CN108101825B - Disubstituted maleimide linker for antibody-drug coupling, preparation method and application thereof - Google Patents

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

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CN108101825B
CN108101825B CN201611093699.6A CN201611093699A CN108101825B CN 108101825 B CN108101825 B CN 108101825B CN 201611093699 A CN201611093699 A CN 201611093699A CN 108101825 B CN108101825 B CN 108101825B
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antibody
compound
group
drug conjugate
synthesis
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CN108101825A (en
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沈竞康
孟韬
马兰萍
王昕�
彭红丽
张永良
于霆
陈驎
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Mayway (shanghai) Biotechnology Co Ltd
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Maiwei Shanghai Biotechnology Co ltd
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Priority to CN201780072626.5A priority patent/CN110088086B/en
Priority to CA3044898A priority patent/CA3044898C/en
Priority to ES17873634T priority patent/ES2921236T3/en
Priority to PCT/CN2017/112958 priority patent/WO2018095422A1/en
Priority to KR1020197017841A priority patent/KR102562760B1/en
Priority to US16/464,211 priority patent/US10987430B2/en
Priority to PL17873634.4T priority patent/PL3546448T3/en
Priority to EP17873634.4A priority patent/EP3546448B1/en
Priority to JP2019548514A priority patent/JP7058666B2/en
Priority to DK17873634.4T priority patent/DK3546448T3/en
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Abstract

The invention provides a disubstituted maleimide linker coupled with an antibody, a preparation method and application thereof, and particularly relates to a novel linker for coupling a strong cytotoxic active substance and a biological macromolecule. The linker can selectively act simultaneously with disulfide chains, thereby greatly improving the material uniformity of the conjugate. The conjugate prepared by the linker of the invention has high inhibitory activity on cell strains expressing corresponding antigens. The invention also provides a preparation method and application of the conjugate.

Description

Disubstituted maleimide 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
Over the years, much research has focused on improving the efficiency of drug or other agent delivery to target cells, tissues or tumors to achieve maximum therapeutic efficacy and minimal toxicity. Although many efforts have been made to develop effective methods for delivering biologically active molecules into cells in vivo and in vitro, none have proven to be entirely satisfactory. It is often difficult or ineffective to optimize binding of a drug to its intracellular target molecule while minimizing intracellular redistribution of the drug (e.g., into neighboring cells).
Most drugs currently administered parenterally to patients are not targeted, which results in systemic delivery of such drugs to cells and tissues of the body where they are not needed, often causing adverse reactions. This can lead to adverse side effects of the drug, often limiting the doses of drug that can be administered (e.g., chemotherapy (anti-cancer), cytotoxic, enzyme inhibitors and antiviral or antimicrobial drugs). In contrast, although oral administration is considered a convenient and economical mode of administration, once the drug is absorbed into the systemic circulation, the same problem of nonspecific toxicity to cells not affected by the disease can occur. Other complications include oral bioavailability problems and retention of the drug in the gut, which exposes the gut to the drug for too long, thereby risking visceral toxicity. Therefore, the main goal of developing (drug delivery) methods is to target drugs specifically to cells and tissues. The benefits of such treatment include avoiding systemic physiological effects of inappropriate delivery of such drugs to other cells and tissues, such as uninfected cells. Intracellular targeting can be achieved by methods, compounds and formulations that allow the accumulation or retention of biologically active substances, i.e., active metabolites, within cells.
The use of antibody-drug conjugates (ADCs), i.e., immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in the treatment of cancer, allows for the targeted delivery of drug moieties to tumors and intracellular accumulation therein, where systemic administration of these unconjugated agents also results in unacceptable levels of toxicity to normal cells in addition to the tumor cells sought to be eliminated. Efforts to improve the therapeutic index of ADCs, i.e., highest efficacy with lowest toxicity, have focused on the selectivity as well as drug conjugation and pharmacodynamic release profiles of polyclonal and monoclonal antibodies (mabs).
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 position of conjugation, which in turn leads to heterogeneity of the resulting antibody drug conjugates. 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 technology has recently become more favored, which controls conjugation between antibody drugs in terms of both 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. Therefore, there is a great need in the art to provide efficient, simple, and practical chemical coupling methods.
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:
Figure DEST_PATH_GDA0001277593270000021
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 or unsubstituted C6-C10Aryl, substituted or unsubstituted 5-12 membered heteroaryl; preferably, Ar is selected from phenyl, halogeno-benzene, C1-C4Alkyl phenyl, C1-C4Alkoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl,
Figure DEST_PATH_GDA0001277593270000022
Wherein W is an amino group R attached to a carbonyl group1，R1Is selected from-NH2、
Figure DEST_PATH_GDA0001277593270000023
Figure DEST_PATH_GDA0001277593270000024
Etc.; wherein: c1-C4The alkylphenyl group is more preferably a 4-methylphenyl group; c1-C4The alkoxyphenyl group is more preferably a 4-methoxyphenyl group.
Ar' is selected from the group consisting of: substituted or unsubstituted C6-C10Arylene, substituted or unsubstituted 5-12 membered heteroarylene; preferably, Ar' is selected from substituted or unsubstituted phenylene. The substitution means that the hydrogen atom on the group is substituted by one or more substituents selected from the group consisting of: halogen, C1-C4Alkyl radical, C1-C4An alkoxy group.
L1is-O (CH) attached to an Ar' group2CH2O)n-, where n is selected from any integer from 1 to 20, preferably from any integer from 1 to 10.
In another preferred embodiment, the linker fragment has a structure selected from the group consisting of:
Figure DEST_PATH_GDA0001277593270000025
Figure DEST_PATH_GDA0001277593270000031
Figure DEST_PATH_GDA0001277593270000041
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, and pharmaceutically acceptable salts or solvates thereof, the structure of which is shown in formula Ib:
Figure DEST_PATH_GDA0001277593270000042
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 through peptide bonds), preferably comprising 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) and the like, and PAB is p-aminobenzyl carbamoyl;
CTD is bonded to L through an amide bond2And/or a drug for treating autoimmune diseases and anti-inflammation.
In another preferred embodiment, the compound of formula Ib is selected from the group consisting of:
Figure DEST_PATH_GDA0001277593270000051
Figure DEST_PATH_GDA0001277593270000061
Figure DEST_PATH_GDA0001277593270000071
Figure DEST_PATH_GDA0001277593270000081
in a third aspect of the present invention, there is provided an antibody-drug conjugate formed by coupling an antibody with a drug conjugate of formula Ib substituted maleimide-based linker as described 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 invention, an antibody-drug conjugate is provided, wherein the conjugate has a structure of formula Ic and/or Id;
Figure DEST_PATH_GDA0001277593270000082
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-D12:
Figure DEST_PATH_GDA0001277593270000091
Figure DEST_PATH_GDA0001277593270000101
in another preferred embodiment, the antibody is an antibody capable of binding to a tumor associated antigen selected from the group consisting of:
Figure DEST_PATH_GDA0001277593270000102
Figure DEST_PATH_GDA0001277593270000111
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 present invention, there is provided a use of the antibody-drug conjugate according to any one of the third aspect of the present invention, for preparing a drug for treating tumor.
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 fifth 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), histidineAcid-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.
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 be limited to space.
Drawings
FIG. 1 shows a comparison of pertuzumab-drug conjugate Hydrophobic Interaction Chromatography (HIC);
FIG. 2 shows a mass spectrum contrast of pertuzumab-drug conjugate;
FIG. 3 shows the binding assay of ADC-2, P-mcVC-MMAE, Pertuzumab mab with NCI-N87 cell surface antigen Her 2;
FIG. 4 shows the proliferation inhibition experiments of ADC-2, P-mcVC-MMAE, Kadcyla, Pertuzumab on human breast cancer cell SK-BR-3;
FIG. 5 shows the proliferation inhibition assay of ADC-2, P-mcVC-MMAE, Kadcyla, Pertuzumab on human breast cancer cell BT-474;
FIG. 6 shows the inhibition experiment of ADC-2, P-mcVC-MMAE, Kadcyla, Pertuzumab on the proliferation of human gastric cancer cell N87;
FIG. 7 shows the proliferation inhibition assay of ADC-2 on SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells, and Panc-1 human pancreatic cancer;
FIG. 8 shows the proliferation inhibition assay of ADC-2 on human breast cancer MCF-7, MDA-MB-231, and MDA-MB-468 cells;
FIG. 9 shows the experiment of inhibiting the proliferation of ADC-4 on human gastric cancer cell N87;
FIG. 10 shows the proliferation inhibition assay of ADC-2, ADC-3 and ADC-4 on Calu-3 human lung cancer cells;
FIG. 11 shows the activity studies of ADC-2, ADC-3, P-mcVC-MMAE, Kadcyla inhibiting human gastric cancer NCI-N87 nude mouse subcutaneous transplantable tumor.
Detailed Description
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 the coupling method has a narrower drug/antibody ratio (DAR) distribution compared with a conventional antibody drug conjugate. Based on the above findings, the inventors have completed the present invention.
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 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 target antigen).
Antibodies useful in 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) mucin 16(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.
Figure DEST_PATH_GDA0001277593270000151
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 choice drugs for antibody drug conjugates.
Figure DEST_PATH_GDA0001277593270000152
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 which can be divided into four regions, Mep (D-N-methylpiperidinecarboxylic acid), Ile (isoleucine), Tuv (tubulvaline, tubulylalanine), and Tup (tubulphenylalanine), as shown in the following formula:
Figure DEST_PATH_GDA0001277593270000161
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:
Figure DEST_PATH_GDA0001277593270000162
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.
Figure DEST_PATH_GDA0001277593270000163
In one aspect, the drug is the camptothecin drug derivative SN-38. SN-38 is a biologically active metabolite of irinotecan hydrochloride (CPT-11), a class of topoisomerase inhibitors. SN-38 causes the strongest inhibition of DNA topoisomerase I, inhibits DNA synthesis dose-and time-dependently, and causes frequent DNA single strand breaks. Wherein the structure of SN-38 is shown as the following formula.
Figure DEST_PATH_GDA0001277593270000164
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 Amanita phillyides (Amanita villoides), a bicyclic octapeptide, which inhibits transcription of eukaryotic RNA polymerase II and RNA polymerase III.
Figure DEST_PATH_GDA0001277593270000165
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):
Figure DEST_PATH_GDA0001277593270000171
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 been shown to be highly potent cytotoxic drugs and may be used as drug candidates for antibody drug conjugates.
Figure DEST_PATH_GDA0001277593270000172
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.
Figure DEST_PATH_GDA0001277593270000173
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-D12 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 tumor cells (bystatder effect) that are in the vicinity of cells that do not express the targeted antigen (antigen-negative cells).
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 conjugate can be used for targeted delivery of drugs to target cell populations, such as tumor cells. The antibody drug conjugate can be specifically bound to a cell surface protein, and the resulting conjugate can then be 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. A "drug" is a highly active drug (see definitions section), 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 the average DAR value is close to the range of the optimal antibody drug conjugate (2-4). 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.
Method for preparing antibody-drug conjugate
The preparation route of the antibody drug conjugate is shown below. The disulfide bond between antibody chains is reduced to generate 8 sulfydryl groups, and the substituted maleimide-based linker drug conjugate is crosslinked with the reduced antibody sulfydryl groups to generate the corresponding antibody drug conjugate, wherein the antibody drug conjugate exists in one or two forms of the following reactions.
Figure DEST_PATH_GDA0001277593270000191
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; the reaction buffer described herein may be a buffer prepared in the following ratio: 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.
Cooling the reaction liquid to 0-10 ℃, if DTT is adopted for reduction, removing excessive DTT by a desalting column or ultrafiltration after the reduction reaction is finished, adding a substituted maleimide compound (10mg/ml is dissolved in Acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) or diethyl acetamide (DMA) in advance), ensuring that the volume ratio of an organic solvent in the reaction liquid is not more than 15%, and carrying out coupling reaction at 0-37 ℃ for 2-4 hours under stirring. If TCEP is adopted for reduction, the substituted maleimide compound can be directly added for coupling without removing residual TCEP.
The coupling reaction mixture was purified by filtration using sodium succinate/NaCl buffer or histidine-acetic acid/sucrose gel using a desalting column, and peak samples were collected according to UV280 UV absorbance. Or ultrafiltering for several times. Then filtering and sterilizing, and storing the obtained product at low temperature. The temperature is preferably from-100 to 60 ℃ and the pore size of the filter unit is preferably from 0.15 to 0.3. mu.m.
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 described in the patent, and 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).
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 particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
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 antibody drug conjugate, 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.
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.
First, compound synthesis and preparation method
The first scheme is as follows:
the substituted maleimide linker fragment molecules represented by formula Ia listed in the first aspect of the present invention can be synthesized by the method in scheme one, and by substitution reaction of n-glycol with fluoronitrobenzene to obtain intermediate B, followed by reduction of nitro group to obtain amino compound C, 2, 3-dibromomaleimide, and aryl thiol to obtain intermediate D, followed by reaction with methyl chloroformate to obtain intermediate E, 2, 3-dibromomaleimide, or by direct reaction with methyl chloroformate to obtain intermediate E ', and intermediate C is reacted with intermediate E or intermediate E' to obtain linker fragment molecule F. The reaction scheme and specific examples are illustrated below:
Figure DEST_PATH_GDA0001277593270000211
example 1 Synthesis and preparation of Compounds of formulae 1-12
1.1 Synthesis of Compound F-1 (formula 1)
1.1.1 Synthesis of intermediate B-1 (step a)
Figure DEST_PATH_GDA0001277593270000221
4-Fluoronitrobenzene (10.0g, 0.071mol), diethylene glycol (75.2g, 0.71mol) and potassium carbonate (14.7g, 0.11mol) were weighed into a 250mL round-bottomed flask and stirred at 80 ℃ for 22 hours under nitrogen. Slowly cooling to room temperature, extracting with dichloromethane, washing with 1mol/L dilute hydrochloric acid, water and saturated saline solution in sequence, drying with anhydrous sodium sulfate, and spin-drying the solvent. Column chromatography (silica gel, 200 mesh, 300 mesh, PE/EtOAC10:1) gave the product as a pale yellow clear liquid (15.1g, 94% yield). LC-MS (M)+) Theoretical value of 227.08, LC-MS (ESI, M + H)+) Found 228.12.
1.1.2 Synthesis of intermediate C-1 (step b)
Figure DEST_PATH_GDA0001277593270000222
Dissolving an intermediate compound B-1(3.0g and 9.52mmol) in acetone (30mL), cooling in an ice-water bath, slowly dropwise adding a fresh Jones preparation reagent (15mL), stirring the reaction mixture for 3 hours at room temperature, cooling in the ice-water bath, slowly dropwise adding isopropanol, continuously stirring in the ice-water bath for 15 minutes, removing the organic solvent by rotary evaporation, extracting the water phase by diethyl ether for 3 times, combining the organic phase, washing with saturated saline water, drying with anhydrous sodium sulfate, and removing the solvent by rotary evaporation to obtain a yellow oily crude product, wherein the intermediate is directly used for the next reaction without purification and separation. The above intermediate was dissolved in tetrahydrofuran (30mL),10% Palladium on carbon (300mg) was added thereto, and the mixture was hydrogenated at 30 ℃ for 6 hours. After the catalyst is removed by suction filtration, the solvent is removed by rotary evaporation to obtain a brown yellow oily crude product. Used in the next reaction without further purification. LC-MS (M)+) Theoretical value of 211.08, LC-MS (ESI, M + H)+) Found 212.05.
1.1.3 Synthesis of intermediate D-1 (step c)
Figure DEST_PATH_GDA0001277593270000223
2, 3-Dibromomaleimide (7.0g, 27.69mmol) was dissolved in methanol (80mL), and sodium acetate (4.5g, 55.4mmol) and thiophenol (11.3mL, 110.7mmol) were added in that order. The reaction mixture was stirred at room temperature for 30 minutes, the organic solvent was removed by rotary evaporation, extracted with dichloromethane, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was dried by rotary drying. The crude product was recrystallized from petroleum ether/ethyl acetate, filtered and dried to give D-1 as a bright yellow solid (6.5g, 75% yield).
1.1.4 Synthesis of intermediate E-1 (step d)
Figure DEST_PATH_GDA0001277593270000231
Intermediate D-1(3.0g, 9.6mmol) and N-methylmorpholine (1.37mL, 12.5mmol) were dissolved in ethyl acetate (40mL), cooled in an ice-water bath, and methyl chloroformate (1.11mL, 14.4mmol) was added slowly dropwise and the reaction mixture stirred at room temperature for 30 min. The reaction mixture was diluted with ethyl acetate, extracted with water, washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, and the solvent was spin-dried. The crude product was recrystallized from petroleum ether/ethyl acetate, filtered and dried to give E-1 as a bright yellow solid (3.5g, 98% yield).
1.1.5 Synthesis of Compound F-1 (step e)
Figure DEST_PATH_GDA0001277593270000232
Intermediate C-1(2.0g, 9.5 mmo)l) was dissolved in anhydrous dichloromethane (40ml), intermediate E-1(3.5g, 9.5mmol) was added, the reaction mixture was stirred at room temperature for 24 hours under nitrogen protection, silica gel (12g, 200 mesh, 300 mesh) was weighed in and the reaction mixture was stirred at room temperature overnight. The solvent was dried and dry column chromatography (silica gel, 200 mesh, 300 mesh, dichloromethane/methanol 10:1) afforded the product F-1 as an orange oil (4.1g, 85% yield). LC-MS (M)+) Theoretical value of 507.08, LC-MS (ESI, M + H)+) Found 508.11.
1.2 Synthesis of Compound F-2 (formula 2)
Figure DEST_PATH_GDA0001277593270000233
The synthesis of compound F-2 was identical to the synthesis of compound F-1 in example 1.1, except that diethylene glycol in step a was changed to triethylene glycol. The product F-2 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 551.11, LC-MS (ESI, M + H)+) Found 552.13.
1.3 Synthesis of Compound F-3 (formula 3)
Figure DEST_PATH_GDA0001277593270000234
The synthesis of compound F-3 was identical to the synthesis of compound F-1 in example 1.1, except that diethylene glycol in step a was changed to tetraethylene glycol. The product F-3 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 595.13, LC-MS (ESI, M + H)+) Found 596.14.
1.4 Synthesis of Compound F-4 (formula 4)
Figure DEST_PATH_GDA0001277593270000241
The synthesis of compound F-3 was identical to the synthesis of compound F-1 in example 1.1, except that the diethylene glycol in step a was changed to pentaethylene glycol. The product F-3 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 639.16, LC-MS (ES)I,M+H+) Found 640.18.
1.5 Synthesis of Compound F-5 (formula 5)
Figure DEST_PATH_GDA0001277593270000242
The synthesis of compound F-3 was identical to the synthesis of compound F-1 in example 1.1, except that diethylene glycol in step a was changed to hexaethylene glycol. The product F-3 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 683.19, LC-MS (ESI, M + H)+) Found 684.21.
1.6 Synthesis of Compound F-6 (formula 6)
Figure DEST_PATH_GDA0001277593270000243
The synthesis of compound F-3 was identical to the synthesis of compound F-1 in example 1.1, except that diethylene glycol in step a was changed to octaethylene glycol. The product F-3 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 771.24, LC-MS (ESI, M + H)+) Found 772.26.
1.7 Synthesis of Compound F-7 (formula 7)
Figure DEST_PATH_GDA0001277593270000244
The synthesis of compound F-3 was identical to the synthesis of compound F-1 in example 1.1, except that diethylene glycol in step a was changed to decaethylene glycol. The product F-3 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 895.29, LC-MS (ESI, M + H)+) Found 896.31.
1.8 Synthesis of Compound F-8 (formula 8)
Figure DEST_PATH_GDA0001277593270000245
Synthesis of Compound F-8 Synthesis of Compound F-3 in example 1.3The procedure is the same except that thiophenol in step c is replaced with p-methylthiothiophenol. The product F-8 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 623.16, LC-MS (ESI, M + H)+) Found 624.18.
1.9 Synthesis of Compound F-9 (formula 9)
Figure DEST_PATH_GDA0001277593270000251
The synthesis of compound F-9 was identical to the synthesis procedure for compound F-3 in example 1.3, except that the thiophenol in step c was changed to p-methoxythiophenol. The product F-9 was obtained as an orange oil after a total of 5 steps. LC-MS (M)+) Theoretical value of 655.15, LC-MS (ESI, M + H)+) Found 656.17.
1.10 Synthesis of Compound F-10 (formula 10)
Figure DEST_PATH_GDA0001277593270000252
Synthesis of Compound F-9 the same procedure was followed as for Compound F-3 in example 1.3, except that the thiophenol in step c was changed to 4- (N-methylformamide) thiophenol. The product F-10 is obtained after 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 709.18, LC-MS (ESI, M + H)+) Found 710.23.
1.11 Synthesis of Compound F-11 (formula 11)
Figure DEST_PATH_GDA0001277593270000253
The synthesis of compound F-10 was identical to the synthesis of compound F-3 in example 1.3, except that the thiophenol in step c was changed to 2-mercaptopyridine. The product F-10 is obtained after 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 597.12, LC-MS (ESI, M + H)+) Found 598.13.
1.12 Synthesis of Compound F-12 (formula 12)
Figure DEST_PATH_GDA0001277593270000254
The synthesis of compound F-11 was performed in the same manner as the synthesis of compound F-3 in example 1.3, except that thiophenol in step c was changed to 2-mercaptopyrimidine. The product F-11 is obtained through 5 steps of reaction and is orange oil. LC-MS (M)+) Theoretical value of 599.11, LC-MS (ESI, M + H)+) Found 600.13.
1.13 Synthesis of Compound F-13 (formula 13)
Figure DEST_PATH_GDA0001277593270000261
Synthesis of Compound F-12 was performed in the same manner as in example 1.3 except that p-fluoronitrobenzene in step a was changed to m-fluoronitrobenzene. The product F-12 was obtained as an orange oil after a total of 5 steps. LC-MS (M)+) Theoretical value of 595.13, LC-MS (ESI, M + H)+) Found 596.15.
1.14 Synthesis of Compound F-14 (formula 14)
Figure DEST_PATH_GDA0001277593270000262
The synthesis of compound F-12 was identical to the synthesis of compound F-3 in example 1.3, except that intermediate E in step E was exchanged for brominated intermediate E'. The product F-13 was obtained as a colorless oil after a total of 4 steps. LC-MS (M)+) Theoretical value of 534.95, LC-MS (ESI, M + H)+) Found 536.01.
1.15 Synthesis of Compound F-15 (formula 15)
Figure DEST_PATH_GDA0001277593270000263
Synthesis of Compound F-15 the same procedure was followed as for Compound F-3 in example 1.3, except that thiophenol in step c was changed to 2-mercapto-1-methylimidazole. After 5 steps of reaction, the mixture is obtainedThe product F-15 was obtained as an orange oil. LC-MS (M)+) Theoretical value of 603.15, LC-MS (ESI, M + H)+) Found 604.14.
1.16 Synthesis of Compound F-16 (formula 16)
Figure DEST_PATH_GDA0001277593270000264
Synthesis of Compound F-16 was performed in the same manner as in example 1.3 except that the thiophenol in step c was changed to 4- (N-morpholinocarboxamide) thiophenol. The product F-16 was obtained as an orange oil after a total of 5 steps. LC-MS (M)+) Theoretical value of 821.23, LC-MS (ESI, M + H)+) Found 822.21.
1.17 Synthesis of Compound F-17 (formula 17)
Figure DEST_PATH_GDA0001277593270000271
Synthesis of Compound F-17 was performed in the same manner as in the Synthesis of Compound F-3 in example 1.3, except that thiophenol in step c was changed to 4- (N-2-methoxyethylcarboxamide) thiophenol. The product F-17 was obtained as an orange oil after a total of 5 steps. LC-MS (M)+) Theoretical value of 797.23, LC-MS (ESI, M + H)+) Found 798.31.
1.18 Synthesis of Compound F-18 (formula 18)
Figure DEST_PATH_GDA0001277593270000272
Synthesis of Compound F-18 was performed in the same manner as in the Synthesis procedure for Compound F-3 in example 1.3, except that thiophenol in step c was changed to 4- (N-methoxy-N-methylformamide) thiophenol. The product F-18 was obtained as an orange oil after a total of 5 steps. LC-MS (M)+) Theoretical value of 769.20, LC-MS (ESI, M + H)+) Found 770.28.
The substituted maleimide linker fragment molecule represented by formula Ia can also be synthesized by the method shown in the following reaction scheme, wherein n glycol and fluoronitrobenzene are subjected to substitution reaction to obtain an intermediate B, and then the intermediate B is subjected to reaction with tert-butyl bromoacetate to obtain an intermediate Z, and the intermediate Z can also be obtained by the substitution reaction of n glycol and tert-butyl bromoacetate and then tert-butyl bromoacetate; reducing the intermediate Z to obtain an intermediate Y; carrying out substitution reaction on 2, 3-dibromomaleimide and aryl thiophenol to obtain an intermediate D, then reacting with methyl chloroformate to obtain an intermediate E, reacting the intermediate E with the intermediate Y to obtain an intermediate X, and removing tert-butyl ester in the intermediate X under an acidic condition to obtain a linker fragment molecule W. The reaction scheme is exemplified as follows:
Figure DEST_PATH_GDA0001277593270000281
scheme II:
the substituted maleimide linker drug conjugates represented by formula Ib (formula 13-formula 19) listed in the second aspect of the invention may be synthesized by the route outlined in scheme II, and the series of molecules G may be obtained by condensation coupling of compound F with cytotoxic drug CTD (D1-D11, a toxic drug molecule commercially available). The reaction scheme and specific examples are illustrated below:
Figure DEST_PATH_GDA0001277593270000282
example 2 Synthesis and preparation of Compounds of formulae 19-25
2.1 Synthesis of Compound G-1 (formula 19)
Figure DEST_PATH_GDA0001277593270000283
Compound F-3(200mg, 0.34mmol) and compound D1(220mg,0.34mmol) were dissolved in N, N-dimethylformamide (10mL), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDCI) (77mg, 0.40mmol) and 1-Hydroxybenzotriazole (HOBT) (54mg,0.40mmol) were added at room temperature, and the reaction mixture was stirred at room temperature overnight. Acetic acid B for reaction solutionAfter ester dilution, water was added for extraction, and the mixture was washed with water and saturated brine in this order, dried over anhydrous sodium sulfate, and the solvent was dried by rotary drying. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol), suction filtered and dried to give G-1 as a yellow amorphous solid (3.5G, 98% yield). LC-MS (M)+) Theoretical value of 1226.40, LC-MS (ESI, M + H)+) Found 1227.42.
2.2 Synthesis of Compound G-2 (formula 20)
Figure DEST_PATH_GDA0001277593270000291
Synthesis of Compound G-2 was performed in the same manner as in example 2.1 except that Compound D1 was replaced with Compound D2. Product G-2 was obtained as a yellow amorphous solid. LC-MS (M)+) Theoretical value of 1294.63, LC-MS (ESI, M + H)+) Found 1295.64.
2.3 Synthesis of Compound G-3 (formula 21)
Figure DEST_PATH_GDA0001277593270000292
Synthesis of Compound G-3 was performed in the same manner as in example 2.1 except that Compound D1 was replaced with Compound D3. Product G-3 was obtained as a yellow amorphous solid. LC-MS (M)+) Theoretical value of 1308.61, LC-MS (ESI, M + H)+) Found 1309.63.
2.4 Synthesis of Compound G-4 (formula 22)
Figure DEST_PATH_GDA0001277593270000293
Synthesis of Compound G-4 was performed in the same manner as in example 2.1 except that Compound D1 was replaced with Compound D9. Product G-4 was obtained as a yellow amorphous solid. LC-MS (M)+) Theoretical value of 1302.41, LC-MS (ESI, M + H)+) Found 1303.43.
2.5 Synthesis of Compound G-5 (formula 23)
Figure DEST_PATH_GDA0001277593270000294
Synthesis of Compound G-5 was performed in the same manner as in example 2.1 except that Compound D1 was replaced with Compound D6. Product G-5 was obtained as a yellow amorphous solid. LC-MS (M)+) Theoretical value of 1290.51, LC-MS (ESI, M + H)+) Found 1291.53.
2.6 Synthesis of Compound G-6 (formula 24)
Figure DEST_PATH_GDA0001277593270000295
Synthesis of Compound G-6 was performed in the same manner as in example 2.1 except that Compound D1 was replaced with Compound D7. Product G-6 was obtained as a yellow amorphous solid. LC-MS (M)+) Theoretical value of 1274.44, LC-MS (ESI, M + H)+) Found 1275.45.
2.7 Synthesis of Compound G-7 (formula 25)
Figure DEST_PATH_GDA0001277593270000301
Synthesis of Compound G-7 was performed in the same manner as in example 2.1 except that Compound D1 was replaced with Compound D11. Product G-7 was obtained as a yellow amorphous solid. LC-MS (M)+) Theoretical value of 1291.38, LC-MS (ESI, M + H)+) Found 1292.40.
The third scheme is as follows:
the substituted maleimide linker drug conjugates represented by formula Ib (formula 22-formula 41) listed in the second aspect of the invention may be synthesized by the route outlined in scheme III, by condensation of compound F with the amino group of a dipeptide/tripeptide-PAB linker (the linker is commercially available), and subsequent condensation coupling of the PAB group with di (p-nitrophenyl) carbonate followed by activation with the cytotoxic drug CTD (D1-D11) to give the series of molecules K. The reaction scheme and specific examples are illustrated below:
Figure DEST_PATH_GDA0001277593270000302
3.1 Synthesis of Compound K-1 (formula 26)
3.1.1 Synthesis of intermediate I-1 (step f)
Figure DEST_PATH_GDA0001277593270000303
Compound F-3(2.0g, 3.36mmol) was dissolved in anhydrous N, N dimethylformamide (25ml), EDCI (963mg, 5.04mmol), HOBt (681mg, 5.04mmol) and N-methylmorpholine (1.11ml, 10.08mmol) were added sequentially under nitrogen, the reaction mixture was stirred at room temperature for 20 minutes under nitrogen, and finally compound H-1(Val-Cit-PAB,1.91g, 5.04mmol) was added. The reaction mixture was stirred at room temperature overnight under nitrogen. The solvent was dried and dry column chromatography (silica gel, 200 mesh 300 mesh, DCM/MeOH10:1) afforded product I-1 as an orange oil (2.0g, 62.2% yield). LC-MS (M)+) Theoretical value of 1631.60, LC-MS (ESI, M + H)+) Found 1632.62.
3.1.2 Synthesis of intermediate J-1 (step g)
Figure DEST_PATH_GDA0001277593270000304
Compound I-1(1.5g, 1.57mmol) was dissolved in anhydrous N, N dimethylformamide (10mL), N-diisopropylethylamine (0.52mL, 3.14mmol) and di (p-nitrophenyl) carbonate (717mg, 2.335mmol) were added sequentially under nitrogen, and the mixture was stirred at room temperature for 15 hours. The solvent was dried and dry column chromatography (silica gel, 200 mesh 300 mesh, DCM/MeOH 20:1) afforded the product J-1 as an orange oil (1.4g, 79.9% yield). LC-MS (M)+) Theoretical value of 1121.35, LC-MS (ESI, M + H)+) Found 1122.37.
3.1.3 Synthesis of Compound K-1 (step h)
Figure DEST_PATH_GDA0001277593270000311
Compound J-1(0.5g, 0.52mmol) was dissolved in dry N, N-dimethylformamide (5mL), N-diisopropylethylamine (0.172mL, 1.04mmol), HOBt (70mg, 0.52mmol) and compound D1(340mg,0.52mmol) were added sequentially under nitrogen and the reaction mixture was stirred at room temperature overnight under nitrogen. The solvent was dried and dry column chromatography (silica gel, 200 mesh 300 mesh, DCM/MeOH10:1) afforded product K-1 as a yellow amorphous solid (310mg, 36.33% yield). LC-MS (M)+) Theoretical value of 1631.60, LC-MS (ESI, M + H)+) Found 1632.62.
3.2 Synthesis of Compound K-2 (formula 27)
Figure DEST_PATH_GDA0001277593270000312
Synthesis of Compound K-2 was performed in the same manner as Compound K-1 in example 3.1, except that Compound D1 in step h was replaced with Compound D2 (monomethyl auristatin E). The product K-2 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1699.83, LC-MS (ESI, M + H)+) Found 1610.85.
3.3 Synthesis of Compound K-3 (formula 28)
Figure DEST_PATH_GDA0001277593270000313
Synthesis of Compound K-3 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound F-3 in step F was changed to F-5, and Compound D1 in step h was changed to Compound D2 (monomethyl auristatin E). The product K-3 is yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1787.88, LC-MS (ESI, M + H)+) Found 1788.90.
3.4 Synthesis of Compound K-4 (formula 29)
Figure DEST_PATH_GDA0001277593270000314
Synthesis of Compound K-4 was performed in the same manner as for Compound K-1 in example 3.1, except that Compound F-3 in step F was changed to F-6, and Compound D1 in step h was changed to Compound D2 (monomethyl auristatin E). The product K-4 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1875.93, LC-MS (ESI, M + H)+) Found 1876.95.
3.5 Synthesis of Compound K-5 (formula 30)
Figure DEST_PATH_GDA0001277593270000321
Synthesis of Compound K-5 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound F-3 in step F was changed to F-7, and Compound D1 in step h was changed to Compound D2 (monomethyl auristatin E). The product K-5 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1963.99, LC-MS (ESI, M + H)+) Found 1965.01.
3.6 Synthesis of Compound K-6 (formula 31)
Figure DEST_PATH_GDA0001277593270000322
Synthesis of Compound K-6 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound H-1 in step f was changed to H-2(Val-Ala-PAB) and Compound D1 in step H was changed to Compound D2 (monomethyl auristatin E). The product K-6 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1613.78, LC-MS (ESI, M + H)+) Found 1614.80.
3.7 Synthesis of Compound K-7 (formula 32)
Figure DEST_PATH_GDA0001277593270000323
Synthesis of Compound K-7 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound H-1 in step f was changed to H-3(Phe-Lys-PAB) and Compound D1 in step H was changed to Compound D2 (monomethyl auristatin E). The product K-7 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1718.84, LC-MS (ESI, M + H)+) Found 1719.86.
3.8 Synthesis of Compound K-8 (formula 33)
Figure DEST_PATH_GDA0001277593270000324
Synthesis of Compound K-8 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound H-1 in step f was changed to H-4(Ala-Ala-Asn-PAB) and Compound D1 in step H was changed to Compound D2 (monomethyl auristatin E). The product K-8 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1699.79, LC-MS (ESI, M + H)+) Found 1700.80.
3.9 Synthesis of Compound K-9 (formula 34)
Figure DEST_PATH_GDA0001277593270000325
Synthesis of Compound K-8 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound H-1 in step f was changed to H-5(D-Ala-Phe-Lys-PAB) and Compound D1 in step H was changed to Compound D2 (monomethyl auristatin E). The product K-9 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1789.88, LC-MS (ESI, M + H)+) Found 1790.90.
3.10 Synthesis of Compound K-10 (formula 35)
Figure DEST_PATH_GDA0001277593270000331
Synthesis of Compound K-10 the same procedure was followed as for Compound K-1 in example 3.1, except that the compound in step h wasObject D1 was changed to compound D3 (monomethyl auristatin F). The product K-10 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1713.81, LC-MS (ESI, M + H)+) Found 1714.83.
3.11 Synthesis of Compound K-11 (formula 36)
Figure DEST_PATH_GDA0001277593270000332
Synthesis of Compound K-11 was carried out in the same manner as for Compound K-1 in example 3.1, except that Compound D1 in step h was replaced by Compound D4 (monomethyldolastatin 10, MMAD). The product K-11 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1752.80, LC-MS (ESI, M + H)+) Found 1753.82.
3.12 Synthesis of Compound K-12 (formula 37)
Figure DEST_PATH_GDA0001277593270000333
Synthesis of Compound K-12 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound D1 in step h was changed to Compound D5(Tubulysin derivative 1). The product K-12 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1506.59, LC-MS (ESI, M + H)+) Found 1507.61.
3.13 Synthesis of Compound K-13 (formula 38)
Figure DEST_PATH_GDA0001277593270000334
Synthesis of Compound K-13 was performed in the same manner as the Synthesis of Compound K-1 in example 3.1, except that Compound D1 in step h was changed to Compound D6(Tubulysin derivative 2). The product K-13 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1695.71, LC-MS (ESI, M + H)+) Found 1696.73.
3.14 Synthesis of Compound K-14 (formula 39)
Figure DEST_PATH_GDA0001277593270000341
The synthesis of compound K-14 was identical to the synthesis procedure for compound K-1 in example 3.1, except that compound D1 in step h was changed to compound D7 (a Cryptophycin derivative). The product K-14 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1679.64, LC-MS (ESI, M + H)+) Found 1680.66.
3.15 Synthesis of Compound K-15 (formula 40)
Figure DEST_PATH_GDA0001277593270000342
Synthesis of Compound K-15 was carried out in the same manner as for Compound K-1 in example 3.1, except that Compound D1 in step h was replaced by Compound D8 (Taltobulin). The product K-15 is brown yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1455.65, LC-MS (ESI, M + H)+) Found 1456.67.
3.16 Synthesis of Compound K-16 (formula 41)
Figure DEST_PATH_GDA0001277593270000343
Synthesis of Compound K-16 was performed in the same manner as for Compound K-1 in example 3.1, except that Compound D1 in step h was changed to Compound D9(PBD dimer). The product K-16 is a brown yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1707.61, LC-MS (ESI, M + H)+) Found 1708.63.
3.17 Synthesis of Compound K-17 (formula 42)
Figure DEST_PATH_GDA0001277593270000344
Synthesis and example of Compound K-173.1 the procedure for the synthesis of Compound K-1 was the same except that Compound D1 in step h was changed to Compound D10(Duocarmycin derivative 1). The product K-17 is obtained through 3 steps of reaction and is a brown yellow amorphous solid. LC-MS (M)+) Theoretical value of 1618.55, LC-MS (ESI, M + H)+) Found 1619.57.
3.18 Synthesis of Compound K-18 (formula 43)
Figure DEST_PATH_GDA0001277593270000351
The synthesis of compound K-18 was performed in the same manner as for compound K-1 in example 3.1, except that compound D1 in step h was changed to compound D11(Duocarmycin derivative 2). The product K-18 is a brown yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1696.58, LC-MS (ESI, M + H)+) Found 1697.60.
3.19 Synthesis of Compound K-19 (formula 44)
Figure DEST_PATH_GDA0001277593270000352
Synthesis of Compound K-19 was performed in the same manner as the Synthesis procedure of Compound K-1 in example 3.1, except that Compound D1 in step h was changed to Compound D12(PNU-159682 derivative). The product K-19 is a brown yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1737.61, LC-MS (ESI, M + H)+) Found 1738.68.
3.20 Synthesis of Compound K-20 (formula 45)
Figure DEST_PATH_GDA0001277593270000353
The synthesis of compound K-20 was identical to the synthesis of compound K-1 in example 3.1 except that intermediate F-3 in step F was changed to intermediate F-14. The product K-20 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1639.64, LC-MS (ESI, M + H)+) Found 1640.61.
3.21 Synthesis of Compound K-21 (formula 46)
Figure DEST_PATH_GDA0001277593270000354
The synthesis of compound K-21 was identical to the synthesis of compound K-1 in example 3.1 except that intermediate F-3 in step F was changed to intermediate F-11. The product K-21 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1701.82, LC-MS (ESI, M + H)+) Found 1702.86.
3.22 Synthesis of Compound K-22 (formula 47)
Figure DEST_PATH_GDA0001277593270000361
The synthesis of compound K-22 was identical to the synthesis of compound K-1 in example 3.1, except that intermediate F-3 in step F was changed to intermediate F-16. The product K-22 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1925.92, LC-MS (ESI, M + H)+) Found 1926.88.
3.23 Synthesis of Compound K-23 (formula 48)
Figure DEST_PATH_GDA0001277593270000362
The synthesis of compound K-24 was performed in the same manner as that of compound K-1 in example 3.1, except that intermediate F-3 in step F was changed to intermediate F-16 and compound D1 in step h was changed to compound D11(Duocarmycin derivative 2). The product K-24 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1848.64, LC-MS (ESI, M + H)+) Found 1849.58.
3.24 Synthesis of Compound K-24 (formula 49)
Figure DEST_PATH_GDA0001277593270000363
The synthesis of compound K-25 was performed in the same manner as the synthesis of compound K-1 in example 3.1, except that intermediate F-3 in step F was changed to intermediate F-16 and compound D1 in step h was changed to compound D13(SN38 derivative). The product K-25 is obtained as yellow amorphous solid after 3 steps of reaction. LC-MS (M)+) Theoretical value of 1714.64, LC-MS (ESI, M + H)+) Found 1715.61.
Second, preparation method and detection of antibody conjugate
The first embodiment is as follows: preparation of ADC-1
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 7.4 reaction buffer diluted to 5mg/mL, added with 6.0-fold excess molar ratio of tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and the reaction solution was stirred at 35 ℃ for 10 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 G-2(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 ℃.
Example two: preparation of ADC-2
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 7.4 reaction buffer diluted to 5mg/mL, added with a 10-fold excess molar ratio of tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and the reaction solution was stirred at 10 ℃ for 4 hours.
The reaction solution was cooled to 5 ℃ and the coupling was carried out without purification by adding a suitable amount of Diethylacetamide (DMA) and then adding 6 times the excess molar ratio of compound K-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 ℃.
Example three: preparation of ADC-3
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 7.4 reaction buffer diluted to 5mg/mL, added with a 20-fold excess molar ratio of tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and the reaction solution was stirred at 15 ℃ for 2 hours.
The reaction solution is cooled to 10 ℃, an appropriate amount of Acetonitrile (ACN) is added without purification, then compound K-3(10mg/ml is dissolved in the ACN in advance) with 6 times of excess molar ratio is added, the volume of the ACN in the reaction system is ensured not to exceed 10%, and the coupling is carried out by stirring for 4 hours at 10 ℃.
Filtering and purifying the coupling reaction mixture by using histidine-acetic acid/sucrose gel with pH of 6.0 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.
Example four: preparation of ADC-4
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 7.4 reaction buffer diluted to 5mg/mL, added with tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in an 8-fold excess molar ratio, and the reaction solution was stirred at 25 ℃ for 48 hours.
The reaction solution was cooled to 0 deg.C, an appropriate amount of Dimethylformamide (DMF) was added without purification, and then compound K-4(10mg/ml pre-dissolved in DMF) was added in an excess molar ratio of 6 times, ensuring that the volume of DMF in the reaction system did not exceed 8%, and the coupling was carried out by stirring at 0 deg.C 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-100 ℃.
Comparing the sample obtained in the second example with pertuzumab by Hydrophobic Interaction Chromatography (HIC) (FIG. 1) and mass spectrometry (FIG. 2), it can be seen that the DAR value distribution of the conjugate obtained by using the linker of the present invention is very narrow, the average DAR value is close to 4, the single-distribution component of the obtained cross-linked product (DAR is 4) accounts for more than 80%, and therefore the uniformity of the product is high; and m is 3.0-4.2 by identification. The same results were obtained for DAR by analyzing the second, third and fourth examples in the same manner as above, and m was in the range of 1-5.0.
Biological detection of antibody conjugates
1. Molecular binding assay detection
The working principle of Biacore instrument for detecting the intermolecular affinity of proteins is based on the Surface Plasmon Resonance (SPR) technique. SPR is an optical physical phenomenon in which, when a beam of P-polarized light is incident on the end face of a prism within a certain angle range, a surface plasmon polariton is generated at the interface between the prism and a metal thin film (Au). When the propagation constant of the incident light wave matches the propagation constant of the surface plasmon wave, free electrons in the metal film are caused to resonate. During analysis, a layer of biomolecule recognition membrane is fixed on the surface of a sensing chip, then a sample to be detected flows on the surface of the chip, if molecules capable of interacting with the biomolecule recognition membrane on the surface of the chip exist in the sample, the refractive index of the surface of the gold membrane changes, and finally SPR angle changes are caused, and information such as the concentration, affinity, kinetic constant, specificity and the like of an analyte is obtained by detecting the SPR angle changes.
Binding assays were performed using Biacore to detect the binding activity of 3 batches of monoclonal samples of Pertuzumab, ADC-2, ADC-4 to Human ErbB 2.
TABLE 13 affinity and kinetic parameters of monoclonal antibody samples with Human ErbB2
Sample (I) ka(1/Ms) kd(1/s) KD(M)
Pertuzumab 1.902E+05 1.239E-03 6.517E-10
ADC-2 2.232E+05 1.294E-04 5.799E-10
ADC-4 1.956E+05 1.310E-04 6.969E-10
The surface plasmon resonance technology is used for characterizing the binding activity of 3 monoclonal antibody samples Pertuzumab, ADC-2 and ADC-4 and Human ErbB2, and the results show that all the samples have binding. Experimental results show that the 3 monoclonal antibody samples have similar affinity with Human ErbB2, and all the affinity is in the range of 0.5-0.7 nM.
2. Affinity determination experiment of cell level ADC-2 and Her2
The experimental materials used in the following experiments were derived from: RPMI1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 XPATAC, 100 XPS was purchased from Gibco, Fluorescein Isothiocyanate (FITC) labeled secondary antibody was purchased from Invitrogen, and NCI-N87 gastric cancer cells were from Kunming cell Bank, Chinese academy of sciences. Other reagents were analytically pure. FACSCalibur flow cytometer (BD).
In this example, the affinity of ADC-2, P-mcVC-MMAE, Pertuzumab for binding to Her2 high expressing cells was studied.
In this example, human gastric cancer NCI-N87 cells with high Her2 expression were selected. NCI-N87 cells are cultured in RPMI1640 medium containing 10% fetal calf serum at 37 ℃ in a 5% CO2 incubator, cells subjected to subculture for 4-5 days are counted, the cells are collected in a 15mL centrifuge tube, the cells are washed for 2 times by using cold PBS (phosphate buffer solution), the temperature condition is 4 ℃, the cells are centrifuged at 1000rpm for 5min, the cells are resuspended by using PBS containing 5% fetal calf serum, the cells are incubated at 37 ℃ for 30min, then the cells are centrifuged at 4 ℃ and 1000rpm for 5min, the supernatant is removed, the cells are resuspended by using cold PBS, the cell concentration is 1 multiplied by 106Cells/1.5 mL are subpackaged into an EP tube, centrifuged for 5min at 4 ℃ and 1000rpm, supernatant is removed, 0.5mL of ADC-2, P-mcVC-MMAE, Pertuzumab and human IgG with different concentrations are added, placed on ice for 40min, centrifuged for 5min at 4 ℃ and 1000rpm, washed for 2 times by 1mL of cold PBS, 200 mu L of secondary antibody labeled by fluorescence label (FITC) is added, placed on ice in a dark place for 40min, centrifuged for 5min at 4 ℃ and 1000rpm, supernatant is removed, washed for 2 times by 1mL of cold PBS, 0.5mL of cold PBS is added for cell resuspension, placed on ice in a dark place, and ADC-2, P-mcVC-MMAE and Pertuzumab with different concentrations are detected by a FACSCalibur flow cytometer to determine the average fluorescence intensity (mean fluorescence intensity, MFI) of the combination of the cells, and the fluorescence intensity of the combination of the human IgG is nonspecific combination.
As shown in FIG. 3, ADC-2, P-mcVC-MMAE and Pertuzumab can be combined with NCI-N87 cell surface antigen Her2, and with the increase of antibody concentration, the combination of ADC-2, P-mcVC-MMAE and Pertuzumab with Her2 receptor is increased, and the three have the same affinity with NCI-N87 cell Her2 without obvious difference.
3. In vitro cell proliferation assay for biological activity
The experimental materials used in the following experiments were derived from: DMEM medium, DMEM/F12K medium, RPMI1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100 × sodium pyruvate, 100 × streptomycin were purchased from Gibco. Sulforhodamine B (SRB) was purchased from Sigma. BT-474 human breast cancer cells, SK-RB-3 human breast cancer cells, MDA-MB-231 human breast cancer cells, NCI-N87 human gastric cancer cells are from Kunming cell banks of Chinese academy of sciences, Panc-1 human pancreatic cancer, MDA-MB-468 human breast cancer cells, MCF-7 human breast cancer cells are from Shanghai Life sciences research institute cell banks of Chinese academy of sciences, SKOV-3 human ovarian cancer cells, and Du-145 human prostate cancer cells are from American Type Culture Collection (ATCC). 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-2, ADC-4, P-mcVC-MMAE, Kadcyla, Pertuzumab on the proliferation of tumor cell lines was studied.
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, SK-RB-3, MDA-MB-231, MDA-MB-468, MCF-7 human breast cancer cells, NCI-N87 human gastric cancer cells, SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells, Panc-1 human pancreatic cancer cells.
BT-474, SK-BR-3 and NCI-N87 cells are cultured in RPMI1640 culture medium containing 10% fetal bovine serum, SKOV-3, Du-145, Panc-1, MCF-7, MDA-MB-231 cells in DMEM medium containing 10% fetal bovine serum, MDA-MB-468 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, culturing at 37 deg.C in 5% CO2 incubator to logarithmic phase, inoculating the cells in logarithmic phase into 96-well culture plate at density of 2 × 103-9 × 103 cells per well, culturing for 24 hr, adding drugs with different concentrations for 5 days, respectively diluting with 3, 4 or 5-fold to prepare 9 concentrations, setting multiple wells for each concentration, and setting corresponding concentration of solvent control and cell-free culture medium wells. After the drug action was complete, the medium was decanted and 100. mu.l of 4 ℃ pre-cooled trichloroacetic acid solution (30%, w/v) was added theretoAfter fixation at 4 ℃ for 1 hour followed by 5 washes with deionized water and drying at room temperature, 100. mu.L of 0.4% (w/v) SRB stain (Sigma, 1% in glacial acetic acid) was added to each well, and after incubation and staining at room temperature for 30min, the unbound dye was removed by washing 4 times with 1% glacial acetic acid and dried at room temperature. After adding 100. mu.L of 10mM Tris solution to each well, incubating and staining at room temperature for 15min, washing with 1% glacial acetic acid five times to remove unbound SRB, drying at room temperature, adding 10mM Tris buffer (pH 10.5) to each well to dissolve the dye bound to the cell protein, measuring the light absorption value (OD value) at the wavelength of 510nm and 690nm using a Synergy 2 microplate reader (Bio-Tek), and obtaining A.OD510-OD690。
Inhibition (%) ═ a control-a dosing)/a control × 100%.
In the experiment, ADC-2, ADC-4, P-mcVC-MMAE, Kadcyla and Pertuzumab are used for researching the in vitro cell culture proliferation of a plurality of Her2 high-expression tumor cell lines, and meanwhile, ADC-2 is used for researching the in vitro cell culture proliferation of a plurality of non-Her 2 high-expression tumor cell lines. As shown in FIGS. 4-6, the high-expression SK-BR-3 and BT-474 human breast cancer cells and NCI-N87 human gastric cancer cells treated by ADC-2, P-mcVC-MMAE and Kadcyla Her2 can obviously inhibit the proliferation of tumor cells, the tumor cell proliferation inhibition activity of ADC-2 and P-mcVC-MMAE is obviously higher than that of Kadcyla, and the tumor cell proliferation inhibition activity of ADC-2 and P-mcVC-MMAE is basically equivalent, wherein the tumor cell proliferation inhibition activity of ADC-2 to BT-474 is slightly higher than that of P-mcVC-MMAE; compared with Pertuzumab naked antibody, the antibody drug conjugate ADC-2, P-mcVC-MMAE and Kadcila have obviously improved tumor cell proliferation inhibition activity. As shown in FIG. 7, ADC-2 also showed good tumor cell proliferation inhibition on SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells and Panc-1 human pancreatic cancer cells with high expression of non-Her 2. For human breast cancer MCF-7, MDA-MB-231 and MDA-MB-468 cells which do not express Her2, the proliferation activity of ADC-2 anti-tumor cells is obviously reduced (figure 8), which shows that the antibody drug conjugate ADC-2 basically has no effect on cells which do not express target antigens, thus indicating that the toxic and side effects of the antibody drug conjugate ADC-2 are obviously reduced. As shown in FIG. 9, ADC-4 showed potent tumor cell proliferation inhibition on human gastric cancer cells of NCI-N87 with high Her2 expression. In addition, as shown in FIG. 10, ADC-2, ADC-3 and ADC-4 showed potent tumor cell proliferation inhibition on Calu-3 human lung cancer cells expressed in Her 2.
4. 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 are inoculated with human gastric cancer NCI-N87 cells subcutaneously until the tumor grows to 100-3Thereafter, the animals were randomly assigned (D0). The dosage and schedule of administration are shown in table 1. Tumor volumes were measured 2-3 times a week, mice weighed, and data recorded. Tumor volume (V) was calculated as:
V＝1/2×a×b2wherein 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; t is0、C0Tumor volume at the beginning of the experiment.
3) Results of the experiment
FIG. 11 shows that ADC2(0.5, 1, 2mg/kg, IV, 1 time per week, 2 times total) significantly inhibited the growth of subcutaneous transplantable tumors in human gastric carcinoma NCI-N87 nude mice dose-dependently at 59%, 94% and 200% tumor inhibition rates, with 3/6 tumor partial regression in the 1mg/kg group and 6/6 tumor complete regression in the 2mg/kg group, respectively; the tumor inhibition rates of ADC3(0.5, 1, 2mg/kg, IV, 1 time per week, 2 times total) on NCI-N87 were 65%, 69% and 185%, respectively, with 1/6 tumor partial regression and 5/6 tumor complete regression in the 2mg/kg group; P-vcCMAE (1mg/kg, IV, 1 weekly, 2 total) showed 94% inhibition of NCI-N87 with 2/6 partial tumor regressions; the tumor inhibition rate of the reference drug Kadcyla (2mg/kg, IV, 1 time per week, 2 times in total) against NCI-N87 was 77% respectively. 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 (23)

1. A substituted maleimide linker fragment of the formula Ia:
Figure FDA0003363949880000011
wherein R is X or ArS-,
x is selected from the group consisting of: halogen;
ar is selected from the group consisting of: phenyl radical, C1-C4Alkyl phenyl, C1-C4Alkoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl,
Figure FDA0003363949880000012
Wherein W is an amino group R attached to a carbonyl group1，R1Is selected from-NH2、
Figure FDA0003363949880000013
Figure FDA0003363949880000014
Ar' is selected from the group consisting of: unsubstituted phenylene;
L1is-O (CH) attached to an Ar' group2CH2O)n-, where n is selected from any one of integers from 1 to 20.
2. The substituted maleimide linker fragment of claim 1 wherein Ar is selected from the group consisting of: phenyl, 4-methylphenyl, 4-methoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl,
Figure FDA0003363949880000015
Wherein W is an amino group R attached to a carbonyl group1，R1Is selected from-NH2、
Figure FDA0003363949880000016
Figure FDA0003363949880000017
3. The substituted maleimide linker fragment of claim 1 or 2 wherein X is bromo or iodo.
4. The substituted maleimide linker fragment of claim 1, wherein said linker fragment has a structure selected from the group consisting of:
Figure FDA0003363949880000018
Figure FDA0003363949880000021
Figure FDA0003363949880000031
5. a substituted maleimide-based linker-drug conjugate comprising a substituted maleimide-based linker fragment of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, having the structure of formula Ib:
Figure FDA0003363949880000032
wherein, R, Ar' and L1As defined in any one of claims 1 to 4;
L2is a chemical bond, or an AA-PAB structure; wherein AA is dipeptide or tripeptide fragment, and PAB is p-aminobenzyloxycarbonyl;
CTD is bonded to L through an amide bond2And/or a drug for treating autoimmune diseases and anti-inflammation.
6. The substituted maleimide-based linker-drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein the AA is selected from the group consisting of: Val-Cit, Val-Ala, Phe-Lys.
7. The substituted maleimide-based linker-drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein the CTD is selected from the group consisting of: tubulin inhibitors, topoisomerase inhibitors, DNA binding agents.
8. The substituted maleimide-based linker-drug conjugate of claim 7, or a pharmaceutically acceptable salt thereof, wherein the tubulin inhibitor is selected from the group consisting of: maytansine, Monomethylauristatin E, Monomethylauristatin F, monomethylpalastatin 10, Tubulysin, Cryptophycin, Taltobulin.
9. The substituted maleimide-based linker-drug conjugate of claim 7, or a pharmaceutically acceptable salt thereof, wherein the DNA binding agent is selected from the group consisting of: PBD, duocarmycin.
10. The substituted maleimide-based linker-drug conjugate of claim 7, or a pharmaceutically acceptable salt thereof, wherein the topoisomerase inhibitor is selected from the group consisting of: adriamycin metabolite PNU-159682 and irinotecan metabolite SN 38.
11. The substituted maleimide-based linker-drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein the CTD has a molecular structure selected from the group consisting of D1-D13:
Figure FDA0003363949880000041
Figure FDA0003363949880000051
12. the substituted maleimide-based linker-drug conjugate of claim 5, or a pharmaceutically acceptable salt thereof, wherein the compound of formula Ib is selected from the group consisting of:
Figure FDA0003363949880000052
Figure FDA0003363949880000061
Figure FDA0003363949880000071
Figure FDA0003363949880000081
Figure FDA0003363949880000091
Figure FDA0003363949880000101
Figure FDA0003363949880000111
13. an antibody-drug conjugate formed by coupling an antibody with the substituted maleimide-based linker-drug conjugate of formula Ib of claim 5 or a pharmaceutically acceptable salt thereof.
14. An antibody-drug conjugate, wherein the conjugate has the structure of formula Ic and/or Id:
Figure FDA0003363949880000112
wherein, Ar' and L1、L2CTD is as defined in claim 5;
m＝1.0～5.0；
ab is selected from an antibody or antibody fragment.
15. The antibody-drug conjugate of claim 13, wherein the antibody is selected from the group consisting of: monoclonal antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies.
16. The antibody-drug conjugate of claim 14, wherein Ab is selected from the group consisting of: monoclonal antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies.
17. The antibody-drug conjugate of claim 13 or 14, 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, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR 2, EGFR, EGFRvIII, Tissue factor, C-MET, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A 2, PSMA, EphA2, AGS-5, GPC-3, C-KIT, RoR 2, PD-L2, CD27 2, 5T 2, Mucin 16, NaPi 22, STEAP, SLIke 2, BCBR, TrMA, ACAAP 2-2, ACALS 2, SLC-16, SLC 3639-SC 3639, SLC 3639-C-2, SLC 2, Clarke 2, SLC-16.
18. The antibody-drug conjugate of claim 13 or 14, wherein the antibody is selected from the group consisting of antibodies that bind to tumor associated antigens selected from the group consisting of: HER2 antibodies, EGFR-vIII antibodies, Tissue factor antibodies, c-Met antibodies, and DLL3 antibodies.
19. The antibody-drug conjugate of claim 18, wherein the HER2 antibody is selected from the group consisting of: trastuzumab and pertuzumab.
20. A pharmaceutical composition, comprising: (a) the antibody-drug conjugate of any one of claims 13-19; and (b) a pharmaceutically acceptable carrier or excipient.
21. The pharmaceutical composition of claim 20, wherein the excipient is a diluent.
22. Use of an antibody-drug conjugate according to any one of claims 13-19 for the manufacture of a medicament for the treatment of a tumour.
23. The method of preparing an antibody-drug conjugate of any one of claims 13-19, 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 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.
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