CN109810039B

CN109810039B - Disubstituted maleimide-based linker for antibody-drug coupling, preparation method and application thereof

Info

Publication number: CN109810039B
Application number: CN201711169847.2A
Authority: CN
Inventors: 沈竞康; 孟韬; 马兰萍; 王昕�; 张永良; 于霆; 陈驎; 彭红丽; 杜志彦; 王英
Original assignee: Maiwei Shanghai Biotechnology Co ltd
Current assignee: Mayway (shanghai) Biotechnology Co Ltd
Priority date: 2017-11-22
Filing date: 2017-11-22
Publication date: 2021-11-12
Anticipated expiration: 2037-11-22
Also published as: CN109810039A

Abstract

The present invention provides a double-substituted maleamide type linker coupled to an antibody and a preparation method and application thereof. Specifically, the present invention couples strong cytotoxic active substances and biological macromolecules through a new type of linker. link. This type of linker can selectively act simultaneously with the disulfide chain, thereby greatly improving the material uniformity and stability of the conjugate. The conjugate prepared by the linker of the present invention has high inhibitory activity on cell lines expressing the corresponding antigen. The present invention also provides preparation methods and uses of the above conjugates.

Description

Disubstituted maleimide-based linker for antibody-drug coupling, preparation method and application thereof

Technical Field

The invention relates to a novel disulfide-bridged crosslinking reagent, a macromolecule, a conjugate for treatment and a synthetic method thereof. More particularly, the invention relates to conjugates obtained by crosslinking cytotoxic drugs and macromolecules with disulfide-bridged crosslinking agents based on substituted maleamides, and to methods for their preparation and use.

Background

As a novel targeted therapeutic Drug, Antibody Drug Conjugate (ADC) has opened a new era in tumor therapy, and its basic design concept was originally derived from the "Magic bullet" (Magic bullets) and Drug targeted delivery (Drug targeting) concept first proposed in 1913 by Paul ehrlichh, i.e., Drug targeted delivery to the diseased site via an appropriate carrier. However, due to the limitations of antibody and highly active cytotoxic drug technology, the first antibody-drug conjugate (Mylotarg) for the treatment of Acute Myeloid Leukemia (AML) was the first to be used until 2000^TM) Is approved by the FDA for marketing. New drug Adcetris developed recently by Seattle Genetics for the treatment of Hodgkin Lymphoma (HL)/recurrent Anaplastic Large Cell Lymphoma (ALCL)^TM(2011) And a new drug Kadcyla developed by Genentech Biotech Co., Ltd, for treating breast cancer^TM(2013) The sequential FDA approval for marketing indicates that the use of antibody-drug conjugates in the field of tumor therapy is in a rapid developmental stage.

Antibody-drug conjugates generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug, and a linker coupling the ligand to the drug. In the structure of antibody-drug conjugates currently in clinical trials, highly active cytotoxic drugs are usually attached via a linker to lysine residues on the surface of the ligand, or cysteine residues in the hinge region of the antibody (reduced by the interchain disulfide moiety), with an optimal drug/ligand ratio (DAR) of 2-4. The large number of lysine residues (over 80) on the antibody surface and the non-selectivity of the conjugation reaction lead to uncertainty in the number and location of conjugation, and in turn to heterogeneity of the resulting antibody-drug conjugate. For example, the DAR value distribution for T-DM1 (average DAR value of 3.5) is 0-8. Similarly, although there are only four pairs of interchain disulfide bonds in the hinge region of an antibody, partial reduction of interchain disulfide bonds is required to achieve the optimum average DAR value (2-4). Since the existing reducing agents (DTT, TCEP, etc.) do not selectively reduce interchain disulfide bonds, the resulting conjugates are also not homogeneous products, consisting of a plurality of components, whose main components have DAR values of 0,2,4,6,8, and the components corresponding to each specific DAR value are present as isomers due to differences in the attachment sites. Heterogeneity of antibody-drug conjugate products can lead to heterogeneity of pharmacokinetic properties, potency, and toxicity among the component parts. For example, components with higher DAR values are cleared more rapidly in vivo and result in higher toxicity.

In order to solve the problem of homogeneity of antibody-drug conjugates, site-directed conjugation techniques have recently gained increased interest, which control the conjugation between antibody drugs both in terms of site and quantity. Although these techniques allow the site and quantity of conjugated drug to be controlled, the antibodies/proteins used are obtained by genetic recombination. The gene recombination technology needs a lot of work and elaborate design to find suitable sites for drug coupling or polyethylene glycol modification, and the expression quantity of the site-directed modified antibody/protein obtained by the existing gene recombination technology is low, so that the time is very long in large-scale preparation and production, and the cost of research and development and final industrialization is very high. The factors such as the in vivo efficacy and safety of the modified antibody/protein also need to be further verified.

Aiming at the problems of the coupling technology, the aim of fixed-point coupling of the existing antibody is fulfilled by a simple chemical method, so that a large amount of manpower, material resources and financial resources are saved, and the method is more attractive. Among them, there have been related studies including: a coupling technique reported by boliteix limited CN 200480019814.4; WO2014197871A2, applied by Igenica Biothereutics; CN201380025774.3 filed by sorento medical limited; patent documents such as CN201310025021.4 filed by shanghai new concept biomedicine science and technology limited. However, the above-mentioned techniques have problems of long synthetic route of the coupling reagent, poor chemical stability of the coupling reagent, and disordered electrophoresis pattern of the antibody conjugate, which suggests that there may be side reactions during the coupling process, and that the prior art does not solve the problems of thiol exchange (reverse michael addition reaction) during the in vivo circulation process.

Therefore, there is an urgent need in the art to provide a highly efficient, simple and practical chemical conjugation method, which can achieve the purpose of site-specific conjugation, and also improve the stability, safety and other properties of the antibody-drug conjugate.

Disclosure of Invention

The object of the present invention is to provide a linker that can be easily coupled to most antibodies.

In a first aspect of the present invention, there is provided a substituted maleimide linker fragment, having the structure of formula Ia:

wherein R is X or ArS-,

x is selected from the group consisting of: halogen, preferably bromine or iodine;

ar is selected from the group consisting of: substituted C6-C10 aryl, substituted or unsubstituted 5-12 membered heteroaryl;

ar' is selected from the group consisting of: substituted C6-C10 arylene; a substituted or unsubstituted 5-12 membered heteroarylene;

L₁is-O (CH) attached to an Ar' group₂CH₂O)_n-, where n is selected from any one of integers from 1 to 20.

In one preferred embodiment, Ar is selected from the group consisting of phenyl, halobenzene, C1-C4 alkylphenyl, C1-C4 alkoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl, and,

Wherein W is an amino group R attached to a carbonyl group¹，R¹Is selected from-NH₂、

Etc.; wherein: C1-C4 alkylphenyl is more preferably 4-methylphenyl; the C1-C4 alkoxyphenyl group is more preferably a 4-methoxyphenyl group.

In a preferred embodiment, Ar' is selected from substituted phenylene or pyridyl, wherein said substitution means that a hydrogen atom on the group is substituted with one or more substituents selected from the group consisting of: halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitrile, amide, and the like.

In a preferred embodiment, n is an integer of 1 to 10.

In another preferred embodiment, the linker fragment has a structure selected from the group consisting of:

in a second aspect of the present invention, there is provided a substituted maleimide-based linker-drug conjugate comprising a linker fragment of formula Ia as described in the first aspect of the present invention, a pharmaceutically acceptable salt thereof, or a solvate thereof, having the structure of formula Ib:

wherein, R, Ar' and L₁The definition of (1) is as above;

L₂is a chemical bond or an AA-PAB structure; wherein AA is dipeptide or tripeptide fragment (i.e., a fragment formed by connecting 2-3 amino acids by peptide bond), preferably includes Val-Cit (valine-citrulline), Val-Ala (valine-glycine), Phe-Lys (phenylalanine-lysine), Ala-Ala-Asn (glycine-asparagine), D-Ala-Phe-Lys (D-glycine-phenylalanine-lysine), etc., and PAB is p-aminobenzyl carbamateAn acyl group;

CTD is bonded to L through an amide bond₂And/or a drug for treating autoimmune diseases and/or anti-inflammation.

In another preferred embodiment, the compound of formula Ib is selected from the group consisting of:

in a third aspect of the present invention, there is provided an antibody-drug conjugate formed by coupling an antibody with a substituted maleimide-drug conjugate of formula Ib as defined in the second aspect of the present invention.

In another preferred embodiment, the conjugate is covalently linked to one or more drug components.

In another preferred embodiment, the conjugate comprises an antibody and a drug covalently coupled (e.g., by separate covalent attachment to a linker).

In another aspect of the present invention, there is provided an antibody-drug conjugate having a structure of formula Ic and/or Id;

wherein, Ar' and L₁、L₂CTD is as defined above;

m is 1.0-5.0, preferably 3.0-4.2;

ab is selected from the group consisting of: is protein, enzyme, antibody fragment, polypeptide.

In another preferred embodiment, the formula Id is the ring-opened product of N-phenylmaleimide in the formula Ic.

In another preferred embodiment, the antibody or Ab is selected from the group consisting of: monoclonal antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, antibody fragments (preferably antibody Fab fragments).

In another preferred embodiment, in the antibody-drug conjugate, a pair of cysteine residues is generated by disulfide chain reduction of the hinge region of the antibody or antibody fragment, and the compound of formula Ib is attached to the antibody or antibody fragment by substitution reaction of a thiol group in the cysteine residue with an aryl thioether in formula Ib.

In another preferred embodiment, the CTD is a cytotoxic small molecule drug, preferably a tubulin inhibitor, a topoisomerase inhibitor or a DNA binding agent.

In another further preferred embodiment, the tubulin inhibitor is selected from maytansine (maytansine) derivatives, monomethyyl auristatin E (MMAE), monomethyyl auristatin F (MMAF), monomethyyl Dolastatin10, Tubulysin derivatives, Cryptophycin derivatives, Taltobulin.

In another further preferred embodiment, said DNA binding agent is selected from the group consisting of PBD like derivatives, duocarmycin like derivatives.

In another further preferred embodiment, the topoisomerase inhibitor is selected from the group consisting of a derivative of the metabolite PNU-159682 of Doxorubicin (Doxorubicin), and a derivative of the metabolite SN38 of irinotecan (CPT-11).

In another preferred embodiment, the CTD has a molecular structure selected from the group consisting of D1-D13:

in another preferred embodiment, the antibody is an antibody capable of binding to a tumor associated antigen selected from the group consisting of:

in another preferred embodiment, the antibody is HER2 antibody, more preferably Trastuzumab (Trastuzumab) or Pertuzumab (Pertuzumab).

In another preferred embodiment, the antibody is an EGFR antibody, more preferably Erbitux or Vectibix.

In another preferred embodiment, the antibody is a Tissue Factor (TF) antibody.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising: (a) an antibody-drug conjugate according to the third aspect of the invention; and (b) a pharmaceutically acceptable diluent, carrier or excipient.

In a fifth aspect of the invention, there is provided a use of an antibody-drug conjugate as described in the third aspect of the invention for the manufacture of a medicament for the treatment of a tumour.

In another preferred embodiment, the tumor is selected from the group consisting of: breast cancer, ovarian cancer, non-hodgkin's lymphoma, acute lymphocytic leukemia, anaplastic large cell lymphoma, multiple myeloma, prostate cancer, non-small cell lung cancer, malignant melanoma, squamous cell carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, colorectal cancer, gastric cancer, glioma, mesothelioma.

In another aspect of the present invention, there is provided a method for treating a tumor, the method comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of an antibody-drug conjugate according to the third aspect of the invention.

In another preferred embodiment, the subject is a mammal, preferably a human.

In a sixth aspect of the present invention, there is provided a method of preparing an antibody-drug conjugate according to the third aspect of the present invention, the method comprising the steps of:

(1) reacting the antibody with a reducing reagent in a buffer solution to obtain a reduced antibody;

(2) and (2) crosslinking the linker-drug conjugate with the reduced antibody obtained in the step (1) in a mixed solution of a buffer solution and a certain amount of organic solvent to obtain the antibody-drug conjugate.

Reducing the antibody in the step (1) by using a reducing agent, so that the interchain disulfide bond of the antibody is reduced to generate a sulfhydryl group.

In another preferred embodiment, the reducing agent is tris (2-carboxyethyl) phosphine hydrochloride (TCEP), beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride, or Dithiothreitol (DTT).

In another preferred embodiment, the buffer is selected from the group consisting of: potassium dihydrogen phosphate-sodium hydroxide (KH)₂PO₄-NaOH)/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA) buffer, disodium hydrogen phosphate-citric acid/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA), boric acid-borax/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA), histidine-sodium hydroxide/sodium chloride (NaCl)/diethyltriaminepentaacetic acid (DTPA), and PBS/diethyltriaminepentaacetic acid (DTPA).

In another preferred embodiment, in the step (2), the volume of the organic solvent in the reaction solution is not more than 15%.

In another preferred embodiment, the organic solvent in step (2) is selected from the group consisting of: acetonitrile (ACN), Dimethylformamide (DMF), Dimethylacetamide (DMA), Dimethylsulfoxide (DMSO).

In another preferred embodiment, in the step (2), the coupling reaction is carried out at 0-37 ℃.

In another preferred embodiment, if the step (1) is reduced by using beta-mercaptoethanol, beta-mercaptoethylamine hydrochloride or DTT, the method further comprises the following steps between the step (1) and the step (2): after the reduction reaction is completed, the product is subjected to desalting column or ultrafiltration to remove the reducing agent.

In another preferred embodiment, the reaction scheme of the method is as follows:

wherein R, Ar', L1 and L2 are as defined above.

In another preferred example, the method comprises the steps of:

1) reduction: diluting the antibody stock solution to 2-10mg/mL by using a reaction buffer solution, adding 140-fold Dithiothreitol (DTT) with an excess molar ratio of 200 times or adding 6.0-20-fold tris (2-carboxyethyl) phosphine hydrochloride (TCEP) with an excess molar ratio, and stirring the reaction solution at 10-35 ℃ for 2-48 hours;

2) coupling: cooling the reaction liquid to 0-10 deg.c, adding substituted maleimide compound and stirring at 0-37 deg.c for 2-5 hr.

In another further preferred embodiment, the method further comprises the steps of: when DTT reduction is adopted in the step 1), after the reduction reaction in the step 1) is completed, the reaction liquid is processed by a desalting column or ultrafiltration to remove excessive DTT;

in another further preferred embodiment, said substituted maleamides are pre-dissolved in an organic solvent, preferably selected from: acetonitrile (ACN), Dimethylsulfoxide (DMSO), Dimethylformamide (DMF), or Diethylacetamide (DMA); further preferably, the substituted maleimide compound and the organic solvent are dissolved at a concentration of 10mg/ml, and the volume ratio of the organic solvent is not more than 15% of the reaction solution.

In another further preferred embodiment, the method further comprises the steps of: after the coupling reaction in step 2) was completed, the reaction mixture was purified by filtration using sodium succinate/NaCl buffer or histidine-acetic acid/sucrose gel, and a peak sample was collected according to UV280 UV absorbance.

In another further preferred embodiment, the method further comprises the steps of: after the coupling reaction in the step 2) is finished, carrying out ultrafiltration on the reaction mixture, then carrying out filtration sterilization, and storing the obtained product at low temperature; further preferably, the preservation temperature is-100 to 60 ℃; further preferably, the aperture of the device used for ultrafiltration is 0.15-0.3 micron.

In another further preferred example, said step 1) is carried out reduction using TCEP. Excess TCEP may not be removed.

In another further preferred embodiment, the reaction buffer in step 1) may be: 50mM potassium dihydrogen phosphate-sodium hydroxide (KH)₂PO₄-NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50mM boric acid-borax/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9; 50mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9 and PBS//1mM diethyltriaminepentaacetic acid (DTPA), pH 6-9.

The obtained antibody-drug conjugate has a uniform drug-antibody conjugate ratio (DAR), and the antibody-drug conjugate with a certain difference in product uniformity can be obtained by adopting different substituted maleimide linkers in the invention, if a sample with better uniformity needs to be obtained, the antibody-drug conjugate can be further separated and purified by the following methods: hydrophobic Interaction Chromatography (HIC), Size Exclusion Chromatography (SEC), Ion Exchange Chromatography (IEC).

In a seventh aspect of the present invention, there is provided a process for preparing the substituted maleimide-based linker (preferred embodiment E of formula Ia) of the first aspect:

intermediate D is obtained through cyclization reaction of intermediate C and 2, 3-dibromo maleic anhydride, and then is subjected to substitution reaction with aryl thiophenol to obtain linker fragment molecule E, wherein the reaction formula is as follows:

wherein R, n is as defined above, X represents halogen, preferably Br, Cl; u, V each independently represents N or C.

In another preferred embodiment, C is obtained by reduction of B, according to the following reaction scheme:

wherein R, n, U and V are the same as above.

In another further preferred embodiment, said B is obtainable by substitution reaction of a with fluoronitrobenzene according to the following reaction scheme:

wherein R, n, U and V are the same as above.

In another further preferred embodiment, said B may be prepared by the formula:

wherein R, n, U and V are the same as above.

In a further preferred embodiment, A is obtained by reacting n-glycol with tert-butyl haloacetate. The reaction formula is as follows:

wherein n and X are the same as above.

In the eighth aspect of the present invention, there is provided a method for preparing the substituted maleimide-based linker-drug conjugate (preferred example of formula Ib, F1 or F' 1) according to the second aspect: condensation of a substituted maleimide linker (preferred example Ea of formula Ia) with the dipeptide/tripeptide-PAB cytotoxic drug CTD gives F1 or F' 1 respectively.

The reaction route is as follows:

wherein R is as defined for formula Ia, Rx represents halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitrile or amide group and Ry represents H or alkyl.

The present inventors have extensively and intensively studied and found a linker structure which can cross-link all or part of the light chain-heavy chain and heavy chain-heavy chain of an antibody, and an antibody-drug conjugate obtained by applying such a coupling method has a narrower distribution of the drug/antibody ratio (DAR) than a conventional antibody-drug conjugate. Based on the above findings, the inventors have completed the present invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Term(s) for

As used herein, unless otherwise specified, the term "C1-C4 alkyl" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

The term "C1-C4 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or the like.

The term "halogen" refers to F, Cl, Br and I.

The term "C6-C10 aryl" refers to aryl groups having 6-10 carbon atoms, such as phenyl, naphthyl, and the like, which may be substituted or unsubstituted.

The terms "5-12 membered heteroaryl", "5-12 membered heteroarylene" refer to heteroaryl or heteroarylene groups, preferably 5-8 membered heteroaryl or heteroarylene groups, having 5-12 carbon atoms and one or more (preferably 1-3) heteroatoms selected from O, S and/or N. The heteroaryl or heteroarylene group may be substituted or unsubstituted.

In the present invention, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.

Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

As used herein, the term "compounds of the invention" refers to compounds of formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

Unless otherwise specified, "amino acid" as used herein is intended to include any conventional amino acid, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine, serine, methionine (methionine), tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, arginine.

When a trade name is used herein, the trade name is intended to include the trade name product formulation, its corresponding imitation drug, and the active pharmaceutical component of the trade name product.

The term "antibody" is used herein in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) Journal of Immunology 170: 4854-4861). The antibody may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding specific antigens (Janeway, c., Travers, p., Walport, m., shmchik (2001) immunology biology,5th ed., Garland Publishing, new york). Target antigens typically have a large number of binding sites, also referred to as epitopes, that are recognized by the CDRs of various antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody. Antibodies include full-long immunoglobulin molecules or immunologically active portions of full-long immunoglobulin molecules, i.e., molecules that contain an antigen or portion thereof that specifically binds to a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species. However, in one aspect, the immunoglobulin is derived from human, murine, or rabbit.

An "antibody fragment" comprises a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include: fab, Fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; minibody (Olafsen et al (2004) Protein Eng. design & Sel.17(4): 315-; fragments prepared from Fab expression libraries; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity determining regions); and an epitope-binding fragment of any of the above that binds in an immunospecific manner to a cancer cell antigen, a viral antigen, or a microbial antigen; a single-chain antibody molecule; and multispecific antibodies formed from antibody fragments.

The antibody constituting the antibody-drug conjugate of the present invention preferably retains its antigen-binding ability in its original wild state. Thus, the antibodies of the invention are capable of, preferably specifically, binding to an antigen. Antigens contemplated include, for example, Tumor Associated Antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (e.g., known or predicted to be functional), lymphokines, cytokines, molecules involved in regulation of cell circulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (e.g., antigens to which antibodies are known to bind may be one or a subset of the above categories, while other subsets include other molecules/antigens with specific properties (as compared to the antigen of interest).

Antibodies useful in the antibody-drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by antibody preparation methods and information well known in the art. In order to develop effective cellular level targets for cancer diagnosis and treatment, researchers have sought transmembrane or other tumor-associated polypeptides. These targets are capable of being specifically expressed on the surface of one or more cancer cells, while expressing little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. The confirmation of such tumor-associated factors can greatly improve the specific targeting property of antibody-based cancer treatment.

Tumor associated antigens include, but are not limited to, tumor associated antigens (1) - (53) listed below. For convenience, antigen-related information well known in the art is labeled as follows, including name, other names, and GenBank accession numbers. Nucleic acid and protein sequences corresponding to tumor associated antigens can be found in public databases, such as Genbank. The antibodies target the corresponding tumor associated antigens including all amino acid sequence variants and homologues, having at least 70%, 80%, 85%, 90%, or 95% homology with the sequences identified in the references, or having biological properties and characteristics that are fully identical to the tumor associated antigen sequences in the cited references.

(1) HER2(Gene ID:2064, human epidermal growth factor receptor 2 (English: human epidermal growth factor receptor 2, abbreviated HER2, also known as Neu, ErbB-2, CD340 (clade 340) or p185) is a protein encoded by the ERBB2 Gene HER2 is one of the members of the epidermal growth factor receptor (EGFR/ErbB) family); (2) HER3(Gene ID:2065, epidermal factor receptor 3(ErbB3/HER3) is one of the members of the epidermal growth factor transmembrane receptor family, recently it was shown that ErbB3/HER3 is closely related to the onset of breast cancer, recurrent metastasis, chemotherapy and the efficacy of endocrine therapy, and has become a very promising candidate target for therapy); (3) CD19(Gene ID: 930); (4) CD20(Gene ID: 931); (5) CD22(Gene ID: 933); (6) CD30(Gene ID: 943); (7) CD33(Gene ID: 945); (8) CD37(Gene ID: 951); (9) CD45(Gene ID: 5788); (10) CD56(Gene ID: 4684); (11) CD66e (Gene ID: 1048); (12) CD70(Gene ID: 970); (13) CD74(Gene ID: 972); (14) CD79b (Gene ID: 974); (15) CD138(Gene ID: 6382); (16) CD147(Gene ID: 682); (17) CD223(Gene ID: 3902); (18) EpCAM (Gene ID: 4072); (19) mucin 1(Gene ID: 4582); (20) STEAP1(Gene ID: 26872); (21) GPNMB (Gene ID: 10457); (22) FGF2(Gene ID: 2247); (23) FOLR1(Gene ID: 2348); (24) EGFR (Gene ID: 1956); (25) EGFRvIII (GenBank: GM 832119.1); (26) tissue Factor (TF) (Gene ID: 2152); (27) c-MET (Gene ID: 4233); (28) nectin 4(Gene ID: 81607); (29) AGS-16; (30) guanyl cyclase C (Gene ID: 2984); (31) mesothelin (Gene ID: 10232); (32) SLC44A4(Gene ID: 80736); (33) PSMA (Gene ID: 2346); (34) EphA2(Gene ID: 1969); (35) AGS-5; (36) GPC-3(Gene ID: 2719); (37) c-KIT (Gene ID: 3815); (38) RoR1(Gene ID: 4919); (39) PD-L1(Gene ID: 29126); (40) CD27L (Gene ID: 970); (41)5T4(Gene ID: 7162); (42) mucin16 (Gene ID: 94025); (43) NaPi2b (Gene ID: 10568); (44) STEAP (Gene ID: 26872); (45) SLITRK6(Gene ID: 84189); (46) ETBR (Gene ID: 1910); (47) BCMA (Gene ID: 608); (48) trop-2(Gene ID: 4070); (49) CEACAM5(Gene ID: 1048); (50) SC-16; (51) SLC39A6(Gene ID: 25800); (52) delta-like protein3(DLL3) (Gene ID: 10683); (53) claudin 18.2(Gene ID: 51208).

As used herein, "drug" broadly refers to any compound having a desired biological activity and having reactive functional groups for the preparation of conjugates of the invention. Desirable biological activities include, diagnosing, curing, alleviating, treating, preventing diseases in humans or other animals. Thus, the term "drug" refers to compounds that include the official national pharmacopoeia, as well as recognized drugs such as the official homeopathic pharmacopoeia of the united states, the official national formulary, or any subsidy thereof, so long as they possess the requisite reactive functional groups. Typical drugs are listed in physician's case medication reference (PDR) and the orange book of the united states Food and Drug Administration (FDA). As new drugs continue to be discovered and developed, the present patent states that these drugs should also be incorporated into the "drugs" of the conjugate drugs described herein.

Preferably, the medicament is: a cytotoxic drug for cancer therapy, or a protein or polypeptide having a desired biological activity, e.g., a toxin such as abrin, ricin a, pseudomonas exotoxin, and diphtheria toxin; other suitable proteins include tumor necrosis factor, alpha-interferon, beta-interferon, neuronal growth factor, platelet derived growth factor, tissue type fibroblast lyso-growth factor, and biological response modifying agents such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor, or other growth factors.

A preferred drug of the invention is maytansine or a maytansinoid. Maytansinoids inhibit cell proliferation by inhibiting tubulin microtubule formation. Maytansinoids are derivatives of maytansine. Both maytansinoids and maytansinoids have highly potent cytotoxicity, but they have great limitations in clinical applications for cancer therapy, mainly due to the low selectivity of such molecules for tumors. However, this high cytotoxicity has prompted them to be the drug moiety of choice for antibody-drug conjugates. The structure of deacetylmaytansine is listed below.

Another preferred drug of the invention is an auristatin peptide drug. The auristatin peptide drug is the analog of Dolastatin10 (Dolastatin10), which is a polypeptide with biological activity separated from sea mollusk sea rabbit. Dolastatin10 inhibits tubulin polymerization by binding to tubulin (the same binding domain as vincristine). Dolastatin10, auristatin peptide PE, auristatin peptide E are all linear polypeptides, containing four amino acids (three of which are unique to dolastatin compounds) and a C-terminal amide group. Two representative auristatin peptide compounds, monomethyl auristatin peptide e (mmae) and monomethyl auristatin peptide f (mmaf), are the first drugs of choice for antibody-drug conjugates.

Another preferred agent of the invention is Tubulysin (Tubulysin). Tubulysins, first isolated by the research group from myxobacterial cultures, are very potent inhibitors of cell growth, acting by inhibiting tubulin polymerization and thereby inducing apoptosis. Tubulysin D, the most potent one, has 10 to 100-fold more activity than most other tubulin modulators, including epothilones, vinblastine and paclitaxel. Paclitaxel and vinblastine are currently used in the treatment of a variety of cancers, while epothilone derivatives are being evaluated for activity in clinical trials. Synthetic derivatives of tubulysin D will provide the necessary information regarding inhibition and key binding interactions and may have superior properties as anticancer agents either as separate entities or as chemical warheads on targeting antibodies or ligands. Tubulysin D is a complex tetrapeptide that can be divided into four regions, Mep (D-N-methylpiperidinecarboxylic acid), Ile (isoleucine), Tuv (tubulivaline) and Tup (tubulphenylalanine), as shown in the following formula:

another preferred agent of the invention is a cryptophycin derivative of microbial origin which inhibits microtubule polymerization. Cryptophycin is a novel antitumor active substance which is separated from a culture of blue algae and can inhibit the generation of microtubules, and has activity on various tumors. Cryptophycin is a fat-soluble compound, contains 2 peptide bonds and 2 ester bonds, and has 5 optical active centers and 1 epoxy group. The dipeptide diester bonds are all in one macrocyclic structure. The Cryptophycin derivatives CP1 and CP2 have the following structures:

another preferred agent of the invention is the novel antimicrotubule agent Taltobulin (HTI-286, SPA-110). Taltobulin inhibits polymerization of purified microtubules, interferes with intracellular microtubule organization, induces mitotic block, and induces apoptosis. Taltobulin is a potent inhibitor of cell proliferation, and has an average IC50 of 2.5nM for 18 human tumor cell lines. In contrast to currently used antimicrotubule agents, Taltobulin is not a suitable substrate for p-glycoprotein, wherein the structure of Taltobulin is shown below.

In one aspect, the drug is the camptothecin drug derivative, Exatecan. Is a synthetic analogue of the topoisomerase I inhibitor camptothecin, has stronger activity than SN-38, can cause the strongest inhibition of DNA topoisomerase I, inhibits DNA synthesis in a dose-dependent and time-dependent manner, and causes frequent DNA single-strand breaks. Wherein the Exatecan structure is shown as follows.

Another preferred agent of the invention is the Amanitin drug (alpha-Amanitin), which has the structure shown below. alpha-Amanitin is a mycotoxin from the mushroom Amanitahalloides gilsonii (Amanitahalloides), a bicyclic octapeptide, which inhibits transcription of eukaryotic RNA polymerase II and RNA polymerase III.

Another preferred agent of the invention is benzodiazole antibiotic (duocarmycins, CC-1065, etc.) and other cyclopropylpyrrol-indol-4-one (CPI) derivatives. Such compounds are effective DNA minor groove binding-alkylating agents. Cyclopropylbenzindol-4-one (CBI) analogues are more stable in chemical structure, more biologically active, and easier to synthesize than their parent compounds containing the natural CPI alkylated subunit. One representative CBI derivative is the phenolic hydroxyl protected derivative CBI, with reduced prodrug toxicity and enhanced water solubility (where the CBI-seco-like general structural formula is shown below):

another preferred agent of the invention is a pyrrolobenzodiazepine (pyrrolo [2,1-c ] [1,4] benzodi-azepines, PBDs) or a PBD dimer (PBD dimers). PBDs are a class of natural products produced by Streptomyces and have the unique property of being able to form non-twisted covalent adducts in the DNA minor groove, specifically at the purine-guanine-purine sequence. The use of PBD as part of a small molecule strategy to target locked DNA sequences and as a novel anti-cancer and anti-bacterial drug has attracted increasing interest. The hydroxyl groups of C8/C8' of two PBD units are connected by a flexible carbon chain, and the obtained dimer has enhanced biological activity. PBD dimers are thought to produce sequence selective DNA damage, such as reverse order 5 '-Pu-GATC-Py-3' interchain cross-linking, resulting in their biological activity. These compounds have proven to be highly potent cytotoxic drugs and may be used as drug candidates for antibody-drug conjugates.

Another preferred drug of the invention is a PNU-159682 derivative, PNU-159682 being the major active metabolite of Nemorubicin in human liver microsomes, with a 3000-fold increase in activity compared to MMDX and doxorubicin.

On the other hand, the drug is not limited to only the above-mentioned classes, but also includes all drugs that can be used for the antibody-drug conjugate. And especially those capable of coordinating through an amide linkage with a linker, such as by having a basic amine group (primary or secondary), for example the structures of cytotoxins D1-D13 shown above.

According to the mechanism of drug release in cells, "linker" or "linker of antibody-drug conjugate" can be divided into two categories: non-cleavable linkers and cleavable linkers.

For antibody-drug conjugates containing a non-cleavable linker, the drug release mechanism is: after the conjugate is combined with antigen and endocytosed by cells, the antibody is enzymolyzed in lysosome to release active molecules consisting of small molecular drugs, linkers and antibody amino acid residues. The resulting structural change in the drug molecule does not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Thus, such active drugs cannot kill adjacent tumor cells that do not express the targeted antigen (antigen negative cells) (bystatder effect).

Cleavable linkers, as the name implies, can cleave within the target cell and release the active drug (small molecule drug itself). Cleavable linkers can be divided into two main classes: chemically labile linkers and enzyme labile linkers. Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, and the like. The pH sensitive linker is often also referred to as an acid cleavable linker. Such a linker is relatively stable in the neutral environment of blood (pH7.3-7.5), but will be hydrolyzed in weakly acidic endosomes (pH5.0-6.5) and lysosomes (pH 4.5-5.0). The first generation of antibody-drug conjugates mostly used such linkers as hydrazones, carbonates, acetals, ketals. Antibody-drug conjugates based on such linkers typically have a short half-life (2-3 days) due to the limited plasma stability of the acid-cleaved linker. This short half-life limits to some extent the use of pH sensitive linkers in the next generation of antibody-drug conjugates.

Linkers that are sensitive to glutathione are also known as disulfide linkers. Drug release is based on the difference between high intracellular glutathione concentrations (millimolar range) and relatively low glutathione concentrations in the blood (micromolar range). This is particularly true for tumor cells, where their low oxygen content leads to enhanced activity of the reductase and thus to higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma.

Enzyme-labile linkers, such as peptide linkers, allow for better control of drug release. The peptide linker can be effectively cleaved by an endolytic protease, such as cathepsin (cathepsin b) or plasmin (the content of such enzymes is increased in some tumor tissues). This peptide linkage is believed to be very stable in the plasma circulation, since proteases are generally inactive due to an undesirable extracellular pH and serum protease inhibitors. In view of higher plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme labile linkers include Val-Cit (VC), Phe-Lys, and the like.

The self-releasing linker is typically either chimeric between the cleavable linker and the active drug or is itself part of the cleavable linker. The mechanism of action of the self-releasing linker is: when the cleavable linker is cleaved under convenient conditions, the self-releasing linker is capable of undergoing a spontaneous structural rearrangement, thereby releasing the active drug attached thereto. Common suicide linkers include para-aminobenzols (PAB) and beta-glucuronides (beta-Glucuronide), among others.

Connector

The linker or coupling agent of the invention comprises diarylthiomaleamide units and a coupling group. The diarylthiomaleamide units are used to crosslink the sulfhydryl groups between antibody chains (after reduction), while the coupling groups are used to couple with small molecule drugs or drug-linker units. These ADCs are homogeneous and more stable than ADCs containing monodentate linkers due to the bidentate binding of the diarylthiomaleamide unit to the two sulfur atoms of the open cysteine-cysteine disulfide bond in the antibody. They will therefore have an increased in vivo half-life, reduce the amount of systemically released cytotoxins, and be safer for pharmaceutical properties than ADCs with monodentate linkers.

In another aspect, the resulting drug-linker unit is conjugated to an antibody via the linker, resulting in a conjugate with partial interchain cross-linking. Compared with the traditional antibody-drug conjugate, the antibody-drug conjugate prepared by the method has narrower drug/antibody ratio (DAR) distribution, thereby greatly improving the product uniformity and the pharmacological property uniformity.

The antibody-drug conjugates can be used for targeted delivery of drugs to target cell populations, such as tumor cells. The antibody-drug conjugate can specifically bind to a cell surface protein, and the resulting conjugate is then endocytosed by the cell. Within the cell, the drug is released in the form of the active drug to produce efficacy. Antibodies include chimeric antibodies, humanized antibodies, human antibodies; an antibody fragment that binds to an antigen; or an antibody Fc fusion protein; or a protein. The "drug" is a highly active drug, which in some cases may be polyethylene glycol.

Antibody-drug conjugates

The antibody-drug conjugate provided by the invention consists of an antibody, a linker and a drug, wherein the linker is a cleavable linker combination or a non-cleavable linker.

Antibodies are globular proteins containing a series of amino acid sites that can be used to couple drug-linkers. Due to their tertiary and quaternary structure, only solvent accessible amino acids are available for coupling. In fact, high yields of coupling usually occur on the epsilon-amino group of a lysine residue or on the sulfhydryl group of a cysteine residue.

The large number of lysine side chains on the surface of the antibody protein results in a large number of sites available for drug conjugation, resulting in the generation of antibody-drug conjugates as a mixture containing different numbers of drug conjugates (drug/antibody ratio, DAR) and conjugation sites.

The coupling product provided by the invention is still a mixture, but has a narrow DAR distribution range compared with the antibody-drug conjugate obtained by the conventional coupling method. The average DAR value is close to 4, and is close to the optimal average DAR value (2-4) range of antibody-drug conjugates. Furthermore, the conjugate product is rarely free of naked antibody (DAR ═ 0), and this fraction does not contribute to cytotoxic activity. Also, the coupling product does not contain a heavy coupling product (DAR ═ 8), and this fraction is cleared rapidly in vivo, relative to the low DAR fraction. Therefore, the heterogeneity of the antibody-drug conjugate product provided by the invention is greatly improved.

Pharmaceutical compositions and methods of administration

Since the antibody-drug conjugate provided by the present invention can be targeted to a specific cell population, and bound to a cell surface specific protein (antigen), so that the drug is released into the cell in an active form by endocytosis or drug infiltration of the conjugate, the antibody-drug conjugate of the present invention can be used for treating a target disease, and the above-mentioned antibody-drug conjugate can be administered to a subject (e.g., human) in a therapeutically effective amount by an appropriate route. The subject in need of treatment can be a patient at risk for, or suspected of having, a condition associated with the activity or expression of a particular antigen. Such patients can be identified by routine physical examination.

Conventional methods, known to those of ordinary skill in the medical arts, may be used to administer a pharmaceutical composition to a subject, depending on the type of disease to be treated or the site of the disease. The composition may also be administered by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by implantation. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Furthermore, it may be administered to the subject of depot injectable or biodegradable materials and methods by administration of an injectable depot route, for example using 1-, 3-, or 6-month depot.

Injectable compositions may contain various carriers such as vegetable oils, dimethylacetamide (dimethyl acetamide), dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycols, and the like). For intravenous injection, the water-soluble antibody may be administered by a drip method, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, ringer's solution, or other suitable excipients. Intramuscular formulations, e.g., sterile formulations of an appropriate soluble salt form of the antibody, may be dissolved and administered with a pharmaceutically acceptable excipient such as a water-change injection, 0.9% saline, or 5% dextrose solution.

When treated with the antibody-drug conjugates of the invention, delivery can be by methods conventional in the art. For example, it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector may be delivered locally by direct injection or by use of an infusion pump. Other methods include the use of various transport and carrier systems through the use of conjugates and biodegradable polymers.

The pharmaceutical composition of the present invention comprises a safe and effective amount of the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In general, the pharmaceutical preparations should be adapted to the mode of administration, and the pharmaceutical compositions of the present invention may be prepared in the form of solutions, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount.

The effective amount of the antibody-drug conjugate of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the antibody-drug conjugate of the present invention is administered at a daily dose of about 0.0001mg to 50mg/kg of animal body weight, preferably 0.001mg to 10mg/kg of animal body weight. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 5 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration, the health of the patient, etc.

Experiments show that the main advantages of the invention are as follows:

1. the novel linker provided by the invention can be coupled with an antibody through a simple chemical method, and compared with the traditional coupling mode, the DAR value distribution of the conjugate obtained by applying the linker is very narrow, so that the uniformity of the generated product is high, and the obtained single-distribution component (DAR is 4) of the cross-linked product accounts for more than 80%.

2. The ratio of the naked antibody to the ADC with low crosslinking degree of the antibody-drug conjugate provided by the invention is almost zero (components with DAR of 0 and 1 can not be detected by mass spectrometry).

3. The applicant proves through a large number of experiments that the antibody-drug conjugate provided by the invention has certain safety and effectiveness in the aspect of treating tumors. The hydrophilicity conferred by the ethylene glycol after coupling can be used to modulate biomolecular properties; the in vitro tumor cell proliferation inhibiting activity of the cross-linked product is improved or maintained in comparison with the traditional mcVC-PAB cross-linked biological activity, drug metabolic stability, safety and other drug properties.

4. The coupling method provided by the invention is suitable for most antibodies, so that the complicated recombination modification of each antibody to introduce a site-specific coupling site can be avoided, and the coupling method has wide application prospect.

5. Compared with the existing coupling method, the advantages of the disulfide chain bridging crosslinking reagent based on the maleamide provided by the invention comprise: has a fast crosslinking speed, and the crosslinking reaction time can be finished within 2-4 hours.

6. The disulfide chain bridging based on the maleic amide has better stability, thiol exchange is not easy to occur in vivo, and meanwhile, the unsubstituted phenyl group with the substituent introduced into the Ar' position can greatly slow down the cyclization secondary hydrolysis reaction after the ring opening of the maleic amide, thereby further enhancing the stability of the antibody-drug conjugate in vitro and in vivo.

Drawings

FIG. 1-1: hydrophobic Interaction Chromatography (HIC) profile of Pertuzumab (Pertuzumab);

FIGS. 1-2: a Hydrophobic Interaction Chromatography (HIC) profile of pertuzumab-drug conjugate ADC-I;

FIGS. 1 to 3: a Hydrophobic Interaction Chromatography (HIC) profile of pertuzumab-drug conjugate ADC-II;

FIGS. 1 to 4: a Hydrophobic Interaction Chromatography (HIC) profile of pertuzumab-drug conjugate ADC-III;

FIGS. 1 to 5: a Hydrophobic Interaction Chromatography (HIC) profile of pertuzumab-drug conjugate ADC-IV;

FIGS. 1 to 6: a Hydrophobic Interaction Chromatography (HIC) profile of pertuzumab-drug conjugate ADC-V;

FIGS. 1 to 7: a Hydrophobic Interaction Chromatography (HIC) profile of pertuzumab-drug conjugate ADC-VI;

FIGS. 1 to 8: a Hydrophobic Interaction Chromatography (HIC) profile of pertuzumab-drug conjugate ADC-VII;

FIG. 2-1: hydrophobic Interaction Chromatography (HIC) profile of Trastuzumab (Trastuzumab);

FIG. 2-2: a Hydrophobic Interaction Chromatography (HIC) profile of trastuzumab-drug conjugate ADC-VIII;

FIG. 3-1: mass spectrometry of Pertuzumab (Pertuzumab);

FIG. 3-2: mass spectrum of pertuzumab-drug conjugate ADC-I;

FIGS. 3-3: mass spectrum of pertuzumab-drug conjugate ADC-II;

FIGS. 3-4: mass spectrum of pertuzumab-drug conjugate ADC-III;

FIGS. 3 to 5: mass spectrum of pertuzumab-drug conjugate ADC-IV;

FIGS. 3 to 6: mass spectrum of pertuzumab-drug conjugate ADC-V;

FIGS. 3 to 7: mass spectrum of pertuzumab-drug conjugate ADC-VI;

FIGS. 3 to 8: mass spectrum of pertuzumab-drug conjugate ADC-VII;

FIG. 4-1: mass spectrum of Trastuzumab (Trastuzumab);

FIG. 4-2: (ii) a mass spectrometry profile of trastuzumab-drug conjugate ADC-VIII;

FIG. 5: shows the trend chart of the secondary hydrolysis product generation measured by LC-MS (Q-TOF) of each ADC control, ADC-I, ADC-II and ADC-VII at room temperature for 0-7 days;

FIG. 6-1: showing the HIC profile corresponding to the control ADC at 0 days room temperature;

FIG. 6-2: the HIC profile corresponding to the control ADC at 2 days room temperature is shown;

FIGS. 6-3: the HIC profile corresponding to the control ADC at 4 days room temperature is shown;

FIGS. 6 to 4: the HIC profile corresponding to the control ADC at 7 days room temperature is shown;

FIG. 7-1: showing the HIC map of ADC-I at 0 days at room temperature;

FIG. 7-2: showing the HIC profile of ADC-I at 2 days at room temperature;

FIGS. 7-3: shows the corresponding HIC pattern of ADC-I at 4 days room temperature;

FIGS. 7-4: shows the corresponding HIC pattern of ADC-I at 7 days room temperature;

FIG. 8-1: showing the HIC map of ADC-II at 0 days at room temperature;

FIG. 8-2: shows the corresponding HIC pattern of ADC-II at 2 days room temperature;

FIGS. 8 to 3: shows the corresponding HIC pattern of ADC-II at 4 days room temperature;

FIGS. 8 to 4: shows the corresponding HIC pattern of ADC-II at 7 days room temperature;

FIG. 9-1: showing the corresponding HIC pattern of ADC-VII at 0 days room temperature;

FIG. 9-2: showing the corresponding HIC profile of ADC-VII at 2 days room temperature;

FIGS. 9-3: shows the corresponding HIC profile of ADC-VII at 4 days room temperature;

FIGS. 9 to 4: shows the corresponding HIC profile of ADC-VII at 7 days room temperature;

FIG. 10: ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, Pertuzumab (Perjeta) to human gastric cancer cell NCI-N87 proliferation inhibition experiment result graph;

FIG. 11: ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, Pertuzumab (Perjeta) to human breast cancer cell BT-474 proliferation inhibition experiment result graph;

FIG. 12: ADC-VIII and Trastuzumab (Herceptin) in the result chart of the proliferation inhibition experiment of human gastric cancer cell NCI-N87;

FIG. 13: ADC-VIII and Trastuzumab (Herceptin) on the result of the proliferation inhibition experiment of human breast cancer cell BT-474;

FIG. 14: activity study graph of P-mcVC-MMAE (1.0mg/kg), control ADC (0.5,1.0mg/kg), ADC-I (1.0mg/kg), ADC-IV (1.0mg/kg), ADC-V (1.0mg/kg), ADC-VI (1.0mg/kg) and ADC-VII (0.5,1.0mg/kg) for inhibiting human gastric cancer NCI-N87 nude mouse subcutaneous transplantation tumor.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example group I, Synthesis and preparation of Compounds

1.1 Synthesis of Compound E-1 (formula Ia-1)

1.1.1 intermediate A-1 (step a)

Triethylene glycol (92g,613mmol) was dissolved in tBuOH (200 ml). KOtBu (22.91g,204mmol) was added to the ice bath and stirred for half an hour, and under argon, a solution of t-butyl bromoacetate (39.8g,204mmol) in tBuOH (40ml) was added dropwise and stirred at room temperature overnight. The next day, the reaction was completed by TLC. After removal of the tert-butanol by rotary evaporation, 400ml of dichloromethane were added to the residue, the organic phase was washed with 400ml of water, the aqueous phase was extracted once with 300ml of dichloromethane, the organic phases were combined and washed once with saturated salt, dried over anhydrous sodium sulfate and evaporated to dryness by rotary evaporation. The crude product was purified by petroleum ether: column chromatography in ethyl acetate 3:1 ═ 1:1 afforded intermediate a-1(24g, 44.5% yield) as a yellow oil.

1.1.2 intermediate B-1 (step B)

Intermediate A-1(7.8g,29.5mmol), 5-fluoro-2-nitrobenzotrifluoride (9.26g,44.3mmol), K₂CO₃(6.12g,44.3mmol) powder was heated to 80 ℃ in a 250mL round bottom reaction flask under nitrogen and stirred for 48 hours, monitored by TLC, and only a small amount of starting material remained. :

cooling to room temperature, extracting with 500 dichloromethane, washing with 400ml of 1N diluted hydrochloric acid once, washing with 400ml of water once, washing with 400ml of saturated saline acid once, drying with anhydrous sodium sulfate, and rotary steaming to dryness. Purifying by column chromatography (200-300 mesh silica gel), and purifying by petroleum ether: ethyl acetate 30:1-10:1 to give intermediate B-1(7.5g, 56.1% yield) as a yellow oil.

1.1.3 intermediate C-1

Intermediate B-1(6g,13.23mmol) was dissolved in 100ml of absolute ethanol and the solution was added to a reaction flask containing 10% Pd-C1.2 g. Hydrogenation reaction for 6 hours (1atm,38 ℃), TLC detection reaction complete. The reaction solution was filtered through celite, the filter cake was rinsed with ethanol, and the filtrate was rotary evaporated to dryness to give intermediate C-1(5g, 89% yield) as a yellow oil.

1.1.4 Compounds E-1

Intermediate C-1(0.8g,1.889mmol) was weighed into a parallel reaction tube, AcOH (3ml) was added under nitrogen protection, and dissolved with stirring. Then 3, 4-dibromomaleic anhydride (0.483g,1.889mmol) was added slowly. The mixture was heated to 110 ℃ under nitrogen and stirred overnight. The reaction was checked by TLC. And cooling the reaction solution to room temperature, evaporating the solvent by rotary evaporation, adding toluene, and evaporating by rotary evaporation twice to obtain a brown oily compound E-1. The product was used in the next reaction without purification.

1.2 Synthesis of Compound E-2 (formula Ia-2) (step E)

Compound E-1(2.0g,1.35mmol) was weighed into a 100ml round-bottomed flask, and dissolved by adding 30ml of anhydrous dichloromethane under nitrogen protection with stirring. 297mg of thiophenol was weighed out and added to the reaction mixture under nitrogen protection, and after dissolution, DIPEA (0.44ml,2.70mmol) was slowly dropped in an ice bath, and after completion, stirring was carried out for 5 minutes, and the ice bath was removed. The mixture was stirred at room temperature for 2 hours under nitrogen protection, and the reaction was completed by TLC.

And (3) after the solvent is evaporated to dryness under reduced pressure, performing column chromatography (200-300 meshes of silica gel) for separation and purification, loading and leaching dichloromethane, then slowly increasing the polarity, leaching from 2% to 10% of methanol, and collecting the evaporated solvent to obtain an orange oily product E-2(0.92g, 79% yield). LC-MS (M)⁺) Theoretical value 595.13, found value 596.15(ESI, M + H)⁺)。

1.3 Synthesis of Compound E-3 (formula Ia-3)

Synthesis of Compound E-3 the same procedure as for the Synthesis of Compound E-2 in example 1.2 was followed, except that the thiophenol in step E was changed to 2-mercaptopyridine to give product E-3 as an orange oil.

1.4 Synthesis of Compound E-4 (formula Ia-4)

Synthesis of Compound E-4 was performed in the same manner as in the Synthesis of Compound E-2 in example 1.2 except that 5-fluoro-2-nitrotrifluorotoluene in step b was changed to 2-methoxy-4-fluoronitrobenzene and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol to give product E-4 as an orange-yellow oil.

1.5 Synthesis of Compound E-5 (formula Ia-5)

Synthesis of Compound E-5 was performed in the same manner as in the Synthesis of Compound E-2 in example 1.2 except that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 1-fluoro-2-methoxy-4-nitrobenzene and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol to give product E-5 as an orange-yellow oil.

1.6 Synthesis of Compound E-6 (formula Ia-6)

Synthesis of Compound E-6 the same procedure as for Compound E-2 in example 1.2 was followed, except that 5-fluoro-2-nitrobenzotrifluoride in step b was replaced by 5-fluoro-2-nitrobenzonitrile and thiophenol in step E was replaced by 4- (N-morpholinecarboxamide) thiophenol to give product E-6 as an orange oil.

1.7 Synthesis of Compound E-7 (formula Ia-7)

Synthesis of Compound E-7 was performed in the same manner as in the Synthesis of Compound E-2 in example 1.2 except that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 2-fluoro-5-nitrobenzonitrile and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol to give product E-7 as an orange-yellow oil.

1.8 Synthesis of Compound E-8 (formula Ia-8)

Synthesis of Compound E-8 was performed in the same manner as the Synthesis procedure of Compound E-2 in example 1.2 except that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 5-fluoro-2-nitrobenzamide and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol to give product E-8 as an orange oil.

1.9 Synthesis of Compound E-9 (formula Ia-9)

Synthesis of Compound E-9 the same procedure as for Compound E-2 in example 1.2 was followed, except that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 4-fluoro-1-nitro-2-trifluoromethylbenzene and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol to give product E-9 as an orange oil.

1.10 Synthesis of Compound E-10 (formula Ia-10)

Synthesis of Compound E-10 was performed in the same manner as in the Synthesis of Compound E-2 in example 1.2 except that 5-fluoro-2-nitrotrifluorotoluene in step b was changed to 1-fluoro-4-nitro-2-trifluoromethylbenzene and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol to give product E-10 as an orange-yellow oil.

1.11 Synthesis of Compound E-11 (formula Ia-11)

1.11.1 intermediate F-11 (step F)

Intermediate A-1(4g,15.13mmol), triethylamine (2.53ml,18.16mmol) and dimethylaminopyridine (0.370g,3.03mmol) were dissolved in 100ml molecular sieve dried dichloromethane in a 250ml round-bottom flask and stirred, p-toluenesulfonyl chloride (3.17g,16.65mmol) was added portionwise under ice bath and stirred overnight at room temperature under argon atmosphere.

The reaction system was extracted with 100ml of dichloromethane, washed once with 200ml of 1N dilute hydrochloric acid, twice with 200ml of water, once with 200ml of saturated brine, dried over anhydrous sodium sulfate and the organic phase evaporated to dryness by rotary evaporation. Loading the column with 200-mesh 300-mesh silica gel, and eluting with PE and EA at a ratio of 5:1-2:1 to perform column chromatography separation. This was rotary evaporated to dryness to afford intermediate F-11(2.8g, yield 44.2%).

1.11.2 intermediate B-11 (step g)

Intermediate F-11(1g,2.389mmol), 2, 6-difluoro-4-nitrophenol (0.315g,1.797mmol) were dissolved in 20ml DMF and K was added₂CO₃(0.497g,3.59mmol), heated to 100 ℃ and stirred for 5 hours. Evaporating the solvent by rotary evaporation, adding 200ml of dichloromethane for dissolvingAnd (3) performing decomposition and extraction, washing once respectively by using 200ml of 1N diluted hydrochloric acid, 200ml of water and 200ml of saturated saline, drying by using anhydrous sodium sulfate, performing rotary evaporation and evaporation, packing a column by using 200-mesh 300-mesh silica gel, and performing PE: purifying by column chromatography with EA-5: 1-3:1, rotary evaporating to dryness to obtain intermediate B-11(600mg, 79% yield)

1.11.3 intermediate C-11

Intermediate B-11(600mg,1.42mmol) was dissolved in 100ml of absolute ethanol and the solution was added to the flask

10% Pd-C120 mg reaction flask. Hydrogenation reaction for 6 hours (1atm,38 ℃), TLC detection reaction complete. The reaction solution was filtered through celite, the filter cake was rinsed with ethanol, and the filtrate was rotary evaporated to dryness to give intermediate C-11(450mg, yield 81%) as a yellow oil.

1.11.4 intermediate D-11

Intermediate C-11(0.40g,1.02mmol) was weighed into a parallel reaction tube, AcOH (3ml) was added under nitrogen protection, and dissolved with stirring. Then 3, 4-dibromomaleic anhydride (0.261g,1.02mmol) was added slowly. The mixture was heated to 110 ℃ under nitrogen and stirred overnight. The reaction was checked by TLC. And cooling the reaction solution to room temperature, evaporating the solvent by rotary evaporation, adding toluene, and evaporating by rotary evaporation twice to obtain a brown oily compound D-11. The product was used in the next reaction without purification.

1.11.5 intermediate E-11

Compound D-11(600mg,0.95mmol) was weighed into a 100ml round-bottomed flask, and 30ml of anhydrous dichloromethane was added under nitrogen protection and stirred to dissolve. 425mg (1.91mmol) of 4- (N-morpholine formamide) thiophenol is weighed out and added into the reaction liquid under the protection of nitrogen, DIPEA (0.36ml,1.91mmol) is slowly dropped into the reaction liquid under ice bath after dissolution, stirring is carried out for 5 minutes after completion, and the ice bath is removed. The mixture was stirred at room temperature for 2 hours under nitrogen protection, and the reaction was completed by TLC.

And (3) after the solvent is evaporated to dryness under reduced pressure, performing column chromatography (200-300 meshes of silica gel) for separation and purification, loading and leaching dichloromethane, then slowly increasing the polarity, leaching from 2% to 10% of methanol, and collecting the evaporated solvent to obtain an orange oily product E-11(0.62g, 76% yield). LC-MS (M)⁺) Theoretical value 857.21, found value 858.23(ESI, M + H)⁺)。

1.12 Synthesis of Compound E-12 (formula Ia-12)

Synthesis of Compound E-12 the same procedure as for the Synthesis of Compound E-11 in example 1.11 was followed, except that 2, 6-difluoro-4-nitrophenol in step g was changed to 3-fluoro-4-nitrophenol to give product E-12 as an orange-yellow oil.

1.13 Synthesis of Compound E-13 (formula Ia-13)

Synthesis of Compound E-13 the same procedure as for the Synthesis of Compound E-11 in example 1.11 was followed, except that 2, 6-difluoro-4-nitrophenol in step g was replaced with 2, 5-difluoro-4-nitrophenol to give product E-13 as an orange-yellow oil.

1.14 Synthesis of Compound E-14 (formula Ia-14)

Synthesis of Compound E-14 the same procedure as for the Synthesis of Compound E-11 in example 1.11 was followed, except that triethylene glycol in step a was replaced by diethylene glycol to give product E-14 as an orange oil.

1.15 Synthesis of Compound E-15 (formula Ia-15)

Synthesis of Compound E-15 the same procedure as for the Synthesis of Compound E-11 in example 1.11 was followed, except that triethylene glycol in step a was replaced by tetraethylene glycol to give product E-15 as an orange oil.

1.16 Synthesis of Compound E-16 (formula Ia-16)

Synthesis of Compound E-16 the same procedure as for the Synthesis of Compound E-11 in example 1.11, except that triethylene glycol in step a is replaced by pentaethylene glycol, gives product E-16 as an orange oil.

1.17 Synthesis of Compound E-17 (formula Ia-17)

Synthesis of Compound E-17 the same procedure as for the Synthesis of Compound E-11 in example 1.11 was followed, except that triethylene glycol in step a was replaced by hexaethylene glycol to give product E-17 as an orange oil.

1.18 Synthesis of Compound E-18 (formula Ia-18)

Synthesis of Compound E-18 was performed in the same manner as for Compound E-11 in example 1.11, except that triethylene glycol in step a was replaced by dodecaethylene glycol to give product E-18 as an orange-yellow oil.

1.19 Synthesis of Compound E-19 (formula Ia-19)

Synthesis of Compound E-19 the same procedure as for the Synthesis of Compound E-11 in example 1.11 was followed, except that 4- (N-morpholinocarboxamide) thiophenol in step E was replaced with 1, 1-thiomorpholine to give product E-19 as an orange oil.

1.20 Synthesis of Compound E-20 (formula Ia-20)

Synthesis of Compound E-20 was performed in the same manner as in the Synthesis of Compound E-11 in example 1.11 except that 4- (N-morpholinocarboxamide) thiophenol in step E was changed to 4- (N-methylformamide) thiophenol to give product E-20 as an orange-yellow oil.

1.21 Synthesis of Compound E-21 (formula Ia-21)

Synthesis of Compound E-21 was carried out in the same manner as for Compound E-2 in example 1.2, except that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 2-nitro-5-fluoropyridine and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol, to give product E-21 as an orange-yellow oil.

1.22 Synthesis of Compound E-22 (formula Ia-22)

Synthesis of Compound E-21 was carried out in the same manner as for Compound E-2 in example 1.2, except that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 2-fluoro-5-nitropyridine and thiophenol in step E was changed to 4- (N-morpholinecarboxamide) thiophenol to give product E-22 as an orange-yellow oil.

Example set 2: synthesis and preparation of formulae Ib-1 to Ib-24

2.1 Synthesis of Compound F1-1 (formula Ib-1)

Compound E1-9(300mg,0.337mmol) was weighed into a 100mL round bottom flask, and after completely dissolving it by adding anhydrous DMF (20mL) under nitrogen, HATU (154mg,0.404mmol) and DIEA (0.11mL,0.674mmol) were weighed into the flask in this order. After stirring at room temperature for 15 minutes, compound D1-1(219mg,0.337mmol) was added and stirred at room temperature overnight under nitrogen. TLC followed by HPLC overnight and starting material E9 disappeared. The solvent was evaporated under reduced pressure for quantitative analysis and purified by reverse phase HPLC to give the product as yellow amorphous powder F1-1(0.350g,0.230mmol, 68.2% yield). LC-MS (M)⁺) Theoretical value 1520.48, found value 1521.51(ESI, M + H)⁺)。

2.2 Synthesis of Compound F1-2 (formula Ib-2)

Synthesis of Compound F1-2 the same procedure was followed as for Compound F1-1 in example 2.1, except that D1-1 was replaced by Compound D1-2, giving product F1-2 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1993.91, found value 1994.93(ESI, M + H)⁺)。

2.3 Synthesis of Compound F1-3 (formula Ib-3)

Synthesis of Compound F1-3 the same procedure was followed as for Compound F1-1 in example 2.1, except that D1-1 was changed to Compound D1-3, giving product F1-3 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 2007.89, found value 2008.91(ESI, M + H)⁺)。

2.4 Synthesis of Compound F1-4 (formula Ib-4)

Synthesis of Compound F1-4 the procedure was the same as that for Compound F1-1 in example 2.1, except that D1-1 was exchangedCompound D1-4 to give product F1-4 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 2046.88, found value 2047.86(ESI, M + H)⁺)。

2.5 Synthesis of Compound F1-5 (formula Ib-5)

Synthesis of Compound F1-5 was carried out in the same manner as for Compound F1-1 in example 2.1, except that D1-1 was replaced by Compound D1-5, to give product F1-5 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1800.67, found value 1801.65(ESI, M + H)⁺)。

2.6 Synthesis of Compound F1-6 (formula Ib-6)

Synthesis of Compound F1-6 the same procedure was followed as for Compound F1-1 in example 2.1, except that D1-1 was changed to Compound D1-6, to give product F1-6 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1989.79, found value 1990.80(ESI, M + H)⁺)。

2.7 Synthesis of Compound F1-7 (formula Ib-7)

Synthesis of Compound F1-7 was carried out in the same manner as in example 2.1 for Compound F1-1, except that Compound D1-1 was changed to Compound D1-7, to give product F1-7 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1973.72, found value 1974.72(ESI, M + H)⁺)。

2.8 Synthesis of Compound F1-8 (formula Ib-8)

Chemical combinationThe synthesis of compound F1-8 was carried out in the same manner as for compound F1-1 in example 2.1, except that D1-1 was replaced by compound D1-8, giving product F1-8 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1973.72, found value 1974.72(ESI, M + H)⁺)。

2.9 Synthesis of Compound F1-9 (formula Ib-9)

Synthesis of Compound F1-9 the same procedure as that for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compounds E1-17 and D1-9, respectively, gave product F1-9 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 2015.72, found value 2016.73(ESI, M + H)⁺)。

2.10 Synthesis of Compound F1-10 (formula Ib-10)

Synthesis of Compound F1-10 the same procedure was followed as for Compound F1-1 in example 2.1, except that D1-1 was replaced by Compound D1-10, to give product F1-10 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1912.63, found value 1913.65(ESI, M + H)⁺)。

2.11 Synthesis of Compound F1-11 (formula Ib-11)

Synthesis of Compound F1-11 the same procedure was followed as for Compound F1-1 in example 2.1, except that D1-1 was replaced by Compound D1-11, to give product F1-11 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1916.63, found value 1917.61(ESI, M + H)⁺)。

2.12 Synthesis of Compound F1-12 (formula Ib-12)

Synthesis of Compound F1-12 was carried out in the same manner as for Compound F1-1 in example 2.1, except that D1-1 was replaced by Compound D1-12, to give product F1-12 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 2031.70, found value 2032.71(ESI, M + H)⁺)。

2.13 Synthesis of Compound F1-13 (formula Ib-13)

Synthesis of Compound F1-13 the same procedure was followed as for Compound F1-1 in example 2.1, except that D1-1 was replaced by Compound D1-13, to give product F1-13 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1711.57, found value 1712.55(ESI, M + H)⁺)。

2.14 Synthesis of Compound F1-14 (formula Ib-14)

Synthesis of Compound F1-14 was carried out in the same manner as for Compound F1-1 in example 2.1, except that D1-1 was replaced by Compound D1-2, giving product F1-14 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1767.82, found value 1768.83(ESI, M + H)⁺)。

2.15 Synthesis of Compound F1-15 (formula Ib-15)

Synthesis of Compound F1-15 was carried out in the same manner as for Compound F1-1 in example 2.1 except that E1-9 and D1-1 were changed to Compound E-19 and D1-2, respectively, to give product F1-15 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 2057.84, found value 2058.87(ESI, M + H)⁺)。

2.16 Synthesis of Compound F1-16 (formula Ib-16)

Synthesis of Compound F1-16 the same procedure as that for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compound E1-20 and D1-2, respectively, gave product F1-16 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1849.85, found value 1850.83(ESI, M + H)⁺)。

2.17 Synthesis of Compound F1-17 (formula Ib-17)

Synthesis of Compound F1-17 was carried out in the same manner as for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compound E1-10 and D1-2, respectively, to give product F1-17 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1993.91, found value 1994.90(ESI, M + H)⁺)。

2.18 Synthesis of Compound F1-18 (formula Ib-18)

Synthesis of Compound F1-18 was carried out in the same manner as for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compound E1-11 and D1-2, respectively, to give product F1-18 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1961.90, found value 1962.91(ESI, M + H)⁺)。

2.19 Synthesis of Compound F1-19 (formula Ib-19)

Synthesis of Compound F1-19 the same procedure as that for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compound E1-5 and D1-2, respectively, gave product F1-19 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1955.93, found value 1956.95(ESI, M + H)⁺)。

2.20 Synthesis of Compound F1-20 (formula Ib-20)

Synthesis of Compound F1-20 was carried out in the same manner as for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compound E1-4 and D1-2, respectively, to give product F1-20 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1955.93, found value 1956.95(ESI, M + H)⁺)。

2.21 Synthesis of Compound F1-21 (formula Ib-21)

Synthesis of Compound F1-21 was carried out in the same manner as for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compound E1-12 and D1-2, respectively, to give product F1-21 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1943.91, found value 1944.90(ESI, M + H)⁺)。

2.22 Synthesis of Compound F1-22 (formula Ib-22)

Synthesis of Compound F1-22 the same procedure as that for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were each exchangedThe compound E1-6 and D1-2 gave the product F1-22 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1950.92, found value 1951.93(ESI, M + H)⁺)。

2.23 Synthesis of Compound F1-23 (formula Ib-23)

Synthesis of Compound F1-23 the same procedure as that for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compounds E1-21 and D1-2, respectively, gave product F1-23 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1926.92, found value 1927.93(ESI, M + H)⁺)。

2.24 Synthesis of Compound F1-24 (formula Ib-24)

Synthesis of Compound F1-24 the same procedure as that for Compound F1-1 in example 2.1, except that E1-9 and D1-1 were changed to Compound E1-22 and D1-2, respectively, gave product F1-24 as a yellow amorphous powder. LC-MS (M)⁺) Theoretical value 1926.92, found value 1927.93(ESI, M + H)⁺)。

EXAMPLE group two, preparation of antibody conjugates

1. Preparation of ADC-I

The antibody stock solution of the patu beads is mixed with 50mM potassium dihydrogen phosphate-sodium hydroxide (KH)₂PO₄NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH7 reaction buffer was diluted to 2mg/mL, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in a 6.0-fold excess molar ratio, and the reaction solution was stirred at 35 ℃ for 2.5 hours.

The reaction solution was cooled to 8 ℃ and, without purification, an appropriate amount of dimethyl sulfoxide (DMSO) was added, followed by addition of compound F1-17(10mg/ml pre-dissolved in DMSO) in a 6-fold molar excess, ensuring that the volume of DMSO in the reaction system does not exceed 15%, and coupling was carried out at 37 ℃ for 3 hours with agitation.

The coupling reaction mixture was purified by filtration through a pH 6.0 histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.15 micron pore size filter unit and stored at-60 ℃.

2. Preparation of ADC-II

The antibody stock solution of the patu beads is mixed with 50mM potassium dihydrogen phosphate-sodium hydroxide (KH)₂PO₄NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH 6 reaction buffer was diluted to 5mg/mL, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in a 10-fold excess molar ratio, and the reaction solution was stirred at 10 ℃ for 40 hours.

The reaction solution was cooled to 5 ℃ and the coupling was carried out without purification by adding the appropriate amount of Diethylacetamide (DMA) and then adding 6 times the excess molar ratio of compound F1-2(10mg/ml pre-dissolved in DMA) ensuring that the volume of DMA in the reaction system did not exceed 10% and stirring at 25 ℃ for 2.5 hours.

The coupling reaction mixture was purified by filtration through a pH 6.0 histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.22 micron pore size filter unit and stored at-80 ℃.

3. Preparation of ADC-III

The stock solution of the antibody against Palesterol was diluted to 5mg/mL with PBS//1mM diethyltriaminepentaacetic acid (DTPA), pH 7.4 reaction buffer, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in a 20-fold excess molar ratio, and the reaction solution was stirred at 15 ℃ for 2 hours.

The reaction solution was cooled to 10 ℃ and the coupling was carried out without purification by adding an appropriate amount of Acetonitrile (ACN) and then by adding 6 times the excess molar ratio of compound F1-20(10mg/ml pre-dissolved in ACN) to ensure that the volume of ACN in the reaction system did not exceed 10% and stirring at 10 ℃ for 4 hours.

Filtering and purifying the coupling reaction mixture by using histidine-acetic acid/sucrose gel with pH of 8.0 by using a desalting column, collecting a peak sample according to a UV280 ultraviolet absorption value, filtering and sterilizing, and storing the obtained product at low temperature; such as sterilization via a 0.20 micron pore size filtration apparatus, storage at-90 deg.C.

4. Preparation of ADC-IV

The antibody stock solution of the patu beads is mixed with 50mM potassium dihydrogen phosphate-sodium hydroxide (KH)₂PO₄NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH7 reaction buffer was diluted to 8mg/mL, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in an 8-fold excess molar ratio, and the reaction solution was stirred at 25 ℃ for 25 hours.

The reaction was cooled to 5 ℃ and appropriate amount of Dimethylformamide (DMF) was added without purification, then compound F1-19(10mg/ml pre-dissolved in DMF) was added in 6 molar excess to ensure that the volume of DMF in the reaction system did not exceed 8%, and the coupling was carried out by stirring at 0 ℃ for 2 hours.

The coupling reaction mixture was purified by filtration through a pH 6.0 histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.3 micron pore size filter unit and stored at-80 ℃.

5. Preparation of ADC-V

The antibody stock solution of the Palestinian antibody was diluted to 6mg/mL with 50mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA) and pH 7.4 reaction buffer, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in an excess molar ratio of 8 times, and the reaction solution was stirred at 35 ℃ for 15 hours.

The reaction was cooled to 10 ℃ and appropriate amount of Dimethylformamide (DMF) was added without purification, then compound F1-22(10mg/ml pre-dissolved in DMF) was added in 6 molar excess to ensure that the volume of DMF in the reaction system did not exceed 8%, and the coupling was carried out by stirring at 0 ℃ for 5 hours.

The coupling reaction mixture was purified by filtration through a pH 6.0 histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.15 micron pore size filter unit and stored at-100 ℃.

6. Preparation of ADC-VI

The antibody stock solution of the patupol was diluted to 10mg/mL with 50mM boric acid-borax/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA) reaction buffer solution of pH 9, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in an excess molar ratio of 8 times, and the reaction solution was stirred at 25 ℃ for 10 hours.

The reaction was cooled to 10 ℃ and appropriate amount of Dimethylformamide (DMF) was added without purification, then compound F1-21(10mg/ml pre-dissolved in DMF) was added in 6 molar excess to ensure that the volume of DMF in the reaction system did not exceed 8%, and the coupling was carried out by stirring at 0 ℃ for 4 hours.

The coupling reaction mixture was purified by filtration through a pH 6.0 histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.2 micron pore size filter unit and stored at-60 ℃.

7. Preparation of ADC-VII

The antibody stock solution of the patu beads is mixed with 50mM potassium dihydrogen phosphate-sodium hydroxide (KH)₂PO₄NaOH)/150mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA), pH8 reaction buffer was diluted to 3mg/mL, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in an 8-fold excess molar ratio, and the reaction solution was stirred at 15 ℃ for 48 hours.

The reaction was cooled to 0 ℃ and the coupling was carried out by adding an appropriate amount of Dimethylformamide (DMF) without purification, followed by addition of 6-fold molar excess of compound F1-18(10mg/ml pre-dissolved in DMF) to ensure that the volume of DMF in the reaction system did not exceed 8%, and stirring at 0 ℃ for 3 hours.

The coupling reaction mixture was purified by filtration through a pH 6.0 histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.3 meter pore size filter unit and stored at-70 ℃.

8. Preparation of ADC-VIII

The stock solution of the antibody to trastuzumab was diluted to 5mg/mL with 50mM disodium hydrogenphosphate-citric acid/150 mM sodium chloride (NaCl)/1mM diethyltriaminepentaacetic acid (DTPA) and pH 7.4 reaction buffer, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was added in an excess molar ratio of 8 times, and the reaction solution was stirred at 25 ℃ for 5 hours.

The reaction solution was cooled to 0 ℃ and appropriate amount of Dimethylformamide (DMF) was added without purification, then compound F1-2(10mg/ml pre-dissolved in DMF) was added in 6 times excess molar ratio to ensure that the volume of DMF in the reaction system did not exceed 8%, and the coupling was carried out by stirring at 0 ℃ for 2 hours.

The coupling reaction mixture was purified by filtration through a pH 6.0 histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Then sterilized by filtration through a 0.3 meter pore size filter unit and stored at-80 ℃.

EXAMPLE III detection and stability Studies of antibody conjugates

Performing HIC analysis on antibody-conjugated drugs enables important information to be obtained, such as the number and location of conjugation sites and drug-to-antibody ratio (DAR). HIC analysis was performed on the ADC products based on the following conditions, and the analysis patterns are shown in FIGS. 1-8 and FIGS. 2-1-2.

Agilent 1290Infinity

Chromatographic column Waters Protein-Pak Hi Res HIC (4.6X 100mm, 2.5 μm)

Mobile phase: 2.5M ammonium sulfate (containing 125mM phosphate buffer): 125mM phosphate buffer: isopropanol (I-propanol)

Flow rate: 0.7mL/min, column temperature: 25 deg.C

In addition, LC-MS technology has been used for ADC drug structure and composition analysis, to evaluate the stability of ADC drug linker, to determine the relative proportions of different DAR components analytically, etc. We performed LCMS analysis on the ADC product based on the following conditions. The analysis pattern is shown in figures 3-1-3-8 and figures 4-1-4-2.

The instrument comprises the following steps: agilent 6520Q-TOF

Chromatographic column of polyhydroxyyethyyl-A (PHEA) (PolyLC, Columbia, MD)2.1mm 200 mm; 5 μm

particles with

pores

Mobile phase 200mM ammonium acetate

The flow rate is 0.1 mL/min;

column temperature 25 deg.C

The disulfide chain bridging based on the maleic amide has better stability, thiol ether exchange is not easy to occur in vivo, and in order to further prove that the introduction of the unsubstituted phenyl group with the substituent group at the Ar' position can greatly slow down the cyclization secondary hydrolysis reaction after the ring opening of the maleic amide and can also enhance the stability of the antibody-drug conjugate. In this experiment, control ADC was prepared by coupling a barbituric antibody with a benzene ring compound (as shown below) in which Ar' is only 1, 4-substituted, and the coupling method was the same as that for ADC-I.

ADC-I, ADC-II and ADC-VII were selected and compared with control ADC, and ADC samples with the same protein concentration (10mg/mL) stored in the preparation buffer were respectively sampled and measured at 25 ℃ for 0,2,4 and 7 days.

And (3) determining corresponding secondary hydrolysis products in each antibody-drug conjugate (ADC) by adopting LC-MS (Q-TOF), and extracting mass spectrum characteristic peaks of the secondary hydrolysis products to obtain peak areas. The trend of the secondary ADC hydrolysate was obtained by comparing the peak area changes from 0 to 7 days, as shown in the data below and in FIG. 5. It can be seen from the data that the control ADC secondary hydrolysate is significantly higher than the secondary hydrolysates in the ADC-I, ADC-II, ADC-VII samples.

In addition, the change conditions of 0,2,4 and 7 days are measured by using the HIC method by using each ADC sample, and as can be seen from the graphs in FIGS. 6-1-6-4, an impurity peak appears at the position of the retention time 6.904 in 7 days of the control ADC sample, while the HIC graphs of the ADC-I, ADC-II and ADC-VII samples are basically not changed obviously from 0 to 7 days, which are respectively shown in FIGS. 7-1-7-4, 8-1-8-4 and 9-1-9-4.

EXAMPLE four biological assays of antibody conjugates

1. In vitro cell proliferation assay for biological activity

The experimental materials used in the following experiments were derived from: DMEM medium, DMEM/F12K medium, RPMI 1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 × sodium pyruvate, 100 × streptomycin were purchased from Gibco. Sulforhodamine B (SRB) was purchased from Sigma. NCI-N87 human gastric cancer cell and BT-474 human breast cancer cell come from Kunming cell bank of Chinese academy of sciences. Other reagents were analytically pure. 96-well flat bottom polystyrene (Corning, cat No. 3599). Synergy 2 microplate reader (Bio-Tek).

In this example, the effect of ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, ADC-VIII on the proliferation of tumor cell lines was investigated.

This example uses sulforhodamine b (srb) colorimetry to evaluate the antiproliferative effect of drug combinations. SRB is a pink anionic dye, is easily soluble in water, and can be specifically combined with basic amino acids which form proteins in cells under an acidic condition; the absorption peak is generated under the wavelength of 510nm, and the absorbance value is in positive linear correlation with the cell quantity, so that the method can be used for quantitative detection of the cell quantity. The cell lines selected in this example were: BT-474 human breast cancer cell and NCI-N87 human gastric cancer cell.

BT-474 and NCI-N87 cells are cultured in RPMI 1640 culture medium containing 10% fetal calf serum at 37 ℃ in a 5% CO2 incubator to logarithmic phase, the cells in the logarithmic phase are respectively inoculated to a 96-well culture plate at the density of 2 × 103-9 × 103 cells per well, each well is 100 μ L, after 24 hours of culture, drugs with different concentrations are added for acting for 5 days, 9 concentrations are respectively prepared by 3,4 or 5-fold dilution, each concentration is provided with multiple wells, and a solvent control with corresponding concentration and a well without cell culture medium are provided. After the drug effect is finished, the culture solution is poured out, 100 mu L of trichloroacetic acid solution (30 percent, w/v) precooled at 4 ℃ is added, the mixture is fixed for 1 hour at 4 ℃, then the mixture is washed for 5 times by deionized water, after drying at room temperature, 100 mu L of SRB dye solution (Sigma, prepared by 1 percent glacial acetic acid) of 0.4 percent (w/v) is added into each hole, after incubation and dyeing at room temperature for 30min, the mixture is washed for 4 times by 1 percent glacial acetic acid, unbound dye is removed, and the mixture is dried at room temperature. Add 10mM Tris solution 100. mu.L per well, incubate staining for 15min at room temperature, rinse five times with 1% glacial acetic acid to wash off noneBound SRB was dried at room temperature, 10mM Tris buffer (pH 10.5) was added to each well to dissolve the dye bound to the cell protein, and the light absorption values (OD values) were measured at wavelengths of 510nm and 690nm using a Synergy 2 microplate reader (Bio-Tek) to obtain A ═ OD₅₁₀-OD₆₉₀。

Inhibition (%) ═ a control-a dosing)/a control × 100%.

ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII and ADC-VIII are used in the experiment to research the proliferation effect of Her2 high-expression tumor cell lines in vitro. As shown in the table below, compared with naked antibodies Perjeta and Herceptin, corresponding ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII and ADC-VIII can treat high-expression NCI-N87 human gastric cancer cells and BT-474 human breast cancer cells of Her2, and can obviously inhibit the proliferation of tumor cells. The corresponding proliferation inhibition curves are shown in FIGS. 10-13.

2. In vivo antitumor efficacy assay

The efficacy of the combination of the invention can be measured in vivo, i.e. implantation of an allograft or xenograft of cancer cells in rodents and treatment of tumors with the combination. Test mice were treated with drug or control and monitored for weeks or more to measure time to tumor doubling, log cell killing, and tumor inhibition.

1) Laboratory animal

BALB/cA-nude mice, 6-7 weeks old, purchased from Shanghai Ling Biotech, Inc. Producing license numbers: SCXK (Shanghai) 2013-0018; animal certification number 2013001815683. A breeding environment: SPF grade.

2) Experimental procedure

Nude mice were inoculated subcutaneously with 6106 human gastric cancer NCI-N87 cells, and after tumors grew to 100-200mm3, animals were grouped according to tumor volume (D0). Intravenous Injection (IV) of mice; the administration volume is 10 mL/kg; solvent group was given the same volume of "solvent" (0.1% BSA saline); specific dosages and schedules are shown in the following table. Tumor volumes were measured 2 times per week, mice were weighed and data recorded.

The experimental index is to examine the influence of the drug on the tumor growth, and the specific index is T/C% or tumor inhibition rate TGI (%).

Tumor diameter was measured twice weekly with a vernier caliper and tumor volume (V) was calculated as:

v-1/2 × a × b2 wherein a and b represent length and width, respectively.

T/C (%) - (T-T0)/(C-C0) x100 where T, C is the tumor volume at the end of the experiment; t0, C0 are tumor volumes at the beginning of the experiment.

Tumor inhibition rate (TGI) (%) 100-T/C (%).

When tumors appeared to regress, tumor inhibition rate (TGI) (%) 100- (T-T0)/T0x 100

Partial tumor regression (PR) is defined if the tumor shrinks from the starting volume, i.e., T < T0 or C < C0; if the tumor completely disappears, it is defined as complete tumor regression (CR).

End of experiment (D21), end of experiment, or tumor volume of 1500mm³The animals were sacrificed under CO2 anesthesia and then the tumors were dissected and photographed.

3) Results of the experiment

The curative effect of the drug on HER2 positive human gastric cancer NCI-N87 nude mouse subcutaneous transplantation tumor is shown in the following table and figure 14; the tumor-bearing mice can well tolerate the medicaments, and symptoms such as weight loss and the like do not occur.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A substituted maleimide-based linker having the structure of formula Ia:

wherein, R is ArS-,

ar is selected from the group consisting of: phenyl, halogenobenzene, C1-C4 alkylphenyl, C1-C4 alkoxyphenyl, 2-pyridyl, 2-pyrimidyl, 1-methylimidazol-2-yl,

Ar' is selected from the group consisting of: substituted phenylene, said substitution meaning that a hydrogen atom on a group is substituted with one or more substituents selected from the group consisting of: halogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitrile and aminoacyl;

L₁is-O (CH) attached to an Ar' group₂CH₂O)_n-, where n is selected from any one of integers from 1 to 6.

2. The substituted maleimide-based linker of claim 1, wherein the linker fragment has a structure selected from the group consisting of:

3. a substituted maleimide-based linker-drug conjugate or a pharmaceutically acceptable salt thereof, having a structure shown in formula Ib:

wherein, R, Ar' and L₁As defined in claim 1;

L₂is a chemical bond, or an AA-PAB structure; wherein AA is dipeptide or tripeptide fragment, and PAB is p-aminobenzyloxycarbonyl;

4. The substituted maleimide-based linker-drug conjugate of claim 3, or a pharmaceutically acceptable salt thereof, wherein the AA is selected from the group consisting of: Val-Cit, Val-Ala, Phe-Lys.

5. The substituted maleimide-based linker-drug conjugate of claim 3, or a pharmaceutically acceptable salt thereof, wherein the CTD is selected from the group consisting of: tubulin inhibitors, topoisomerase inhibitors, DNA binding agents.

6. The substituted maleimide-based linker-drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein the tubulin inhibitor is selected from the group consisting of: maytansine derivatives, monomethyl auristatin E, monomethyl auristatin F, monomethyl dolastatin10, microtubule inhibitor Tubulysin derivatives, Cryptophycin derivatives, and Taltobulin.

7. The substituted maleimide-based linker-drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein the DNA binding agent is selected from the group consisting of: PBD derivatives and duocarmycin derivatives.

8. The substituted maleimide-based linker-drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein the topoisomerase inhibitor is selected from the group consisting of: adriamycin metabolite PNU-159682 derivative and irinotecan.

9. The substituted maleimide-based linker-drug conjugate of claim 3, or a pharmaceutically acceptable salt thereof, wherein the CTD has a structure selected from the group consisting of:

10. the substituted maleimide-based linker-drug conjugate of any one of claims 3 to 9, wherein formula Ib is selected from the group consisting of:

11. an antibody-drug conjugate formed by coupling an antibody with the substituted maleimide-based linker-drug conjugate of any one of claims 3 to 10, or a pharmaceutically acceptable salt thereof.

12. The antibody-drug conjugate of claim 11, wherein the conjugate has a structure of formula Ic and/or Id;

wherein, Ar' and L₁、L₂CTD is as defined in claim 3;

m＝1.0～5.0；

ab is an antibody or antibody fragment.

13. The antibody-drug conjugate of claim 11, wherein the antibody is selected from the group consisting of: a monoclonal antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, or an antibody fragment of any of the foregoing.

14. The antibody-drug conjugate of claim 11, wherein the antibody is an antibody capable of binding to a tumor associated antigen selected from the group consisting of: HER2, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD79 74, CD138, CD147, CD223, EpCAM, Mucin1, STEAP 74, GPNMB, FGF 74, FOLR 74, EGFR, EGFRvIII, Tissuefactor, C-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A 74, PSMA, EphA 74, AGS-5, GPC-3, C-KIT, RoR 74, PD-L74, CD27 74, 5T 74, Mucin 74, NaPi2 74, ETSTEAP, SLICK 74, TRBR, TrACAMA-74, ACALC-L74, SLC-16, Clalis 74, SLC-16, Clarke 74, SLC-16, SLC-74, and Clarke 74.

15. The antibody-drug conjugate of claim 14, wherein the HER2 antibody is selected from the group consisting of: trastuzumab and pertuzumab.

16. A pharmaceutical composition, comprising: (a) the antibody-drug conjugate of any one of claims 11-15; and (b) a pharmaceutically acceptable diluent, carrier or excipient.

17. Use of an antibody-drug conjugate according to any one of claims 11-15 for the manufacture of a medicament for the treatment of a tumour.

18. The method of preparing an antibody-drug conjugate of any one of claims 11-15, comprising the steps of:

(2) and (2) crosslinking the linker-drug conjugate and the reduced antibody obtained in the step (1) in a mixed solution of a buffer solution and an organic solvent to obtain the antibody-drug conjugate.