)-L-valineyl)-N 6 N 6 -Dipropyl-L-lysine tert-butyl ester A5-5

[0550] Compounds A5-3 (1.1 g, 3.84 mmol) and A5-4 (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine (1.3 g, 3.84 mmol) were dissolved in N,N-dimethylformamide (5 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1.1 g, 5.76 mmol) and 1-hydroxybenzotriazole (777 mg, 5.76 mmol) were added. The mixture was stirred at room temperature for 4 hours. Ethyl acetate (50 mL) was added to the reaction solution, and the mixture was washed successively with dilute hydrochloric acid (0.5 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give compound A5-5 (2 g, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =608.8.

[0551] Step 4: N 2 -(L-valinel)-N 6 N 6 -Dipropyl-L-lysine tert-butyl ester A5-6

[0552] Compound A5-5 (2 g, 3.29 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by slurrying with methyl tert-butyl ether to give compound A5-6 (1.2 g, yield: 95%). LC-MS: ESI-MS (m / z): [M+H] + =386.6.

[0553] Step 5: N 2 -((6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-L-valineyl)-N 6 N 6 -Dipropyl-L-L-Lysine A5-8

[0554] Compounds A5-6 (1.2 g, 3.1 mmol) and A5-7 (1.43 g, 4.6 mmol) were dissolved in dry dimethyl sulfoxide (15 mL), and N,N-diisopropylethylamine (645 mg, 5.0 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was lyophilized to obtain an oily crude product. The crude product was dissolved in dry dichloromethane (10 mL), and trifluoroacetic acid (2 mL) was added. The reaction mixture was removed under reduced pressure. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A5-8 (1.05 g, yield: 65%). LC-MS: ESI-MS (m / z): [M+H] + =523.7.

[0555] Step 6: (S)-N-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-5,8,11-triazatridecane-13-yl)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-6-(dipropylamino)hexanoamide A5

[0556] Compounds A5-8 (26 mg, 0.0503 mmol) and A5-9 (30 mg, 0.0387 mmol) were dissolved in N,N-dimethylformamide (1 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (15 mg, 0.075 mmol) and 1-hydroxybenzotriazole (10 mg, 0.075 mmol) were added. The mixture was stirred at room temperature for 4 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A5 (24 mg, yield: 64%). LC-MS: ESI-MS (m / z): [M+H] + =998.3.

[0557] Preparation of intermediate A5-9

[0558] Step 1: ((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R))-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-5,8,11-triazatridecane-13-yl)carbamate (9H-fluorene-9-yl)methyl ester B5-2

[0559] Compound MMAE (100 mg, 0.139 mmol) and B5-1 (((9H-fluorene-9-yl)methoxy)carbonyl)glycine (50 mg, 0.167 mmol) were dissolved in N,N-dimethylformamide (2 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (48 mg, 0.251 mmol) and 1-hydroxybenzotriazole (34 mg, 0.251 mmol) were added. The mixture was stirred at room temperature for 4 hours. Ethyl acetate (50 mL) was added to the reaction mixture, and the solution was washed successively with dilute hydrochloric acid (0.5 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give compound B5-2 (125 mg, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =998.3.

[0560] Step 2: (S)-2-((S)-2-(2-amino-N-methylacetamido)-3-methylbutyrylamino)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)-N,3-dimethylbutyramide A5-9

[0561] Compound B5-2 (125 mg, 0.125 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (0.3 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 80 / 20) to give compound A5-9 (78 mg, yield: 80%). LC-MS: ESI-MS (m / z): [M+H] + =776.0.

[0562] Example A6

[0563] ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-(I-1-methoxy-3-oxo-3-(((1S,2R)-1-phenyl-1-(phosphonooxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester (A6)

[0564] Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (9H-fluorene-9-yl)methyl ester A6-7

[0565] Compound A6-5 (4-aminophenyl)methanol (2 g, 16.2 mmol) and A6-6 (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionic acid (6.6 g, 16.2 mmol, supplier Adamas) were dissolved in dichloromethane (50 mL) and methanol (10 mL). N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (6 g, 24.3 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Dichloromethane (100 mL) was added to the reaction mixture, and the mixture was filtered to obtain a grayish-white solid. The grayish-white solid was washed several times with dichloromethane to give compound A6-7 (7.5 g, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =516.25.

[0566] Step 2: (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)-3-methylbutyramide A6-8

[0567] Compound A6-7 (1 g, 1.94 mmol) was dissolved in N,N-dimethylformamide (5 mL), and diethylamine (1 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by slurrying with methyl tert-butyl ether to give compound A6-8 (456 mg, yield: 80%). LC-MS: ESI-MS (m / z): [M+H] + =294.1.

[0568] Step 3: 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl))amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)hexanoamide A6-9

[0569] Compounds A6-8 (456 mg, 1.56 mmol) and A5-7 (720 mg, 2.34 mmol) were dissolved in dry dimethyl sulfoxide (5 mL), and N,N-diisopropylethylamine (400 mg, 3.12 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A6-9 (450 mg, yield: 59%). LC-MS: ESI-MS (m / z): [M+H] + =487.2.

[0570] Step 4: Carbonic acid 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester (4-nitrophenyl) ester A6-10

[0571] Compound A6-9 (450 mg, 0.924 mmol) and bis(4-nitrophenyl) carbonate (420 mg, 1.39 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (357 mg, 2.77 mmol) was added dropwise. The mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A6-10 (516 mg, yield: 90%). LC-MS: ESI-MS (m / z): [M+H] + =652.3.

[0572] Step 5: ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-(I-1-methoxy-3-oxo-3-(((1S,2R)-1-phenyl-1-(phosphonooxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester A6

[0573] Compounds A6-10 (49 mg, 0.075 mmol), A6-4 (MMAE-phosphate, 30 mg, 0.038 mmol), and 1-hydroxybenzotriazole (10 mg, 0.075 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (29 mg, 0.225 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A6 (10 mg, yield: 20%). LC-MS: ESI-MS (m / z): [M+H] + =1310.7.

[0574] Preparation of intermediate A6-4

[0575] Step 1: ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R))-3-(((1S,2R)-1-((bis(benzyloxy)phosphoryl)oxy)-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (9H-fluorene-9-yl)methyl ester A6-2

[0576] Compound A3-2 (419 mg, 0.445 mmol) and A6-1 dibenzyldiisopropylphosphonamide (307 mg, 0.891 mmol) were dissolved in dry acetonitrile (5 mL), and 1H-tetrazole (70 mg, 1 mmol) and N,N-diisopropylethylamine (128 mg, 1 mmol) were added. The mixture was stirred at room temperature for 4 hours. Ethyl acetate (50 mL) was added to the reaction solution, and the mixture was washed successively with water (50 mL) and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 98 / 2) to give compound A6-2 (373 mg, yield: 70%). LC-MS: ESI-MS (m / z): [M+H] + =1201.5.

[0577] Step 2: ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(((1S,2R)-1-phenyl-1-(phosphonooxy)propane-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (9H-fluorene-9-yl)methyl ester A6-3

[0578] Compound A6-2 (373 mg, 0.31 mmol) was dissolved in tetrahydrofuran and methanol (5 mL, 1 / 1 v / v), and a catalytic amount of glacial acetic acid (0.1 mL) and palladium on carbon (10% wt, 50 mg) was added. The mixture was stirred at room temperature for 6 hours under hydrogen balloon pressure. The palladium on carbon was filtered through diatomaceous earth, and the resulting reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 85 / 15) to give compound A6-3 (218 mg, yield: 69%). LC-MS: ESI-MS (m / z): [M+H] + =1021.2.

[0579] Step 3: Dihydrogen phosphate (1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-1-phenylpropyl ester A6-4

[0580] Compound A6-3 (218 mg, 0.21 mmol) was dissolved in N,N-dimethylformamide (2 mL), and diethylamine (0.2 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A6-4 (125 mg, yield: 75%). LC-MS: ESI-MS (m / z): [M+H] + =799.5.

[0581] Example A7

[0582] ((2S)-1-(((2S)-1-(((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-(((1S,2R))-1-((hydroxy(phosphono)phosphoyl)oxy)-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl- 1-Oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester (A7)

[0583] Step 1: ((2S)-1-(((2S)-1-(((3R,4S,5S)-1-((2S)-2-((1R,2R))-3-(((1S,2R)-1-((((bis(benzyloxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (9H-fluorene-9-yl)methyl ester A7-1

[0584] Compound A6-3 (200 mg, 0.196 mmol) and A6-1 dibenzyldiisopropylphosphonamide (135 mg, 0.392 mmol) were dissolved in dry acetonitrile (2 mL). 1H-tetrazole (27 mg, 0.392 mmol) and N,N-diisopropylethylamine (51 mg, 0.392 mmol) were added, and the mixture was stirred at room temperature for 6 hours. Ethyl acetate (20 mL) was added to the reaction mixture, and the solution was washed successively with water (20 mL) and saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 90 / 10) to give compound A7-1 (150 mg, yield: 60%). LC-MS: ESI-MS (m / z): [M+H] + =1281.6.

[0585] Step 2: ((2S)-1-(((2S)-1-(((3R,4S,5S)-1-((2S)-2-((1R,2R))-3-(((1S,2R)-1-((hydroxy(phosphono)phosphoyl)oxy)-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (9H-fluorene-9-yl)methyl ester A7-2

[0586] Compound A7-1 (150 mg, 0.117 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (10 mL, 1 / 1 v / v), and catalytic amounts of glacial acetic acid (0.1 mL) and palladium on carbon (10% wt, 40 mg, 1.7 mmol) were added. The mixture was stirred at room temperature for 2 hours under hydrogen balloon pressure. The palladium on carbon was filtered through diatomaceous earth, and the resulting reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 70 / 30) to give compound A7-2 (83 mg, yield: 65%). LC-MS: ESI-MS (m / z): [M+H] + =1100.5.

[0587] Step 3: (1S,2R)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidine-2-yl)-3-methoxy-2-methylpropionylamino)-1-phenylpropyltrihydropyrophosphate A7-3

[0588] Compound A7-2 (83 mg, 0.075 mmol) was dissolved in N,N-dimethylformamide (1 mL), and diethylamine (0.2 mL) was added. The mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A7-3 (46 mg, yield: 70%). LC-MS: ESI-MS (m / z): [M+H] + =878.4.

[0589] Step 4: ((2S)-1-(((2S)-1-(((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-(((1S,2R))-1-((hydroxy(phosphono)phosphoyl)oxy)-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5- Methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester A7

[0590] Compounds A6-10 (59 mg, 0.0912 mmol), A7-3 (MMAE-pyrophosphate, 40 mg, 0.0456 mmol), and 1-hydroxybenzotriazole (12 mg, 0.0912 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (23 mg, 0.18 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A7 (9 mg, yield: 15%). LC-MS: ESI-MS (m / z): [M+H] + =1390.7.

[0591] Example A8

[0592] ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 3-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)acetamido)-4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)benzyl ester (A8)

[0593] Step 1: (2-((2-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)-5-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-2-oxoethyl)tert-butyl carbamate A8-2

[0594] Compound A8-1 (500 mg, 1.15 mmol, referencing patents WO2020043129 A1 and WO2024067841 A1) and bis(4-nitrophenyl) carbonate (524 mg, 1.72 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (297 mg, 2.3 mmol) was added dropwise. The mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A8-2 (627 mg, yield: 91%). LC-MS: ESI-MS (m / z): [M+H] + =601.2.

[0595] Step 2: ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 3-(2-((tert-butoxycarbonyl)amino)acetamido)-4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)benzyl ester A8-3

[0596] Compound A8-2 (627 mg, 1.05 mmol), monomethylaurestatin E (MMAE, 527 mg, 0.74 mmol), and 1-hydroxybenzotriazole (140 mg, 1.05 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (270 mg, 2.1 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 96 / 4) to give compound A8-3 (628 mg, yield: 72%). LC-MS: ESI-MS (m / z): [M+H] + =1179.7.

[0597] Step 3: ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 3-(2-aminoacetamido)-4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)benzyl ester A8-4

[0598] Compound A8-3 (628 mg, 0.533 mmol) was dissolved in dry dichloromethane (5 mL), and trifluoroacetic acid solution (1 mL) was added dropwise at 0 °C. The mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 90 / 10) to give compound A8-4 (511 mg, yield: 89%). LC-MS: ESI-MS (m / z): [M+H] + =1079.6.

[0599] Step 4: ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 3-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)acetamido)-4-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)benzyl ester A8

[0600] Compound A8-4 (511 mg, 0.474 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (218 mg, 0.71 mmol) were dissolved in dry dimethyl sulfoxide (5 mL), and N,N-diisopropylethylamine (137 mg, 1.07 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A8 (500 mg, yield: 83%). LC-MS: ESI-MS (m / z): [M+H] + =1272.7.

[0601] Example A9

[0602] (2S,3S,4S,5R,6S)-6-(4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2 (-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (A9)

[0603] Step 1: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A9-2

[0604] Compound A1-1 (5 g, 12.6 mmol, supplier: Adamas-beta) and A9-1 4-hydroxy-3-nitrobenzaldehyde (527 mg, 3.2 mmol) were dissolved in anhydrous acetonitrile (30 mL), and Ag₂O (3 g, 12.8 mmol) was added at 0 °C. The mixture was heated to 25 °C and stirred in the dark for 24 hours. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A9-2 (1.0 g, yield: 65%). LC-MS: ESI-MS (m / z): [M+H] + =484.1.

[0605] Step 2: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A9-3

[0606] Compound A9-2 (1.0 g, 2.07 mmol) was dissolved in ethyl acetate (50 mL), and palladium on carbon (10 wt%, 100 mg) and triethylamine (42 mg, 0.41 mmol) were added. The reaction mixture was purged three times with hydrogen and stirred at room temperature for 24 hours under a hydrogen atmosphere. The reaction solution was filtered through a diatomaceous earth layer, and the filtrate was concentrated under reduced pressure to give compound A9-3 (895 mg, yield: 95%). LC-MS: ESI-MS (m / z): [M+H] +=456.1.

[0607] Step 3: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-(3-((tert-butoxycarbonyl)amino)propionylamino)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A9-5

[0608] Compound A9-3 (895 mg, 1.97 mmol) and A9-4 3-((tert-butoxycarbonyl)amino)propionic acid (446 mg, 2.36 mmol) were dissolved in dichloromethane (5 mL) and methanol (1 mL). N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (741 mg, 3.0 mmol) was added, and the mixture was stirred at room temperature for 16 hours. Dichloromethane (50 mL) was added to the reaction mixture, and the solution was washed successively with dilute hydrochloric acid (0.5 M, 50 mL), water (50 mL), and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A9-5 (851 mg, yield: 69%). LC-MS: ESI-MS (m / z): [M+H] + =627.2.

[0609] Step 4: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-(3-((tert-butoxycarbonyl)amino)propionylamino)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester A9-6

[0610] Compound A9-5 (200 mg, 0.319 mmol) and bis(4-nitrophenyl) carbonate (145 mg, 0.479 mmol) were dissolved in dry N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (82 mg, 0.638 mmol) was added dropwise. The mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give compound A9-6 (219 mg, yield: 87%). LC-MS: ESI-MS (m / z): [M+H] + =792.2.

[0611] Step 5: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-(3-((tert-butoxycarbonyl)amino)propionylamino)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester A9-7

[0612] Compound A9-6 (219 mg, 0.276 mmol), monomethylaurestatin E (MMAE, 139 mg, 0.194 mmol), and 1-hydroxybenzotriazole (37 mg, 0.276 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (53 mg, 0.414 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 97 / 3) to give compound A9-7 (189 mg, yield: 72%). LC-MS: ESI-MS (m / z): [M+H] + =1370.7.

[0613] Step 6: (2S,3S,4S,5R,6S)-6-(2-(3-aminopropionamid)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A9-8

[0614] Compound A9-7 (189 mg, 0.139 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (1 / 1, 6 mL, v / v). Under ice bath conditions, lithium hydroxide solution (20 mg, 0.834 mmol, 1 mL water) was slowly added dropwise over 10 minutes. The reaction mixture was stirred at room temperature for 1 hour. Glacial acetic acid was added and stirred for 30 minutes. The solvent was removed by vacuum distillation. The crude product was dissolved in dry dichloromethane (5 mL), and trifluoroacetic acid (1 mL) was added dropwise at 0 °C, with stirring at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A9-8 (77 mg, yield: 50%). LC-MS: ESI-MS (m / z): [M+H] + =1130.6.

[0615] Step 7: (2S,3S,4S,5R,6S)-6-(4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl (-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A9

[0616] Compound A9-8 (40 mg, 0.0358 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (17 mg, 0.0538 mmol) were dissolved in dry dimethyl sulfoxide (15 mL), and N,N-diisopropylethylamine (10 mg, 0.0807 mmol) was added in an ice bath. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A9 (32 mg, yield: 70%). LC-MS: ESI-MS (m / z): [M+H] + =1323.7.

[0617] Example A10

[0618] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-(I-1-methoxy-3-oxo-3-((I-1-phenylprop-2-yl)amino)propyl)pyrrolo-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate (A10)

[0619] Step 1: (S)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((I-1-phenylpropan-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyramide A10-1

[0620] MMAE (200 mg, 0.278 mmol) was dissolved in methanol (10 mL). Catalytic amounts of glacial acetic acid (0.1 mL) and palladium on carbon (10% wt, 50 mg) were added to the reaction system. The reaction system was stirred under a hydrogen atmosphere for 4 hours. The palladium on carbon was filtered through a diatomaceous earth layer. The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give compound A10-1 (173 mg, yield: 89%). LC-MS: ESI-MS (m / z): [M+H] + =702.5.

[0621] Step 2: ((S)-1-(((S)-1-(((3R,4S,5S)-3-methoxy-1-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((I-1-phenylpropan-2-yl)amino)propyl)pyrrolidine-1-yl)-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)(methyl)carbamate 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)benzyl ester A10

[0622] Compounds A6-10 (55 mg, 0.0856 mmol), A10-1 (MMAE analog, 30 mg, 0.0428 mmol), and 1-hydroxybenzotriazole (11 mg, 0.0856 mmol) were dissolved in dry N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (17 mg, 0.128 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A10 (20 mg, yield: 40%). LC-MS: ESI-MS (m / z): [M+H] + =1214.7.

[0623] Example A11

[0624] (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-(I-1-methoxy-3-oxo-3-((I-1-phenylprop-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl (A11)-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6)-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0625] Step 1: Triacetic acid (2S,3R,4S,5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R) ,2R)-1-methoxy-2-methyl-3-oxo-3-((1-1-phenylpropan-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester A11-1

[0626] Compounds A1-5 (216 mg, 0.214 mmol), A10-1 (100 mg, 0.142 mmol), and 1-hydroxybenzotriazole (28 mg, 0.214 mmol) were dissolved in dry N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (41 mg, 0.321 mmol) was added dropwise. The mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give compound A11-1 (163 mg, yield: 73%). LC-MS: ESI-MS (m / z): [M+H] + =1575.8.

[0627] Step 2: (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((I-1-phenylpropan-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A11-2

[0628] Compound A11-1 (163 mg, 0.103 mmol) was dissolved in a mixture of tetrahydrofuran and methanol (1 / 1 v / v, 6 mL). Under ice bath conditions, lithium hydroxide solution (15 mg, 0.618 mmol, 1 mL water) was slowly added dropwise over 30 minutes. The reaction was brought to room temperature and stirred for 1 hour. Glacial acetic acid was added and stirred for 30 minutes. The solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A11-2 (74 mg, yield: 60%). LC-MS: ESI-MS (m / z): [M+H] + =1213.7.

[0629] Step 3: (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-(2-((S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-((I-1-phenylpropan-2-yl)amino)propyl)pyrrolidine-1-yl)-2-oxoethyl )-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6)-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)propionylamino)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid A11

[0630] Compound A11-2 (30 mg, 0.0247 mmol) and 2,5-dioxopyrrolidine-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid 2,5-dioxopyrrolidine-1-yl ester (10 mg, 0.03 mmol) were dissolved in dry dimethyl sulfoxide (1 mL), and N,N-diisopropylethylamine (6 mg, 0.045 mmol) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 2 hours. The crude product was purified by silica gel column chromatography (C18 column, gradient from acetonitrile / water = 1 / 10 to pure acetonitrile, 0.05% formic acid as additive) to give compound A11 (20 mg, yield: 59%). LC-MS: ESI-MS (m / z): [M+H] + =1406.8.

[0631] Preparation of linker toxin NT2

[0632] Synthesis of 15-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-((S)-1-(((S)-1-(((3-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-11-yl)propoxy)methyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12-tetraoxapentadecanamide (NT2)

[0633] Step 1: Zn(OAc)₂ (16 mg, 0.088 mmol) was added to a solution of D1 (50 mg, 0.11 mmol) and compound 8 (79 mg, 0.25 mmol) in 1,4-dioxane (10 mL) at room temperature. The reaction mixture was stirred at 80 °C for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM / MeOH = 50:1) to give compound 13 as a yellow solid (55 mg, 0.062 mmol, 57% yield). LC-MS: ESI-MS (m / z): [M+H] + =876.3.

[0634] Step 2: Compound 13 (55 mg, 0.062 mmol) was dissolved in DCM (5 ml), and then diethylamine (2 ml) was added to the solution. The reaction mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM / MeOH = 10:1) to give compound 14 as a yellow solid (37 mg, 0.057 mmol, 92% yield). LC-MS: ESI-MS (m / z): [M+H] + =654.3.

[0635] Step 3: Triethylamine (11 mg, 0.114 mmol) was added to a solution of compound 14 (37 mg, 0.057 mmol) and Mal-Peg4-NHS ester (30 mg, 0.068 mmol) in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (DCM / MeOH = 20:1) to give NT2 as a white solid (24 mg, 0.024 mmol, 42% yield). LC-MS: ESI-MS (m / z): [M+H] + =981.4.

[0636] Example B. Antibody Expression

[0637] Example B1. Antibody expression targeting HER2 and CEACAM5

[0638] Antibodies targeting HER2 and CEACAM5 were expressed for subsequent use.

[0639] HER2 antibodies include the following types: mAb1, mAb2, mAb3, mAb4, and mAb5. mAb2 is a Thiomab protein with a cysteine ​​residue at position 239 of the heavy chain; each antibody has two cysteine ​​mutation sites. mAb3 has a cysteine ​​residue at position 239 of one heavy chain; each antibody has one cysteine ​​mutation site. mAb4 and mAb5 both have a cysteine ​​residue at position 160 of one light chain; each antibody has one cysteine ​​mutation site for conjugation. Furthermore, to prevent mismatch between the heavy and light chains, in vitro recombination using a "knob into hole" method is employed for expression. The difference between these two antibodies is that mAb5 has two light chains of λ and k, respectively, while mAb4 has two light chains of λ.

[0640] CEACAM5 antibodies include the following types: mAb6, mAb7, mAb8, mAb9, and mAb10. mAb7 is created by mutating amino acid position 160 of both light chains of the mAb6 antibody to cysteine. mAb8 is created by mutating amino acid position 160 of one light chain of the mAb6 antibody to cysteine, and performing a knock-in-hole mutation on the heavy chain (Knob: S354C, T366W; Hole: Y349C, T366S, L368A, Y407V). mAb9 is created by mutating amino acid position 239 of one heavy chain of the mAb6 antibody to cysteine, and also performing a knock-in-hole mutation. mAb10 is created by mutating amino acid position 239 of the heavy chain of the mAb6 antibody to cysteine.

[0641] mAb11 is based on the negative control protein anti-GP120. The amino acid at position 160 of one light chain of the antibody is mutated to cysteine, and the heavy chain is knob-in-hole and LALA is mutated. The ADC conjugated with this antibody is used as the negative control group in this study.

[0642] In addition, it also expressed the antibodies mAb12, mAb13, mAb14, and mAb15 used by SAR4078701 targeting CEACAM5.

[0643] The specific relationships and sequences mentioned above are shown in the table below:

[0644] Using ExpiCHO TM The expression system (Gibco, A29133) produces proteins. Specifically, it involves passage of ExpiCHO-S according to the required transfection volume. TM The cells (Gibco) were adjusted to a density of 3.5 × 10⁻⁶ cells the day before transfection.6 Cells / ml. On the day of transfection, the cell density was adjusted to 6 × 10⁶ cells / ml. 6 Cells / ml. Take a 50ml centrifuge tube and add OptiPRO transfection buffer at 8% of the transfection cell volume. TM SFM (Gibco, 12309019) was used to calculate the total required amount of plasmid for cell transfection at a rate of 0.8 μg / ml. Monoclonal antibodies (mAb2, mAb6, mAb7, mAb10, mAb12, mAb13, mAb14, mAb15) and parental antibodies (mAb4, mAb8) were premixed at a light chain:heavy chain plasmid mass ratio of 1:1; mAb3 and mAb9 were premixed at a light chain:heavy chain 1:heavy chain 2 = 1:1:1. The transfection buffer containing DNA plasmids was filtered through a 0.22 μm filter into a new 50 ml centrifuge tube. Reagent was added to the filtered mixture at a DNA:Reagent ratio of 1:4. (Polyplus, 101000019) Mix thoroughly and incubate at room temperature for 10 min. Then, immediately and slowly add the transfection reagent and plasmid DNA mixture to the cells while gently shaking the flask. Incubate the transfected cells at 37°C in a shaker with 8% CO2. After 18-22 h, add 6 μl / ml of Enhancer (Gibco, 100033019) and 300 μl / ml of Feed (Gibco, A29101-01) at 37°C, 120 rpm, and 8% CO2. Continue culturing until day 7 or when cell viability is ≤70%, collect the cell culture. Mix the cell culture with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L of cell culture), filter using a 0.22 μm disposable vacuum filter, and use the supernatant for subsequent affinity purification.

[0645] Affinity chromatography purification of the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the affinity chromatography column, followed by 10-20 column volumes of distilled water to wash the tubing and the column. The packing column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell material was passed through the column, and the packing column was washed with 10 column volumes of 1×PBS to remove non-specifically bound proteins. The packing column was washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), the eluent was collected, the pH was adjusted to 6.0 with 2M Tris, and the solution was filtered for sterilization.

[0646] The samples obtained after affinity purification of the parental antibodies required for mAb4, mAb5, and mAb8 require the addition of 50 mM MEA. After reacting overnight at room temperature, the samples are re-copied onto a HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) column. The column is equilibrated with 5-10 column volumes of PBS + 50 mM MEA, and then equilibrated again with 5-10 column volumes of PBS to remove excess reducing agent MEA. Finally, the packing material is washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluent is collected, the pH is adjusted to 6.0 with 2 M Tris, and the solution is filtered for sterilization.

[0647] In vitro reductive oxidation of mAb4, mAb5, and mAb8: The purified parental lines were mixed in a 1:1 molar ratio, and an appropriate amount of GSH was added. The pH of the reaction was adjusted to 8.0 with 1M arginine. The mixture was incubated overnight at room temperature. The reaction mixture was then transferred to PBS and stored at 4°C for later use.

[0648] Ion exchange chromatography purification of antibodies: A Mono S 5 / 50GL ion exchange chromatography column (from GE Healthcare) was used and placed in an AKTApure system (from GE Healthcare). Endotoxins were removed from the AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column using 0.5M NaOH for 2 hours. The system and column were then washed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20mM NaPO4, pH 6.0) until conductivity and pH stabilized. The protein obtained from affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was reequilibrated with 5 column volumes of loading buffer. Linear elution was performed using a gradient of 0-40% elution buffer (20mM NaPO4, 1M NaCl, pH 6.0) for a total of 30 column volumes. Samples were collected based on UV absorbance.

[0649] The purity of samples collected from each fraction was determined using size exclusion chromatography (SEC). Samples from fractions with a purity greater than 95% were pooled based on the SEC results. Antibody concentration was then determined. Further qualitative and quantitative analysis of antibody purity and impurities was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0650] Example B2. Antibody expression targeting B7H3 and Trop2

[0651] Antibodies targeting B7H3 and Trop2 were expressed for subsequent use.

[0652] There are two types of B7H3 antibodies: Ifinatamab and Ifinatamab_LLC160KIH. The Ifinatamab antibody is derived from Daiichi's Ifinatamab deruxtecan (Ifinatamab is prepared according to the description in WO2022102695). Ifinatamab_LLC160KIH is made by replacing the constant regions of both k light chains of Ifinatamab with λ type and mutating the 160th amino acid of one light chain to cysteine. In addition, to prevent mismatch between light and heavy chains, it is expressed by in vitro recombinant knob into hole (Knob: S354C, T366W; Hole: Y349C, T366S, L368A, Y407V).

[0653] There are two types of Trop2 antibodies: hRS7 and hRS7_LLC160_KIH. The hRS7 antibody is derived from Immunomedics' sacituzumab govitecan (hRS7 was prepared according to the description in US 7,238,785 B2). hRS7_LLC160_KIH is created by replacing the constant regions of both κ light chains of hRS7 with λ-type antibodies and mutating amino acid position 160 of one light chain to cysteine. In addition, to prevent mismatch between the light and heavy chains, a knob-to-hole in vitro recombination method is used for expression.

[0654] The specific relationships and sequences mentioned above are shown in the table below:

[0655] Antibodies were expressed according to the method in Example B1.

[0656] Using ExpiCHO TM The expression system (Gibco, A29133) produces proteins. Specifically, it involves passage of ExpiCHO-S according to the required transfection volume. TM The cells (Gibco) were adjusted to a density of 3.5 × 10⁻⁶ cells the day before transfection. 6 Cells / ml. On the day of transfection, the cell density was adjusted to 6 × 10⁶ cells / ml. 6 Cells / ml. Take a 50ml centrifuge tube and add OptiPRO transfection buffer at 8% of the transfection cell volume. TMSFM (Gibco, 12309019) was used to calculate the total required plasmid amount for cell transfection at a rate of 0.8 μg / ml. The monoclonal antibody Ifinatamab and the hRS7 parental antibody were premixed at a light chain:heavy chain plasmid mass ratio of 1:1, and Ifinatamab_LLC160_KIH and hRS7_LLC160_KIH were premixed at a light chain:heavy chain 1:heavy chain 2 mass ratio of 1:1:1. The transfection buffer containing the DNA plasmid was filtered through a 0.22 μm filter into a new 50 ml centrifuge tube. DNA:Reagent was added to the filtered mixture at a ratio of 1:4. (Polyplus, 101000019) Mix thoroughly and incubate at room temperature for 10 min. Then, immediately and slowly add the transfection reagent and plasmid DNA mixture to the cells while gently shaking the flask. Incubate the transfected cells at 37°C in a shaker with 8% CO2. After 18-22 h, add Enhancer (Gibco, 100033019) at 6 μL / ml cell volume and Feed (Gibco, A29101-01) at 300 μL / ml cell volume. Continue culturing at 37°C, 120 rpm, and 8% CO2. Collect the cell culture medium on day 7 or when cell viability is ≤70%. Mix the cell culture medium with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L cell culture medium) and filter using a 0.22 μm disposable vacuum filter. Use the supernatant for subsequent affinity purification.

[0657] Affinity chromatography purification of the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the affinity chromatography column, followed by 10-20 column volumes of distilled water to wash the tubing and the column. The packing column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell material was passed through the column, and the packing column was washed with 10 column volumes of 1×PBS to remove non-specifically bound proteins. The packing column was washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), the eluent was collected, the pH was adjusted to 6.0 with 2M Tris, and the solution was filtered for sterilization.

[0658] The samples obtained after affinity purification of the parental antibodies required for Ifinatamab_LLC160_KIH and hRS7_LLC160_KIH require the addition of 50mM MEA. After reacting overnight at room temperature, the samples are re-copied onto a HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) column. The column is equilibrated with 5-10 column volumes of PBS + 50mM MEA, and then equilibrated again with 5-10 column volumes of PBS to remove excess reducing agent MEA. Finally, the packing material is washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), the eluent is collected, the pH is adjusted to 6.0 with 2M Tris, and the solution is filtered for sterilization.

[0659] In vitro reductive oxidation of Ifinatamab_LLC160_KIH and hRS7_LLC160_KIH: The two purified parents were mixed in a 1:1 molar ratio, and an appropriate amount of GSH was added. The pH of the reaction was adjusted to 8.0 with 1M arginine. The mixture was incubated overnight at room temperature. The reaction mixture was then transferred to PBS and stored at 4°C for later use.

[0660] Ion exchange chromatography purification of antibodies: A Mono S 5 / 50GL ion exchange chromatography column (from GE Healthcare) was used and placed in an AKTApure system (from GE Healthcare). Endotoxins were removed from the AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column using 0.5M NaOH for 2 hours. The system and column were then washed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20mM NaPO4, pH 6.0) until conductivity and pH stabilized. The protein obtained from affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was reequilibrated with 5 column volumes of loading buffer. Linear elution was performed using a gradient of 0-40% elution buffer (20mM NaPO4, 1M NaCl, pH 6.0) for a total of 30 column volumes. Samples were collected based on UV absorbance.

[0661] The purity of samples collected from each fraction was determined using size exclusion chromatography (SEC). Samples from fractions with a purity greater than 95% were pooled based on the SEC results. Antibody concentration was then determined. Further qualitative and quantitative analysis of antibody purity and impurities was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0662] Example C. Preparation and Characterization of Antibody-Drug Conjugates

[0663] Example C1. Preparation and characterization of bitoxin ADC1 and monotoxin ADC2

[0664] A single-toxin ADC2 was prepared using Thiomab conjugation. A dual-toxin ADC1 was prepared using a sequential conjugation method: first, A1 was linked to the engineered cysteine ​​residue of the antibody light chain using Thiomab conjugation; then, the interchain disulfide bonds of the antibody were opened using conventional conjugation methods, and (e.g., eight) NT3 residues were conjugated to obtain the dual-toxin ADC1. The specific conjugation steps are as follows:

[0665] (a) Dissolve antibody mAb4 in histidine buffer (20 mM, pH 6.5, Sigma, Lot#K5376054201). Add the reducing agent TCEP aqueous solution and react the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio is 10–20; (iii) the optimal reaction temperature is 25 °C; and (iv) the optimal reaction pH is 6.0–8.0. After reduction, remove the reducing agent by desalting, ultrafiltration, or dialysis. Add the oxidizing agent dehydroascorbic acid (dhAA, dissolved in DMSO) and oxidize in a water bath for 2–3 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal dhAA / mAb molar ratio is 20–40; (iii) the optimal reaction temperature is 20–37 °C; and (iv) the optimal reaction pH is 6.0–8.0. An excess of linker toxin A1 (dissolved in DMSO) was added to the mixture, with the volume ratio of DMSO in the solution being 10%. The reaction mixture was left at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of A1 / mAb was 2–6, and (ii) the optimal reaction temperature was 20–37 °C. The resulting crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain monotoxin ADC2.

[0666] (b) Add an aqueous solution of TCEP, a reducing agent, to the product of step (a), and react the reaction mixture at 20–37°C for 3 hours, wherein: (i) the optimal concentration of ADC2 is 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb is 8–20; and (iii) the optimal pH of the reaction is 6.0–8.0. Add excess NT3 (dissolved in DMSO) to the mixture, and allow the reaction mixture to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT1 / mAb is 10–20; and (ii) the optimal temperature of the reaction is 20–37°C, thereby obtaining crude ADC product. The obtained crude ADC product is purified by spin desalting, ultrafiltration, or dialysis to obtain the dual-toxin ADC1.

[0667] The DAR values ​​of A1 and NT3 for the dual toxin ADC1, and the DAR value for ADC2, were determined using RP-HPLC. The DAR values ​​were calculated based on the peak areas at UV 280 nm. Figure 1a shows the RP plot used for the analysis and calculation of the A1 and NT3 DAR values ​​for ADC1. For the DAR value calculation of A1: where LC... hole With LC hole +NT1*1 (NT1 is the toxin of NT3) represents the light chain component that is not linked to drug A1, LC knob +A1*1+NT1*1 represents the light chain component of one A1 drug atom coupled together. For the DAR value calculation of NT1: where LC... hole Represents the light chain component without a drug linker, LC hole +NT1*1 and LC knob +A1*1+NT1*1 represents the light chain component of one NT1 drug coupled to it, HC hole +NT1*1、HC hole +NT1*2、HC knob +NT1*2、HC knob +NT1*3 and HC hole +NT1*3 represent the heavy chain components of 1, 2, and 3 NT1 drugs coupled together, respectively. The calculated DAR values ​​for A1 of ADC1 are 0.99 and NT1 are 7.74, while the DAR value for A1 of ADC2 is 0.99.

[0668] The purity of the ADCs was determined using SEC-HPLC. The purity of the ADC was obtained based on the peak area ratio of monomers, aggregates, and oligomers at UV 280 nm. Figure 1b shows the purity analysis of ADC1. It can be seen that ADC1 contains only a small amount of oligomers, with most components being homogeneous monomers, and a purity of 97.56%. The SEC purity of ADC2 was 100%.

[0669] Example C2. Preparation and characterization of bitoxin ADC3

[0670] The dual-toxin ADC3 was prepared using a method similar to that in Example C1, except that antibody mAb4 was replaced with mAb5. The DAR value and purity of ADC3 were determined using RP-HPLC and SEC-HPLC methods, and the results are shown in Figures 2a and 2b. The calculated DAR value for ADC3 was 0.94, the DAR value for NT3 was 7.85, and the SEC purity was 94.59%.

[0671] Example C3. Preparation and characterization of bitoxin ADC4 and monotoxin ADC5

[0672] The dual-toxin ADC4 and the monotoxin ADC5 were prepared using a method similar to that in Example C1.

[0673] (a) Dissolve antibody mAb3 in phosphate buffer (20 mM, pH 7.0; Na2HPO3, Sigma 30435-500G; NaH2PO3, Sigma V900060-6X500G). Add the reducing agent TCEP aqueous solution and incubate the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio is 10–20; (iii) the optimal reaction temperature is 25 °C; and (iv) the optimal pH value is 6.0–8.0. After reduction, remove the reducing agent by desalting, ultrafiltration, or dialysis. Add the oxidant dehydroascorbic acid (dhAA, dissolved in DMSO), and oxidize in a water bath for 2–3 h, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal molar ratio of dhAA / mAb is 20–40; (iii) the optimal reaction temperature is 20–37 °C; and (iv) the optimal pH value of the reaction is 6.0–8.0. Add excess linker toxin A1 (dissolved in DMSO) to the mixture, with the volume ratio of DMSO in the solution being 10%. Let the reaction mixture stand at room temperature for 2–3 h, wherein: (i) the optimal molar ratio of A1 / mAb is 2–6; and (ii) the optimal reaction temperature is 20–37 °C. The obtained crude ADC product is purified by spin desalting, ultrafiltration, or dialysis to obtain the monotoxin ADC5.

[0674] (b) Add an aqueous solution of TCEP, a reducing agent, to the product of step (a), and react the reaction mixture at 20–37°C for 3 hours, wherein: (i) the optimal concentration of ADC5 is 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb is 10–20; (iii) the optimal reaction temperature is 25°C; and (iv) the optimal pH value of the reaction is 6.0–8.0. Add excess NT3 (dissolved in DMSO) to the mixture, and allow the reaction mixture to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT1 / mAb is 10–20; and (ii) the optimal reaction temperature is 20–37°C, thereby obtaining crude ADC product. The obtained crude ADC product is purified by spin desalting, ultrafiltration, or dialysis to obtain the dual-toxin ADC4.

[0675] The DAR values ​​of A1 and NT3 for the dual toxins ADC4 and ADC5 were determined using RP-HPLC. The DAR values ​​were calculated based on the peak areas at UV 280 nm. Figure 3a shows the RP-HPLC plot used for the analysis and calculation of A1 and NT3 DAR values ​​for ADC4. For the DAR value calculation of A1: where HC... hole +NT1*1、HChole +NT1*2 and HC hole +NT1*3 represents the heavy chain component without the A1 drug linkage, HC knob +A1*1+NT3*1、HC knob +A1*1+NT1*2 and HC knob +A1*1+NT1*3 represents the heavy chain component coupled with one A1 drug. For the DAR value calculation of NT1: LC represents the light chain component without drug attachment, LC+NT3*1 represents the light chain component coupled with one NT1 drug, and HC... hole +NT1*1、HC knob +A1*1+NT1*1、HC hole +NT1*2、HC knob +A1*1+NT1*2、HC hole +NT1*3 and HC knob +A1*1+NT1*3 represent the heavy chain components of the 1st, 2nd, and 3rd NT3 drugs coupled together. The calculated DAR values ​​are 0.94 for A1 and 7.52 for NT3 of ADC4, and 0.94 for A1 of ADC5.

[0676] The purity of the ADCs was determined using SEC-HPLC. The purity of the ADCs was obtained based on the peak area ratio of monomers, aggregates, and oligomers at UV 280 nm. Figure 3b shows the purity analysis of ADC4. It can be seen that ADC4 contains only a small amount of oligomers and aggregates, with most components being homogeneous monomers, and a purity of 97.55%. The SEC purity of ADC5 was 97.1%.

[0677] Example C4. Preparation and characterization of bitoxin ADC6 and monotoxin ADC7

[0678] The dual-toxin ADC6 and the monotoxin ADC7 were prepared using a method similar to that in Example C1.

[0679] (a) Dissolve antibody mAb2 in phosphate buffer (20 mM, pH 7.0). Add the reducing agent TCEP aqueous solution and react the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio is 10–20; (iii) the optimal reaction temperature is 25 °C; and (iv) the optimal reaction pH is 6.0–8.0. After reduction, remove the reducing agent by desalting, ultrafiltration, or dialysis. Add the oxidizing agent dehydroascorbic acid (dhAA, dissolved in DMSO) and oxidize in a water bath for 2–3 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal dhAA / mAb molar ratio is 20–40; (iii) the optimal reaction temperature is 20–37 °C; and (iv) the optimal reaction pH is 6.0–8.0. An excess of linker toxin A1 (dissolved in DMSO) was added to the mixture, with the volume ratio of DMSO in the solution being 10%. The reaction mixture was left at room temperature for 2–3 hours. The optimal molar ratio of A1 / mAb was (i) 3–8, and the optimal reaction temperature was 20–37 °C. The crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain the monotoxin ADC7.

[0680] (b) Add an aqueous solution of TCEP, a reducing agent, to the product of step (a), and react the reaction mixture at 20–37°C for 3 hours, wherein: (i) the optimal concentration of ADC7 is 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb is 10–20; (iii) the optimal reaction temperature is 25°C; and (iv) the optimal pH value of the reaction is 6.0–8.0. Add excess NT3 (dissolved in DMSO), and allow the reaction mixture to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT1 / mAb is 10–20; and (ii) the optimal reaction temperature is 20–37°C, thereby obtaining crude ADC product. The obtained crude ADC product is purified by spin desalting, ultrafiltration, or dialysis to obtain the dual-toxin ADC6.

[0681] The DAR values ​​of A1 and NT3 of the dual-toxin ADC6 were determined using RP-HPLC. The DAR values ​​were calculated based on the peak areas at UV 280 nm. Figure 4a shows the RP plot used for the analysis and calculation of A1 and NT1 DAR values ​​for ADC6. For the A1 DAR value calculation: HC+NT1*1, HC+NT1*2, and HC+NT1*3 represent heavy chain components without A1 drug linkage, while HC+A1*1+NT3*1, HC+A1*1+NT1*2, and HC+A1*1+NT1*3 represent heavy chain components coupled with one A1 drug linkage. For the DAR value calculation of NT3: LC represents the light chain component without a drug, LC+NT1*1 represents the light chain component with one NT3 drug attached, and HC+NT1*1, HC+A1*1+NT1*1, HC+NT1*2, HC+A1*1+NT1*2, HC+NT1*3, and HC+A1*1+NT1*3 represent the heavy chain components with 1, 2, and 3 NT1 drugs attached, respectively. The calculated DAR value for A1 of ADC6 is 1.78, and the DAR value for NT3 is 7.60; the DAR value for A1 of ADC7 is 1.78.

[0682] The purity of the ADC was determined using SEC-HPLC. The purity of the ADC was determined based on the peak area ratio of monomers, aggregates, and oligomers at UV280 nm. Figure 4b shows the purity analysis of ADC6. It can be seen that ADC6 contains only a small amount of oligomers and aggregates, with most components being homogeneous monomers, achieving a purity of 97.11%.

[0683] Example C5. Preparation and characterization of monotoxin ADC8

[0684] mAb1 was dissolved in 20 mM histidine buffer, and a 20-fold molar ratio of TCEP aqueous solution was added. The reaction mixture was allowed to react at room temperature for 2 hours. Then, excess linker toxin (NT3, dissolved in DMSO) was added, and the reaction was continued at room temperature for another 1–2 hours. Finally, Zeba was used. TM A desalting column (Thermo Scientific, Zeba Spin Desalting Columns 40KMWCO) was used to remove unreacted small molecule drugs and other impurities, and the ADC was then displaced into a 20 mM histidine solution at pH 5.5 for storage. ADC8 was analyzed using RP-HPLC and SEC-HPLC, yielding a DAR value of 7.9 and an SEC purity of 96.8%.

[0685] Example C6. Preparation and characterization of monotoxin ADCs

[0686] The monotoxin ADC9 was prepared using a method similar to that in Example C3, step (a), except that A1 was replaced with mc-vc-PAB-MMAE. The DAR value and purity of ADC9 were analyzed using RP-HPLC and SEC-HPLC. Figure 5a shows the RP plot for the DAR value analysis and calculation of MMAE used for ADC9, where mAb represents the antibody component without drug conjugation, and mAb+MMAE*1 represents the antibody component conjugated with one MMAE drug. The calculated DAR value was 0.93. Figure 5b is the purity analysis plot of ADC9. It can be seen that ADC9 contains only a small amount of aggregates, with most components being homogeneous monomers, and a purity of 99.63%.

[0687] The single toxins ADC10, ADC11, ADC12 and ADC13 were prepared using a method similar to that in Example C3, step (a), except that the linker toxin A1 was replaced accordingly according to Table 1 below.

[0688] The single toxins ADC14, ADC15, and ADC16 were prepared using a method similar to that in Example C1, step (a), except that the linker toxin A1 was replaced accordingly according to Table 1 below.

[0689] DAR value and purity were analyzed using RP-HPLC and SEC-HPLC methods, and the results are shown in Table 1.

[0690] Table 1. Characterization of ADCs

[0691] Example C7. Preparation and characterization of ADCs targeting CEACAM5

[0692] Monotoxins ADC20, ADC21, and ADC22 were prepared using a method similar to that in Example C1, step (a), except that the antibody was replaced with mAb8, and A1 (for ADC20), A3 (for ADC21), and A4 (for ADC22) were used as linker toxins, respectively.

[0693] Using the preparation method of Example C1, mAb8 antibody was sequentially conjugated with A1 and NT3 to obtain dual-toxin ADC17.

[0694] Using the preparation method of Example C1, mAb8 antibody was sequentially conjugated with A3 and NT3 to obtain dual-toxin ADC18.

[0695] Using the preparation method of Example C1, mAb8 antibody was sequentially conjugated with A4 and NT3 to obtain dual-toxin ADC19.

[0696] The DAR value and SEC purity of the ADCs were determined using RP-HPLC and SEC-HPLC, and the results are shown in Table 2.

[0697] Figure 6a is the RP plot used for DAR value analysis and calculation of ADC17. Regarding the DAR value calculation for A1, LC represents the component without drug linkage, and LC+A1 represents the component coupled with one A1 drug. Regarding the DAR value calculation for NT1, LC represents the light chain component without drug linkage, LC+NT1 and LC+A1+NT1 represent the light chain components coupled with one NT1 drug, and HC+NT1*1, HC+NT1*2, and HC+NT1*3 represent the heavy chain components coupled with 1, 2, and 3 NT1 drugs, respectively. The calculated DAR values ​​are: A1 DAR value of 0.91 and NT1 DAR value of 7.5 for ADC17; A3 DAR value of 0.90 and NT1 DAR value of 7.64 for ADC18; and A4 DAR value of 0.87 and NT1 DAR value of 7.37 for ADC19.

[0698] Figure 6b is a purity analysis diagram of ADC17. It can be seen that ADC17 contains only a small amount of oligomers, with most components being homogeneous monomers, and a purity of 96.32%.

[0699] Table 2. Characterization of ADCs

[0700] Example C8. Preparation and characterization of monotoxin ADC23

[0701] The mAb6 antibody was dissolved in 20 mM histidine buffer, and a 20-fold molar ratio of TCEP reducing agent aqueous solution was added. The reaction mixture was incubated at room temperature for 2 hours, then an excess of linker toxin (NT3, dissolved in DMSO) was added, and the reaction was continued at room temperature for another 1-2 hours. Finally, Zeba was used. TM Unreacted small molecule drugs and other impurities were removed using a desalting centrifuge column, and the ADC was then displaced into a 20 mM histidine solution at pH 5.5 for storage. The DAR value and SEC purity of the ADC were determined using RP-HPLC and SEC-HPLC, and the results are shown in Table 3.

[0702] Example C9. Preparation and characterization of monotoxin ADC24 and bitoxin ADC25

[0703] (a) Antibody mAb7 was dissolved in histidine buffer (20 mM, pH 6.5). A reducing agent, TCEP aqueous solution, was added, and the reaction mixture was incubated at room temperature for 2 hours. The antibody concentration was 5 mg / mL, and the TCEP / mAb molar ratio was 20. After reduction, the reducing agent was removed by desalting, ultrafiltration, or dialysis. Oxidizing agent dhAA was added, and oxidation was carried out at 37°C for 3 hours. The antibody concentration was 5 mg / mL, and the dhAA / mAb molar ratio was 40. An excess of linker toxin (A1, dissolved in DMSO) was added to the mixture, with a DMSO volume ratio of 10%. The reaction mixture was incubated at room temperature for 1–2 hours, with an A1 / mAb molar ratio of 6, and the reaction temperature was 20–37°C. The resulting crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC24.

[0704] (b) Add a 20-fold molar ratio of the reducing agent TCEP aqueous solution to the ADC24 obtained in step (a), and react the reaction mixture at 20–37°C for 3 hours, wherein the concentration of ADC24 is 5 mg / mL, and the optimal pH for the reaction is 6.0–8.0. Add excess NT3 (dissolved in DMSO) to the mixture, and allow the reaction mixture to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT1 / mAb is 10–16, and (ii) the optimal reaction temperature is 20–37°C, thereby obtaining crude ADC25. The obtained crude ADC25 is purified by spin desalting, ultrafiltration, or dialysis to obtain the dual-toxin ADC25.

[0705] The DAR values ​​and SEC purity of ADC24 and ADC25 were determined using RP-HPLC and SEC-HPLC. The analysis showed that the DAR value of ADC24 was 1.91, with a purity of 99.34%; the DAR value of A1 for ADC25 was 1.88, the DAR value of NT3 was 7.83, and the SEC purity was 97.71%. The results are shown in Table 3.

[0706] Example C10. Preparation and characterization of monotoxin ADC26 and bitoxin ADC27

[0707] (a) Dissolve antibody mAb9 in histidine buffer (20 mM, pH 6.5). Add the reducing agent TCEP aqueous solution and incubate the reaction mixture at room temperature for 2 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio is 10–20; (iii) the optimal reaction temperature is 25 °C; and (iv) the optimal reaction pH is 6.0–8.0. After reduction, remove the reducing agent by desalting, ultrafiltration, or dialysis. Add dhAA oxidant and oxidize in a water bath for 2–3 hours, wherein: (i) the optimal antibody concentration is 5–15 mg / mL; (ii) the optimal dhAA / mAb molar ratio is 20–40; (iii) the optimal reaction temperature is 20–37 °C; and (iv) the optimal reaction pH is 6.0–8.0. An excess of linker toxin (A1, dissolved in DMSO) was added to the mixture, with the volume ratio of DMSO in the solution being 10%. The reaction mixture was left at room temperature for 1–2 hours, wherein (i) the optimal molar ratio of A1 / mAb was 3–6, and (ii) the optimal reaction temperature was 20–37 °C. The resulting crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC26.

[0708] (b) Add a 20-fold molar ratio of the reducing agent TCEP aqueous solution to the ADC26 obtained in step (a), and react the reaction mixture at 20–37°C for 3 hours, wherein: (i) the optimal concentration of ADC26 is 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb is 8–20; and (iii) the optimal pH of the reaction is 6.0–8.0. Add excess NT3 (dissolved in DMSO) to the mixture, and allow the reaction mixture to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of NT1 / mAb is 10–16; and (ii) the optimal temperature of the reaction is 20–37°C, thereby obtaining crude ADC27. The obtained crude ADC27 is purified by spin desalting, ultrafiltration, or dialysis to obtain the dual-toxin ADC27.

[0709] The DAR values ​​and SEC purity of ADC26 and ADC27 were determined using RP-HPLC and SEC-HPLC. Analysis showed that the DAR value of ADC26 was 0.93, and the SEC purity was 96.39%; the DAR value of A1 in ADC27 was 0.93, the DAR value of NT3 was 7.7, and the SEC purity was 96.15%. The results are shown in Table 3.

[0710] Example C11. Preparation and characterization of positive control molecule M9140 and negative control molecule ADC28

[0711] M9140 ADC is a positive control ADC targeting CEACAM5, and its antibody sequence is derived from WO2022048883A1 (antibody SO8G4, heavy chain SEQ ID NO:13, light chain: SEQ ID NO:14). The antibody was dissolved in 20 mM histidine buffer, and a 20-fold molar ratio of reducing agent TCEP aqueous solution was added. The reaction mixture was incubated at room temperature for 2 hours, then an excess of the linker toxin Mal-Gly-PAB-ethatecan-D-glucuronic acid (MCE, HY-153179) was added, and the reaction was continued at room temperature for 1-2 hours. Finally, Zeba was used. TM Unreacted small molecule drugs and other impurities were removed using a desalting centrifuge column, and the ADC was then displaced into a 20 mM histidine solution at pH 5.5 for storage. The DAR value and SEC purity of the ADC were determined using RP-HPLC and SEC-HPLC, and the results are shown in Table 3.

[0712] The negative control molecule ADC28 was prepared according to the method in Example C1, except that mAb11 was used as the negative control antibody. The DAR value and purity of ADC28 were determined using RP-HPLC and SEC-HPLC methods, and the results are shown in Table 3.

[0713] Example C12. Preparation and Characterization of ADCs

[0714] ADC29

[0715] Antibody mAb13 was dissolved in histidine buffer (20 mM, pH 6.5). A TCEP aqueous solution was added as a reducing agent, and the reaction mixture was incubated at room temperature for 2 hours, wherein: (i) the optimal antibody concentration was 5–15 mg / mL; (ii) the optimal TCEP / mAb molar ratio was 10–20; (iii) the optimal reaction temperature was 25 °C; and (iv) the optimal reaction pH was 6.0–8.0. After reduction, the reducing agent was removed by desalting, ultrafiltration, or dialysis. dhAA oxidizing agent was added, and oxidation was carried out in a water bath for 2–3 hours, wherein: (i) the optimal antibody concentration was 5–15 mg / mL; (ii) the optimal dhAA / mAb molar ratio was 20–40; (iii) the optimal reaction temperature was 20–37 °C; and (iv) the optimal reaction pH was 6.0–8.0. An excess of a DMSO solution of mc-vc-PAB-MMAE (MCE, HY-15575) was added to the mixture, with a DMSO volume ratio of 10%. The reaction mixture was allowed to stand at room temperature for 1–2 hours. The optimal molar ratio of linker toxin to mAb was (i) 3–6, and the optimal reaction temperature was (ii) 20–37 °C. The crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC29. RP-HPLC and SEC-HPLC analysis showed that the SEC purity of ADC29 was 96.83%, and the DAR was 1.75.

[0716] ADC30

[0717] Antibody mAb15 was dissolved in histidine buffer (20 mM, pH 6.5). A reducing agent, TCEP aqueous solution, was added, and the reaction mixture was incubated at room temperature for 2 hours. The optimal antibody concentration was 5–15 mg / mL; the optimal TCEP / mAb molar ratio was 10–20; the optimal reaction temperature was 25 °C; and the optimal pH was 6.0–8.0. After reduction, excess linker toxin (NT2, dissolved in DMSO solution) was added to react with the antibody. The volume ratio of DMSO in the solution was 10%. The reaction mixture was incubated at room temperature for 1–2 hours. The optimal linker-toxin / mAb molar ratio was 4.0–8.0; and the optimal reaction temperature was 20–37 °C. The crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC30. RP-HPLC and SEC-HPLC analysis showed that the SEC purity of ADC30 was 97.54%, and the DAR was 3.50.

[0718] ADC31

[0719] ADC29 was prepared as described above, the only difference being that mAb13 was replaced with mAb14. A TCEP aqueous solution was added to ADC29, and the reaction mixture was allowed to react at room temperature for 2 hours, wherein: (i) the optimal concentration of ADC29 was 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb was 10–20; (iii) the optimal reaction temperature was 25°C; and (iv) the optimal pH was 6.0–8.0. After reduction, excess linker toxin (NT2, dissolved in DMSO solution) was added to react with the antibody, with the DMSO volume ratio in the solution being 10%. The reaction mixture was allowed to stand at room temperature for 1–2 hours, wherein: (i) the optimal molar ratio of linker toxin / mAb was 4.0–8.0; and (ii) the optimal reaction temperature was 20–37°C. The resulting crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC31. RP-HPLC and SEC-HPLC analyses showed that the SEC purity of ADC31 was 98.46%, the DAR of MMAE was 1.75, and the DAR of NT2 was 4.00.

[0720] ADC32

[0721] Antibody mAb12 was dissolved in histidine buffer (20 mM, pH 6.5). A reducing agent, TCEP aqueous solution, was added, and the reaction mixture was incubated at room temperature for 2 hours. The optimal concentrations of the antibody were: (i) 5–15 mg / mL; (ii) the optimal molar ratio of TCEP / mAb was 10–20; (iii) the optimal reaction temperature was 25 °C; and (iv) the optimal pH was 6.0–8.0. After reduction, excess linker toxin (NT2, dissolved in DMSO solution) was added to react with the antibody. The volume ratio of DMSO in the solution was 10%. The reaction mixture was incubated at room temperature for 1–2 hours. The optimal molar ratio of linker toxin / mAb was: (i) 8.0–12.0; and (ii) the optimal reaction temperature was 20–37 °C. The crude ADC product was purified by spin desalting, ultrafiltration, or dialysis to obtain ADC32. RP-HPLC and SEC-HPLC analysis showed that the SEC purity of ADC32 was 99.37%, and the DAR was 7.60.

[0722] ADC33

[0723] Following a similar preparation method to ADC32, the wild-type antibody against GP120 (the unmutated wild-type antibody corresponding to mAb11) was conjugated with NT2 to obtain ADC33. RP-HPLC and SEC-HPLC analyses showed that ADC33 had a DAR value of 7.33 and a SEC purity of 100%.

[0724] Table 3. DAR values ​​and SEC purity of ADCs

[0725] Example C13. Preparation and characterization of the dual toxin ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 and the single toxin ifinatamab LLC160KIH-A1-DAR1

[0726] The dual-toxin ADC was prepared according to the method described in Example C1.

[0727] ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 and monotoxin ADC

[0728] The difference with ifinatamab_LLC160_KIH-A1-DAR1 is that antibody mAb4 is replaced with...

[0729] ifinatamab_LLC160_KIH. The determination was performed using RP-HPLC and SEC-HPLC methods.

[0730] The DAR values ​​and purities of ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 and ifinatamab_LLC160_KIH-A1-DAR1 were calculated. The DAR value of A1 in ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 is 0.84, the DAR value of NT3 is 7.70, and the SEC purity is 92.85%. The DAR value of A1 in ifinatamab_LLC160_KIH-A1-DAR1 is 0.84, and the SEC purity is 94.34%.

[0731] Example C14. Preparation and characterization of the monotoxin ifinatamab-NT3-DAR8

[0732] The single toxin ifinatamab-NT3-DAR8 was prepared using a method similar to that in Example C5. ifinatamab was dissolved in 20 mM histidine buffer, and a 20-fold molar ratio of the reducing agent TCEP aqueous solution was added. After reacting the reaction mixture at room temperature for 2 hours, an excess of the linker toxin (NT3, dissolved in DMSO) was added, and the reaction was continued at room temperature for 1–2 hours. Then, it was reacted with Zeba… TMA desalting column (Thermo Scientific, Zeba Spin Desalting Columns 40K MWCO) was used to remove unreacted small molecule drugs and other impurities, and the ADC was displaced into a 20 mM histidine solution at pH 5.5 for storage. ifinatamab-NT3-DAR8 was analyzed by RP-HPLC and SEC-HPLC, yielding a DAR value of 7.82 for NT3 and an SEC purity of 99.59%.

[0733] Example C15. Preparation and characterization of the dual toxin hRS7_LLC160_KIH-A1-DAR1+NT3-DAR8 and the single toxin hRS7_LLC160_KIH-A1-DAR1

[0734] A dual-toxin ADC, hRS7_LLC160_KIH-A1-DAR1+NT3-DAR8, and a single-toxin ADC, hRS7_LLC160_KIH-A1-DAR1, were prepared according to the method described in Example C1, except that antibody mAb4 was replaced with hRS7_LLC160_KIH. The DAR values ​​and purities of hRS7_LLC160_KIH-A1-DAR1+NT3-DAR8 and hRS7_LLC160_KIH-A1-DAR1 were determined using RP-HPLC and SEC-HPLC methods. The calculated DAR value of A1 for hRS7_LLC160_KIH-A1-DAR1+NT3-DAR8 was 0.82, the DAR value of NT3 was 7.66, and the SEC purity was 95.48%. The DAR value of A1 in hRS7_LLC160_KIH-A1-DAR1 is 0.82, and the SEC purity is 97.81%.

[0735] Example C16. Preparation and characterization of monotoxin hRS7-NT3-DAR8

[0736] The monotoxin hRS7-NT3-DAR8 was prepared using a method similar to that in Example C5. hRS7 was dissolved in 20 mM histidine buffer, and a 20-fold molar ratio of the reducing agent TCEP aqueous solution was added. After reacting the reaction mixture at room temperature for 2 hours, an excess of the linker toxin (NT3, dissolved in DMSO) was added, and the reaction was continued at room temperature for 1–2 hours. Then, it was treated with Zeba... TMA desalting column (Thermo Scientific, Zeba Spin Desalting Columns 40K MWCO) was used to remove unreacted small molecule drugs and other impurities, and the ADC was displaced into a 20 mM histidine solution at pH 5.5 for storage. hRS7-NT3-DAR8 was analyzed using RP-HPLC and SEC-HPLC, yielding a DAR value of 7.55 and an SEC purity of 98.38%.

[0737] Example D. Biological Part

[0738] The cytotoxic activity, tumor growth inhibitory activity, and safety of the ADCs of this invention were verified in different cell lines and animal models. In this study, human gastric cancer cells NCI-N87, SNU601, human breast adenocarcinoma cells SKBR3, human breast cancer cells JIMT1, human breast cancer cells HCC1954, human ovarian cancer cells SKOV3, human colon cancer cells HT55, human gastric cancer cells MKN45, human pancreatic adenocarcinoma cells HPAF-II, human colon cancer tumor cells LS174T, human pharyngeal squamous cell carcinoma cells FADU, and human bladder cancer cells RT112 were all obtained from Nanjing Kebai Biotechnology Co., Ltd. Human malignant melanoma cells A375 were obtained from ATCC.

[0739] RPMI 1640 medium (22400-071), DMEM medium (11965-092), McCoy's 5a medium (16600-082), MEM Alpha medium (12561-056) and FBS (10091-148) were obtained from Gibco unless otherwise indicated.

[0740] Example D1. In vitro toxicity test of HER2-targeting ADCs on NCI-N87, SKBR3 and JIMT1 cells

[0741] 10% FBS was added to RPMI 1640 and mixed thoroughly to serve as the growth medium for NCI-N87 cells. 10% FBS was added to McCoy's 5a and mixed thoroughly to serve as the growth medium for SKBR3 cells. 10% FBS was added to DMEM and mixed thoroughly to serve as the growth medium for JIMT1 cells. The cell concentration was adjusted to 20,000 cells / mL with the growth medium, and 50 μL / well (1000 cells / well) was seeded into 96-well white-background plates (136101, Thermo). 200 μL of PBS (pH 7.4) was added to each well to seal the plates, and the plates were incubated overnight at 37°C. ADCs were diluted with the medium, with 500 nM as the highest concentration, and serially diluted 5-fold for a total of 9 dilutions. 50 μL / well of the diluted ADC solution or blank medium (control) was added to the cell plate, with a total volume of 100 μL. The cells were incubated at 37°C for 5 days. Equilibrate the 96-well plate and Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of CTG solution to each well. Shake at 300 rpm for 30 min on a plate shaker to fully lyse the cells. Detect the luminescence value at all wavelengths using a microplate reader (Spectra MAX i3x, Molecular Device).

[0742] The cell proliferation inhibition rate was calculated using the following formula: Cell viability (%) = a / b * 100, where a: average luminescence value of the experimental sample wells; b: average luminescence value of the control culture medium wells. The IC50 of the drug was calculated by fitting data with GraphPad Prism. 50 .

[0743] Figure 7 shows the cell survival percentages of ADCs in NCI-N87 (A), SKBR3 (B), and JIMT-1 (C) cells, respectively. It can be seen that the ADCs of this invention exhibit significant tumor cell killing activity in the test cells, particularly significantly superior to the single-toxin ADC8. Furthermore, in NCI-N87 cells, the inhibitory IC50 of ADC1... 50 The IC value is 0.1734 nM, while the suppression IC of ADC8 is... 50 The inhibitory effect of ADC1 on tumor growth was 1.005 nM, and it was 5.8 times stronger than that of ADC8. In SKBR3 cells, the inhibitory IC50 of ADC1 was [missing value]. 50 The IC value is 0.5657 nM, while the suppression IC of ADC8 is... 50 The inhibitory effect of ADC1 on tumor growth was 202.7 nM, and it was 358.3 times stronger than that of ADC8. In JIMT1 cells, the inhibitory IC50 of ADC3 was... 50 The IC value is 2.487 nM, while the suppression IC of ADC8 is... 50The concentration was 289.9 nM, and the tumor growth inhibitory effect of ADC3 was 116.5 times stronger than that of ADC8. This indicates that A1 plays a significant role in cell killing, and that the dual-toxin ADCs of this invention have a clear advantage over single-toxin ADCs.

[0744] Example D2. In vitro toxicity test of HER2-targeting ADCs on NCI-N87 and HCC1954 cells

[0745] Add 10% FBS to RPMI 1640 and mix well to prepare the growth medium for NCI-N87 and HCC1954 cells. Adjust the cell concentration to 20,000 cells / mL with the growth medium, and seed 50 μL / well (1000 cells / well) into 96-well white-background plates (136101, Thermo). Seal each well with 200 μL of PBS (pH 7.4) and incubate overnight at 37°C. Dilute ADCs with the medium, with 500 nM as the highest concentration, and perform 5-fold serial dilutions for a total of 9 dilutions. Add 50 μL / well of the diluted ADC solution or blank medium (control) to the cell plate, for a total volume of 100 μL. Incubate at 37°C for 5 days. Equilibrate the cultured 96-well plates and Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of CTG solution to each well. The cells were shaken at 300 rpm for 30 minutes on a plate shaker to ensure complete cell lysis. The luminescence values ​​were then measured at all wavelengths using a microplate reader (Spectra MAX i3x, Molecular Device).

[0746] Enhertu is an antibody-drug conjugate (ADC) that combines the anti-HER2 antibody trastuzumab with the topoisomerase inhibitor deruxtecan via a linker. It has been approved in several countries and regions for the treatment of adult patients with unresectable or metastatic HER2-positive breast cancer.

[0747] As described in Example D1, cell survival (%) and IC50 inhibition of ADCs were measured. 50 The results are shown in Figure 8 and Table 4. It can be seen that the ADC4 of the present invention exhibits significant tumor cell killing activity and excellent inhibitory IC50 in the tested cells. 50 In particular, it is significantly superior to single-toxin ADC8 and the benchmark molecule Enhertu, indicating that A1 plays a significant role in the killing of tumor cells, and the dual-toxin ADCs of the present invention have obvious advantages over single-toxin ADCs.

[0748] Table 4. Suppression IC of ADCs 50

[0749] Example D3. In vitro toxicity test of HER2-targeting ADCs on NCI-N87, JIMT1 and SKOV3 cells

[0750] 10% FBS was added to RPMI 1640 and mixed thoroughly to serve as the growth medium for NCI-N87 cells. 10% FBS was added to DMEM and mixed thoroughly to serve as the growth medium for JIMT1 cells. 10% FBS was added to McCoy's 5a and mixed thoroughly to serve as the growth medium for SKOV3 cells. The cell concentration was adjusted to 20,000 cells / mL with growth medium, and 50 μL / well (1000 cells / well) was seeded into 96-well white-background plates (136101, Thermo). 200 μL of PBS (pH 7.4) was added to each well to seal the plates, and the plates were incubated overnight at 37°C. ADCs were diluted with culture medium, with 500 nM as the highest concentration, and serially diluted 5-fold for a total of 9 dilutions. 50 μL / well of diluted ADC solution or blank medium (control) was added to the cell plate, with a total volume of 100 μL. The plates were incubated at 37°C for 5 days. Equilibrate the cultured 96-well plates and Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of CTG solution to each well. Shake at 300 rpm for 30 min on a plate shaker to fully lyse the cells. Detect the luminescence values ​​at all wavelengths using a microplate reader (Spectra MAX i3x, Molecular Device).

[0751] As described in Example D1, cell survival (%) and IC50 inhibition of ADCs were measured. 50 The results are shown in Figure 9 and Table 5. It can be seen that the ADC of the present invention exhibits significant tumor cell killing activity and excellent inhibitory IC50 in the tested cells. 50 It even significantly outperformed the benchmark molecule Enhertu. Specifically, in NCI-N87 cells, ADC6 showed superior maximal killing power and IC50. 50 Both were significantly superior to the baseline molecule Enhertu; in JIMT1 cells, ADC6 exhibited nearly 90% cell killing activity, while Enhertu showed almost no killing effect on JIMT1 cells, indicating that ADC6 was significantly superior to the baseline molecule; in SKOV3 cells, the inhibitory IC50 of ADC6 was significantly higher than that of Enhertu. 50 It is also stronger than Enhertu.

[0752] Table 5. Suppression IC of ADCs 50

[0753] Example D4. In vitro toxicity test of HER2-targeting ADCs on NCI-N87 cells

[0754] Add 10% FBS to RPMI 1640 and mix well to prepare the growth medium for NCI-N87 cells. Adjust the cell concentration to 20,000 cells / mL with the growth medium, and seed 50 μL / well (1000 cells / well) into 96-well white-background plates (136101, Thermo). Seal each well with 200 μL of PBS (pH 7.4) and incubate overnight at 37°C. Dilute the ADCs with the medium, with 500 nM as the highest concentration, and perform 5-fold serial dilutions for a total of 9 dilutions. Add 50 μL / well of the diluted ADC solution or blank medium (control) to the cell plate, for a total volume of 100 μL. Incubate at 37°C for 5 days. Equilibrate the cultured 96-well plates and Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of CTG solution to each well. Cells were fully lysed by shaking at 300 rpm for 30 minutes on a plate shaker. The luminescence values ​​were then measured at all wavelengths using a microplate reader (Spectra MAX i3x, Molecular Device).

[0755] As described in Example D1, cell survival (%) and IC50 inhibition of ADCs were measured. 50 The results are shown in Figure 10 and Table 6. It can be seen that the ADCs coupled with the linker toxin of this invention exhibited significant tumor cell killing activity and excellent inhibitory IC50 in the tested cells. 50 .

[0756] Table 6. Suppression IC of ADCs 50

[0757] Example D5. In vitro toxicity test of HER2-targeting ADCs on NCI-N87 and SKBR3 cells

[0758] Add 10% FBS to RPMI 1640 and mix well to use as the growth medium for NCI-N87 cells. Add 10% FBS to McCoy's 5a and mix well to use as the growth medium for SKBR3 cells. Adjust the cell concentration to 20,000 cells / mL with the growth medium, and seed 50 μL / well (1000 cells / well) into 96-well white-background plates (136101, Thermo). Seal each well with 200 μL of PBS (pH 7.4) and incubate overnight at 37°C. Dilute ADCs with the medium, with 500 nM as the highest concentration, and perform 5-fold serial dilutions for a total of 9 dilutions. Add 50 μL / well of the diluted ADC solution or blank medium (control) to the cell plate, for a total volume of 100 μL. Incubate at 37°C for 5 days. Equilibrate the cultured 96-well plates and Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of CTG solution to each well. Shake at 300 rpm for 30 min on a plate shaker to fully lyse the cells. Detect the luminescence values ​​at all wavelengths using a microplate reader (Spectra MAX i3x, Molecular Device).

[0759] As described in Example D1, the cell survival (%) and IC50 of the ADCs were measured. 50 The results are shown in Figure 11 and Table 7. It can be seen that the ADCs of the present invention exhibit significant tumor cell killing activity in the tested cells. In particular, A1, A6, and A7 showed considerably strong ADC killing activity in NCI-N87 and SKBR3 cells.

[0760] Table 7. Suppression IC of ADCs 50

[0761] Example D6. In vitro toxicity test of CEACAM5-targeting ADCs on HT55 and MKN45 cells.

[0762] Add 10% FBS (SH30406.05, HYCLONE) to MEM Alpha and mix well to use as the growth medium for HT55 cells. Add 10% FBS (SH30406.05, HYCLONE) to RPMI 1640 (22400-089, Gibco) and mix well to use as the growth medium for MKN45 cells. Adjust the cell concentration to 20,000 cells / mL with the medium and seed 50 μL / well (1000 cells / well) into 96-well white plates (167008, NUNC) and incubate overnight at 37°C. Dilute the ADCs with the medium in a 5-fold serial dilution series (9 dilutions in total). Add 50 μL of diluted ADC solution or control (blank medium) to each well, for a total volume of 100 μL. Seal the wells with 200 μL of PBS (pH 7.4) and incubate at 37°C for 5 days. The cultured 96-well plates and CTG (VKEY-BIO TECHNOLOGIES, AJC06A) were equilibrated to room temperature. 100 μL of CTG solution was added to each well. The plates were shaken on a plate for 5 min to fully lyse the cell clusters, and then incubated at room temperature for 5 min to stabilize the luminescence signal. Cell viability was calculated using a microplate reader (Spectra MAX i3x, Molecular Divices) at A450. Data were fitted using GraphPad Prism, and the inhibitory IC50 of the drug was calculated. 50 .

[0763] The results are shown in Figure 12 and Table 8. It can be seen that the ADCs of the present invention exhibit significant tumor cell killing activity and excellent inhibitory IC50 in the tested cells. 50 .

[0764] Table 8. Suppression IC of ADCs 50

[0765] Example D7. In vitro toxicity test of CEACAM5-targeting ADCs on HPAF-II and HT55 cells.

[0766] Add 10% FBS (SH30406.05, HYCLONE) to RPMI 1640 and mix well to use as the growth medium for HPAF-II cells. Add 10% FBS (SH30406.05, HYCLONE) to MEM Alpha and mix well to use as the growth medium for HT55 cells. Adjust the cell concentration to 20,000 cells / mL with the growth medium, and seed 50 μL / well (1000 cells / well) into 96-well white plates (167008, NUNC) and incubate overnight at 37°C. Dilute the ADCs with the medium, performing 5-fold serial dilutions for a total of 9 dilutions. Add 50 μL of the diluted ADC solution or control (blank medium) to each well, for a total volume of 100 μL. Seal the wells with 200 μL of PBS (pH 7.4) and incubate at 37°C for 5 days. Equilibrate the cultured 96-well plates and CTG (VKEY-BIO TECHNOLOGIES, AJC06A) to room temperature. Add 100 μL of CTG solution to each well. Shake on a plate for 5 min to fully lyse the cell clusters, then incubate at room temperature for 5 min to stabilize the luminescence signal. Detect using a microplate reader (Spectra MAX i3x, Molecular Divices), reading at A450, and calculate cell viability percentage.

[0767] The cell proliferation inhibition rate was calculated using the following formula: Cell proliferation inhibition rate (%) = a / b * 100, where a: the average value of the experimental sample wells; b: the average value of the control culture medium added wells. The IC50 of the drug was calculated by fitting the data with GraphPad Prism. 50 The inhibitory IC50 of the drug was calculated by fitting data using GraphPad Prism. 50 .

[0768] The results are shown in Figure 13 and Tables 9a to 9b. It can be seen that the ADCs of the present invention exhibit significant tumor cell killing activity in the tested cells. In particular, ADC26 and ADC27 both showed strong cytotoxicity in HT55 cells.

[0769] Table 9a. Suppression IC of ADCs 50

[0770] Table 9b. Suppression IC of ADCs 50

[0771] Example D8. In vitro toxicity test of CEACAM5-targeting dual toxins HPAF-II and LS174T cells.

[0772] HT55 cell (Kobco) growth medium was prepared by adding 10% FBS (SH30406.05, HYCLONE) to MEM Alpha (12561-056, Gibco) and mixing thoroughly. HPAF-II and LS174T cell (Kobco) growth media were prepared by adding 10% FBS (SH30406.05, HYCLONE) to RPMI 1640 (22400-071, Gibco) and mixing thoroughly. The cell concentration was adjusted to 20,000 cells / mL using the growth medium, and 50 μL / well (1000 cells / well) was seeded into 96-well white plates (167008, NUNC) and incubated overnight at 37°C. The ADC was diluted with the medium in a 5-fold serial dilution series (9 dilutions in total). 50 μL of diluted ADC solution or blank medium (control) was added to each well, for a total volume of 100 μL. The well edges of the plates were sealed with 200 μL of PBS (pH 7.4). The plates were incubated at 37°C for 5 days. The cultured 96-well plates and CTG (VKEY-BIO TECHNOLOGIES, AJC06A) were then equilibrated to room temperature. An equal volume of 100 μL CTG solution was added to each well. The plates were shaken on a plate for 5 min to ensure complete cell lysis, and then incubated at room temperature for 5 min to stabilize the luminescence signal. Cell viability was calculated using a microplate reader (Spectra MAX i3x, Molecular Divices) at A450. Data were fitted using GraphPad Prism, and the inhibitory IC50 of the drug was calculated. 50 .

[0773] The results are shown in Figure 14 and Table 10. It can be seen that in HPAF-II cells, the cytotoxic activity of ADC31 was similar to that of ADC29, but much stronger than that of ADC30 (Figure 14A). In LS174T cells, the cytotoxic effect of ADC31 was stronger than that of both ADC29 and ADC30 (Figure 14B). This demonstrates the superiority of the dual-toxin ADCs.

[0774] Table 10. Suppression IC of ADCs 50

[0775] Example D9. Therapeutic effect of ADC drugs in HT55 tumor-bearing mouse model

[0776] This experiment used HT55 cells to inoculate CB17 SCID mice to determine the antitumor effect of the present invention.

[0777] CB17 SCID mice: Female CB17 SCID mice were purchased from Beijing Viton Lever Laboratory Animal Technology Co., Ltd. They were SPF grade mice. The mice were acclimatized for 3 days after arrival before the study began.

[0778] Cells: HT55 cells were obtained from Nanjing Kebai (CAT#: CBP60012) and were passaged strictly according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25 × 10⁶ cells / mL. 6 Cells / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB17 SCID mice to establish an HT55 tumor-bearing mouse model.

[0779] Drug administration: Seven days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from approximately 80.80 to 203.70 mm² were selected. 3 Mice were divided into groups of 5 mice each according to tumor volume. The dosage and administration method are shown in Table 11. h-IgG (purchased from EQUITECH-BIO) was used as a negative control. Mice were administered the drug on day 7 post-inoculation, and tumor volume and body weight were monitored twice weekly. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on day 32 post-inoculation using the following formula: TGI% = 100% * (Tumor volume of control group – Tumor volume of treatment group) / (Tumor volume of control group – Tumor volume of control group before administration). Tumor volume was measured using calipers, with the maximum long axis (L) and maximum wide axis (W) of the tumor measured. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0780] The tumor inhibition rate results are shown in Figure 15(A) and Table 11: On day 32 post-inoculation, the tumor inhibition rates of ADC17 (3 mg / kg), ADC18 (3 mg / kg), and ADC19 (3 ​​mg / kg) were 95%, 60%, and 71%, respectively. ADC17 was more potent than ADC18 and ADC19. ADC17 showed a significant inhibitory effect on HT55 tumors.

[0781] Meanwhile, we measured the body weight of the mice, and the results are shown in Figure 15(B). It can be seen that during the experimental observation period, the weight loss in tumor-bearing mice treated with the ADCs of this invention was acceptable, comparable to the negative control, indicating that the ADCs of this invention exhibit good tolerability and safety while exerting effective tumor-killing activity.

[0782] Table 11. Experimental design and tumor inhibition rate on day 32

[0783] Example D10. Therapeutic effect of ADC drugs in HT55 tumor-bearing mouse model

[0784] This experiment used HT55 cells to inoculate CB17 SCID mice to determine the antitumor effect of the ADC1 of this invention.

[0785] CB17 SCID mice: Female CB17 SCID mice were purchased from Zhejiang Viton Lever Laboratory Animal Technology Co., Ltd. They were SPF grade, and the quality control unit was Beijing Viton Lever Laboratory Animal Technology Co., Ltd. The mice were acclimatized for 3 days after arrival before the study began.

[0786] Cells: HT55 cells were obtained from Nanjing Kebai (CAT#: CBP60012) and were passaged strictly according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in sterile PBS, and the cell density was adjusted to 25 × 10⁶ cells / mL. 6 Cells / ml. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of CB17 SCID mice to establish an HT55 tumor-bearing mouse model.

[0787] Drug administration: Six days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from approximately 64.8 to 191.7 mm were selected. 3 Mice were divided into groups of 6 mice each, based on average tumor volume. The dosage and administration method are shown in Table 12. h-IgG (purchased from EQUITECH-BIO) served as a negative control and was administered on day 6 post-inoculation. Tumor volume and body weight were monitored twice weekly. Body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on day 31 post-inoculation using the following formula: TGI% = 100% * (Tumor volume of control group – Tumor volume of treatment group) / (Tumor volume of control group – Tumor volume of control group before administration). Tumor volume was measured using calipers, measuring the maximum long axis (L) and maximum wide axis (W). The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0788] The tumor inhibition rate results are shown in Figure 16(A) and Table 12: On day 31 post-inoculation, the tumor inhibition rates of ADC17 (3 mg / kg), ADC20 (3 mg / kg), and ADC28 (3 mg / kg) were 89%, 68%, and 15%, respectively. ADC17 showed a significant inhibitory effect on HT55 tumors. Simultaneously, we measured the mouse body weight, and the results are shown in Figure 16b. It can be seen that during the experimental observation period, the body weight change curves of tumor-bearing mice administered the ADCs of this invention were comparable to those of the negative control, especially the body weight decrease in the ADC17 group was superior to that of the negative control, indicating that the ADCs of this invention have good tolerability and safety.

[0789] Table 12. Experimental design and tumor inhibition rate on day 31

[0790] Example D11. In vitro toxicity test of B7H3-targeting ADCs on NCI-N87, FADU and A375 cells

[0791] Add 10% FBS to RPMI 1640 and mix well to use as the growth medium for NCI-N87 and FADU cells. Add 10% FBS to DMEM and mix well to use as the growth medium for A375 cells. Adjust the NCI-N87 cell concentration to 40,000 cells / mL with the growth medium, and seed 50 μL / well (2000 cells / well) into 96-well white-background plates (136101, Thermo). Seal each well with 200 μL of PBS (pH 7.4) around its perimeter and incubate overnight at 37°C. Adjust the FADU cell concentration to 20,000 cells / mL using growth medium, and seed 50 μL / well (1000 cells / well) into 96-well white-bottomed plates (136101, Thermo). Seal each well with 200 μL of PBS (pH 7.4) around the perimeter and incubate overnight at 37°C. Adjust the A375 cell concentration to 60,000 cells / mL using growth medium, and seed 50 μL / well (3000 cells / well) into 96-well low-absorption round-bottomed white-bottomed plates (CLS7007-24EA, Costa). Seal each well with 200 μL of PBS (pH 7.4) around the perimeter and incubate overnight at 37°C. Dilute the ADCs with culture medium, with 500 nM as the highest concentration, and perform 5-fold serial dilutions for a total of 9 concentrations. Add 50 μl / well of diluted ADC solution or blank culture medium (control) to the cell plate, for a total volume of 100 μL. Incubate at 37°C for 5 days. Equilibrate the NCI-N87 cells and FADU cells in a 96-well plate and Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of CTG solution to each well. Equilibrate the A375 cells in a 96-well plate and 3D Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of 3D CTG solution to each well. Shake at 300 rpm for 30 min on a plate shaker to ensure complete cell lysis. Detect the luminescence values ​​at all wavelengths using a microplate reader (Spectra MAX i3x, Molecular Device).

[0792] The cell proliferation inhibition rate was calculated using the following formula: Cell viability (%) = a / b * 100, where a: average luminescence value of the experimental sample wells; b: average luminescence value of the control culture medium wells. The IC50 of the drug was calculated by fitting data with GraphPad Prism. 50 .

[0793] Figure 17 shows the cell survival percentage of ADCs in NCI-N87(A), FADU(B), and A375(C) cells, respectively. It can be seen that the ADCs of the present invention exhibit significant tumor cell killing activity in the test cells, particularly significantly superior to single toxins. Furthermore, in NCI-N87 cells, the inhibitory IC50 of ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 was significantly reduced. 50 The inhibitory effect was 42.71 nM, while ifinatamab-NT3 showed almost no inhibitory effect. In FADU cells, the inhibitory IC50 of ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 was 42.71 nM, while ifinatamab-NT3 showed almost no inhibitory effect. 50 The inhibition IC is 13.37 nM, while that of ifinatamab_LLC160_KIH-A1-DAR1 is 13.37 nM. 50 The IC50 value of ifinatamab-NT3-DAR8 was 24.70 nM, with a concentration of 20.13 nM. The tumor growth inhibitory effect of ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 was 1.528 times stronger than that of ifinatamab_LLC160_KIH-A1-DAR1 and 1.85 times stronger than that of ifinatamab-NT3-DAR8. In A375 cells, the inhibitory IC50 value of ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 was [not specified in the original text]. 50 The value is 0.266 nM, while the inhibition IC of ifinatamab_LLC160_KIH-A1-DAR1 is 0.266 nM. 50 At a concentration of 0.777 nM, ifinatamab_LLC160_KIH-A1-DAR1+NT3-DAR8 exhibited 2.921 times stronger tumor growth inhibition than ifinatamab_LLC160_KIH-A1-DAR1, and demonstrated a significant enhancement in maximum cytotoxic activity. This indicates that the dual-toxin ADCs of the present invention have advantages over single-toxin ADCs.

[0794] Table 13: Inhibitory IC50 of B7H3 ADCs on different cell lines 50

[0795] Example D12. In vitro toxicity test of Trop2-targeting ADCs on SNU601 and RT112 cells

[0796] Add 10% FBS to RPMI 1640 and mix well to use as the growth medium for SNU601 cells. Add 10% FBS to MEM Alpha and mix well to use as the growth medium for RT112 cells. Adjust the cell concentration to 20,000 cells / mL with growth medium, and seed 50 μL / well (1000 cells / well) into 96-well white-background plates (136101, Thermo). Seal each well with 200 μL of PBS (pH 7.4) and incubate overnight at 37°C. Dilute ADCs with culture medium, with 500 nM as the highest concentration, and perform 5-fold serial dilutions for a total of 9 dilutions. Add 50 μL / well of diluted ADC solution or blank medium (control) to the cell plate, for a total volume of 100 μL. Incubate at 37°C for 5 days. Equilibrate the 96-well plate and Cell Titer-Glo (G7572, Promega) to room temperature. Add 100 μL of CTG solution to each well. Shake at 300 rpm for 30 min on a plate shaker to fully lyse the cells. Detect the luminescence value at all wavelengths using a microplate reader (Spectra MAX i3x, Molecular Device).

[0797] The cell proliferation inhibition rate was calculated using the following formula: Cell viability (%) = a / b * 100, where a: average luminescence value of the experimental sample wells; b: average luminescence value of the control culture medium wells. The IC50 of the drug was calculated by fitting the data with GraphPad Prism.

[0798] Figure 18 shows the cell survival percentage of ADCs in SNU601 and RT112 cells, respectively. It can be seen that the ADCs of this invention exhibit significant tumor cell killing activity in the test cells, and are superior to single-toxin ADCs. In SNU601 cells, the inhibitory IC50 of hRS7_LLC160_KIH-A1-DAR1+NT3-DAR8 was 0.1219 nM, while the IC50 of hRS7_LLC160_KIH-A1-DAR1 was 0.3163 nM, indicating that the dual-toxin ADC has stronger killing activity than the single-toxin ADC. In RT112 cells, the inhibitory IC50 of hRS7_LLC160_KIH-A1-DAR1+NT3-DAR8 was 0.1668 nM, while that of hRS7-NT3-DAR8 was 0.2341 nM. hRS7_LLC160_KIH-A1-DAR1+NT3-DAR8 exhibited stronger tumor growth inhibition than hRS7-NT3-DAR8 and demonstrated greater maximum killing effect. This indicates that the dual-toxin ADCs of the present invention have significant advantages over single-toxin ADCs.

[0799] Table 14: Inhibitory IC50 of Trop2 ADCs on different cell lines 50

[0800] sequence list

[0801] 1. Constant region sequence variants

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof: T a1 -L a2 -L a3 -L a4 -D1 (I) in, D1 has the structure of formula (Ia): in, R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose, hexuronic acid, of which R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring; R d2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkyl groups, pentoses, penturonic acid, hexoses, hexuronic acid; The wavy line represents the T value of the molecule. a1 -L a2 -L a3 -L a4 - Partial connection; T a1 It is a connector unit or H; L a2 There is no or a bridging spacer; L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C14, C24, C34, C4 ... 1-6 Substituents in alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3, or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection, C end and L a4 Connection or when L a2 If it does not exist, connect it to D1; L a4 It either does not exist or is a cuttable connector or a self-destructing connector.

2. The compound of claim 1 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein D1 has the structure of formula (Ib): in, Each variable is defined as described in claim 1.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein... R d1 It is H, OH, Where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl groups, preferably H; Preferably, R d1 It is H, OH, (For example )or (For example ), where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl groups, preferably H; Furthermore, R d1 It is H, OH, (For example ), where R dm R dn and R dp Each is independently H or C 1-6 Alkyl groups such as C 1-4 Alkyl groups, preferably H; Furthermore, R d1 It is H, OH, Most preferably, R d1 It is H, OH, 4. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein... R d2 and R d3 Independently speaking, they are: H and C 1-6 Alkyl, C 1-6 Alkoxy, Preferably, R d2 and R d3 Each is C independently 1-6 Alkoxy, (For example )or (For example )； Furthermore, R d2 and R d3 Each is C independently 1-6 Alkoxy groups, such as C 1-4 Alkyl groups, preferably methoxy, ethoxy, propoxy, or butoxy; most preferably, R d2 and R d3 Each is an methoxy group independently.

5. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein: T a1 yes Where Lg is the leaving group, and the wavy line represents the interaction with the -L group in the molecule. a2 -L a3 -L a4 -D1 partial connection; Preferably, T a1 yes Where Lg is the leaving group, and the wavy line represents the interaction with the -L group in the molecule. a2 -L a3 -L a4 -D1 partial connection; Furthermore, T a1 yes For example The wavy line represents the -L of the molecule. a2 -L a3 -L a4 -D1 partial connection; Preferably, lg is a halogen, C 1-6 alkylsulfonyl, C 1-6 Halogenated alkyl sulfonyl, optionally C 1-6 Alkyl-substituted C 6-10 Aryl-sulfonyl, C 1-6 Alkylsulfonyloxy, C 1-6 Halogenated alkyl sulfonyloxy, optionally C 1-6 Alkyl-substituted C 6-10 aryl-sulfonyloxy, (C 1-6 Alkyl)3N + Or a diazonium base; for example, Lg is a halogen or C 1-6 Alkyl sulfonyl; for example, Lg is F, Cl, Br or methanesulfonyl.

6. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein L a2 Bridge spacers that do not exist or are selected from the following: *-(CR p R q ) s1 -(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -L M -(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR p R q ) s1 -(OCH2CH2) t -(CR m R n ) s2 -CO-**; in, *Terminal and T a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl, L M It is -NH-CO-, -CO-NH-, -CO-, -NH- or -O, and s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L a2 Bridge spacers that do not exist or are selected from the following: *-(CR p R q ) s1 -(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -NH-CO-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -CO-NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -CO-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -NH-(CH2CH2O) t -(CR m R n ) s2 -CO-**; *-(CR p R q ) s1 -O-(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR p R q ) s1 -(OCH2CH2) t -CO-**; Among them, * end and T a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl groups, s1, s2, and t are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; Furthermore, L a2 Bridge spacers that do not exist or are selected from the following: *-(CH2) s1 -(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -NH-CO-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -CO-(CH2CH2O) t -(CH2) s2 -WHAT-**; *-(CH2) s1 -NH-(CH2CH2O) t -(CH2) s2 -CO-**; *-(CH2) s1 -O-(CH2CH2O) t -(CH2) s2 -CO-**; and *-(CH2) s1 -(OCH2CH2) t -WHAT-**; Among them, * end and T a1 Connect, and the **end is connected to L a3 Connect, where s1, s2, and t are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; Furthermore, L a2 The bridging spacer either does not exist or is selected from the following: *-(CR p R q ) s1 -CO-**;*-(CH2CH2O) t -(CR m R n ) s2 -CO-**; and *-(CR) p R q ) s1 -(OCH2CH2) t -CO-**; where the * terminator is related to T. a1 Connect, and the **end is connected to L a3 Connect, and where R p R q R m and R n Each is independently H or C 1-6 Alkyl groups, preferably H or C 1-4 Alkyl groups, s1, s2, and t are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; Furthermore, L a2 The bridging spacer either does not exist or is selected from the following: *-(CH2) s1 -CO-**;*-(CH2CH2O) t -(CH2) s2 -CO-**; and *-(CH2) s1 -(OCH2CH2) t -CO-**; where the * terminator is related to T. a1 Connect, and the **end is connected to L a3 Connect , where s1, s2 and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

7. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein L a2 It does not exist or is the formula *-(CR) p R q ) s1 -CO-** bridging spacers, where, *Terminal and T a1 Connect, and the **end is connected to L a3 Connection, R p and R q Each is independently H or C 1-6 Alkyl, preferably H or C 1-4 Alkyl group, and s1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L a2 It does not exist or is expressed as *-(CH2). s1 -CO-** bridging spacer, where the * end is connected to T a1 Connect, and the **end is connected to L a3 The connection, and s1 are 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6 or 7, more preferably 5 or 6; Furthermore, L a2 The following are possible values: -CO-, *-(CH2)-CO-**, *-(CH2)2-CO-**, *-(CH2)3-CO-**, *-(CH2)4-CO-**, *-(CH2)5-CO-**, *-(CH2)6-CO-**, *-(CH2)7-CO-**, or *-(CH2)8-CO-**, preferably *-(CH2)5-CO-**, wherein the * terminus is related to the T terminus. a1 Connect, and the **end is connected to L a3 connect.

8. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein T a1 -L a2 -yes Where s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and the wavy line represents L. a3 connect; Preferably, T a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the wavy line represents L. a3 connect.

9. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein L a3 Yes: (i) a short chain containing 1-8 amino acid residues, such as a single amino acid residue or a peptide containing 2, 3, 4, 5, 6, 7 or 8 amino acids, preferably 2, 3 or 4 amino acids, wherein said amino acid is optionally bounded by one or more (e.g. 1, 2, 3 or 4, preferably 1 or 2) C 1-6 Alkyl substitution; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3 or 4, preferably 1 or 2). 1-6 Alkyl substituents; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1.

10. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein each of the amino acids is independently selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His), and threonine (Thr), wherein the amino acid is optionally decomposed by one or more C 1-6 Alkyl substitution; Preferably, the amino acid is selected from valine, alanine, glycine, lysine, citrulline, glutamine, glutamic acid, phenylalanine, and leucine, wherein the amino acid is optionally converted by one or more C... 1-6 Alkyl substitution; Preferably, the amino acid is selected from valine, alanine, glycine, lysine, citrulline, and glutamine, wherein the amino acid (e.g., lysine) is optionally oxidized by one or more C14 groups. 1-6 Alkyl substitution; Preferably, the amino acid is selected from valine, alanine, glycine, and lysine, wherein the lysine is optionally oxidized by one or more C2O groups. 1-6 Alkyl substitution, preferably N-substitution.

11. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein L a3 It includes Gly, Val-Ala, Val-Cit, Phe-Lys, Val-Lys, Leu-Cit, Val-Lys-Gly, Val-(N6,N6-C1-6 alkyl-Lys)-Gly, and -NH-(CH2). 2-6 -CO- or absent, L is preferred. a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly, or -NH-(CH2). 2-6 -CO-, and L is preferred a3 It is Gly, Val-Ala, Val-Lys-Gly, Val-(N6,N6-diisopropyl-Lys)-Gly or -NH-(CH2)2-CO-; where L a3 The N-terminus and L a2 -Connection or when L a2 When it does not exist and T a1 Connect, and C end is connected to L a4 Connection or when L a4 If it does not exist, connect it to D1.

12. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein L a3 yes (Gly) Or -NH-(CH2)2-CO-, where L a3 The N-terminus and L a2 Connection or when L a2 When it does not exist and T a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1.

13. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein L a4 It does not exist or is -NH-CH2-. Preferably, L a4 Does not exist or or The right side of the group is connected to D1, and the left side is connected to the T group of the molecule. a1 -L a2 -L a3 - Partial connection.

14. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein, R L Selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose, hexuronic acid, disaccharide, trisaccharide, and (optionally selected by one or more of C) 1-6 5-8 membered heteroaryl groups substituted with alkyl and nitro groups)-(C 1-4 (alkylene)-O-; Preferably, R L It is H. Furthermore, R L It is H. (like )、 (like ),or Most preferably, R L It is H. (like ),or 15. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein L a4 Does not exist or Terminal C is connected to D1, and terminal N is connected to L. a3 connect; Preferably, L a4 Does not exist or Terminal C is connected to D1, and terminal N is connected to L. a3 connect; Furthermore, L a4 yes For example Terminal C is connected to D1, and terminal N is connected to L. a3 connect.

16. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein... R d1 Indicates valence bond, L a4 yes R L yes Where R L 1st bit and R d1 Connect and 2 bits with L a4 Connect, and where L a4 The C terminal is connected to D1, and the N terminal is connected to L. a3 connect; Preferably, R d1 Indicates valence bond, L a4 express For example Where L a4 Bit 1 is connected to D1, and bit 2 is connected to R. d1 Connect in a ring, and 3 bits with L a3 connect.

17. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein T a1 -L a2 -L a3 -L a4 - Selected from: in, The wavy line indicates a connection to D1, and * indicates that the price key is connected to R. d1 connect.

18. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein... D1 has the structure of formula (Ib): in, R d1 It is H, OH, (For example )； R d2 and R d3 Each is C independently 1-4 Alkyl groups, preferably methoxy groups; The wavy line represents the T value of the molecule. a1 -L a2 -L a3 -L a4 - Partial connection; T a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the wavy line represents -L. a3 -L a4 -D1 partial connection; L a3 yes (Gly)、 Or -NH-(CH2)2-CO-, where the N-terminus is associated with the L-terminus. a2 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1; L a4 Does not exist or (For example )、 (For example ),or Preferred is For example Where L a4 When present, terminal C is connected to D1, and terminal N is connected to L. a3 connect; Or, R d1 Indicates valence bond, L a4 express For example Where L a4 Bit 1 is connected to D1, and bit 2 is connected to R. d1 Connect in a ring, and 3 bits with L a3 connect.

19. The compound of claim 1 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein the compound is selected from: Or its pharmaceutically usable salts or esters, solvates or isotopic labels.

20. Antibody-drug conjugates of formula (II) or their pharmaceutically acceptable salts or esters, solvates or isotopic labels: in, Ab is an antibody or its antigen-binding fragment; D1 has the structure of formula (Ia): in, R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose, hexuronic acid, of which R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring; R d2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkyl groups, pentoses, penturonic acid, hexoses, hexuronic acid; The wavy line represents L. a4 connect; L a1 It is a connector unit; L a2 There is no or a bridging spacer; L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C14, C24, C34, C4 ... 1-6 Substituents in alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3, or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1; L a4 There are no non-existent, or the connectors are either cuttable or self-destructive; and m can be 1, 2, 3, 4, 5, or 6.

21. The antibody-drug conjugate of claim 20, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein the antibody-drug conjugate has the structure of formula (IIa): Each of the variables is defined as described in claim 20.

22. The antibody-drug conjugate according to claim 20 or 21, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein L a2 L a3 L a4 And D1 as defined in any one of claims 2-4, 6-7 and 9-18.

23. The antibody-drug conjugate according to any one of claims 20-22, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein... L a1 yes: One bit is connected to Ab, and two bits are connected to L. a2 connect; Preferably, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect; Furthermore, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect; Most preferably, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

24. The antibody-drug conjugate according to any one of claims 20-23, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein -L a1 -L a2 -yes Wherein s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, and t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and the left side of the group is connected to Ab, and the right side is connected to L. a3 connect; Preferably, -L a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and the left side of the group is connected to Ab, and the right side is connected to L. a3 connect.

25. The antibody-drug conjugate of claim 20, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein the antibody-drug conjugate has the following subform: in, Ab and m are as defined in claim 20.

26. The antibody-drug conjugate according to any one of claims 20-25, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein m is 1, 2, 3 or 4, for example m is 1 or m is 2.

27. An antibody-drug conjugate according to any one of claims 20-26, or a pharmaceutically acceptable salt or ester, solvate or isotope label thereof, wherein the conjugate is in the form of a composition comprising one or more of the antibody-drug conjugate according to any one of claims 20-26, or a pharmaceutically acceptable salt or ester, solvate or isotope label thereof, and said composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2, for example an average DAR of about 1, or for example an average DAR of about 2.

28. The antibody-drug conjugate according to any one of claims 20-27, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein the Ab comprises a natural or cysteine-mutant cysteine; preferably, wherein the -L a1 -L a2 -L a3 -L a4 The -D1 moiety introduces a cysteine ​​covalent coupling of cysteine ​​with the thiol group of Ab.

29. The antibody-drug conjugate according to any one of claims 20-28, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein the Ab comprises a heavy chain constant region and / or a light chain constant region, for example, a heavy chain constant region and a light chain constant region, for example, the Ab contains... (i) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160, and covalently coupled via the thiol group of the mutated cysteine. (ii) One or two heavy chain constant regions having a cysteine ​​mutation at position 239, and covalently coupled via the thiol group of the mutated cysteine. (iii) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160 and one heavy chain constant region with a cysteine ​​mutation at position 239, covalently coupled via the thiol group of the mutated cysteine; or (iv) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160 and two heavy chain constant regions with a cysteine ​​mutation at position 239, and covalently coupled via the thiol group of the mutated cysteine. (v) One or two light chain constant regions with a cysteine ​​mutation at position 205, such as the Kappa light chain constant region, and covalently coupled via the thiol group of the mutated cysteine. (vi) One or two light chain constant regions, such as the Kappa light chain constant region, with cysteine ​​mutations at positions 164 and 205 and a 214S light chain constant region at position 214, and one or two heavy chain constant regions, with cysteine ​​mutations at position 170 and a 220S heavy chain constant region at position 220, covalently coupled via the thiol group of the mutated cysteine; or (viii) One or two light chain constant regions, such as the Kappa light chain constant region, have a cysteine ​​mutation at position 164 and a 214S light chain constant region at position 214, and one or two heavy chain constant regions, have a cysteine ​​mutation at position 170 and a 220S heavy chain constant region at position 220, and are covalently coupled via the thiol group of the mutated cysteine. Preferably, the Ab comprises two heavy chain constant regions and two light chain constant regions, wherein (i) The two heavy chain constant regions each contain a cysteine ​​mutation at position 239, and the two light chain constant regions do not contain a cysteine ​​mutation (e.g., the light chain constant region is a Kappa light chain constant region), and are covalently coupled via the thiol group of the mutated cysteine. (ii) One heavy chain constant region contains a cysteine ​​mutation at position 239, and another heavy chain constant region does not contain a cysteine ​​mutation at position 239 (or another heavy chain constant region does not contain a cysteine ​​mutation), and two light chain constant regions do not contain a cysteine ​​mutation (e.g., the light chain constant region is a Kappa light chain constant region), and is covalently coupled via the thiol group of the mutated cysteine. (iii) The two light chain constant regions are Lambda light chains, and one of the light chain constant regions contains a cysteine ​​mutation at position 160, while the other light chain constant region does not contain a cysteine ​​mutation at position 160, and is covalently coupled via the thiol group of the mutated cysteine. (iv) One light chain constant region is the Lambda light chain constant region containing a cysteine ​​mutation at position 160, and the other light chain constant region is the Kappa light chain constant region not containing a cysteine ​​mutation at position 160, and is covalently coupled via the thiol group of the mutated cysteine. (v) The two light chain constant regions are Lambda light chains, and the two light chain constant regions contain a cysteine ​​mutation at position 160, and are covalently coupled via the thiol group of the mutated cysteine. (vi) The two light chain constant regions are Lambda light chains, and both light chain constant regions contain a cysteine ​​mutation at position 160, and one heavy chain constant region contains a cysteine ​​mutation at position 239, and the other heavy chain constant region does not contain a cysteine ​​mutation at position 239 (or the other heavy chain constant region does not contain a cysteine ​​mutation), and is covalently coupled via the thiol group of the mutated cysteine. (vii) The two light chain constant regions are Lambda light chains, and the two light chain constant regions each contain a cysteine ​​mutation at position 160, and the two heavy chain constant regions each contain a cysteine ​​mutation at position 239, and are covalently coupled via the thiol group of the mutated cysteine. (viii) The two light chain constant regions are Kappa light chains, and the two light chain constant regions each contain a cysteine ​​mutation at position 205, and are covalently coupled via the thiol group of the mutated cysteine. (ix) The two light chain constant regions are Kappa light chains, and each light chain constant region contains a cysteine ​​mutation at positions 164 and 205, respectively, and a 214S mutation at position 214; the two heavy chain constant regions each contain a cysteine ​​mutation at position 170 and a 220S mutation at position 220, respectively, and are covalently coupled via the thiol group of the mutated cysteine; or (x) The two light chain constant regions are Kappa light chains, and the two light chain constant regions contain a cysteine ​​mutation at position 164 and a 214S mutation at position 214, respectively; the two heavy chain constant regions contain a cysteine ​​mutation at position 170 and a 220S mutation at position 220, respectively, and are covalently coupled via the thiol group of the mutated cysteine.

30. Antibody-drug conjugates of formula (III) or their pharmaceutically acceptable salts or esters, solvates or isotopic labels: in: Ab represents an antibody or its antigen-binding fragment; D1 has the structure of formula (Ia): in, R d1 It is H, OH, C 1-6 Alkyl, C 1-6 Alkoxy, Pentoose, penturonic acid, hexose, hexuronic acid, of which R dm R dn and R dp Each is independently H or C 1-6 Alkyl; or R d1 Represents the valence bond, and is related to L. a2 L a3 and L a4 Any one of them is connected to form a loop, preferably with L. a4 Connect them into a ring; R d2 and R d3 Each is independently H, OH, C 1-6 Alkyl, C 1-6 Alkyl groups, pentoses, penturonic acid, hexoses, hexuronic acid; The wavy line represents L. a4 connect; L a1 It is a connector unit; L a2 There is no or a bridging spacer; L a3 Yes: (i) a short chain containing 1-10 amino acid residues, wherein said amino acid residues are optionally selected from C14, C24, C34, C4 ... 1-6 Substituents in alkyl and polyol groups; (ii)-NH-(CH2) 2-6 -CO-, which is optionally selected from C by one or more (e.g., 1, 2, 3, or 4). 1-6 Substituents of alkyl and polyol groups; or (iii) none; wherein L a3 When present, N-terminus and L a2 Connection or when L a2 When it does not exist and L a1 Connection, C end and L a4 Connection or when L a4 If it does not exist, connect it to D1; L a4 The connectors either do not exist or are cuttable or self-destructive connectors. L b It is a connector; D2 is the drug component, preferably the anti-tumor drug component; m is 1, 2, 3, 4, 5, or 6; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

31. The antibody-drug conjugate of claim 30 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein said antibody-drug conjugate has the structure of (IIIa): Each of the variables is defined as described in claim 30.

32. The antibody-drug conjugate according to claim 30 or 31, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein L a2 L a3 L a4 And D1 as defined in any one of claims 2-4, 6-7 and 9-18.

33. The antibody-drug conjugate according to any one of claims 30-32, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein... L a1 yes: One bit is connected to Ab, and two bits are connected to L. a2 connect; Preferably, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect; Furthermore, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect; Most preferably, L a1 yes One bit is connected to Ab, and two bits are connected to L. a2 connect.

34. The antibody-drug conjugate according to any one of claims 30-33, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein -L a1 -L a2 -yes Where s1 and s2 are each independently 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6, t is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, 6, 7 or 8, and one bit is connected to Ab, and two bits are connected to L. a3 connect; Preferably, -L a1 -L a2 -yes Where s1 is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 5, and one bit is connected to Ab, and two bits are connected to L. a3 connect.

35. The antibody-drug conjugate according to any one of claims 30-34, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein the pharmaceutical portion D2 has the following structure: -Q-L 2 -L 1 -D 2a in: Q is -O-, -S-, or -NR 7 -; L 2 It does not exist, *-(C 1-10 alkylene)-C(O)N(R 5 )- or *-(C 1-10 alkylene)-N(R 5 )C(O)-; where * indicates that the end is covalently connected to Q, and R 5 Is it H or C? 1-6 alkyl; L 1 Is it non-existent or -(C) 1-10 alkylene); D 2a It has the structure shown in equation (D-1): Among them, R 1 and R 6 Selected from H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 1-6 Haloalkyl, C 2-6 Haloalkenyl and C 2-6 Halogenated alkynyl group; or R 1 and R 6 Together with the carbon atoms they are attached to, they form 5-9 membered rings, such as 5-8 membered rings; R 2 It is H, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, -OR 4 or -SR 4 ; R 3 It is H, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl or -OR 4 ; Or R 2 and R 3 Together they form -O(CH2) p O- or -O(CF2) p O-, where p is 1 or 2; R 4 Is it H or C? 1-4 Alkyl; and R 7 Is it H or C? 1-6 alkyl.

36. The antibody-drug conjugate of claim 35 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein D 2a It has the structure shown in formula (D-1a) or (D-1b): in, Each symbol is as defined in claim 35.

37. The antibody-drug conjugate of claim 35 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein D 2a It has the structure shown in equation (D-2): in, Each symbol is as defined in claim 35.

38. The antibody-drug conjugate of claim 35 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein D 2a It has the structure shown in equation (D-2a) or (D-2b): in, Each symbol is as defined in claim 35.

39. The antibody-drug conjugate according to any one of claims 35-38, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein R 1 and R 6 For H, R 2 C 1-6 Alkyl or C 1-6 Alkoxy, R 3 Halogen, preferably -F.

40. The antibody-drug conjugate according to any one of claims 35-39, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein -L 2 -L 1 -is-(C 1-6 alkylene)-, *-(C 1-6 alkylene)-C(O)N(R 5 )-(C 1-6 alkylene)- or *-(C 1-6 alkylene)-N(R 5 )C(O)-(C 1-6 (alkylene)-, where * indicates that the terminus is covalently connected to Q; and R 5 Is it H or C? 1-6 alkyl.

41. The antibody-drug conjugate according to any one of claims 35-39, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein -QL 2 -L 1 - can be -OCH2-CH2-CH2-CH2-, -OCH2-CH2-CH2-, or -NH-, wherein the left side of this group is adjacent to L. b Connect, right side with D 2a connect.

42. The antibody-drug conjugate according to any one of claims 30-34, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein the pharmaceutical portion D2 is selected from:

43. The antibody-drug conjugate according to any one of claims 30-42, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein the linker L b It has the following structure: -ZEX- in: Connect Z to Ab, and connect X to D; Z is selected from Where m a1 and m a2 Independently selected integers from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, or 8; The carbonyl group on the right end of Z is covalently connected to E; E is a short chain containing 1-10 amino acids, such as a single amino acid residue or a peptide residue containing 2-10 amino acids, wherein the short chain is optionally selected by one or more (e.g., 2, 3, or 4) from C. 1-6 Group substitution of alkyl and polyol groups, wherein the N-terminus of the short chain is covalently linked to Z; and X is selected from non-existent, -NH-CH2-, or... Where R a1 Selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, pentose, penturonic acid, hexose, hexuronic acid, disaccharide and trisaccharide; the left end of the group is connected to E and the right end is connected to D2.

44. The antibody-drug conjugate of claim 43 or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein Z is in, m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, or 8, preferably 5; or Z is... Where, m a2 It is an integer selected from 0 to 20, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, preferably 4, and m is an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8, preferably 2.

45. The antibody-drug conjugate of claim 43 or 44 or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein E is a single amino acid residue or a peptide residue comprising 2, 3 or 4 amino acids.

46. ​​The antibody-drug conjugate of claim 45 or a pharmaceutically acceptable salt or ester, solvate or isotope-labeled form thereof, wherein the amino acid is selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine and leucine, wherein the glutamine or glutamic acid is optionally substituted with one polyol group and optionally with one C 1-6 Alkyl substitution; preferably, the polyol group is 47. The antibody-drug conjugate of claim 46 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein the substituted glutamine or glutamic acid has the following structure: Preferred Where R 8 Is it H or C? 1-6 alkyl.

48. The antibody-drug conjugate of claim 43 or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein E is -Gln-Val-Ala-, -Gly-Val-Ala-, -Gln-Phe-Ala-, -Gly-Phe-Ala-, -Gly-Gly-Phe-Gly-, -Val-Ala-, or Where R 8 Is it H or C? 1-6 alkyl.

49. The antibody-drug conjugate according to any one of claims 43-48, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein X is -NH-CH2- or R a1 Selected from: H, Preferred R a1 yes 50. The antibody-drug conjugate of claim 49 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein X is -NH-CH2-, 51. The antibody-drug conjugate according to any one of claims 30-42, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein the linker L b It has the following structure:

52. The antibody-drug conjugate according to any one of claims 30-34, or its pharmaceutically acceptable salt or ester, solvate or isotopic label, wherein -L b The -D2 part is: in, The wavy line indicates a connection to Ab.

53. The antibody-drug conjugate of claim 30, or a pharmaceutically acceptable salt or ester, solvate, or isotope-labeled thereof, wherein the antibody-drug conjugate has the following structure: in, Ab represents an antibody or its antigen-binding fragment; m is 1, 2, 3, 4, 5, or 6; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

54. The antibody-drug conjugate according to any one of claims 30-53, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, wherein m is 1, 2, 3 or 4, preferably 1 or 2; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, preferably n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, more preferably n is 4 or 8.

55. An antibody-drug conjugate according to any one of claims 30-54, or a pharmaceutically acceptable salt or ester, solvate or isotope label thereof, wherein the conjugate is in the form of a composition comprising one or more of the antibody-drug conjugate according to any one of claims 30-54, or a pharmaceutically acceptable salt or ester, solvate or isotope label thereof, and wherein the composition has an average DAR of 0.6-6, preferably 0.8-4.0, more preferably 0.8-2.2, even more preferably 1.0±0.4 or 2.0±0.4 for drug fraction D1, and an average DAR of 2.0-14.0, preferably 4.0-12.0, more preferably 6.0-10.0, for example 4.0±0.4 or 8.0±0.4 for drug fraction D2.

56. The antibody-drug conjugate according to any one of claims 30-55, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein the Ab comprises a natural or cysteine-mutant cysteine; preferably, the -L a1 -L a2 -L a3 -L a4 The -D1 portion introduces a cysteine ​​mutation in Ab to introduce a thiol covalent coupling of cysteine, and / or the -L portion. b -D2 is covalently coupled to the thiol group formed by the reduction of interchain disulfide bonds of Ab.

57. The antibody-drug conjugate according to any one of claims 30-56, or a pharmaceutically acceptable salt or ester, solvate, or isotopic label thereof, wherein the Ab comprises a heavy chain constant region and / or a light chain constant region, for example, a heavy chain constant region and a light chain constant region, for example, the Ab contains... (i) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160, and covalently coupled via the thiol group of the mutated cysteine. (ii) One or two heavy chain constant regions having a cysteine ​​mutation at position 239, and covalently coupled via the thiol group of the mutated cysteine. (iii) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160 and one heavy chain constant region with a cysteine ​​mutation at position 239, covalently coupled via the thiol group of the mutated cysteine; or (iv) One or two Lamda light chain constant regions with a cysteine ​​mutation at position 160 and two heavy chain constant regions with a cysteine ​​mutation at position 239, and covalently coupled via the thiol group of the mutated cysteine. (v) One or two light chain constant regions with a cysteine ​​mutation at position 205, such as the Kappa light chain constant region, and covalently coupled via the thiol group of the mutated cysteine. (vi) One or two light chain constant regions, such as the Kappa light chain constant region, with cysteine ​​mutations at positions 164 and 205 and a 214S light chain constant region at position 214, and one or two heavy chain constant regions, with cysteine ​​mutations at position 170 and a 220S heavy chain constant region at position 220, covalently coupled via the thiol group of the mutated cysteine; or (viii) One or two light chain constant regions, such as the Kappa light chain constant region, have a cysteine ​​mutation at position 164 and a 214S light chain constant region at position 214, and one or two heavy chain constant regions, have a cysteine ​​mutation at position 170 and a 220S heavy chain constant region at position 220, and are covalently coupled via the thiol group of the mutated cysteine. Preferably, the Ab comprises two heavy chain constant regions and two light chain constant regions, wherein (i) The two heavy chain constant regions each contain a cysteine ​​mutation at position 239, and the two light chain constant regions do not contain a cysteine ​​mutation (e.g., the light chain constant region is a Kappa light chain constant region), and are covalently coupled via the thiol group of the mutated cysteine. (ii) One heavy chain constant region contains a cysteine ​​mutation at position 239, and another heavy chain constant region does not contain a cysteine ​​mutation at position 239 (or another heavy chain constant region does not contain a cysteine ​​mutation), and two light chain constant regions do not contain a cysteine ​​mutation (e.g., the light chain constant region is a Kappa light chain constant region), and is covalently coupled via the thiol group of the mutated cysteine. (iii) The two light chain constant regions are Lambda light chains, and one of the light chain constant regions contains a cysteine ​​mutation at position 160, while the other light chain constant region does not contain a cysteine ​​mutation at position 160, and is covalently coupled via the thiol group of the mutated cysteine. (iv) One light chain constant region is the Lambda light chain constant region containing a cysteine ​​mutation at position 160, and the other light chain constant region is the Kappa light chain constant region not containing a cysteine ​​mutation at position 160, and is covalently coupled via the thiol group of the mutated cysteine. (v) The two light chain constant regions are Lambda light chains, and the two light chain constant regions contain a cysteine ​​mutation at position 160, and are covalently coupled via the thiol group of the mutated cysteine. (vi) The two light chain constant regions are Lambda light chains, and both light chain constant regions contain a cysteine ​​mutation at position 160, and one heavy chain constant region contains a cysteine ​​mutation at position 239, and the other heavy chain constant region does not contain a cysteine ​​mutation at position 239 (or the other heavy chain constant region does not contain a cysteine ​​mutation), and is covalently coupled via the thiol group of the mutated cysteine. (vii) The two light chain constant regions are Lambda light chains, and the two light chain constant regions each contain a cysteine ​​mutation at position 160, and the two heavy chain constant regions each contain a cysteine ​​mutation at position 239, and are covalently coupled via the thiol group of the mutated cysteine. (viii) The two light chain constant regions are Kappa light chains, and the two light chain constant regions each contain a cysteine ​​mutation at position 205, and are covalently coupled via the thiol group of the mutated cysteine. (ix) The two light chain constant regions are Kappa light chains, and each light chain constant region contains a cysteine ​​mutation at positions 164 and 205, respectively, and a 214S mutation at position 214; the two heavy chain constant regions each contain a cysteine ​​mutation at position 170 and a 220S mutation at position 220, respectively, and are covalently coupled via the thiol group of the mutated cysteine; or (x) The two light chain constant regions are Kappa light chains, and the two light chain constant regions contain a cysteine ​​mutation at position 164 and a 214S mutation at position 214, respectively; the two heavy chain constant regions contain a cysteine ​​mutation at position 170 and a 220S mutation at position 220, respectively, and are covalently coupled via the thiol group of the mutated cysteine.

58. A pharmaceutical composition comprising one or more compounds of any one of claims 1-19 or their pharmaceutically acceptable salts or esters, solvates or isotopic labels and / or antibody-drug conjugates of any one of claims 20-57 or their pharmaceutically acceptable salts or esters, solvates or isotopic labels, and one or more pharmaceutically acceptable carriers.

59. Use of the compound of any one of claims 1-19 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof in the preparation of an antibody-drug conjugate; preferably, the antibody-drug conjugate is as defined in any one of claims 20-57.

60. Use of the antibody-drug conjugate of any one of claims 20-57 or its pharmaceutically acceptable salt or ester, solvate or isotopic label in the preparation of a medicament for treating or preventing tumors or cancer; preferably, the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer or ovarian cancer.

61. A method for treating or preventing tumors or cancer, the method comprising administering to a patient in need of the antibody-drug conjugate of any one of claims 20-57 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof; preferably, the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer or ovarian cancer.

62. An antibody-drug conjugate of any one of claims 20-57 for the treatment or prevention of tumors or cancer, or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof; preferably, the cancer is gastric cancer, colon cancer, pancreatic cancer, breast cancer or ovarian cancer.

63. A pharmaceutical combination comprising a compound of any one of claims 1-19 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, and / or an antibody-drug conjugate of any one of claims 20-57 or a pharmaceutically acceptable salt or ester, solvate or isotopic label thereof, and one or more common pharmaceuticals, such as an antitumor drug.

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