Erianin-containing antibody drug conjugate as well as preparation method and application thereof

Abstract

The present invention provides an antibody drug conjugate that is covalently conjugated by at least one erianin-loaded linker and a tumor-targeting antibody, wherein the erianin-loaded linker comprises erianin as a cytotoxic agent, an antibody linking moiety, a cleavable bond, or may comprise a self-consuming linker. The antibody drug conjugate can effectively deliver erianin to tumor cells in a targeted mode, the treatment effect is enhanced, and meanwhile the off-target side effect is reduced.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 598,041, filed on November 10, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the fields of biotechnology and pharmaceutical science. More specifically, the present invention relates to an antibody drug conjugate comprising Erianin. Background Art

[0003] Cancer is a health issue that is taken seriously around the world. In 2020 alone, it claimed the lives of nearly 10 million people, making it one of the leading causes of death in the world. Although chemotherapy is a common method of treating cancer, it also has certain limitations. For example, many traditional anticancer drugs lack the ability to specifically target cancer cells, so the therapeutic window is narrow, often causing adverse off-target effects and dose-dependent toxicity, leading to serious systemic toxicity. In order to solve these problems, there is an urgent need to develop new anticancer drugs in this field that can effectively target tumor sites while reducing toxicity to healthy cells.

[0004] The emergence of antibody-drug conjugates (ADCs) has attracted a lot of attention in the field of anticancer drug research and development. Antibody-drug conjugates contain monoclonal antibodies that can target tumor-specific antigens. The antibodies are covalently bound to cytotoxic drugs through chemical linkages. By utilizing the highly specific targeting ability of antibodies and the cytotoxic properties of drugs, antibody-drug conjugates can directly deliver cytotoxic drugs to tumor sites, expanding the therapeutic window for treating tumors, and have considerable potential in treating tumors.

[0005] The U.S. Food and Drug Administration has currently approved 14 antibody-drug conjugates, and more than 100 antibody-drug conjugates are in different stages of clinical trials. However, current antibody-drug conjugates face a fundamental challenge, namely that their drug payloads are often highly cytotoxic and lack selectivity, affecting both cancer cells and normal cells, and may cause serious side effects due to their binding to targeted antigens on non-cancerous cells. Therefore, the development of new, safe and effective payload drugs is crucial to promoting the development of antibody-drug conjugates, while ensuring the targeting of cancer treatment and reducing cytotoxic side effects.

[0006] For thousands of years, traditional Chinese medicine and natural herbal medicine have been effective in treating many types of cancer and are considered a safe alternative anti-cancer therapy due to their fewer adverse reactions. The uniqueness of traditional Chinese medicine in treating cancer is the use of natural compounds, such as Erianin, instead of using highly cytotoxic payload drugs from the 14 antibody-drug conjugates approved by the U.S. Food and Drug Administration.

[0007] Erianin is a natural herbal compound that is considered a potential anticancer agent. It has strong anticancer activity against a variety of cancers, and at the same time, it has high safety and is economically affordable. Previous studies have shown that Erianin has a significant inhibitory effect on a variety of human cancers (including nasopharyngeal carcinoma, hepatocellular carcinoma, oral squamous cell carcinoma and lung cancer) without inhibiting normal cells or causing toxicity to major organs.

[0008] However, Erianin faces some challenges in terms of therapeutic efficiency because it has poor water solubility and is rapidly metabolized and cleared by the human body, resulting in low bioavailability. This also means that high doses are required to achieve effective therapeutic effects, which increases the burden on patients taking the drug. If these problems can be solved, the therapeutic potential of Erianin can be enhanced, making it a more effective option in the field of cancer prevention.

[0009] Therefore, the present invention is directed to addressing this need. Summary of the invention

[0010] The object of the present invention is to provide a compound, a composition or a use to solve the above technical problems.

[0011] According to the first aspect of the present invention, an antibody-drug conjugate is provided. More specifically, the antibody-drug conjugate comprises: Tumor-targeting antibodies; and At least one linker carrying Erianin.

[0012] According to one embodiment of the present invention, the tumor targeting antibody and the at least one Erianin-loaded linker are bound to each other via a covalent bond; the Erianin-loaded linker comprises Erianin, an antibody linking portion, and a cleavable bond, and may further selectively comprise or not comprise a self-consumable linker.

[0013] According to one embodiment of the present invention, the tumor targeting antibody comprises at least one thiol group or at least one amino group.

[0014] According to one embodiment of the present invention, the antibody linking moiety comprises a thiol-reactive Michael acceptor group or an amino-reactive N-hydroxysuccinimide group.

[0015] According to one embodiment of the present invention, the tumor targeting antibody can be bound to the loaded drug via a non-cleavable linker, rather than the self-consumable linker and the cleavable bond.

[0016] According to one embodiment of the present invention, the at least one thiol group undergoes a Michael addition reaction with the Michael acceptor group of the Erianin-loaded linker to combine the Erianin-loaded linker and the tumor-targeting antibody.

[0017] According to one embodiment of the present invention, the at least one amino group undergoes a condensation reaction with the N-hydroxysuccinimide group of the Erianin-loaded linker to combine the Erianin-loaded linker and the tumor-targeting antibody.

[0018] According to one embodiment of the present invention, the at least one Erianin-carrying linker has the following chemical formula: The antibody connecting part is a maleimide group, the cleavable bond is a Val-Cit unit cleavable by cathepsin B, and the self-consumable linker is a self-consumable p-aminobenzyl and biscarbamate linker.

[0019] According to one embodiment of the present invention, the cleavable bond can be any enzyme cleavable unit, acid cleavable unit or glutathione cleavable unit.

[0020] According to one embodiment of the present invention, the antibody-drug conjugate has 1 to 8 Erianin-loaded linkers.

[0021] According to one embodiment of the present invention, the tumor-related receptor targeted by the tumor-targeting antibody can be selected from HER2, TROP2, Nectin4, folate receptor α, epidermal growth factor receptor (EGFR), HER3 or c-MET.

[0022] According to one embodiment of the present invention, the tumor targeting antibody is trastuzumab.

[0023] According to a second aspect of the present invention, a method for preparing the above-mentioned antibody-drug conjugate is provided. More specifically, the method comprises: synthesizing the Erianin-loaded linker; and The Erianin-loaded linker is coupled to the tumor-targeting antibody.

[0024] According to one embodiment of the present invention, the method further comprises subjecting the tumor-targeting antibody to a reduction treatment before coupling to reduce its interchain disulfide bonds and form free thiol groups on the tumor-targeting antibody.

[0025] According to one embodiment of the present invention, the tumor targeting antibody comprises at least one thiol group or at least one amino group.

[0026] According to one embodiment of the present invention, the antibody linking moiety comprises a thiol-reactive Michael acceptor group or an amino-reactive N-hydroxysuccinimide group.

[0027] According to one embodiment of the present invention, the at least one thiol group undergoes a Michael addition reaction with the Michael acceptor group of the Erianin-loaded linker to combine the Erianin-loaded linker and the tumor-targeting antibody.

[0028] According to one embodiment of the present invention, the at least one amino group undergoes a condensation reaction with the N-hydroxysuccinimide group of the Erianin-loaded linker to combine the Erianin-loaded linker and the tumor-targeting antibody.

[0029] According to the third aspect of the present invention, a composition is provided. More specifically, the composition comprises the above antibody-drug conjugate and a pharmaceutically acceptable additive.

[0030] According to one embodiment of the present invention, the pharmaceutically acceptable additives include excipients, stabilizers, carriers, diluents and solubilizers.

[0031] According to a fourth aspect of the present invention, there is provided a use of the above-mentioned antibody-drug conjugate in preparing a composition for treating cancer. More particularly, the method comprises: A pharmaceutically effective dose of the above antibody-drug conjugate composition is administered to a subject.

[0032] According to one embodiment of the present invention, the cancer includes breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer, melanoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, glioblastoma, astrocytoma, medulloblastoma, meningioma, sarcoma, bone cancer, head and neck cancer, testicular cancer, oral cancer, anal cancer, mesothelioma, neuroblastoma, retinoblastoma and bile duct cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Embodiments of the present invention will be described in more detail below with reference to the accompanying drawings, in which:

[0034] Figure 1 The structure of the antibody drug conjugate of trastuzumab and Erianin according to one embodiment of the present invention is shown;

[0035] Figure 2 The synthetic pathways of Erianin precursor and Erianin-loaded linker are shown;

[0036] Figure 3 The method for preparing the antibody-drug conjugate of trastuzumab and Erianin according to one embodiment of the present invention is shown;

[0037] Figures 4A-4G The characterization of the antibody drug conjugate of trastuzumab and Erianin prepared according to one embodiment of the present invention is shown, wherein Figure 4A Shown is a reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis (SDS-PAGE) of trastuzumab (monoclonal antibody) and a conjugate of trastuzumab and Erianin (i.e., antibody-drug conjugate); Figure 4B UV-visible absorption spectra of antibody drug conjugate, monoclonal antibody and Erianin-loaded linker 5 are shown; Figure 4C The reverse phase high performance liquid chromatography (RP-HPLC) chromatograms of trastuzumab (tris(2-carbonylethyl)phosphine hydrochloride TCEP reduced or not TCEP reduced) and antibody drug conjugates are shown; Figure 4D Deconvoluted mass spectra showing light and heavy chain fragments in an antibody drug conjugate; Figure 4E Size exclusion chromatograms (SEC) of trastuzumab and antibody drug conjugate are shown; Figure 4F Showing hydrophobic interaction chromatograms (HIC) of trastuzumab and antibody drug conjugates; and Figure 4G Shows the extent of binding of monoclonal antibodies and antibody-drug conjugates to HER2 protein detected by enzyme-linked immunosorbent assay (ELISA);

[0038] Figures 5A-5I The in vitro test results of the antibody drug conjugate of trastuzumab and Erianin are shown, Figure 5A Displays binding activity analysis of antibody drug conjugates, monoclonal antibodies, and IgG control antibodies, and Figure 5B The corresponding flow cytometry quantification data are shown; Figure 5C Show internalization analysis of antibody drug conjugates and monoclonal antibodies, and Figure 5D The corresponding flow cytometry quantification data are shown; Figure 5E Confocal microscopy images of antibody-drug conjugates in different cell lines; Fig. 5F The co-localization image of the antibody-drug conjugate and the lysosomal green fluorescent probe (LysoTracker Green) is shown; Figure 5G Shows the results of in vitro antiproliferative assays of antibody-drug conjugates; Figure 5H In vitro antiproliferative assay results showing trastuzumab; and Fig.5I In vitro antiproliferative test results showing Erianin; and

[0039] Figures 6A-6EThe in vivo test results of the antibody drug conjugate of trastuzumab and Erianin are shown, wherein Fig. 6A Schematic diagram of the in vivo experimental process; Figure 6B Shows the tumor growth curves of tumor-bearing SK-OV-3 mice treated with Erianthrin, trastuzumab, antibody-drug conjugate, and the control group; Figure 6C Shows tumor images 28 days after treatment; Fig.6D shows the tumor weight of mice at 28 days; and Fig. 6E Shown are the body weight changes of mice over 28 days. DETAILED DESCRIPTION

[0040] In the following description, compounds, compositions and / or methods of antibody drug conjugates with Erianin are listed as preferred embodiments. It is obvious to those skilled in the art that modifications including additions and / or substitutions may be made without departing from the scope and spirit of the invention. Specific details may be omitted hereinafter to avoid obscuring the invention; however, this article is written to enable those skilled in the art to practice the technical content disclosed herein without excessive experimentation.

[0041] The term "antibody drug conjugate" as used herein refers to a targeted cancer treatment method that combines tumor-specific targeted antibodies and cytotoxic drugs (also known as loaded drugs), wherein the antibody can recognize and bind to specific antigens on the surface of cancer cells, while the cytotoxic drug is responsible for killing cancer cells. Once the antibody drug conjugate binds to its targeted antigen, the antibody drug conjugate will be internalized by the cancer cell, thereby releasing the cytotoxic drug, causing the cancer cell to die. Because antibody drug conjugates can specifically target cancer cells, they can reduce damage to normal healthy cells and reduce side effects compared to traditional chemotherapy.

[0042] According to the first aspect of the present invention, an antibody drug conjugate is provided. The antibody drug conjugate can deliver a potent anti-tumor agent (Erianin) directly to tumor cells to minimize the systemic toxicity of the drug and enhance the therapeutic effect. The antibody drug conjugate is covalently bonded to a tumor-targeting antibody by at least one linker loaded with Erianin, wherein these linkers play a key role in the function of the antibody drug conjugate, and are responsible for binding Erianin to the antibody and releasing Erianin in the tumor environment.

[0043] The Erianin-loaded linker is a multi-component structure consisting of Erianin, an antibody linking moiety, a cleavable bond, or a self-consumable linker. The cleavable bond is particularly sensitive to tumor-specific conditions to release Erianin at the targeted tumor site, and the tumor-targeting antibody can selectively bind to receptors overexpressed on the surface of tumor cells, thereby further enhancing tumor specificity.

[0044] In this article, the terms "cleavable bond" and "self-consumable linker" refer to chemical linkers that connect cytotoxic drugs (loaded drugs) to monoclonal antibodies, and when they reach the target cancer cells, these linkers will trigger decomposition or "self-consumption" to release the effective drug in active form. When the antibody drug conjugate enters the blood, the monoclonal antibody portion will bind to a specific antigen on the surface of the cancer cell, where the specific antigen is usually a protein that is only overexpressed on cancer cells.

[0045] Once the antibody drug conjugate binds to its targeted antigen, the conjugate is engulfed into the cancer cell through endocytosis. Inside the cell, the antibody drug conjugate is transported to the lysosome. Since the lysosome is an organelle that contains degraded proteins and other molecules, the cleavable bond will be cleaved in response to specific stimuli in the lysosome or other specific cellular environments, including: (1) Enzymatic cleavage: Specific enzymes in the lysosome (such as cathepsin) will cleave the linker. (2) Reduction reaction: Some linkers (such as disulfide bonds) respond to the reducing environment in the cell (such as the presence of glutathione in the environment). (3) Acidic pH: The acidic environment of lysosomes can also trigger cleavage.

[0046] After initial cleavage, the self-consumable linker rapidly undergoes spontaneous degradation through a series of intramolecular reactions to completely dissociate the linker and release the cytotoxic drug in its active form.

[0047] After being released, cytotoxic drugs interact with their targets (e.g. DNA, tubulin) within the cell, leading to cell death. Therefore, this design ensures that the drug is released only within the target cancer cells to minimize damage to healthy tissues.

[0048] The tumor targeting antibody in the antibody-drug conjugate may contain at least one thiol group or at least one amino group to provide multiple attachment points for the linker loaded with Erianin. Accordingly, the antibody linking portion is specifically configured to include a thiol-reactive Michael acceptor group or an amino-reactive N-hydroxysuccinimide group; when the antibody has a thiol group, the thiol group will produce a Michael addition reaction with the Michael acceptor group of the linker loaded with Erianin to effectively connect Erianin to the antibody; or, if the antibody contains an amino group, the amino group can undergo a condensation reaction with the N-hydroxysuccinimide group of the linker to combine the linker loaded with Erianin and the antibody.

[0049] In one embodiment of the present invention, the linker carrying Erianin has the following structural formula:

[0050] The linker carrying Erianin has a specific chemical structure. In one embodiment, the antibody linking portion is a maleimide group that can react with the sulfhydryl group on the antibody; the cleavable bond of the linker is a Val-Cit unit that can be cleaved by cathepsin B and is stable in the blood, so it can be cleaved in the lysosomal environment of tumor cells. This cleavage process can further trigger the self-consumable linker, thereby releasing Erianin in the tumor cells and exerting its cytotoxic effect, wherein the self-consumable linkers include p-aminobenzyl and biscarbamate linkers.

[0051] The antibody drug conjugate can flexibly adjust the drug loading according to the treatment needs. In one embodiment, the antibody drug conjugate can carry 1 to 8 linkers loaded with Erianin. Tumor-targeted antibodies can target a variety of tumor-related receptors, making the antibody drug conjugate suitable for a variety of cancer types, including but not limited to HER2, TROP2, Nectin4, folate receptor α, EGFR, HER3 or c-MET. In a preferred embodiment, the tumor-targeted antibody is trastuzumab, which is a monoclonal antibody against the HER2 receptor, which is often overexpressed in certain types of breast cancer.

[0052] In some embodiments, tumor-associated receptors further include, but are not limited to, PD-L1, VEGF, CD20, CD19, CD22, CD33, PSMA, MUC1, CD30, CD38, mesothelin, EpCAM, c-MET, RANKL, glycosaminoglycan-3, and CD47.

[0053] In addition to trastuzumab, other antibodies can also be used in the antibody-drug conjugates of the present invention. The following lists relevant examples of such antibodies and their target antigens: Brentuximab (Adcetris): Antibody: Brentuximab monoclonal antibody (anti-CD30). Target antigen: CD30. Indications: Mainly used for Hodgkin's lymphoma and anaplastic large cell lymphoma. Ogalotuzumab (Besponsa): Antibody: Inotuzumab monoclonal antibody (anti-CD22). Target antigen: CD22. Indications: For relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Trontuximab (Zynlonta): Antibody: Loncastuximab monoclonal antibody (anti-CD19). Target antigen: CD19. Indications: Large B-cell lymphoma. Gosartumomab (Trodelvy): Antibody: Sacituzumab monoclonal antibody (anti-TROP-2). Target antigen: TROP-2. Indications: Metastatic triple-negative breast cancer and urothelial carcinoma. Ogagiizumab (Mylotarg): Antibody: Gemtuzumab monoclonal antibody (anti-CD33). Target antigen: CD33. Indications: Acute myeloid leukemia. Viagra (Padcev): Antibody: Enfortumab monoclonal antibody (anti-Nectin-4). Target antigen: Nectin-4. Indications: Locally advanced or metastatic urothelial carcinoma. Vempotuzumab (Polivy): Antibody: Polatuzumab monoclonal antibody (anti-CD79b). Target antigen: CD79b. Indications: Diffuse large B-cell lymphoma. Vectisolutuzumab (Tivdak): Antibody: Tisotumab monoclonal antibody (anti-tissue factor). Target antigen: tissue factor. Indications: Cervical cancer and other solid tumors, including liver cancer. Sumituximab (Elahere): Antibody: Mirvetuximab (anti-folate receptor α). Target antigen: Folate receptor alpha. Indications: Ovarian, fallopian tube and peritoneal cancer. Glypican-3(GPC3)-targeted antibody drug conjugates: Antibodies: Experimental antibodies targeting GPC3. Target antigen: Glypican-3. Indications: GPC3 is a cell surface protein that is often overexpressed in liver cancer and has become a potential target for the development of antibody-drug conjugates. Cetuximab Sarotalocan: Antibody: Cetuximab (anti-EGFR). Target antigen: epidermal growth factor receptor. Indications: Unresectable locally advanced and recurrent head and neck cancer. Nimotuzumab: Antibody: Nimotuzumab (anti-EGFR). Target antigen: epidermal growth factor receptor. Indications: Nasopharyngeal carcinoma and other head and neck cancers, especially in areas where nasopharyngeal carcinoma is common. Dato-DXd: Antibody: Datopotamab monoclonal antibody (anti-TROP-2). Target antigen: TROP-2. Indications: It is being studied for non-small cell lung cancer and other solid tumors. Rybrevant: Antibodies: Amituzumab (bispecific, anti-epidermal growth factor receptor and anti-mesenchymal epidermal transition factor receptor). Targeted antigens: epidermal growth factor receptor and mesenchymal epidermal transition factor receptor. Indications: Approved for non-small cell lung cancer with epidermal growth factor receptor exon 20 insertion mutation, combined with epidermal growth factor receptor and mesenchymal epidermal transformation factor receptor targeted therapy. Zenidatuzumab (ZW25): Antibody: Zanidatamab monoclonal antibody (bispecific anti-HER2). Target antigen: HER2. Indications: It is being studied in a variety of HER2-overexpressing cancers, including gastroesophageal, breast, and potentially lung cancer. Patritumab Deruxtecan: Antibody: Patritumab monoclonal antibody (anti-HER3). Target antigen: HER3. Indications: Locally advanced or metastatic non-small cell lung cancer with EGFR mutation. Telisotuzumab Vedotin: Antibody: Telisotuzumab monoclonal antibody (anti-c-MET). Target antigen: c-MET. Indications: EGFR wild-type non-squamous NSCLC with advanced or metastatic disease and high levels of c-MET overexpression.

[0054] In some embodiments, the cleavable bond can be selected from the following group: a protease-sensitive peptide linker, a disulfide bond, and an acid-sensitive bond; the self-consumable linker can be selected from the following group: p-aminobenzyl alcohol, p-hydroxybenzyl alcohol, and a disulfide bond-type self-consumable linker.

[0055] The antibody-drug conjugates of the present invention utilize the specificity of tumor-targeting antibodies with the potent anti-tumor activity of Erianin and deliver them in a controllable and targeted manner through a linker system, providing a powerful and effective treatment option for cancer patients.

[0056] According to the second aspect of the present invention, a method for preparing the above-mentioned antibody-drug conjugate is provided, which includes multiple key steps to ensure the successful synthesis of the linker loaded with Erianin and can be smoothly combined with the tumor-targeting antibody. First, the cytotoxic drug Erianin is loaded onto a suitable linker molecule through a chemical reaction to synthesize the linker loaded with Erianin, and the linker is conducive to the combination with the antibody; wherein, the selection of the linker is very important, and it must have the necessary functional groups to be able to bind to the antibody while maintaining the biological activity of Erianin.

[0057] Once the Erianin-loaded linker is synthesized, the next step is to bind the linker to the tumor-targeting antibody; in some embodiments, in order to optimize the binding ability of the antibody, the antibody can be reduced to reduce the interchain disulfide bonds in the antibody, thereby forming free thiol groups on the antibody, which are conducive to subsequent binding. For example, the reduction treatment includes exposing the antibody to a reducing agent (such as tris (2-carbonylethyl) phosphine hydrochloride) to reduce the interchain disulfide bonds of the antibody to generate free thiol groups.

[0058] Tumor-targeting antibodies themselves contain at least one thiol group or at least one amino group as a potential binding site. The specific binding mode depends on the functional groups on the antibody. For example, if the antibody contains a thiol group, the binding effect can be achieved by reacting with the thiol-reactive Michael acceptor group on the linker carrying Erianin. This reaction is called Michael addition reaction, which helps to form a stable bond between the thiol group on the antibody and the Michael acceptor group on the linker, thereby effectively binding the Erianin drug to the antibody.

[0059] On the other hand, if the antibody contains an amino group, the binding process can be carried out through the amine-reactive N-hydroxysuccinimide group on the linker loaded with Erianin. The amino group on the antibody will undergo a condensation reaction with the N-hydroxysuccinimide group of the linker to form a stable amide bond, thereby firmly binding the Erianin-loaded linker to the antibody.

[0060] The linker synthesized through the above steps effectively connects the Erianin drug to the tumor-targeting antibody to prepare an antibody-drug conjugate, thereby specifically targeting and delivering Erianin to tumor cells, maximizing the therapeutic effect while reducing off-target effects.

[0061] The method of the present invention provides a robust and versatile method for manufacturing antibody drug conjugates containing Erianin, which can optimize the binding process according to the specific functional groups present on the tumor targeting antibody. By selecting and synthesizing appropriate Erianin-loaded linkers and optimizing the binding conditions, the method ensures that the prepared antibody drug conjugates have high efficiency and targeted effects in cancer treatment.

[0062] According to a third aspect of the present invention, a composition is provided, comprising the above-mentioned antibody drug conjugate and a pharmaceutically acceptable additive. Pharmaceutically acceptable additives are not only fillers, but also are essential for the functionality and stability of the antibody drug conjugate in the composition. Additives may include various ingredients, such as excipients, stabilizing additives, carriers, diluents and solubilizing agents. Each of these ingredients plays a key role in ensuring the effective delivery and efficacy of the antibody drug conjugate and maintaining the long-term stability of the formulation.

[0063] According to a fourth aspect of the present invention, there is provided a use of an antibody drug conjugate in preparing a composition for treating cancer. More particularly, the method comprises administering a pharmaceutically effective dose of the antibody drug conjugate composition to a subject.

[0064] The antibody-drug conjugate is designed to selectively target tumor cells, minimize damage to healthy tissue, and reduce the side effects commonly associated with traditional chemotherapy. By delivering potent cytotoxic drugs such as Erianin directly to cancer cells via tumor-targeting antibodies, the antibody-drug conjugate can enhance the therapeutic effect and ensure that the drug is concentrated where it is most needed - within the tumor microenvironment.

[0065] This use is applicable to a variety of cancers, making it a versatile and valuable tool in oncology. Cancers that can be treated by this use include, but are not limited to, breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, and endometrial cancer. In addition, this use is also effective for kidney cancer, bladder cancer, thyroid cancer, melanoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, and various types of leukemia (such as glioblastoma, astrocytoma, medulloblastoma, and meningioma). This use also extends its treatment range to sarcoma, bone cancer, head and neck cancer, as well as testicular cancer, oral cancer, and anal cancer. It also shows promise in the treatment of mesothelioma, neuroblastoma, retinoblastoma, and bile duct cancer. This use covers a wide range of cancer types, highlighting its potential as a comprehensive treatment option in the field of oncology.

[0066] When implemented, the use involves administering an antibody drug conjugate at a pharmacologically effective dose, i.e., a dose sufficient to achieve the desired therapeutic effect (reduction or elimination of cancer cells in the subject's body). The specific dose and dosing schedule will be tailored to the individual patient's condition, such as the type and stage of cancer, the patient's overall health, and the presence of other medical conditions.

[0067] The present invention will be further illustrated by the following non-limiting examples.

[0068] Example

[0069] Materials and methods

[0070] Preparation of activated carbonate 1

[0071] 150 mg (0.47 mmol) of Erianin, 190 mg (0.94 mmol) of p-nitrophenyl chloroformate and 150 μL (1.88 mmol) of pyridine were dissolved in 10 mL of dichloromethane. After stirring at room temperature for 4 hours, the solvent was evaporated under reduced pressure and then purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate, volume ratio 3:1) to obtain product 1 with a yield of 220 mg (96%). H NMR spectrum 1 H NMR (600MHz,CDCl3):δ8.31(d,J=9.0Hz,2H),7.49(d,J=9Hz,1H),7.03-7.04(m,2H),6.93(d,J=8.4Hz,1H),6.33(s,1H),3.88(s,3H),3.82(s,3H),3.81(s,6H),2.83-2.90(m,4H). 13 C NMR (150 MHz, CDCl3): δ 155.67, 153.22, 150.75, 149.16, 145.65, 139.56, 137.14, 136.37, 134.61, 127.74, 125.5, 122.16, 121.83, 112.69, 105.54, 61.01, 56.29, 56.18, 38.32, 37.06. High resolution mass spectrometry HRMS (electrospray ionization): C 25 H 26 NO9 + [M+H] + Calculated m / z: 484.1608; found: 484.1617.

[0072] Preparation of compound 2

[0073] Dissolve 1 (220 mg, 0.46 mmol), methyl (2-(methylamino)ethyl) carbamic acid tert-butyl ester (171 mg, 0.91 mmol) and triethylamine (0.25 mL, 1.82 mmol) in 10 mL of dichloromethane, stir at room temperature overnight, evaporate the solvent under reduced pressure, and purify by silica gel column chromatography (eluent: n-hexane / ethyl acetate, volume ratio 3:1) to obtain compound 2 (240 mg, 99%). H NMR spectrum 1 H NMR (600MHz,CDCl3):δ6.84-6.97(m,3H),6.37(s,1H),3.83(s,6H),3.82(s,3H),3.80(s,3H),3.43-3.60(m,4H),3.04-3.14(m,3H),2.94(s,3H),2.83(s,4H),1.46(s,9H). 13 C NMR (150 MHz, CDCl3): δ 154.60, 153.18, 150.00, 137.66, 136.23, 134.29, 126.26, 123.49, 123.35, 115.70, 112.43, 112.16, 105.46, 61.01, 56.19, 56.03, 38.40, 37.19, 35.64, 34.94, 28.58. High resolution mass spectrometry (electrospray ionization): C 28 H 41 N2O8 + [M+H] + Calculated m / z: 533.2863; found: 533.2861.

[0074] Preparation of Erianin Precursor 3

[0075] Compound 2 was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2 mL) was added, and after stirring at room temperature for 1 hour, the solvent was removed in vacuo. The crude product 3 was used directly in subsequent experiments without purification.

[0076] Preparation of compound 4

[0077] 3 (200 mg, 0.46 mmol), Fmoc-Val-Cit-PAB-PNP (180 mg, 0.23 mmol) and N,N-diisopropylethylamine (0.82 mL, 4.69 mmol) were dissolved in dimethylformamide (5 mL) and stirred at room temperature overnight. The solvent was then evaporated under reduced pressure and purified by silica gel column chromatography (eluent: chloroform / methanol, volume ratio 50:1) to obtain compound 4 (192 mg, 77%). H NMR spectrum 1H NMR (600MHz, DMSO-d6): δ10.08(s,1H),8.14(d,J=7.2Hz,1H),7.89(d,J=7.8Hz,2H),7.73-7.76(m,2H ),7.33-7.58(m,3H)7.28-7.31(m,6H),6.94-7.03(m,3H),6.48-6.53(m,2H),5.97(s,1H),5.42(s,2H ),5.02(s,2H),4.30(s,1H),4.22-4.23(m,3H),3.93(t,J=7.8Hz,1H),3.69-3.72(m,9H),3.60(s,3H),3.42-3.53(m,4H),2.77-3.02(m,12H),2.00-2.03(m,1H),1.35-1.70(m,4H),0.84-0.88(m,6H). 13 C NMR (150MHz, DMSO-d6): δ171.29,170.59,158.90,156.13,155.65,155.49,155.38,153.83,153.64,152.64,149.59,143.92,143.78,140.72,1 39.87,139.73,138.59,137.24,137.15,135.56,133.91,133.81,131.7 8,131.55,128.45,128.34,128.25,127.65,127.08,125.98,125.38,12 3.13,120.09,119.01,112.53,112.42,112.31,105.60,79.19,65.69,60.08,59.94,55.76,55.71,53.10,46.70,46.47,46.29,46.03,45.92,45.52,38.60,37.56,37.45,36.14,36.07,34.74,34.67,34.60,34.53,34.19,34.06,30.47,29.49,26.80,19.23,1819.28. High resolution mass spectrometry (electrospray ionization): C 70 H 71 N7O 13 C 57 H 71 N7O 13 + [M+H] + m / z calculated value: 1060.5032; actual value: 1060.5020.

[0078] Preparation of Erianin-loaded Linker 5

[0079] Compound 4 (172 mg, 0.16 mmol) was dissolved in 5 mL of dimethylformamide containing 20% ​​piperidine, reacted at room temperature for 30 minutes, and then the solvent was removed under vacuum. After the intermediate was reacted with 6-(maleimido)hexanoic acid succinimidyl ester (100 mg, 0.32 mmol) and triethylamine (0.11 mL, 0.81 mmol) in 5 mL of dimethylformamide for 2 hours, the solvent was evaporated under reduced pressure, and purified by silica gel column chromatography (eluent: chloroform / methanol, volume ratio 20:1) to obtain 5 (130 mg, 77%). H NMR spectrum 1 H NMR (600MHz, DMSO-d6): δ9.99(s,1H),8.09(d,J=7.2Hz,1H),7.81(d,J=8.4Hz,1H),7.50-7.58(m, 2H),7.21-7.35(m,2H),6.93-7.03(m,5H),6.48-6.52(m,2H),5.97(s,1H),5.41(s,2H),5.01(s,2H ),4.36-4.37(m,1H),3.72(t,J=7.8Hz,1H),3.70-3.73(m,9H),3.60(s,3H),3.41-3.49(m,6H),2.77-3.02(m,12H),2.12-2.16(m,2H),2.00-2.03(m,1H),1.04-1.48(m,10H),0.81-0.85(m,6H). 13 C NMR (150 MHz, DMSO-d6): δ 172.72, 171.75, 171.54, 171.04, 159.33, 153.09, 150.04, 139.06, 136.01, 134.91, 128.93, 126.42, 123.60, 119.44, 106.06, 79.65, 60.40, 58.01, 56.49, 56.17, 53.56, 37.47, 35.39, 31.17, 30.85, 29.76, 28.23, 27.28, 26.25, 25.69, 19.71, 19.03, 18.65. High resolution mass spectrometry (electrospray ionization): C 70 H 71 N7O 13 C 57 H 71 N7O 13 + [M+H] +m / z calculated: 1031.5090; found: 1031.5092.

[0080] Preparation and characterization of trastuzumab-eranine antibody-drug conjugates

[0081] Trastuzumab (MCE, 2.5 mg / mL) was dissolved in a borate buffer at pH 8.0, tris(2-carbonylethyl)phosphine hydrochloride (10 equivalents) was added, and the reduction of disulfide bonds was completed within 1.5 hours. Then, the Erianin-loaded linker 5 (12 equivalents) dissolved in a 2% dimethyl sulfoxide solution was added to the reaction mixture and stirred for 2 hours. The synthesized antibody-drug conjugate was purified using a desalting centrifugal column (Thermo Fisher, 7K MWCO), and the protein concentration was subsequently determined by BCA protein quantification, with a measured yield of 87%, and analyzed by Thermo Scientific μDrop and reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis (12% acrylamide and 4% stacking gel).

[0082] Antibodies and antibody drug conjugates were analyzed at a flow rate of 0.3 ml / min on a MAbPac reverse phase HPLC column (4 μm, 3 mm×50 mm), and electrospray ionization mass spectra were recorded using an Agilent 6546 LC / Q-TOF mass spectrometer. The analysis conditions were: mobile phase A = 0.1% formic acid in deionized water, mobile phase B = 0.1% formic acid in acetonitrile; gradient: 80% A+20% B in the first 0.5 min, changed to 40% A+60% B in 3 min, held for 5 min, then changed back to 80% A+20% B in 0.5 min, held for 1 min.

[0083] The drug / antibody ratio was determined by the chromatographic peak area in the deconvoluted mass spectra. In addition, the antibody and antibody drug conjugate were analyzed by size exclusion chromatography on a Zenix-C SEC-300 column (3 μm, 7.8 mm×300 mm) with an isocratic mobile phase (150 mM sodium phosphate, pH 7.0) at 1.0 mL / min.

[0084] In MAbPac TM Hydrophobic interaction chromatography was performed on a HIC-Butyl HPLC column (5 μm, 4.6 mm×100 mm) at a flow rate of 1.0 ml / min. The analysis conditions were: mobile phase A = 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 / isopropanol (95:5 v / v), mobile phase B = 50 mM sodium phosphate, pH 7.0 / isopropanol (80:20 v / v); gradient: 100% A+0% B in 1 minute, changed to 0% A+100% B in 14 minutes, maintained for 5 minutes, then changed back to 100% A+0% B in 1 minute, maintained for 4 minutes.

[0085] Flow cytometry

[0086] Different cells (5x 10 5 Cells / tube) were suspended in FACS staining buffer (i.e., 2% fetal bovine serum in phosphate-buffered saline) and placed in a 1.5 mL microcentrifuge tube. After trastuzumab, antibody-drug conjugates, or IgG control antibodies were cultured with cell suspensions (100 μL) at 4°C for 1 hour, the cell suspensions were centrifuged at 1000 rpm for 3 minutes at 4°C, and washed twice with 1 mL staining buffer to remove unbound antibodies; the cells were then cultured with secondary antibodies at 4°C for 1 hour, centrifuged again, and washed twice with buffer, then resuspended in staining buffer, and the mean fluorescence intensity (MFI) of the cells was subsequently measured using a Beckman Cytoflex S flow cytometer. For the cell internalization assay, cells were treated with trastuzumab or antibody-drug conjugates at 4°C for 1 hour, cultured at 37°C for different time periods, and stained with secondary antibodies. The degree of internalization was judged by the percentage decrease in the mean fluorescence intensity of cells cultured at 37°C compared with the 4°C control group, where the internalization efficiency was calculated according to the following formula: Internalization efficiency (%) = [(average fluorescence intensity of cells at 4°C - average fluorescence intensity of cells incubated at 37°C) / average fluorescence intensity of cells at 4°C] × 100%.

[0087] Confocal microscopy

[0088] Different cells (1x10 5 After 24 hours of culture, the culture medium was removed and washed with ice-cold PBS, and the cells were placed on ice; then the cells were incubated with Alexa Fluor-labeled TM Antibody drug conjugates with 647NHS ester (Invitrogen) were co-incubated at 4°C for 1 hour, then the solution was removed and the cells were rinsed twice with ice-cold PBS and observed using a Zeiss LSM900 confocal microscope. To observe the internalization process, the cells were cultured at 37°C for 4 hours or 24 hours, and after another 24 hours, lysosomes were further stained with a lysosomal green fluorescent probe (LysoTracker Green DND-26).

[0089] Example 1. Preparation of antibody-drug conjugates of trastuzumab and Erianin

[0090] Because trastuzumab has a high binding affinity and specificity for HER2, trastuzumab has been widely studied in the development of antibody drug conjugates and has been approved for targeting a variety of HER2-overexpressing cancers. Currently, Fam-trastuzumab deruxtecan-nxki and Ado-trastuzumab emtansine are two HER2-specific antibody drug conjugates approved by the U.S. Food and Drug Administration for the treatment of early and metastatic breast cancer. Therefore, the present invention selects trastuzumab as a tumor-targeting antibody and conjugates it to Erianin ( via a Val-Cit dipeptide unit cleavable by cathepsin B and a short self-consumable linker. Figure 1 ). After the interchain disulfide bonds of the antibody are reduced, the linker is linked to the cysteine ​​residue of the antibody with the maleimidocaproyl moiety through Michael addition reaction. When the antibody-drug conjugate binds to the HER2 receptor on the surface of cancer cells, it can be internalized into the lysosome through receptor-mediated endocytosis and then digested by cathepsin B, resulting in the cleavage of the linker and the release of the drug.

[0091] Figure 2 The synthetic pathways of Erianin precursor and Erianin-loaded linker are shown. Due to the simple chemical structure of Erianin, it can be easily modified through its phenolic hydroxyl group. First, Erianin was converted into an activated carbonate 1, which was then condensed with ethylenediamine with tert-butyloxycarbonyl protection to give compound 2; it was then deprotected with acid to give Erianin precursor 3, which was coupled with Fmoc-Val-Cit-PAB-PNP to generate compound 4; then the Fmoc protection was removed and condensed with 6-(maleimido)hexanoic acid succinimidyl ester to give Erianin-loaded linker 5.

[0092] Finally, trastuzumab was coupled to linker 5 loaded with Erianin using a conventional cysteine ​​coupling method ( Figure 3 ), wherein tris(2-carbonylethyl)phosphine hydrochloride was used to reduce the four interchain disulfide bonds of the antibody to obtain the thiol groups of the free cysteine ​​residues for Michael addition reaction with the linker 5 loaded with Erianin. After the unreacted 5 was removed by Zeba desalting centrifugal column (7kDa MWCO), the antibody drug conjugate of trastuzumab and Erianin was obtained, and its protein concentration was determined by the standard bicinchoninic acid BCA method, and its yield was determined to be 87%.

[0093] Example 2. Characterization and evaluation of the antibody drug conjugate of trastuzumab and Erianin

[0094] The antibody-drug conjugate was comprehensively analyzed by multiple methods. Figure 4AAs shown in , it can be seen from the analysis of reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis that after trastuzumab is coupled with eranine, the migration positions of its light chain and heavy chain fragments are both in a larger kilodalton range; Figure 4B The UV-visible absorption spectrum of the antibody drug conjugate shown in FIG. 4 shows the absorption peaks of Erianin and Trastuzumab; through reversed-phase high-performance liquid chromatography, since both the light chain and heavy chain fragments of the antibody are coupled to hydrophobic molecules, their retention times are prolonged ( Figure 4C ); further mass spectrometry analysis revealed a drug / antibody ratio of 8, which is the theoretical maximum drug loading number for conventional interchain cysteine ​​conjugation ( Figure 4D ); Under native non-denaturing conditions, no significant aggregation of the antibody-drug conjugate was observed in the size exclusion chromatogram ( Figure 4E ), and the drug was further confirmed to be uniformly distributed by hydrophobic interaction chromatography ( Figure 4F ); and by enzyme-linked immunosorbent assay, the binding of antibody-drug conjugates to HER2 protein also showed dose-dependency, with the half-maximal effect concentration (EC 50 value) was 0.73±0.03nM, which was not significantly different from the half-maximal effect concentration value of trastuzumab (0.29±0.02nM), indicating that the chemical modification of the antibody did not significantly affect the original binding affinity of the antibody to the HER2 protein.

[0095] Example 3. In vitro test of antibody drug conjugates of trastuzumab and Erianin

[0096] Selective binding and internalization of the antibody drug conjugates were examined by flow cytometry and confocal microscopy using two HER2-overexpressing cell lines: SK-BR-3 breast cancer cells and SK-OV-3 ovarian cancer cells, and two HER2-negative cell lines: MDA-MB-231 breast cancer cells and MCF-10A mammary epithelial cells. 3 cells / well) were inoculated in a 96-well microplate and cultured overnight; then different concentrations of Erianin, trastuzumab and antibody-drug conjugates were added to the wells and cultured for 6 days; after removing the culture medium, the cells were fixed with phosphate-buffered saline containing formaldehyde (3%; 50μL) and stained with NBB reagent (0.05%, 0.1M sodium acetate, 9% acetic acid; 50μL) overnight; the cells in the wells were gently washed three times with deionized water, and then dissolved in sodium hydroxide solution (50mM; 100μL), and finally the absorbance of each well was measured by a microplate analyzer at a wavelength of 620nm to detect cell activity. The above experiment was repeated three times, and the half inhibitory concentration (IC 50 Values) are expressed as mean ± standard deviation.

[0097] like Figure 5A and Figure 5B As shown in the figure, the antibody drug conjugate and trastuzumab will only specifically bind to HER2-positive cancer cells at 4°C, and will not bind to HER2-negative cells. After incubation at 37°C, the internalization efficiency of the antibody drug conjugate in HER2-positive cancer cells was detected and semi-quantitatively analyzed by flow cytometry. The results showed that the internalization efficiency of the antibody and the antibody drug conjugate was similar, and the internalization rate reached 80% after 24 hours ( Figure 5C-5D ). The location of the antibody drug conjugate in the cell was further examined by Alexa 647 labeling ( Figure 5E ), where at 4°C, the antibody drug conjugate was mainly located on the cell membrane of HER2-positive cancer cells and did not appear in HER2-negative cell lines; however, when cultured at 37°C, receptor-mediated endocytosis was promoted, so the antibody drug conjugate appeared as a red dot structure in HER2-positive cancer cells; further lysosomal green fluorescent probe (LysoTracker Green) staining ( Fig. 5F ), the results showed that some of the red dots representing the antibody drug conjugates were located in the lysosome, indicating that the antibody drug conjugates will enter the lysosomal degradation pathway after internalization into HER2-positive cancer cells. In addition, the antiproliferative activities of the antibody drug conjugates, trastuzumab and Erianin were compared ( Figure 5G-5I ), the results showed that Erianin exhibited extremely high cytotoxicity against all four cell lines, with half-inhibitory concentration values ​​ranging from 21 to 25 nM, while trastuzumab had no significant growth inhibitory effect on all cells. In contrast, the antibody drug conjugate had a significant cytotoxic effect on SK-OV-3 and SK-BR-3 cancer cells that overexpressed HER2, with half-inhibitory concentration values ​​of 0.6 μM and 0.9 μM, respectively, but had less effect on HER2-negative cell lines (Table 1).

[0098] Table 1. Half-inhibitory concentrations of antibody drug conjugates, trastuzumab and Erianin against different cell lines

[0099] Example 4. In vivo testing of the antibody-drug conjugate of trastuzumab and Erianin

[0100] To investigate the antitumor activity of antibody drug conjugates in the SK-OV-3 human ovarian cancer (HER2-positive) xenograft model ( Fig. 6A ), all animal experiments were performed in accordance with the guidelines approved by the University of Hong Kong Committee CULATR. Briefly, 5-8 week-old female BALB / cAnN-nu (nude mice) were housed in the Hong Kong University Laboratory Animal Center with free access to food and water. SK-OV-3 ovarian cancer cells (1×10 7100 μL) were suspended in PBS and injected subcutaneously into the right groin of each mouse once the tumor volume reached approximately 50 mm 3 At the time of the study, the tumor-bearing mice were divided into 5 groups (n = 5 in each group), namely: (A) PBS control group, (B) Erianin (50 mg / kg), (C) Trastuzumab (20 mg / kg), (D) Antibody Drug Conjugate (10 mg / kg) and (E) Antibody Drug Conjugate (20 mg / kg). The mice were treated with tail vein injection on days 0, 4, 8 and 12, for a total of four doses. Tumor size and body weight were continuously monitored during the treatment period. After 28 days, the mice were killed and the tumor weight was measured.

[0101] As described above, tumor-bearing nude mice were given 10 mg / kg or 20 mg / kg of the antibody-drug conjugate by intravenous injection for a total of four times, once every four days, and mice treated with trastuzumab (20 mg / kg) and Erianin (50 mg / kg) served as controls. Figure 6B As shown in the results, compared with the PBS control group, four injections of Erianin had no significant effect on tumor growth inhibition. The reason may be that Erianin is rapidly metabolized and cleared. Other studies have suggested the need for high doses and intraperitoneal injection of Erianin. Although trastuzumab (20 mg / kg) showed significant antitumor activity, treatment with 20 mg / kg of antibody-drug conjugates more significantly inhibited tumor growth, with an inhibition rate of about 70% ( Figure 6C and Fig.6D ), in addition, the body weight of mice did not change significantly during the treatment period, indicating that the treatment did not show obvious toxic side effects ( Fig. 6E ).

[0102] As used herein, the terms "approximately", "substantially", "essentially", "about" and "about" are used to describe and explain a small change. When used in conjunction with an event or situation, the term can refer to the situation where the event or situation occurs exactly, as well as the situation where the event or situation occurs approximately. For example, when used in conjunction with a numerical value, these terms can cover a range of variation less than or equal to ±10% of the numerical value, such as a range of variation less than or equal to ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1% or ±0.05%. The term "approximately" as used herein with respect to a given value or range generally refers to within the range of ±10%, ±5%, ±1% or ±0.5% of a given value or interval, which range can be understood herein as from one endpoint to another or between two endpoints. Unless otherwise stated, all ranges disclosed in this disclosure include endpoints.

[0103] In addition, unless otherwise explicitly stated, the terms "a" and "an" used in this article should be understood to include the meaning of one or more; in addition, the terms "first", "second" and "third" are used only as labels and are not intended to give numerical meanings or rankings according to their importance.

[0104] The above description has been presented to illustrate and describe the present invention, but is not intended to be exhaustive or to limit the present invention to the precise form disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0105] The above embodiments were chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention for various embodiments and with various modifications as are suited to the particular use contemplated.

Claims

1. An antibody-drug conjugate, characterized in that: include: Tumor-targeting antibodies; as well as at least one linker loaded with Erianin; The tumor targeting antibody and the at least one Erianin-loaded linker are bound by a covalent bond; wherein the Erianin-loaded linker comprises Erianin, an antibody linking portion, a cleavable bond, or may contain a self-consumable linker.

2. The antibody-drug conjugate of claim 1, wherein the tumor targeting antibody comprises at least one sulfhydryl group or at least one amino group.

3. The antibody drug conjugate of claim 2, wherein the antibody linking moiety comprises a thiol-reactive Michael acceptor group or an amino-reactive N-hydroxysuccinimide group. 4 . The antibody drug conjugate according to claim 3 , wherein the at least one thiol group reacts with the Michael acceptor group of the Erianin-loaded linker to produce a Michael addition reaction to bind the Erianin-loaded linker to the tumor targeting antibody. 5 . The antibody drug conjugate according to claim 3 , wherein the at least one amino group undergoes a condensation reaction with the N-hydroxysuccinimide group of the Erianin-loaded linker to bind the Erianin-loaded linker to the tumor targeting antibody.

6. The antibody drug conjugate according to claim 1, wherein the at least one Erianin-loaded linker has the following chemical formula: in, The antibody linking part is a maleimide group, the cleavable bond is a Val-Cit unit cleavable by cathepsin B, and the self-consumable linker is a self-consumable p-aminobenzyl and a biscarbamate linker. 7 . The antibody drug conjugate according to claim 1 , wherein the antibody drug conjugate has 1 to 8 Erianin-loaded linkers.

8. The antibody-drug conjugate according to claim 1, wherein the tumor-associated receptor targeted by the tumor-targeting antibody can be selected from HER2, TROP2, Nectin4, folate receptor α, epidermal growth factor receptor (EGFR), HER3 or c-MET.

9. The antibody-drug conjugate according to claim 1, wherein the tumor-targeting antibody is trastuzumab.

10. A method for preparing the antibody-drug conjugate according to claim 1, characterized in that: include: synthesizing the Erianin-loaded linker; as well as The coupling of the Erianin-loaded linker to the tumor-targeting antibody. 11 . The method according to claim 10 , wherein the method further comprises subjecting the tumor-targeting antibody to a reduction treatment before the conjugation to reduce its interchain disulfide bonds and form free thiol groups on the tumor-targeting antibody.

12. The method of claim 10, wherein the tumor targeting antibody comprises at least one sulfhydryl group or at least one amino group.

13. The method of claim 12, wherein the antibody linking moiety comprises a thiol-reactive Michael acceptor group or an amino-reactive N-hydroxysuccinimide group. 14 . The method according to claim 13 , wherein the at least one thiol group undergoes a Michael addition reaction with a Michael acceptor group of the Erianin-loaded linker to bind the Erianin-loaded linker to the tumor targeting antibody. 15 . The method according to claim 13 , wherein the at least one amino group undergoes a condensation reaction with an N-hydroxysuccinimide group of the Erianin-loaded linker to bind the Erianin-loaded linker to the tumor-targeting antibody.

16. A composition, characterized in that include: The antibody-drug conjugate according to claim 1; as well as Pharmaceutically acceptable additives.

17. The composition according to claim 16, wherein the pharmaceutically acceptable additives include excipients, stabilizers, carriers, diluents and solubilizing agents.

18. Use of the antibody-drug conjugate according to claim 1 in preparing a composition for treating cancer, characterized in that: include: A pharmaceutically effective dose of the antibody-drug conjugate composition according to claim 1 is administered to a subject.

19. The method of claim 18, wherein the cancer comprises breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, liver cancer, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer, melanoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, glioblastoma, astrocytoma, medulloblastoma, meningioma, sarcoma, bone cancer, head and neck cancer, testicular cancer, oral cancer, anal cancer, mesothelioma, neuroblastoma, retinoblastoma and bile duct cancer.

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