Topoisomerase ii-based antibody conjugate drugs and uses thereof

Abstract

The present application belongs to the technical field of medicine, and more particularly relates to topoisomerase II-based antibody conjugated drugs and application thereof. The present application provides a novel antibody conjugated drug based on a tumor topoisomerase II-based toxic warhead, which is coupled with a HER2 targeting antibody through a cleavable linker, so as to simultaneously have HER2 targeting recognition ability and Topo II catalytic inhibition effect. The ADC of the present application can reduce the side effect of DNA double-strand break induced by conventional Topo II inhibitors, while realizing efficient killing of HER2 high-expression tumor cells, and has a better therapeutic index.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceuticals, and more specifically, relates to antibody-drug conjugates based on topoisomerase II and their applications. Background Technology

[0002] Cancer remains a leading cause of death worldwide. Traditional cytotoxic chemotherapy drugs, lacking tumor selectivity, often cause dose-limiting toxicities such as myelosuppression and peripheral neuropathy, limiting their clinical benefits. Antibody-drug conjugates (ADCs), by using antibodies to precisely deliver highly active toxins to tumors, can improve the therapeutic index. Currently, several ADCs targeting HER2 (such as T-DM1 and DS-8201a) have been approved for indications such as breast cancer and gastric cancer.

[0003] However, the following common problems still exist in clinical practice: 1. Heterogeneity in HER2 expression and downstream signaling compensation lead to primary or acquired drug resistance, ultimately leaving patients with no available drugs.

[0004] 2. Most existing ADCs use "DNA breakage type" topoisomerase II (TOPO II) toxins (such as DXd and MMAE). Their toxic mechanism of inducing DNA double-strand breaks can easily activate tumor repair pathways and is associated with dose-related hematologic toxicity and drug resistance mutations.

[0005] 3. The single mechanism of action of the warhead makes it difficult to achieve multi-point intervention on key survival signal networks such as PI3K / AKT / mTOR, which limits the further improvement of efficacy.

[0006] Therefore, efficiently and stably conjugating Topo II catalytic inhibitors to anti-HER2 antibodies and precisely releasing them within tumor cells has become a key bottleneck in improving efficacy and safety. Currently, there are no reports, either domestically or internationally, of HER2-targeted ADCs using Topo II catalytic inhibitors as payloads, and systematic research on warhead-connector structure optimization and conjugation process parameters is also lacking. There is an urgent need to develop novel conjugation strategies to meet unmet clinical needs. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, the primary objective of this invention is to provide an antibody-drug conjugate.

[0008] The second objective of this invention is to provide a method for preparing antibody-drug conjugates.

[0009] A third object of the present invention is to provide a pharmaceutical composition.

[0010] A fourth object of the present invention is to provide the use of antibody-drug conjugates or pharmaceutical compositions in the preparation of medicaments for the treatment or prevention of tumors.

[0011] A fifth object of the present invention is to provide a topoisomerase IIA inhibitor or a pharmaceutically acceptable salt thereof.

[0012] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for an antibody-drug conjugate comprising: an antibody, a linker, and a drug molecule, wherein the drug molecule is coupled to the antibody via the linker; The antibody-drug conjugate has the structure shown in formula (II): Ab-(LD)n(II) In the formula, Ab is the antibody element, L is the linker, D is the drug molecule, and n is 2-8; The drug molecule has the structure shown in formula (I): (I) In the formula, R1 contains groups selected from the following group: , or ; The connector L is a divalent connector with the structure shown in equation (III): -L1-L2-L3-(III) In the formula, L1 is the first linker element connected to the antibody; L2 is the second linker element, which may be absent or connected to both L1 and L3; and L3 is the third linker element connected to the drug molecule. Wherein, the first connecting element L1 contains: a substituted or unsubstituted maleimide group; The second linker element L2 is selected from at least one of the following groups: PEG linker, C1-C10 alkylene group, phenyl linker, heteroaromatic ring linker, and adipic acid group; The third connecting element L3 is selected from: .

[0013] Preferably, the antibody is an antibody targeting tumor-associated antigens; the tumor-associated antigens are selected from: HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, glycoprotein NMB, guanylate cyclase C, CEA, AXL, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Ephrin-A, LAMP1, MUC1, PDL1, NY-ESO-1, WT1, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, At least one of EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFRvIII, VEGFR2, or MAGE-A3.

[0014] Preferably, the antibody is an antibody that targets HER2.

[0015] More preferably, the antibody targeting HER2 is selected from at least one of trastuzumab, pertuzumab, inetuzumab, trastuzumab emtansine, detrastuzumab, mastuzumab, and MGAH22. More preferably, the antibody targeting HER2 is selected from trastuzumab.

[0016] Preferably, in the structure of formula (II), the linker-drug molecule LD is selected from the structures shown in formulas (V) and (VI) below:

[0017] (V)(VI) Where n is 1-8. Preferably, n is 3-6.

[0018] Furthermore, this invention claims protection for a method for preparing an antibody-drug conjugate, comprising the following steps: (1) Provides a linker-drug molecule LD having the structures shown in formulas (V) and (VI); (2) The linker-drug intermediate LD is coupled with the antibody element to form the antibody-drug conjugate.

[0019] Preferably, in step (1), the linker-drug molecule LD is prepared by a method comprising the following steps: (1) A linker precursor is provided, the linker precursor comprising a maleimide group, a dipeptide sequence, a self-eliminating spacer group (PAB) and a reactive ester group; preferably, the reactive ester group is p-nitrophenyl carbonate or a derivative thereof; (2) The linker precursor is condensed with a drug molecule having the structure of formula (I) in the presence of a base to obtain the intermediate linker-drug molecule LD.

[0020] Furthermore, the present invention claims protection for a pharmaceutical composition comprising: (a) the antibody-drug conjugate or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier thereof.

[0021] Furthermore, the present invention seeks protection for the use of the antibody-drug conjugate or the pharmaceutical composition in the preparation of medicaments for treating or preventing tumors.

[0022] Furthermore, the present invention claims protection for a topoisomerase IIA inhibitor or a pharmaceutically acceptable salt thereof, comprising: (a) the antibody-drug conjugate or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier thereof.

[0023] Preferably, the tumor is selected from at least one of breast cancer, gastric cancer, ovarian cancer, lung cancer, and colorectal cancer.

[0024] Compared with the prior art, the present invention has the following beneficial effects: Through extensive and in-depth research, screening, and experimentation, the inventors have unexpectedly developed a novel antibody-drug conjugate (ADC) based on a topoisomerase II toxic warhead (i.e., compound (I)) and its application in tumor therapy. The ADC provided by this invention uses a topoisomerase II (Topo II) inhibitor as its toxic warhead and is conjugated to an antibody-targeting antibody via a cleavable linker, thus simultaneously possessing antibody-targeting recognition capability and Topo II catalytic inhibition activity. This invention's ADC can achieve highly efficient killing of HER2-overexpressing tumor cells while reducing the DNA double-strand breaks easily induced by traditional Topo II inhibitors, resulting in a better therapeutic index. Attached Figure Description

[0025] Figure 1 The coupling rate of EP-1-PAB-Val-Cit-Mc varies under different conditions.

[0026] Figure 2 The spectrum is the detection result of Trastuzumab-Mc-Val-Cit-PAB-EP-1.

[0027] Figure 3 This is a scratch test for antibody-drug conjugates.

[0028] Figure 4 DNA loosening experiment for Topo II.

[0029] Figure 5 The effect of antibody-drug conjugates on caspase-3 / 7 activity. Detailed Implementation

[0030] The present invention will be further described below with reference to the specification and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] the term Unless otherwise defined, all technical and scientific terms used in this embodiment have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] Unless otherwise specified, the term “amino acid” as used in this article is intended to include any common amino acid, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine, serine, methionine, tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, arginine, etc.

[0033] As used herein, the term "amino acid residue" refers to the group formed by removing an H atom from the N-terminal -NH2 group and removing an OH atom from the C-terminal -COOH group of an amino acid. Generally, the segment of an amino acid (residue) including the N-terminus and C-terminus is called the main chain, while the portion that determines the specific type of amino acid is called the side chain. Unless otherwise defined, amino acids herein include both native and non-native amino acids, including D-type and / or L-type amino acids; preferably, selected from L-type amino acids. Examples of amino acids include, but are not limited to, Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V). Preferably, in this document, the amino acid is selected from the group consisting of: L-glycine (L-Gly), L-alanine (L-Ala), β-alanine (β-Ala), L-glutamic acid (L-Glu), L-aspartic acid (L-Asp), L-histidine (L-His), L-arginine (L-Arg), L-lysine (L-Lys), L-valine (L-Val), L-serine (L-Ser), and L-threonine (L-Thr); furthermore, when the amino acid has two or more amino groups and / or two or more carboxyl groups, the term also includes groups formed by removing one H from -NH2 and -OH from -COOH on different carbon atoms, such as the divalent group -C(O)-(CH2)2-C(COOH)-NH- formed by removing one H from -NH2 and non-α-COOH of glutamic acid respectively.

[0034] In this invention, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0035] In this invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the chosen therapeutic agent and / or combination of therapeutic agents. Therefore, pre-specifying an accurate effective amount is useless. However, for a given condition, the effective amount can be determined using routine experiments, and a clinician can judge it accordingly.

[0036] Unless otherwise specified, all compounds mentioned in this invention are intended to include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds (i.e., racemates). In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0037] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the compounds of the present invention with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; 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, benzenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0038] When a trade name is used in this document, it is intended to include the product formulation, its generic counterpart, and the active pharmaceutical ingredient of the product.

[0039] Antibody As used herein, the term "antibody element" includes an antibody or the antigen-binding domain of said antibody. Preferred antibody elements include antibodies (such as intact antibodies, single-chain antibodies, antibody fragments, etc.), especially antibodies against tumor cell markers (such as tumor markers located on the surface of tumor cells).

[0040] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0041] As used herein, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheeted and linked by three CDRs forming a linking loop, and in some cases, partially folded structures. The CDRs in each chain are tightly packed together via the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0042] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively designated as α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0043] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0044] In this invention, the polypeptide element may include not only the complete antibody, but also fragments of an immunologically active antibody (such as Fab or (Fab')2 fragments; antibody heavy chains; or antibody light chains) or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0045] Preferably, the antibody is an antibody targeting HER2. More preferably, the antibody targeting HER2 is selected from at least one of: trastuzumab, pertuzumab, inetuzumab, trastuzumab emtansine, detrastuzumab, mastuzumab, and MGAH22. More preferably, the antibody targeting HER2 is selected from trastuzumab.

[0046] Preferably, the light chain variable region (VL) amino acid sequence of the trastuzumab contains the amino acid sequence shown in SEQ ID NO.: 1; and / or the heavy chain variable region (VH) amino acid sequence of the trastuzumab contains the amino acid sequence shown in SEQ ID NO.: 2.

[0047] SEQ ID NO.: Amino acid sequence shown in 1: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0048] SEQ ID NO.: Amino acid sequence shown in 2: DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0049] Antibody-drug conjugate (ADC) The present invention also provides immunoconjugates based on the antibodies of the present invention, preferably antibody-drug conjugates (ADCs).

[0050] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0051] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0052] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0053] Typically, antibody-drug conjugates (ADCs) contain a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0054] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0055] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0056] Preferably, the preferred drug of the present invention is the topoisomerase IIA inhibitor described in the present invention or a pharmaceutically acceptable salt thereof.

[0057] Specifically, the topoisomerase IIA inhibitor has a structure as shown in formula (I): (I) Wherein, R1 contains groups selected from the group consisting of: , or .

[0058] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0059] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0060] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0061] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0062] In some embodiments, the antibody-drug conjugate has the structure shown in formula (II): Ab-(LD)n(II) in, Ab is an antibody element; L is a linker; D is a drug molecule represented by formula (I); n ≥ 1; preferably, n is 2-8.

[0063] drug As used herein, "drug" refers to any compound having the desired biological activity and possessing reactive functional groups (such as amino (-NH2), hydroxyl (-OH), etc.) for the preparation of the conjugates described herein or for covalent connection with the linkers described herein. The desired biological activity includes the diagnosis, cure, relief, treatment, and prevention of diseases in humans or other animals. Therefore, the term "drug" refers to compounds identified in the Chinese Pharmacopoeia, as well as those confirmed by, for example, the United States Pharmacopeia of Allotherapy, the National Formulary of America, or any of its supplements, provided they possess the necessary reactive functional groups. Typical drugs are listed in the Physician's Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). It should be understood that as new drugs are discovered and developed, these drugs should also be included in the term "drug" in the conjugates described herein.

[0064] The drug that can be used to form the ADC of the present invention is a topoisomerase IIA inhibitor or a pharmaceutically acceptable salt thereof.

[0065] Preferably, the cytotoxic agent (or toxic warhead) of the present invention has a compound structure as shown in formula (I) above.

[0066] connector The linker of the present invention is preferably a breakable linker, more preferably an enzyme-unstable linker (such as the Val-Cit dipeptide linker).

[0067] In one embodiment of the present invention, the connector has the structure of formula (III): -L1-L2-L3-(III) In the formula, L1 is the first linker element linked to the antibody; L2 is a second linker element, either absent or linked to both L1 and L3; and L3 is the third linker element linked to the drug molecule. Preferably, L1 is a maleimide group for reacting with the thiol group of the antibody; and L3 is preferably a Val-Cit-PAB structure for cleavage by cathepsins in lysosomes to release the drug.

[0068] Pharmaceutical Composition The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the above-described ADC and a pharmaceutically acceptable carrier. Although the pH value may vary depending on the nature of the formulated substance and the condition to be treated, generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0069] The pharmaceutical compositions of the present invention can be directly used to bind to TAAs such as HER2 protein molecules, and therefore can be used for the prevention and treatment of diseases such as tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0070] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0071] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0072] For antibody-drug conjugates (ADCs), because the antibody-drug conjugates provided by this invention can target specific cell populations and bind to cell surface-specific proteins (antigens), thereby releasing the drug into the cells in its active form through conjugate endocytosis or drug infiltration, the antibody-drug conjugates of this invention can be used to treat target diseases. The aforementioned antibody-drug conjugates can be administered to subjects (e.g., humans) in therapeutically effective amounts via appropriate routes. Subjects requiring treatment may be patients at risk or suspected of having a condition related to the activity or expression level of a specific antigen. Such patients can be identified through routine physical examinations.

[0073] When treated with the antibody-drug conjugate of the present invention, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump.

[0074] Pharmaceutical Compositions and Administration Because the linker used in the antibody-drug conjugate (antibody-drug conjugate) provided by this invention can remain stable outside the tumor environment and be efficiently cleaved by cathepsin L after entering the tumor environment, thereby releasing specific drugs (such as cytotoxic drugs) within the tumor site or other target sites, the conjugate or antibody-drug conjugate of this invention can be used to treat tumors or inflammation (such as bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and leukemia, etc.).

[0075] The aforementioned drug-drug conjugates or antibody-drug conjugates can be administered to subjects (e.g., humans) in therapeutically effective amounts via appropriate routes. Subjects requiring treatment may be patients at risk or suspected of having a condition related to the activity or expression level of a specific antigen. Such patients can be identified through routine physical examinations.

[0076] Those skilled in the art can administer the pharmaceutical composition to a subject using methods conventional in the art, depending on the type or site of the disease to be treated. This pharmaceutical composition can also be administered via other conventional routes, such as parenteral administration. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Furthermore, it can be administered via injectable repositories, such as repositories with 1-month, 3-month, or 6-month durations of injectable or biodegradable materials and methods.

[0077] Injectable pharmaceutical compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered via infusion, thereby delivering a pharmaceutical formulation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% glucose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, such as sterile preparations in the form of a suitable soluble salt of the antibody, may dissolve and administer pharmaceutical excipients such as water-based injections, 0.9% saline, or 5% glucose solutions.

[0078] When treated with the conjugate or antibody-drug conjugate of the present invention, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump. Other methods include various transport and carrier systems using conjugates and biodegradable polymers.

[0079] The pharmaceutical compositions of the present invention contain a safe and effective amount of the conjugate or antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated as solutions, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. The pharmaceutical compositions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount.

[0080] The effective amount of the conjugate drug or antibody-drug conjugate described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the antibody-drug conjugate, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the antibody-drug conjugate of this invention is administered daily at a dose of approximately 0.0001 mg to 50 mg / kg animal body weight (preferably 0.001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0081] Dosage forms of the compounds of this invention for local administration include injections, lyophilized formulations, etc. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0082] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0083] When using the pharmaceutical composition, a safe and effective amount of the antibody-drug conjugate of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 5–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0084] application The present invention also provides uses of the ADC of the present invention, such as for the preparation of diagnostic agents or for the preparation of medicaments for the prevention and / or treatment of tumors (preferably HER2-related diseases).

[0085] The main advantages of this invention include: (1) The functional groups (such as amino, carboxyl, alkynyl, thiol, etc.) provided by the present invention can be used for coupling, which facilitates the construction of drug release structures with Val-Cit cleavable connectors; (2) The drug-antibody conjugate provided by the present invention has a stable molecular structure and maintains chemical integrity under reduction, coupling and storage conditions, avoiding breakage or degradation. (3) The drug-antibody conjugate provided by the present invention maintains high topoisomerase IIA inhibitory activity while having effective killing power and the ability to be taken up by cells. (4) The drug-antibody conjugate provided by the present invention has appropriate physicochemical properties, which facilitates purification, preparation and industrial scale-up.

[0086] (5) The antibody conjugate of the present invention uses a novel topoisomerase IIA inhibitor as a warhead, which has a novel mechanism of action and overcomes the limitations of existing warhead types; (6) The antibody conjugate of the present invention can maintain high cytotoxic activity even under low DAR value conditions, which helps to improve the stability of the formulation and its safety in vivo. (7) The antibody conjugate of the present invention uses a specific Val-Cit linker to achieve highly stable and highly selective release of toxic warheads; (8) The antibody conjugate of the present invention, coupled with an antibody targeting HER2 and a Topo IIA inhibitor, provides a synergistic anti-tumor effect.

[0087] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0088] Experimental methods: 1. Synthesis of arylvinylquinoline compounds 1.1 Synthetic Route (1) Synthetic route of series I The synthesis of series I compounds requires the introduction of a nitro group to obtain intermediate EI-1 (9-ethyl-6-nitrocarbazole-3-aldehyde), which is then condensed with piperidine, intermediates BI-3, and BI-4 under high temperature conditions to obtain intermediate EI-2. Stannous chloride is then used as a reducing agent to reduce the nitro group to an amino group, allowing the introduction of an amine reaction site. After the introduction of the aromatic amine, it further reacts with Boc-protected glycine, as shown below. Three compounds, EP-1, EP-2, and EP-2-Boc, are obtained.

[0089]

[0090] 1.2 General Method for Synthesizing Arylenylquinoline-based Projectiles Adding intermediates BI-3 and BI-4 to the starting aldehyde yields arylvinylquinoline compounds; specifically, the general synthetic method is shown below:

[0091] 2. General Synthesis Method of Arylenylquinoline-based Projectiles and Linkers The linker should contain a group capable of covalently binding to the antibody. Currently, maleimide-based coupling exhibits the inherent advantage of thiol specificity and is widely used in ADC design. Most clinically applied ADCs currently use a combination of cathepsin B-cleavable Val-Cit dipeptides and N-alkylmaleimides as linkers. When the ADC is transported to the lysosome, the linker is cleaved, releasing the free drug. This article selects N-alkylmaleimides and peptide linkers commonly used in clinically developed ADCs. Both linkers utilize maleimide double bonds as cysteine ​​reaction sites for antibody coupling, enabling Michael addition reactions with the free thiol groups generated during antibody reduction, thereby constructing ADCs.

[0092] Based on the analysis of the overall synthetic route of antibody conjugation, as shown below, a group capable of reacting with cysteine ​​residues needs to be introduced to carry out the conjugation reaction with the antibody. Therefore, we first need to bind the warhead to a linker containing maleimide.

[0093]

[0094] The general synthetic route for the linker involves the direct amide condensation reaction of the adaptor compound's reactive handle with the active ester of maleimide. There are two routes to obtain the drug-linker, as shown below, differing in whether a thiol reactive group is attached first to obtain the complete linker or the linker-linker product is obtained first. Route one involves reacting the adaptor-modified compound with the linker, which requires accumulating a certain amount of compound and is time-consuming. Route two involves obtaining the linker first and then reacting it with the linker in the final step; however, the linker increases consumption and is expensive. Considering economic factors, this paper chooses route one to synthesize the drug-linker, directly reacting the adaptor-modified linker with the carboxyl group or active ester contained in the peptide linker, utilizing its reactive handle.

[0095]

[0096] Example 1: Synthesis of intermediates of arylvinylquinoline compounds 1.1 Synthesis of intermediate BI-1

[0097] Aniline (20 mmol, 1 eq) and ethyl acetoacetate (20 mmol, 1 eq) were added to a 250 mL round-bottom flask, followed by polyphosphoric acid (PPA, 100 mmol, 5 eq). The flask was sealed with a balloon, and the mixture was heated to 90 °C. The reaction flask was shaken to ensure thorough mixing of the reactants. After the reaction was complete, the mixture was cooled, and the pH was adjusted to neutral with saturated sodium hydroxide. The mixture was then filtered to obtain a white solid, intermediate BI-1, in 73.3% yield. 1 H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 8.1 Hz, 1H), 7.60 (t,J = 7.7 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 5.90 (s,1H), 2.33 (s,3H).

[0098] 1.2 Synthesis of intermediate BI-2

[0099] Intermediate BI-1 (20 mmol, 1 eq) was placed in a round-bottom flask, and phosphorus oxychloride (130 mmol, 6.5 eq) was slowly added. The mixture was heated and stirred at 120 °C. After the reaction was completed, the mixture was cooled, and ice water was slowly added to the reaction system. Finally, the pH was slowly adjusted to 5-6 with saturated sodium hydroxide solution, and a white solid precipitated. The solid was filtered, dried at room temperature, and purified by column chromatography to obtain a white solid, intermediate BI-2, with a yield of 74.3%. 1 H NMR (400 MHz, CDCl3) δ 8.18 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.74 (t, J = 8.5, 7.0, 1.6 Hz, 1H), 7.62-7.54 (t,1H), 7.40 (s, 1H), 2.73 (s, 3H).

[0100] 1.3 Synthesis of intermediates BI-3 and BI-4

[0101] Intermediate BI-2 (2 mmol, 1 eq), acetonitrile (6 mmol, 3 eq), and iodomethane (6 mmol, 3 eq) were added to a pressure-resistant tube. After the reaction was completed, the tube was cooled in a fume hood until the excess iodomethane had completely evaporated. Then, diethyl ether was added to wash the solid. After filtration and drying at room temperature, the solid could be directly added to the next step, yielding an off-white solid. Yield: 37.1%.

[0102] 1.4 Synthesis of intermediate DI-1

[0103] 3-Methyl-9H-carbazole (1 mmol, 1 eq) and 2,3-dichloro-5,6-dicyanobenzoquinone (3 mmol, 3 eq) were added to a flask, followed by methanol (10 mL) and water (1 mL). The mixture was stirred at room temperature, and the reaction solution gradually changed from dark blue to dark red. After the reaction was complete, methanol was removed by rotary evaporation, and the solution was extracted with ethyl acetate and saturated sodium bicarbonate solution. The extract was purified by column chromatography to give a white solid, intermediate DI-1, in 65% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.86 (s, 1H), 10.04 (s, 1H), 8.74 (d, J = 1.6 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.94 (dd, J = 8.5, 1.7Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.50 – 7.44 (m,1H), 7.27 (t, J = 7.5 Hz, 1H).

[0104] 1.5 Synthesis of intermediate EI-2

[0105] N-ethylcarbazole-3-carbaldehyde (intermediate DI-1, 4.47 mmol, 1 eq) was added to a round-bottom flask, dissolved in 10 mL of acetic acid at room temperature, followed by nitric acid (0.01 mmol, 0.0022 eq). The mixture was first heated to 35 °C and reacted for 40 min, then cooled to 25 °C. After the reaction was complete, the mixture was filtered, the filter cake was washed with acetic acid, and excess acetic acid was washed with water. The dark green solid, air-dried at room temperature, was purified by column chromatography to give a yellow solid, intermediate EI-1, with a yield of 65%. 1 H NMR (400 MHz, Chloroform-d) δ 10.14 (d, J = 1.9 Hz, 1H), 9.05 (s, 1H), 8.65 (s, 1H), 8.47-8.41 (m, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.50 (d, J= 9.0 Hz, 1H), 4.47 (q, J = 7.4 Hz, 2H), 1.55-1.49 (m, 3H).

[0106] 1.6 Synthesis of intermediate EI-2

[0107] Intermediates BI-3 and BI-4 (4 mmol, 1 eq) were first added to a pressure-resistant tube, and an appropriate amount of n-butanol was added as a solvent. Piperidine (6 mmol, 1.5 eq) was added, and the mixture was stirred at room temperature for 5 min. Then, intermediate EI-1 (4 mmol, 1 eq) was added, and the temperature was raised to 120 °C. The reaction solution gradually changed from purple to yellow, and a solid precipitated. After 12 h, the reaction was completed. After cooling, water was added, and the mixture was filtered. Column chromatography yielded a yellow solid, intermediate EI-2, with a yield of 68%. 1 H NMR (400 MHz, DMSO-d6) δ9.21 (s, 1H), 9.03 (s, 1H), 8.41 (d, J = 9.1 Hz, 1H), 8.29 (d, J = 8.8 Hz,1H), 8.12 (d, J = 8.6 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 8.04 (q, J = 9.5,7.8 Hz, 2H), 7.89 (d, J = 8.9 Hz, 2H), 7.80 (d, J = 15.8 Hz, 1H), 7.74 (t, J= 8.0 Hz, 1H), 7.52 (s, 1H), 4.59 (d, J = 7.3 Hz, 2H), 4.28 (s, 3H), 3.78 (s,4H), 1.82 (d, J = 26.0 Hz, 6H), 1.39 (t, J = 7.1 Hz, 3H).

[0108] Example 2: Synthesis of a series of compounds 2.1 Synthesis of compound EP-1

[0109] Intermediate EI-2 (1.40 mmol) and SnCl2 (14 mmol) were added to a pressure-resistant tube, followed by 4 mL of concentrated hydrochloric acid. The reaction solution turned dark purple. The temperature was raised to 95 °C, and the reaction was stopped after 30 min. The mixture was then cooled to room temperature, and the pH was adjusted to approximately 8 with saturated sodium hydroxide solution. The solution was extracted with sodium iodide solution, and column chromatography yielded a dark red solid. Purity: 95.7%, Yield: 73.1%. 1H NMR (500MHz, DMSO-d6) δ 8.49 (s, 1H), 8.25 (d, J = 8.9 Hz, 1H), 8.11 (d, J = 16.0 Hz, 1H), 8.08 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 8.7 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 15.7 Hz, 1H), 7.59 (d, J = 8.6Hz, 1H), 7.49 (s, 1H), 7.36 (s, 1H), 7.34 (d, J = 2.6 Hz, 2H), 6.88 (dd, J =8.5, 2.3 Hz, 1H), 5.00 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 4.25 (s, 3H), 3.73(m, J = 5.2 Hz, 4H), 1.80 (d, J = 38.7 Hz, 6H), 1.30 (t, J = 7.0 Hz, 3H). 13CNMR (126 MHz, DMSO-d6) δ 159.46, 154.34, 144.93, 141.10, 140.83, 133.49,133.38, 126.45, 125.92, 125.88, 125.23, 122.34, 121.95, 119.75, 119.08,115.88, 115.48, 110.08, 109.34, 104.62, 104.33, 52.80, 37.98, 37.16, 25.45,23.57, 13.87.ESI-HRMS [M]+ m / z = 461.2712, calcd for C 31 H 33 N4 + , requires 461.2700.

[0110] 2.2 Synthesis of compound EP-2-Boc

[0111] Compound EP-1 (1 mmol), N-tert-butoxycarbonylglycine (1.2 mmol), HATU (1.2 mmol), and DIPEA (3 mmol) were added to a reaction flask, and a yellow solid was obtained. Purity: 96%, Yield: 54.8%.1 1H NMR (500 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.65 (s, 1H), 8.63 (s, 1H), 8.26 (d, J = 8.9 Hz, 1H), 8.10 (s, 1H), 8.08 (d, J = 6.1 Hz, 1H), 8.00 (t, J = 8.3 Hz, 2H), 7.74 (d, J = 6.5 Hz, 1H), 7.72 (t, J = 4.4 Hz, 2H), 7.62 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 7.2 Hz, 2H), 7.09 (t, J = 6.1 Hz, 1H), 4.46 (q, J = 7.2 Hz, 2H), (s, 3H), 3.79 (d, J = 6.1 Hz, 2H), 3.74 (t, J = 5.4 Hz, 4H), 1.82 (d, J = 34.8 Hz, 6H), 1.42 (s, 9H), 1.34 (t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 167.92, 159.53, 156. .00, 154.27, 144.52, 141.34, 140.86, 136.62, 133.55, 131.83, 129.65, 126.72, 126.50, 126.11, 125.90, (s, 3H), 122.60, 122.04, 1 .59, 119.72, 119.34, 119.07, 116.33, 111.32, 109.76, 104.37, 78.06, 52.79, 43.80, 37.98, 37.32, 28.25, 25.46, 23.57, 13.86. ESI-HRMS [M]+ m / z = 618.3412, calcd for C 38 H 43 N5O3 + , requires 618.3439.

[0112] 2.3 Synthesis of Compound EP-2

[0113] It should be noted that there seems to be an incomplete or incorrect part in the original text (the "(s, 3H)" marked in the translation of ID=1). Please check and correct the original text for a more accurate translation.EP-2-Boc (0.65 mmol), dichloromethane (4 mL), and trifluoroacetic acid (0.8 mL) were added to the reaction system, and a yellow solid was obtained. Purity: 92%, Yield: 62.3%. 1 H NMR (500 MHz, DMSO-d6) δ 10.00 (s, 1H), 8.65 (s, 1H), 8.63 (s, 1H), 8.26 (d, J = 8.9 Hz, 1H), 8.10 (s, 1H), 8.08 (d,J = 6.1 Hz, 1H), 8.00 (t, J = 8.3 Hz, 2H), 7.74 (d, J = 6.5 Hz, 1H), 7.72 (t,J = 4.4 Hz, 2H), 7.62 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 7.2 Hz, 2H), 7.09 (t,J = 6.1 Hz, 1H), 4.46 (q, J = 7.2 Hz, 2H), 4.25 (s, 3H), 3.79 (d, J = 6.1 Hz, 2H), 3.74 (t, J = 5.4 Hz, 4H), 1.82 (d, J = 34.8 Hz, 6H), 1.42 (s, 9H), 1.34(t, J = 7.0 Hz, 3H). 13C NMR (126 MHz, DMSO-d6) δ 165.30, 159.56, 154.28,141.38, 140.90, 133.61, 126.56, 126.27, 125.95, 122.48, 122.10, 121.89,119.74, 119.30, 119.14, 116.52, 111.29, 109.99, 109.91, 104.39, 52.82, 41.53,38.03, 37.37, 25.48, 23.58, 13.88. ESI-HRMS [M]+ m / z = 518.2917, calcd forC 33 H 36 N5O + , requires 518.2914.

[0114] Example 3: Synthesis of the warhead-connector EP-1-PAB-Val-Cit-Mc

[0115] EP-1-PAB-Val-Cit-Mc (E)-2-(2-(6-((((4-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido))-3-methylbutanamido)-5-ureidopen tanamido)benzyl)oxy)carbonyl)amino)-9-ethyl-9H-carbazol-3-yl)vinyl)-1-methyl-4-(piperidin-1-yl)quinolin-1-ium iodide.

[0116] (E)-2-(2-(6-((((4-(2-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-3-methylbutyryl)-5-ureidovaleryl)phenyl)oxy)carbonyl)amino)-9-ethyl-9H-azaanthracene-3-yl)ethylene)-1-methyl-4-(piperidin-1-yl)quinoline-1-ammonium iodide.

[0117] Weigh out 0.1 mmol of the starting material Mc-Val-Cit-PABC-PNP (CAS No.: 159857-81-5) and place it in a pressure-resistant tube, then dissolve it in 800 μL of dry DMF. Weigh out 1.1 mmol of the starting material compound EP-1 poison warhead, dissolve it in 800 μL of dry DMF, and then slowly add it dropwise to the reaction system under ice bath conditions. Finally, add 0.6 mmol of pyridine, and after the addition is complete, move the mixture to room temperature and continue the reaction. After the reaction was monitored by TLC until complete, the mixture was cooled to room temperature, and an appropriate amount of diethyl ether was added until an orange solid precipitated. The mixture was centrifuged at 3500 rpm for 15 min, washed three times with diethyl ether, and the supernatant was discarded. The precipitate was dried under vacuum and dissolved completely in methanol (2 mL) and dichloromethane (1 mL) at 100 °C. The precipitate was then allowed to cool naturally at room temperature to precipitate a solid. The solid was filtered, and the filter cake was collected and purified by column chromatography to obtain the target product, warhead-linker EP-1-PAB-Val-Cit-Mc. Purity: 93%, Yield: 21%. 1H NMR (500 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.80 (s, 1H), 8.63 (s, 1H),8.28 (d, J = 8.9 Hz, 1H), 8.12 (d, J = 5.6 Hz, 1H), 8.09 (d, J = 7.7 Hz, 2H),8.03 (d, J = 3.8 Hz, 1H), 8.02 – 7.99 (m, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.75(d, J = 5.9 Hz, 1H), 7.72 (dd, J = 8.4, 3.2 Hz, 2H), 7.65 (d, J = 8.3 Hz,2H), 7.60 (d, J = 8.7 Hz, 1H), 7.51 (s, 1H), 7.46 (dd, J = 8.8, 2.1 Hz, 1H),7.40 (d, J = 8.2 Hz, 2H), 7.00 (d, J = 3.5 Hz, 1H), 6.99 (s, 2H), 5.99 (t, J= 5.7 Hz, 1H), 5.42 (s, 2H), 5.13 (s, 2H), 4.46 (q, J = 6.9 Hz, 2H), 4.38 (q,J = 7.8 Hz, 1H), 4.26 (s, 3H), 4.18 (t, J = 7.8 Hz, 1H), 3.75 (t, J = 5.4 Hz,4H), 3.38 – 3.36 (m, 2H), 3.01 (m, J = 6.7 Hz, 1H), 2.94 (m, J = 6.3 Hz, 1H),2.14 (m J = 27.8, 14.1, 6.9 Hz, 2H), 1.99 – 1.93 (m, 1H), 1.89 – 1.75 (m,6H), 1.71 – 1.57 (m, 2H), 1.47 (m, J = 16.9, 9.2, 8.0 Hz, 6H), 1.33 (t, J =7.2 Hz, 3H), 1.20 – 1.15 (m, 2H), 0.83 (dd, J = 16.2, 6.7 Hz, 6H). 13C NMR(126 MHz, DMSO-d6) δ 172.31, 171.31, 171.08, 170.66, 159.54, 158.90, 154.32,153.83, 144.63, 141.34, 140.88, 138.82, 136.34, 134.45, 133.57, 131.97, 131.53, 128.98, 127.51, 126.66, 126.64, 126.53, 126.07, 125.93, 1225.55, 121.76, 119.75, 119.12, 118.99, 116.26, 109.86, 109.75, 104.38, 65.45, 57.61, 53.11, 52.81, 38.03, 37.32, 37.01, 34.94, 30.36, 29.28, 27.77, 26.84, 25.79,25.47, 24.92, 23.58, 19.26, 18.22, 13.86. ESI-HRMS m / z: calcd for C60 H70N10O8+, [M]+ 1059.5403, found 1059.5451. .

[0118] Example 4: Antibody Coupling Reaction (1) At 25°C, add the prepared EDTA solution (20 mM) and the prepared TCEP solution (5 mM) to the Histidine buffer solution (pH=6.0, 20 mM Histidine-2 mM EDTA buffer solution, 3 mg / mL) containing 25 mg / mL HER2 trastuzumab (Shanghai Institute of Biological Products), and let it stand in a water bath at 25°C for 2 h.

[0119] (2) After the reduction reaction is completed, the warhead-linker EP-1-PAB-Val-Cit-Mc dissolved in DMSO is added and coupled at 25°C for 2 h.

[0120] (3) After the coupling reaction is completed, the reaction solution is ultrafiltered and replaced with 10 mM Histidine (pH= 5.5) solution using a 30 kD ultrafiltration tube to remove residual toxin molecules. The purified sample is then stored at -80℃ in the dark.

[0121] (4) Perform quality analysis on the sample.

[0122] Example 5: Evaluation of in vitro cytotoxic activity of adaptability-modified compounds This embodiment selected the CCK-8 assay based on cellular metabolic activity to determine the cytotoxic activity of the compounds. Six cell lines were selected for cytotoxicity testing: HER2-overexpressing SKBR-3 and BT474 breast cancer cells, SKOV-3 ovarian cancer cells, HER2-low expressing MDA-MB-453 and MCF-7 breast cancer cells, and BT549 triple-negative breast cancer cells. The test compounds are shown in Table 1, with QEC29 as a positive control. After overnight cell adhesion, serially diluted compounds were added, and cells were cultured for the corresponding time. Chemiluminescence values ​​or OD values ​​were measured at 450 nm. Table 1 shows the in vitro cytotoxic activity evaluation results of arylvinylquinoline compounds in multiple cancer cell lines.

[0123] Table 1

[0124] The results in Table 1 show that arylvinylquinoline compounds exhibit significant growth-inhibiting activity against multiple cancer cell lines. Among them, the parent compound EP-1 showed the highest IC50 values ​​against four cancer cell lines. 50 All concentrations are below 1 μM, with the optimal concentration reaching 0.28 μM. Therefore, this invention considers the deBoc product and EP-1 as candidate toxic warheads. Furthermore, the warhead-connector is inactive, suggesting that the warhead relies on intracellular release to function. This also indicates that even if the warhead-connector is released in the bloodstream, it possesses a certain degree of plasma safety, reducing systemic toxicity.

[0125] Example 6: Evaluation of cellular uptake of adaptive modified compounds Cellular uptake experimental methods (1) Take logarithmically proliferating cells, digest them with trypsin, and then use 1×10⁻⁶ cells. 5 Cells were seeded at a density of 6 cells / well in 6-well culture plates and cultured overnight until the cells adhered. The old culture medium was then discarded, and a certain concentration of the test compound was added. The DMSO group was used as a control. The cells were incubated at 37°C for 4.5 h.

[0126] (2) Cell collection: Remove the culture medium, wash twice with PBS, collect cells using a cell scraper, transfer to a 1.5 mL EP tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add an appropriate amount of cell lysis buffer, and incubate at room temperature for 0.5 h to lyse the cells. Use the cell lysis buffer as a solvent to dilute different drugs at a certain concentration gradient for the detection of the standard curve.

[0127] The measurements were taken using a UV spectrophotometer. Table 2 shows the evaluation results of the uptake of the adaptogenic compounds in SKOV-3 cells.

[0128] Table 2

[0129] The results in Table 2 show that after incubation of the compound with SKOV-3 cells for 4.5 h, the uptake rate of EP-1 was close to that of the positive control drug QEC29, approximately 11%, and its IC50 was similar. 50 They are basically the same. Overall, the cellular uptake rate of the warhead itself is not high.

[0130] Example 7: Optimization of ADC Coupling Process Analysis of the conjugation process of EP-1-PAB-Val-Cit-Mc: The antibody dosage in the table is 25 mg / mL. By adjusting the concentration of DMSO, the equivalent of TCEP, and the equivalent of Linker-Payload in Example 4, the effect of the conjugation process on the conjugation rate of the warhead-linker EP-1-PAB-Val-Cit-Mc was clarified. Here, DMSO (%) refers to the concentration of DMSO in the reaction solution.

[0131] Table 3

[0132] like Figure 1 As shown in Table 3, the DAR value first increases and then decreases with increasing TCEP equivalent. When the reducing agent equivalent is 1 TCEP, the coupling DAR value is only close to 0.5. As the reducing agent equivalent increases, the coupling rate increases, reaching a peak at 4 TCEP equivalent with a DAR value of around 4. Further increasing the reducing agent equivalent to 5 and 6 times results in a gradual decrease in the DAR value to around 2. This indicates that the guanidinium group of the citrulline side chain in Val-Cit has high polarity, significantly improving the overall hydrophilicity of the molecule. Even without the addition of a hydrophilic polyethylene glycol side chain, it can mitigate the intermolecular repulsion effect caused by the charged warhead to some extent. The decrease in DAR value due to excessively high TCEP equivalent may be because the antibody is over-reduced, causing conformational changes that affect coupling.

[0133] Through process exploration in this experimental section, the conditions for preparing ADCs were finally determined to be 3 times the equivalent of TCEP, 10 times the equivalent of the warhead connector EP-1-PAB-Val-Cit-Mc, and 10% of DMSO in the reaction solution.

[0134] Example 8: Quality Analysis of ADC In this embodiment, the quality analysis of the prepared ADC includes the detection of DAR value, aggregation degree and endotoxin. The SEC method is a commonly used technique for analyzing the aggregation degree of the conjugate.

[0135] (1) Mass spectrometry for detecting DAR value Experimental Method: Mass spectrometry can provide a relatively intuitive way to obtain the molecular weight of different DAR values ​​of ADCs. In this embodiment, a Q-TOF mass spectrometer (quadrupole-time-of-flight mass spectrometer) based on an ESI source (electrospray ionization) was used to analyze the DAR value of the conjugate. The principle of this method is that the sample solution is ejected through a glass capillary under high pressure, forming charged droplets. After the solvent evaporates, the droplets shrink, and the antibody conjugate forms multiply charged ions due to charge enrichment. These multiply charged ions enter the detection range of the mass spectrometer, are transmitted to the analyzer through the quadrupole, and the mass-to-charge ratio (m / z) is determined by calculating the time of flight. Finally, after deconvolution processing, the true molecular weight is obtained.

[0136] Experimental results: The ADC in this embodiment is Trastuzumab-Mc-Val-Cit-PAB-EP-1. Its structure, number, and corresponding DAR value are shown in Table 4. This indicates that the warhead-connector Mc-Val-Cit-PAB-EP-1 can be coupled with trastuzumab to form a stable structure.

[0137] Table 4

[0138] (2) SEC method for detecting clustering Experimental method: SEC method, or size exclusion chromatography, utilizes the characteristic that large molecules cannot enter porous gel particles, while small molecules can. Large molecules can be preferentially eluted due to their short retention time. It is a method for detecting the purity of antibody-drug conjugates. In this embodiment, the aggregation degree of the obtained ADC was detected.

[0139] Figure 2 The spectrum of the detection results for Trastuzumab-Mc-Val-Cit-PAB-EP-1 is shown. The test results are as follows... Figure 2 As shown, its SEC purity is 97.26%.

[0140] (3) Endotoxin detection In this embodiment, the gel electrophoresis method was used to detect the endotoxin content of the obtained ADCs. The sample to be tested was mixed with Limulus amebocyte lysate (LAL) reagent and incubated at 37°C for a certain period of time. If endotoxin is present in the sample, it will activate the coagulation proenzyme in the LAL reagent, causing the mixture to form a firm gel (the gel does not slide when the test tube is inverted 180 degrees). Table 5 shows the endotoxin content detection results for different ADCs.

[0141] Table 5

[0142] The results in Table 5 show that the endotoxin content of each ADC did not exceed the standard.

[0143] Example 9: Determination of the binding affinity between antibody conjugates and antigens 1) Experimental method: ELISA, the specific operation is as follows: (1) Protein coating: Add 100 μL of His-labeled HER2-ECD protein diluted with Coating Buffer to each well in a coating plate, cover with plastic wrap, and incubate overnight at 4°C.

[0144] (2) Washing: Wash 5 times with 1×Washing Buffer, 300 μL per well, and leave for 1 min each time.

[0145] (3) Blocking: Add 200 μL of blocking buffer (10% skim milk powder) to each well and incubate at 37°C for 2 h to block the remaining protein binding sites in the well.

[0146] (4) Discard the blocking solution and add the sample to be tested: Add antibody samples of different concentration gradients diluted with PBS, 100 μL per well, and incubate at 37°C for 1.5 h.

[0147] (5) Washing the plate: Repeat the above steps.

[0148] (6) Add secondary antibody: Add 100 μL of HRP-conjugated anti-human IgG secondary antibody diluted with 5% skim milk powder and incubate at 37°C for 1 h.

[0149] (7) Washing the plate: Repeat the above steps.

[0150] (8) Color development: Add 100 μL of TMB to each well and incubate at 37°C in the dark for 2 min.

[0151] (9) Termination: Add 100 μL of Stop Solution to each well to terminate the reaction.

[0152] Ten minutes after termination, the A content in each well was measured using a microplate reader. 450 The antigen target was immobilized on an immunosorbent plate, and antibody samples were added and incubated to form antigen-antibody complexes. Enzyme-labeled secondary antibody was then added, followed by the enzyme substrate. The substrate reacted under the catalysis of the enzyme, and quantitative analysis was performed by detecting the reaction products. HER2-ECD protein was used as the target antigen, Isotype control IgG1 was used as a negative control to exclude non-specific binding, and trastuzumab was used as a positive control. Table 6 shows the KD values ​​of trastuzumab and Trastuzumab-Mc-Val-Cit-PAB-EP-1.

[0153] Table 6

[0154] The results are shown in Table 6. Compared with the positive control trastuzumab, the binding ability of the ADC (Trastuzumab-Mc-Val-Cit-PAB-EP-1) conjugated with the small molecule was not significantly affected with the HER2 protein. The KD value was 1.67 nM, which is close to that of trastuzumab (KD value 0.91 nM), indicating that the binding ability of the antibody to the antigen was not affected by the small molecule after conjugation.

[0155] Example 10 Detection of the degree of endocytosis of antibody-conjugates 1) Experimental methods (1) Plating: SKBR-3 cells in logarithmic growth phase were seeded into 96-well plates at a density of 35,000 cells / well and the cell suspension volume was 50 μL / well. The cells were cultured overnight to adhere to the plate.

[0156] (2) Sample preparation: Prepare antibody sample dilution solution with a concentration of 80 nM.

[0157] (3) Labeling antibody: In each group in step 2, Zenon™ working solution was added at a volume ratio of 1:1 and incubated at room temperature for 5 min to form the labeling complex.

[0158] (4) Labeling adherent cells: Add 50 µL of the antibody-fluorescent complex from step 3 to each well and incubate at 4°C for 1 h. Wash twice slowly with PBS to remove non-specifically bound antibodies, add culture medium, and incubate at 37°C for 15 h.

[0159] (5) Nuclear staining: Remove the culture medium, wash slowly with 200 μL PBS, add Hoechst 33342 for staining, and observe after incubation at room temperature for 3-10 min.

[0160] (6) Washing: Remove the dye, add 200 μL PBS, let stand for a while, remove the PBS, and repeat twice.

[0161] Observation: Observation was performed on the instrument. This study used an endocytosis-specific indicator (Zenon™ pHrodo™ iFL Red Human IgG Labeling Reagent) to detect the degree of endocytosis of the conjugate. This reagent is a pH-sensitive dye that does not fluoresce outside the cell. The Fab fragment it contains can bind to the Fc fragment of intact IgG to form a labeling complex. When the formed Fab-antibody complex is endocytosed into the lysosome, it emits bright red fluorescence in an acidic environment. The intensity of the red fluorescence of the cells was detected by a high-content system. Finally, the proportion of cells with high red fluorescence intensity to the total number of cells was analyzed using R language, which represents the endocytosis rate.

[0162] The groups in this experiment included ADC samples, DS-8201a, and the positive drug trastuzumab. The cells used included SKBR-3 cells with high HER2 expression and MCF-7 cells with low HER2 expression. The incubation time for samples and cells was 15 h.

[0163] Table 7

[0164] The results are shown in Table 7. In HER2-high expressing SKBR-3 cells, the endocytosis rate after incubation with Trastuzumab-Mc-Val-Cit-PAB-EP-1 for 15 h was approximately 10%, slightly lower than the endocytosis rate of DS-8201a (15%). In HER2-low expressing MCF-7 cells, endocytosis was significantly reduced, below 5%, demonstrating that the endocytosis of antibody-drug conjugates is mediated by the binding of antibodies and antigens. These results indicate that ADCs have the ability to target and enter cells, carrying a target to exert their therapeutic effect.

[0165] Example 11 In vitro cytotoxicity evaluation of antibody-drug conjugate Trastuzumab-Mc-Val-Cit-PAB-EP-1 1) Experimental methods This experiment selected 11 cell lines with different HER2 expression levels for cytotoxic activity testing, including HER2-high expressing ovarian cancer cells SKOV-3, breast cancer cells SKBR-3 and BT474, gastric cancer cells NCI-N87 and NUGC4, HER2-low expressing breast cancer cells MDA-MB-453 and MCF-7, human non-small cell lung cancer cells A549 and human colon cancer cells HCT116, and HER2-negative breast cancer cells MDA-MB-468 and BT549. The experimental groups included naked antibody (trastuzumab), Trastuzumab-Mc-Val-Cit-PAB-EP-1, positive control drug DS-8201a, and toxic warhead EP-1. The CCK-8 assay was used for measurement. (1) Take cells in the logarithmic growth phase. Digest with trypsin, centrifuge, dilute with culture medium and count the cells. Seed the cells at a density of 3000 cells per well in a 96-well plate and incubate overnight to allow them to adhere and grow.

[0166] (2) The prepared compound stock solution was slowly diluted in a gradient with culture medium and serially diluted in a 96-well plate. 100 μL of compound dilution solution was added to each well and the plate was incubated in a 37°C cell culture incubator for 120 h.

[0167] (3) Add 10 μL of CCK-8 reagent to each well and incubate in a 37℃ cell culture incubator for about 2-3 h.

[0168] The absorbance at 450 nm was measured using an ELISA reader. After subtracting the absorbance of the control group from the experimental group absorbance value, the cell viability was calculated as (ODdose - ODblank) / (ODcontrol - ODblank) * 100%. The IC50 value was fitted using GraphPad Prism 9.

[0169] Table 8

[0170] The results are shown in Table 8. In A549, HCT116, MDA-MB-453, and MCF-7 cells with low HER2 expression, the cytotoxic activity of Trastuzumab-Mc-Val-Cit-PAB-EP-1 was weakened compared to that in cells with high HER2 expression, demonstrating that the cytotoxic activity of antibody-drug conjugates depends on the expression level of HER2 on the surface of cancer cells. Notably, the cytotoxic activity of ADCs in MCF-7 cells was 20-30 times higher than that of the positive control drug DS-8201a, indicating a stronger killing effect on tumor cells with low HER2 expression. This suggests that ADCs have a greater advantage in the treatment of tumors with low HER2 expression and have the potential to overcome the limitations of traditional therapies in this field.

[0171] In HER2-negative BT549 cells, at the same concentration (x-axis consistent), neither Trastuzumab-Mc-Val-Cit-PAB-EP-1 nor DS-8201a exhibited cytotoxic activity, indicating that ADCs cannot bind to HER2 to undergo endocytosis and release the warhead to exert their effect. Compared with other HER2-positive cells, this confirms that the cytotoxic activity of ADCs is mediated by the HER2 antigen.

[0172] In summary, the warhead itself has poor membrane permeability and low bioavailability, exhibiting low cytotoxic activity. However, in cells with high and low HER2 expression, ADCs carry the warhead into the cell, causing it to accumulate. In synergy with HER2 antibodies, the cytotoxicity is increased by hundreds to thousands of times. However, it has no activity in cells that do not express HER2, indicating that small molecules can exert their effects precisely by binding to antibodies and leveraging the targeting of antibodies. Furthermore, the conjugates depend on binding to the HER2 antigen to exert their effects.

[0173] Example 12: Antibody conjugates can inhibit the migration ability of tumor cells. 1) Experimental method of scratch test (1) Collect SKOV-3 cells in good growth condition by digestion with trypsin, count them, and measure them at 1×10⁻⁶. 6 Cells were seeded at a density of 1 cell per well in 12-well plates and cultured overnight until the cells adhered.

[0174] (2) Use a 10 μL micro-volume sterile pipette tip to make a uniform scratch in the center of each well (vertical to the bottom of the plate), rinse gently twice with 1×PBS to thoroughly remove the detached cell debris, add serum-free culture medium, and the final volume is 1.5 mL / well.

[0175] (3) Prepare different concentrations of the drug and add them to the well plates for co-incubation with the cells. Cell migration was observed and photographed using a microscope at 0 h, 24 h, 48 h, and 72 h after drug administration. The migration process of cells from the scratch edge to the central empty area was observed using microscopic imaging to assess their migration ability. If tumor cells migrated towards the scratch area, the scratch area decreased, and the degree of area reduction was positively correlated with migration ability.

[0176] This embodiment investigates the effect of antibody-drug conjugates on the migration ability of SKOV-3 cells using a scratch assay. A monolayer of SKOV-3 cells was seeded in 12 wells for the assay.

[0177] Figure 3 This is a scratch test for antibody-drug conjugates. (By...) Figure 3 It was observed that, without drug administration, the edges of the scratched cells gradually blurred and the scratch area significantly decreased over time, indicating cell migration. When the concentration of Trastuzumab-Mc-Val-Cit-PAB-EP-1 was 5 nM, the scratch area remained essentially unchanged, indicating that cell migration was inhibited, demonstrating that this concentration of ADC has a strong inhibitory effect on migration. With increasing time, the scratch area reduction was less pronounced in the ADC-treated groups compared to the control group. After 72 h, only a few cells in the ADC group migrated, indicating that ADC has the ability to inhibit SKOV-3 cell migration.

[0178] Example 13: Study on the mechanism of action of antibody-drug conjugates 1) This experiment is used to demonstrate that the warhead has Topo II inhibitory activity. The specific experimental method is as follows: Topo II-mediated DNA loosening assay (1) Prepare 8% agarose gel and 5×Topo II buffer, then dilute the compound with deionized water and prepare 0.1 μg / μL pBR322 DNA solution.

[0179] (2) Dilute with deionized water to obtain a suitable concentration of Topo II solution and place it on ice.

[0180] (3) Add 2 μL of pBR322 DNA solution, 4 μL of Topo II solution and compound solution and 4 μL of 5 × Topo II buffer to the reaction system, and make up the volume with deionized water.

[0181] (4) Incubate the reaction mixture at 37°C for 30 min.

[0182] (5) Add Loading Buffer to the sample and mix. Add 6.5 μL of sample to the agarose gel well and electrophoresis at 75 V for 1 h.

[0183] After the reaction was complete, the sample was stained with ethidium bromide (0.5 μg / mL) for 60 minutes. The DNA bands were observed under ultraviolet light and photographed using a gel imaging system.

[0184] The electrophoretic velocity of DNA in agarose gel is related to its spatial structure; tightly packed supercoiled DNA has the fastest electrophoretic velocity, while loosely packed DNA has the slowest. Because the warhead EP-1 exhibits significant cytotoxic activity, it was chosen for the detection of Topo II enzyme inhibitory activity.

[0185] Figure 4 DNA loosening assay for Topo II. Results are as follows. Figure 4 As shown, compared with the positive control drug VP-16, EP-1 exhibited slightly weaker enzyme inhibitory activity, but still maintained inhibitory activity at 6 μM. This indicates that aryl vinyl compounds, after adaptation modification, can still exert Topo II inhibitory activity. Combined with the above transcriptomic and proteomic analyses, the small molecule, after entering the cell with the aid of antibody targeting, also demonstrated Topo II inhibitory activity.

[0186] 2) This experiment demonstrates that antibody-drug conjugates can activate caspase 3 / 7. The specific experimental method is as follows: 1) Experimental methods (1) Take SKBR-3 cells in the logarithmic growth phase. Digest with trypsin, centrifuge, dilute with culture medium and count, seed in 96-well plates at a density of 5000 cells per well, and let them adhere and grow overnight in an incubator.

[0187] (2) The drug was serially diluted in a 96-well plate with culture medium. 100 μL of drug diluent was added to each well and the plate was incubated at 37°C for 48 h.

[0188] (3) Prepare the working solution in advance according to the kit instructions.

[0189] (4) Add 50 μL of working solution to each well of the orifice plate.

[0190] (5) Gently mix the contents of the micropores for 30 s using a plate shaker at a speed of 300-500 rpm. Incubate at room temperature for 1 h.

[0191] The luminescence intensity was measured at all wavelengths using an ELISA reader. The luminescence value is directly proportional to the activity of caspase. By measuring the luminescence intensity, the activity of caspase-3 / 7 induced by the drug can be determined, thereby detecting the drug-induced apoptosis.

[0192] This experiment included a control group, a group receiving small molecule EP-1, trastuzumab, a group receiving a combination of small molecule EP-1 and trastuzumab, and a group receiving Trastuzumab-Mc-Val-Cit-PAB-EP-1. The ADCs were administered at concentrations of 2 nM, 0.5 nM, 0.13 nM, and 0.03 nM, with corresponding trastuzumab concentrations. The small molecule EP-1 was administered at concentrations of 80 nM, 20 nM, 5 nM, and 1.3 nM.

[0193] Figure 5 The effect of antibody-drug conjugates on caspase-3 / 7 activity. Results are as follows: Figure 5 As shown, neither the EP-1 and trastuzumab groups nor the combination group induced caspase-3 / 7 activity in SKBR-3 cells. However, the antibody conjugate at a concentration of 2 nM could activate caspase-3 / 7 activity. Among them, at the same concentration, the caspase-3 / 7 activity of Trastuzumab-Mc-Val-Cit-PAB-EP-1 gradually increased with increasing concentration, and this increased activity was concentration-dependent.

[0194] This indicates that, through the targeting action of antibodies, extremely low concentrations of small molecules can enter cells and exert their effects effectively.

[0195] The above results suggest that the conjugate may activate the activity of Caspase 3 / 7 by inhibiting cell mitosis, resulting in the failure of sister chromosomes to separate, thereby initiating the Caspase cascade reaction and triggering apoptosis.

[0196] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. An antibody-drug conjugate, characterized in that, include: Antibody, linker, and drug molecule, wherein the drug molecule is coupled to the antibody via the linker; The antibody-drug conjugate has the structure shown in formula (II): Ab-(LD)n(II) In the formula, Ab is the antibody element, L is the linker, D is the drug molecule, and n is 2-8; The drug molecule has the structure shown in formula (I): (I) In the formula, R1 contains groups selected from the following group: , or ; The connector L is a divalent connector with the structure shown in equation (III): -L1-L2-L3-(III) In the formula, L1 is the first linker element connected to the antibody; L2 is the second linker element, which may be absent or connected to both L1 and L3; and L3 is the third linker element connected to the drug molecule. Wherein, the first connecting element L1 contains: a substituted or unsubstituted maleimide group; The second linker element L2 is selected from at least one of the following groups: PEG linker, C1-C10 alkylene group, phenyl linker, heteroaromatic ring linker, and adipic acid group; The third connecting element L3 is selected from: 。 2. The antibody-drug conjugate according to claim 1, characterized in that, The antibody is an antibody targeting tumor-associated antigens; the tumor-associated antigens are selected from: HER2, CD19, CD20, EGFR, CD22, CD3, TROP2, glycoprotein NMB, guanylate cyclase C, CEA, AXL, PSMA, ENPP3, Mesothelin, CD138, NaPi2b, CD56, CD74, FOLR1, DLL3, CEACAM5, CD142, SLAMF7, CD25, SLTRK6, CD37, CD70, AGS-22, C4.4A, FGFR2, Ly6E, MUC16, BCMA, pCadherin, Ephrin-A, LAMP1, MUC1, PDL1, NY-ESO-1, WT1, CD23, ROR1, CD123, CD33, CD44v6, CD174, CD30, CD133, cMet, FAP, At least one of EphA2, GD2, GPC3, IL-13Ra2, LewisY, SS1, CD171, EGFRvIII, VEGFR2, or MAGE-A3.

3. The antibody-drug conjugate according to claim 2, characterized in that, The antibody is an antibody that targets HER2.

4. The antibody-drug conjugate according to claim 3, characterized in that, The antibody targeting HER2 is selected from at least one of the following: trastuzumab, pertuzumab, inetuzumab, trastuzumab emtansine, detrastuzumab, matozumab, and MGAH22.

5. The antibody-drug conjugate according to claim 1, characterized in that, In the structure of formula (II), the linker-drug molecule LD is selected from the structures shown in formulas (V) and (VI) below: (V)(VI) Where n is 1-8.

6. The method for preparing the antibody-drug conjugate according to claim 5, characterized in that, Includes the following steps: (1) Provides a linker-drug molecule LD having the structures shown in formulas (V) and (VI); (2) The linker-drug intermediate LD is coupled with the antibody element to form the antibody-drug conjugate.

7. A pharmaceutical composition, characterized in that, include: (a) The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5; and (b) its pharmaceutically acceptable carrier.

8. A topoisomerase IIA inhibitor or a pharmaceutically acceptable salt thereof, characterized in that, include: (a) The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5; and (b) its pharmaceutically acceptable carrier.

9. The use of the antibody-drug conjugate of any one of claims 1-5 or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating or preventing tumors.

10. The application according to claim 9, characterized in that, The tumor is selected from at least one of breast cancer, gastric cancer, ovarian cancer, lung cancer, and colorectal cancer.

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