Antibody-drug complexes and pharmaceutical components including these antibody-drug complexes.

Abstract

The invention relates to a compound and an antibody-drug complex comprising such a compound, in the field of biomedical science. The invention relates to a pharmaceutical composition comprising this antibody-drug complex. Specifically, this antibody-drug complex is an antibody-drug complex against HER3. The antibody-drug complex exhibits good stability and biological activity.

Description

Compound and antibody-drug conjugate containing the compound

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202311037231.5 and application date August 16, 2023, and claims its priority. The entire content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present invention belongs to the field of biopharmaceuticals and relates to a compound and an antibody-drug conjugate containing the compound. The present invention also relates to a pharmaceutical composition containing the antibody-drug conjugate and its use. Specifically, the antibody-drug conjugate is an anti-HER3 antibody-drug conjugate. Background Art

[0004] Cancer is the number one killer that endangers human life and health. Traditional treatments include surgery, chemotherapy, and radiotherapy, but these methods usually have toxic side effects on normal cells while killing cancer cells, resulting in a narrow treatment window. Immunotherapy is an important cancer treatment method that usually uses the body's own immune mechanism to fight cancer cells. Due to its unique advantages of high targeting and few side effects, it is the preferred treatment option for cancer patients with small early lesions. However, for advanced solid tumors, immunotherapy has limited effects and needs to be combined with other methods to fight cancer cells (Li Yunfeng, Xin Jie, Li Jing, et al. Research progress of antibody-drug conjugate technology [J]. Chinese Journal of New Drugs and Clinical Medicine, 2022, 41(12):10.).

[0005] Antibody-drug conjugates (ADCs) are emerging large-molecule targeted drugs that combine the potent killing effects of traditional small-molecule chemotherapies with the tumor-targeting properties of antibody-based drugs. ADCs consist of three main components: an antibody that selectively recognizes antigens on the surface of cancer cells, a payload responsible for killing cancer cells, and a linker that connects the antibody and payload. Once ADCs enter the bloodstream, the antibody portion specifically binds to antigens on the surface of tumor cells. The resulting complex enters the cell via receptor-mediated endocytosis (which can be categorized into four types: clathrin-mediated endocytosis, cave-like invagination, macropinocytosis, and clathrin- and caveolin-independent endocytosis). Cleavable linkers are sensitive to environmental factors within tumor cells and are cleaved by specific pH levels, proteases, or certain chemicals. ADCs carrying non-cleavable linkers are digested by lysosomes, releasing the drug. The small molecules of certain ADC drugs can penetrate the cell membrane and further kill surrounding cancer cells, which is the bystander killing effect (Khongorzul, Puregmaa, et al. "Antibody-Drug Conjugates: A Comprehensive Review." Mol Cancer Res 1 (2020).;Kostova Vesela, Désos Patrice, Starck et al.The Chemistry Behind ADCs.[J].Pharmaceuticals(Basel),2021,14:undefined.).

[0006] Payload accumulation within tumors is limited, so tumor killing is required at subnanomolar concentrations. Furthermore, they must possess excellent stability to maintain structural integrity and activity in the circulatory system and lysosomes. Common payloads include tubulin inhibitors and DNA-damaging agents. Tubulin inhibitors kill tumor cells and inhibit their rapid proliferation by inhibiting the production and aggregation of tubulin. Tubulin inhibitors commonly used in ADC construction include maytansines, auristatins, and tubulysins. DNA-damaging agents are divided into three major categories based on their mechanisms of action: DNA double-strand breakers, DNA intercalators, and DNA alkylating agents. DNA is crucial in the growth and proliferation of cells; damaging it can effectively kill tumor cells and inhibit their rapid proliferation. DNA damaging agents commonly used in ADC construction include pyrrole benzazepines and indolechlorobenzazepines (such as pyrrolo[2,1-c][1,4]benzodiazepine, PBD), dukamycin, camptothecin (CPT) and its derivatives (such as topoisomerase I inhibitors Dxd and SN-38), calicheamicin, etc. (Dan Nirnoy, Setua Saini, Kashyap Vivek K et al. Antibody-Drug Conjugates for Cancer Therapy: Chemistry to Clinical Implications. [J]. Pharmaceuticals (Basel), 2018, 11: undefined.).

[0007] HER3 is a member of the human epidermal growth factor receptor (HER) family. It consists of three components: an extracellular ligand-binding domain, an α-helical transmembrane domain, and an intracellular tyrosine kinase domain. HER3 is overexpressed in a variety of aggressive tumors, including breast tumors, non-small cell lung cancer (NSCLC), metastatic colon cancer, head and neck cancer, pancreatic cancer, ovarian cancer, clear cell sarcoma, gastric cancer, and skin cancer. HER3 binds to its ligand, neuregulin (NRG). Although it lacks intrinsic tyrosine kinase activity, HER3 can form heterodimers with HER2, inducing cross-phosphorylation of highly conserved intracellular kinase residues. This allows one receptor to phosphorylate specific tyrosine residues on the other receptor, thereby recruiting and activating downstream proteins, directly activating the PI3K / AKT pathway and triggering a signaling cascade that promotes tumor development and progression.

[0008] In summary, the development of ADC drugs targeting HER3 with significant efficacy and minimal toxic side effects has broad prospects.

[0009] Summary of the Invention

[0010] Through in-depth research and creative work, the present inventors have developed a compound that can serve as a linker and payload, and further developed an antibody-drug conjugate. The resulting antibody-drug conjugate exhibits excellent stability and / or biological activity. This invention provides the following:

[0011] One aspect of the present invention relates to a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises: a compound of formula III, n alanines, and a compound of formula IV connected in sequence,

[0012] in,

[0013] n is 2, 3, 4, 5 or 6;

[0014] The compound of formula III, n alanines, and the compound of formula IV are independently connected directly (eg, via a chemical bond) or via a chemical group.

[0015] Pharmaceutically acceptable salts of the compounds of the present invention include conventional salts formed with pharmaceutically acceptable inorganic or organic acids, or inorganic or organic bases. Examples of suitable acid addition salts include salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, tannic acid, etc. Examples of suitable base addition salts include salts formed with sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine.

[0016] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof has a structural formula as shown in Formula I below:

[0017] in,

[0018] A represents alanine (alanine residue);

[0019] m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0020] n is 2, 3, 4, 5 or 6.

[0021] In some embodiments of the present invention, the compound or pharmaceutically acceptable salt thereof is as shown in the following formula II:

[0022] Another aspect of the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof, a linker and a payload,

[0023] Wherein, the linker and the effective load are any one of the compounds of the present invention or a pharmaceutically acceptable salt thereof;

[0024] Preferably, the linker is linked to the antibody or antigen-binding fragment thereof via one or more thioether bonds;

[0025] Preferably, the linker forms a thioether bond with a sulfur atom at the disulfide bond position of the antibody hinge region.

[0026] Without being bound by theory, the maleimide-N-group undergoes an alkylation reaction with the sulfhydryl group in the antibody to form a stable thioether bond.

[0027] In some embodiments of the present invention, the antibody-drug conjugate is an IgG antibody, preferably an IgG1 antibody.

[0028] In some embodiments of the present invention, the antibody-drug conjugate comprises an anti-HER3 antibody.

[0029] In some embodiments of the present invention, the antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region.

[0030] in,

[0031] The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3;

[0032] The light chain variable region comprises LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6.

[0033] In some embodiments of the present invention, the antibody-drug conjugate, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 9.

[0034] In some embodiments of the present invention, in the antibody-drug conjugate, the heavy chain constant region of the antibody is Ig gamma-1 chain C region or Ig gamma-4 chain C region; and the light chain constant region is Ig kappa chain C region.

[0035] In some embodiments of the present invention, the antibody-drug conjugate, wherein the amino acid sequence of the heavy chain constant region of the antibody is as shown in SEQ ID NO: 11; and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 13.

[0036] In some embodiments of the present invention, the antibody-drug conjugate has an average number of linker-payloads conjugated to each antibody molecule of 1-8, 2-8, 3-8, 4-8, 5-8, 6-8 or 7-8.

[0037] In some embodiments of the present invention, the antibody-drug conjugate, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or diabody.

[0038] In some embodiments of the present invention, the antibody-drug conjugate, wherein the heavy chain constant region of the antibody is the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably the human IgG1 heavy chain constant region; the light chain constant region is the Ig kappa chain C region.

[0039] In some embodiments of the present invention, the antibody-drug conjugate has ADCC activity.

[0040] In some embodiments of the present invention, the antibody-drug conjugate, wherein,

[0041] "The EC of the antibody-drug conjugate binding to its targeted antigen 50 " and "the antibody or antigen-binding fragment thereof contained in the antibody-drug conjugate binds to the same antigen EC 50 "The same or substantially the same. The meaning of "substantially the same" here can be, for example:

[0042] The EC of the antibody-drug conjugate combined with its targeted antigen 50 " and "the antibody or antigen-binding fragment thereof contained in the antibody-drug conjugate binds to the same antigen EC 50” within ±10%, within ±15%, within ±20%, within ±25%, within ±30%, within ±35%, within ±40% or within ±45%.

[0043] The antibody-drug conjugate of the present invention can be used to treat or prevent tumors;

[0044] Preferably, the tumor is a HER3-positive tumor;

[0045] Preferably, the tumor is selected from one or more of lung cancer, clear cell sarcoma, skin cancer, kidney cancer, urothelial carcinoma, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer and penile cancer;

[0046] Preferably, the lung cancer is non-small cell lung cancer (NSCLC);

[0047] Preferably, the colon cancer is metastatic colon cancer.

[0048] Antibody-drug conjugates can be prepared by reacting a compound with an antibody or its reactive derivative and conjugating the compound of the present invention or a pharmaceutically acceptable salt thereof to the antibody via formation of a thioether bond in the disulfide bond portion of the hinge region of the antibody.

[0049] Another aspect of the present invention relates to a pharmaceutical composition comprising the antibody-drug conjugate according to any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0050] In another aspect, the present invention relates to use of the antibody-drug conjugate according to any one of the present invention in the preparation of a drug for treating or preventing tumors;

[0051] Preferably, the tumor is a HER3-positive tumor;

[0052] Preferably, the tumor is selected from one or more of lung cancer, clear cell sarcoma, skin cancer, kidney cancer, urothelial carcinoma, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer and penile cancer;

[0053] Preferably, the lung cancer is non-small cell lung cancer (NSCLC);

[0054] Preferably, the colon cancer is metastatic colon cancer.

[0055] Another aspect of the present invention relates to a method for treating or preventing tumors, comprising the step of administering an effective amount of the antibody-drug conjugate according to any one of the present invention to a subject in need thereof;

[0056] Preferably, the tumor is a HER3-positive tumor;

[0057] Preferably, the tumor is selected from one or more of lung cancer, clear cell sarcoma, skin cancer, kidney cancer, urothelial carcinoma, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer and penile cancer;

[0058] Preferably, the lung cancer is non-small cell lung cancer (NSCLC);

[0059] Preferably, the colon cancer is metastatic colon cancer.

[0060] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0061] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0062] As used herein, the term "no ADCC activity" means that there is no ADCC activity, ADCC activity cannot be detected, or the ADCC activity exceeds the detection limit of the instrument or detection method as detected by existing instruments or detection methods (such as the method described in Example 9).

[0063] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The assignment of amino acids to various regions or domains follows the definition of Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0064] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions, called complementarity-determining regions (CDRs). The heavy chain (H) CDRs comprise HCDR1, HCDR2, and HCDR3, while the light chain (L) CDRs comprise LCDR1, LCDR2, and LCDR3. In this invention, CDRs are defined using the IMGT numbering system. See Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF, and MhcSF [J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0065] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0066] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but it can also be obtained by using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0067] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature 1986; 321: 522 525; Reichmann et al., Nature, 1988; 332: 323 329; Presta, Curr. Op. Struct. Biol. 1992; 2: 593-596; and Clark, Immunol. Today 2000; 21: 397 402. In some cases, the antigen-binding fragments of antibodies are diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448 and Poljak RJ et al., Structure 1994; 2: 1121-1123).

[0068] As used herein, the term "single chain antibody (ScFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker. The VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 15) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 16) can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al, Protein Eng. 1995; 8: 725-731, Choi et al, Eur. J. Immunol. 2001; 31: 94-106, Hu et al, Cancer Res. 1996; 56: 3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293: 41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59.

[0069] As used herein, the term "antibody portion" refers to the antibody portion of the antibody-drug conjugate, which, in certain specific embodiments, is connected to the intermediate linker portion through a specific functional group, and the antibody portion can specifically bind to the antigen.

[0070] As used herein, the term "linker" refers to the part that connects the antibody and the cytotoxin, which is divided into cleavable and non-cleavable types. Cleavable linkers are mainly sensitive chemical bonds that can promote the cleavage of linkers and drugs according to the specific chemical species (such as glutathione, acidity and alkalinity, etc.) or enzyme concentrations of the body's internal environment, mainly through hydrazone bonds, disulfide bonds, and polypeptide forms. Non-cleavable linkers do not have built-in chemical bonds that can trigger cleavage. Antibodies need to be converted into amino acids through a proteolytic mechanism in cancer cells, and then release cytotoxic drugs with linkers and amino acid fragments, mainly through thioether forms.

[0071] As used herein, the term "payload" refers to the cytotoxin molecule in the antibody-drug conjugate that is primarily responsible for executing the cell-killing function. It is chemically synthesized and connected to the intermediate linker portion, enters the tumor cell through internalization, and releases the cytotoxin molecule under the action of lysosomes, thereby exerting an anti-tumor effect. Common targets are DNA in the cell nucleus and microtubules in the cytoplasm.

[0072] As used herein, the term "DAR (drug-to-antibody ratio)" refers to the average number of small molecule drugs conjugated per antibody molecule and is a key quality attribute of ADCs. Common characterization methods include UV-visible spectrophotometry, hydrophobic chromatography, reversed-phase chromatography, and mass spectrometry (MS), depending on the chemical nature of the linker and small molecule cytotoxin and the conjugation method (lysine-conjugated, cysteine-conjugated, or glycosyl-conjugated).

[0073] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0074] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0075] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0076] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The antigen is bound with an affinity (KD) of M or less.

[0077] As used herein, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies bind with a KD of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 The dissociation equilibrium constant (KD) of the antigen is 24 M or less. KD can be determined using methods known to those skilled in the art, such as using a Fortebio molecular interaction instrument.

[0078] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0079] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0080] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0081] Advantageous Effects of the Invention

[0082] The present invention achieves one or more of the following technical effects:

[0083] (1) Antibody-drug conjugates prepared using the compounds of the present invention, such as the anti-HER3 antibody-drug conjugate KA0013, have good biological activity.

[0084] (2) Antibody-drug conjugates prepared using the compounds of the present invention, such as the anti-HER3 antibody-drug conjugate KA0013, can effectively release the payload portion.

[0085] (3) The anti-HER3 antibody-drug conjugate KA0013 of the present invention can effectively bind to HER3.

[0086] (4) Antibody-drug conjugates prepared using the compounds of the present invention, such as the anti-HER3 antibody-drug conjugate KA0013, have internalization activity.

[0087] (5) Antibody-drug conjugates prepared using the compounds of the present invention, such as the anti-HER3 antibody-drug conjugate KA0013, have good stability in plasma.

[0088] (6) Antibody-drug conjugates prepared using the compounds of the present invention, such as the anti-HER3 antibody-drug conjugate KA0013, can specifically kill tumor cells.

[0089] (7) Antibody-drug conjugates prepared using the compounds of the present invention, such as the HER3 antibody-drug conjugate KA0013, have ADCC activity.

[0090] (8) Antibody-drug conjugates prepared using the compounds of the present invention, such as the anti-HER3 antibody-drug conjugate KA0013, can effectively inhibit tumor growth in vivo.

[0091] (9) Antibody-drug conjugates prepared using the compounds of the present invention, such as the HER3 antibody-drug conjugate KA0013, have good safety.

[0092] (10) Antibody-drug conjugates prepared using the compounds of the present invention, such as the HER3 antibody-drug conjugate KA0013, have a bystander killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1: Binding activity test results of anti-HER3 antibody-drug conjugate KA0013 to HER3 on the surface of SK-BR-3 cells expressing HER3 on the cell surface.

[0094] Figure 2: Internalization activity test results of the anti-HER3 antibody-drug conjugate KA0013 in MDA-MB-453 cells expressing HER3 on the cell surface.

[0095] Figure 3: Stability test results of nonspecific killing caused by cleavage of the linker-payload portion of the anti-HER3 antibody-drug conjugate KA0013.

[0096] Figure 4: Plasma stability test results of anti-HER3 antibody-drug conjugates KA0013 and KA0011.

[0097] FIG5 : In vitro detection of linker-payload efficiency of KA0013 and KA0011 by cathepsin B.

[0098] Figure 6: Indirect ELISA method was used to determine the binding activity of anti-HER3 antibody-drug conjugates KA0013 and KA0011 with the antigen human HER3-his.

[0099] Figure 7: Kinetic characteristic parameter detection results of anti-HER3 antibody-drug conjugate KA0013.

[0100] Figure 8: Kinetic characteristic parameter detection results of anti-HER3 antibody-drug conjugate KA0011.

[0101] Figure 9: Specific cell killing activity test results of anti-HER3 antibody-drug conjugates.

[0102] Figure 10: ADCC activity test results of anti-HER3 antibody-drug conjugates.

[0103] Figure 11: In vivo efficacy results of anti-HER3 antibody-drug conjugates in the HCC827 human lung cancer cell xenograft tumor model in CB-17 SCID mice.

[0104] Figure 12: Effects of anti-HER3 antibody-drug conjugates on body weight in the HCC827 human lung cancer cell transplantation model in CB-17 SCID mice. DETAILED DESCRIPTION

[0105] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0106] In the following experimental examples of the present invention, the positive control ADC drug KA0011 used is the Her3 antibody conjugate drug U3-1402 developed by Daiichi Sankyo Pharmaceutical Co., Ltd. of Japan, and its sequence information and preparation method are referenced from Chinese patent document CN 106163559 B.

[0107] In the following experimental examples of the present invention, the isotype control antibodies used, namely the IgG (i.e., anti-HEL) components in IgG-Dxd, IgG-KA0012, and IgG-KA0010, as well as hIgG1 and anti-HEL are all antibodies targeting human hen egg lysosomes (HEL). The variable region sequences of these antibodies are derived from Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1): 130-46.) published by Acierno et al., and their constant region fragments use Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region; IgG-DXd or IgG-KA0012, hIgG1, and anti-HEL were all produced in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0108] Preparation Example 1: Preparation of anti-HER3 antibody Patritumab

[0109] Amino acid sequence of the heavy chain variable region of Patritumab:

[0110] Nucleic acid sequence encoding the heavy chain variable region of Patritumab:

[0111] Amino acid sequence of the light chain variable region of Patritumab:

[0112] Nucleic acid sequence encoding the light chain variable region of Patritumab:

[0113] The heavy chain constant region of Patritumab uses the Ig gamma-1 chain C region; the light chain constant region uses the Ig kappa chain C region.

[0114] Amino acid sequence of the heavy chain constant region of Patritumab:

[0115] Nucleic acid sequence of the Patritumab heavy chain constant region:

[0116] Amino acid sequence of the light chain constant region of Patritumab:

[0117] Nucleic acid sequence of the light chain constant region of Patritumab:

[0118] The heavy and light chain cDNAs of Patritumab were cloned into the pcDNA3.1 vector to generate recombinant expression plasmids for the antibody Patritumab. The recombinant plasmids were transfected into 293F cells. The 293F cell culture fluid was purified and then tested.

[0119] The anti-HER3 monoclonal antibody Patritumab was produced.

[0120] According to the IMGT numbering system, the six CDRs of the anti-HER3 monoclonal antibody Patritumab are as follows:

[0121] HCDR1: GGSFSGYY (SEQ ID NO: 1)

[0122] HCDR2: INHSGST (SEQ ID NO: 2)

[0123] HCDR3:ARDKWTWYFDL(SEQ ID NO:3)

[0124] LCDR1:QSVLYSSSNRNY(SEQ ID NO:4)

[0125] LCDR2:WAS (SEQ ID NO:5)

[0126] LCDR3: QQYYSTPRT (SEQ ID NO: 6).

[0127] Preparation Example 2: Preparation of Mc-AAA-Dxd (KA0012)

[0128] Mc-AAA-Dxd is (maleimide-N-yl)-CH2CH2CH2CH2CH2-C(=O)-AAA-NH-CH2-O-CH2-C(=O)-(NH-DX), wherein the CAS number of Dxd is: 1599440-33-1.

[0129] Mc-AAA-Dxd is named KA0012 in the present invention, and its structural formula is shown in the following formula II:

[0130] Wherein Mc is a maleimide-N-group, and its structure is shown in the following formula III:

[0131] Wherein the structural formula of -(NH-DX) is shown in the following formula IV:

[0132] The synthesis steps of KA0012 are as follows: Step 1 to Step 10.

[0133] Step 1: Preparation of intermediate HM-1315_6B_2

[0134] The raw material HM-1315_6B_1 (450.0 g, 1.44 mol, 1 eq) was suspended in DCM (dichloromethane) (4.5 L, 10 V) and the temperature was controlled at 0-5°C. HOSU (182.9 g, 1.59 mol, 1.1 eq) and EDCI (304.8 g, 1.59 mol, 1.1 eq) were added sequentially. After complete addition, the reaction solution dissolved until clear. The reaction was allowed to react at 10-20°C for 3 hours. Semi-saturated brine (1.0 L) was added to the reaction solution, stirred, and allowed to stand for separation. The organic phase was then washed once with semi-saturated brine (1.0 L) and twice with saturated brine (1.0 L x 2). The mixture was dried over anhydrous sodium sulfate and concentrated to yield 579.0 g of a white solid in a 98% yield.

[0135] 1 H NMR (400MHz, DMSO) δ8.13(d,J=7.4Hz,1H),7.89(d,J=7.5Hz,2H),7.71(t,J=6.7Hz,2H),7.42(t,J=7.3Hz,2H),7.33(t,J=7.4Hz ,2H),4.52(t,J=7.4Hz,1H),4.41-4.17(m,3H),2.79(d,J=15.5Hz,4H),2.59(s,1H),1.46(d,J=7.4Hz,3H).LCMS[M+23]:431.1.

[0136] Step 2: Preparation of intermediate HM-1315_1

[0137] The raw material HM-1315_6B_2 (579.0 g, 1.42 mol, 1.0 eq) was dissolved in acetonitrile (2895 mL, 5 v), followed by the addition of glycine (117.2 g, 1.56 mol, 1.1 eq) and water (2895 mL, 5 v). The mixture was cooled to -5-0°C, and DIPEA (N,N-diisopropylethylamine) (275.3 g, 2.13 mol, 1.5 eq) was slowly added. After complete addition, the reaction mixture was allowed to react at 20-25°C for 3 hours. Ethyl acetate (1.0 L) was added to dilute the reaction mixture, and the pH was adjusted to 3-4 with dilute hydrochloric acid (0.5 N). Ethyl acetate (500 mL) was then added to separate the two phases. The aqueous phase was extracted twice with ethyl acetate (1.0 L x 2). The organic phases were combined, washed once with saturated brine (1.5 L), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was slurried with methyl tert-butyl ether / ethyl acetate (2895 mL / 289.5 mL, 5 v / 0.5 v) for 2 h, filtered, and the filter cake was pumped dry with an oil pump to obtain 447.2 g of a white powdery solid with a yield of 85%.

[0138] 1 H NMR (400MHz, DMSO) δ8.12(t,J=5.5Hz,1H),7.89(d,J=7.5Hz,2H),7.73(t,J=7.1Hz,2H),7.54(d,J=7.8Hz,1H),7.37(dt,J=34.6,7.1Hz,4H ),4.24(dd,J=15.6,5.9Hz,3H),4.13-4.05(m,1H),3.75(dd,J=14.9,5.7Hz,3H),1.23(d,J=7.2Hz,3H).LCMS[M+l]:369.1,[M+23]:392.1.

[0139] Step 3: Preparation of intermediate HM-1315_2

[0140] The raw material HM-1315_1 (474.0 g, 1.29 mol, 1.0 eq) was dissolved in ultra-dry DMF (2370 mL, 5 v) under nitrogen. Lead tetraacetate (1143.9 g, 2.58 mol, 2.0 eq), anhydrous copper acetate (234.3 g, 1.29 mol, 1.0 eq), and acetic acid (170.5 g, 2.84 mol, 2.2 eq) were added. After addition, the reaction temperature was raised to approximately 50°C. The reaction mixture was placed in an oil bath at 50-55°C for 0.5 h. The reaction mixture was cooled, and ethyl acetate (2370 mL, 5 v) was added, followed by ice water (2370 mL, 5 v). A black, viscous solid precipitated. The black insoluble matter was filtered out using a Buchner funnel, and the filtrate was separated using a separatory funnel. The aqueous layer was extracted twice with ethyl acetate (2.0 L × 2), and the organic phases were combined, washed twice with water (1.0 L × 2), and then washed three times with saturated brine (1.0 L × 3), dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was slurried with methyl tert-butyl ether (2370 mL) at room temperature for 2 h, filtered, and the filter cake was pumped dry with an oil pump to obtain 367.7 g of white solid product with a yield of 74%.

[0141] 1 H NMR (400MHz, DMSO) δ8.90 (s, 1H), 7.89 (d, J = 7.5Hz, 2H), 7.73 (t, J = 6.6 Hz,2H),7.59(d,J=7.6Hz,1H),7.42(t,J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),5.10(d,J=6.7Hz,2H),4. 25(dd,J=20.3,6.5Hz,3H),4.12-4.01(m,1H),1.99(s,3H),1.22(d,J=7.2Hz,3H).LCMS[M+23]:405.0.

[0142] Step 4: Preparation of intermediate HM-1315_4

[0143] Dissolve the raw material HM-1315_2 (326.0 g, 0.85 mol, 1.0 eq) in THF (3260.0 mL, 10 v) and add HM-1315_3 (211.9 g, 1.27 mol, 1.5 eq). After addition, cool the reaction mixture to 0-10°C and slowly add a solution of lithium hydroxide (24.4 g, 1.02 mol, 1.2 eq) in water (163.0 mL, 0.5 v). After addition, warm the reaction mixture to room temperature (15-25°C) and allow to react for 1 hour. Ethyl acetate (1.5 L) and water (1.5 L) were added to the reaction solution, and the layers were separated. The aqueous layer was extracted once with ethyl acetate (1.0 L). The organic phases were combined, washed twice with saturated brine (1.0 L × 2), and dried over anhydrous sodium sulfate. The crude product was filtered and concentrated, and then purified by column chromatography with petroleum ether / ethyl acetate = 1 / 1 to elute the product. The product was collected, concentrated, and pumped dry with an oil pump to obtain 311.8 g of a white solid product with a yield of 74%.

[0144] 1 H NMR (400MHz, DMSO) δ8.74(s,1H),7.89(d,J=7.5Hz,2H),7.72(t,J=7.9Hz,2H),7.59(d,J=7.3Hz,1H),7.47-7.25(m,9H),5.13 (s,2H),4.64(d,J=6.7Hz,2H),4.31-4.18(m,3H),4.15(s,2H),4.07-3.95(m,1H),1.23(d,J=7.2Hz,3H).LCMS[M+23]:511.1.

[0145] Step 5: Preparation of intermediate HM-1315_7

[0146] The raw material HM-1315_4 (60.00 g, 122.82 mmol, 1.0 eq) was dissolved in DMAc (N,N-dimethylacetamide) (600.0 mL, 10 v), cooled to 0-5°C, and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (18.70 g, 122.82 mmol, 1.0 eq) was slowly added. After the addition, the mixture was reacted at 0-5°C for 1 hour. The reaction mixture was cooled to -5-0°C. PPTS (4-methylbenzenesulfonate) (30.86 g, 122.82 mmol, 1.0 eq), EDCI (23.54 g, 122.82 mmol, 1.0 eq), HOBT (1-hydroxybenzotriazole) (16.59 g, 122.82 mmol, 1.0 eq), and HM-1315_6 (42.27 g, 110.54 mmol, 0.9 eq) were added sequentially. The mixture was allowed to react at -5-0°C for 16 hours. After adding n-hexane (1200 mL, 20 vol), the mixture was allowed to stand for separation. The DMAc layer was washed four times with n-hexane (1.2 L x 4) and then poured into water (1800 mL, 30 vol). A large amount of white solid precipitated. The mixture was filtered using a Buchner funnel, and the filter cake was dried to obtain a crude white solid. Acetonitrile (300.0 mL, 5 v) was added to the reaction flask, and the white crude product obtained in the previous step was added, slurried for 1 hour, filtered, and the filter cake was pumped dry to obtain 56.2 g of white solid product with an 80% yield.

[0147] 1 H NMR (400MHz, DMSO) δ8.68(t,J=6.7Hz,1H),7.99(dd,J=15.4,7.2Hz,2H),7.89(d,J=7.5Hz,2H),7.72(t,J=7.0Hz,2H),7.54(d,J=7.4Hz,1H),7.46-7 .28(m,9H),5.14(s,2H),4.63(d,J=7.7Hz,2H),4.32-4.16(m,5H),4.13(s ,2H),4.09-4.01(m,1H),1.21(dd,J=7.0,2.6Hz,9H).LCMS[M+23]:654.3.

[0148] Step 6: Preparation of intermediate HM-1315_8

[0149] The raw material HM-1315_7 (2.59 g, 4.10 mmol, 1.0 eq) was suspended in THF (tetrahydrofuran) (51.8 mL, 20 v) and water (5.2 mL, 2 v). The temperature was cooled to 0-10°C, and 10% palladium on carbon (0.39 g, 0.15 w / w) was added. The atmosphere was replaced with hydrogen three times, and the reaction was carried out at 0-10°C for 16 hours. The palladium on carbon was removed by filtration, and the filter cake was rinsed with acetonitrile / water (10 mL / 10 mL). The filtrate was extracted with 2-methyltetrahydrofuran (50 mL). The organic phase was washed once with water (50 mL) and once with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to yield 2.0 g of a crude white solid. The crude product (2.0 g, 3.70 mmol, 1.0 eq) was suspended in dichloromethane (20.0 mL, 10 v). DBU (0.56 g, 3.70 mmol, 1.0 eq) was slowly added dropwise at 20-25°C. After the addition, the reaction solution partially cleared. The reaction was allowed to react at 20-25°C for 16 hours, during which time a white solid precipitated. Methyl tert-butyl ether (40.0 mL, 20 v) was added to the reaction solution, resulting in the precipitation of more white solid. The mixture was filtered, and the filter cake was transferred to a single-necked flask, acetonitrile (20.0 mL) was added, and the mixture was slurried for 2 hours. The mixture was filtered, and the filter cake was pumped dry using an oil pump to yield 0.5 g of a white solid product in a 38% yield.

[0150] 1 H NMR (400MHz, D2O) δ4.73 (q, J = 10.8Hz, 1H), 4.43-4.21 (m, 1H), 4.04-3.82 (m, 1H), 1.47 (d, J = 7.0Hz, 2H), 1.42 (d, J = 7.2Hz, 3H). LCMS [M+l]: 319.1.

[0151] Step 7: Preparation of intermediate HM-1315_9

[0152] The raw material HM-1315_8 (300 mg, 0.94 mmol, 1.0 eq) was dissolved in acetonitrile (3.0 mL, 10 v) and water (4.5 mL, 15 v), and HM-297D_9 (290 mg, 0.94 mmol, 1.0 eq) was added. The mixture was cooled to 0-10°C, and DIPEA (121 mg, 0.94 mmol, 1.0 eq) was added. After reacting at 0-10°C for 16 hours, the product (290 mg, 60% yield) was prepared and isolated as a white solid.

[0153] 1H NMR (400MHz, DMSO) δ8.61(t,J=6.7Hz,1H),7.97(dd,J=7.1,3.4Hz,2H),7.91(d,J=7.3Hz,1H),7.00(s,2H),4.66-4.52(m,2H),4.2 9-4.12(m,3H),3.95(s,2H),3.44-3.35(m,5H),2.08(t,J=7.4Hz,2H),1.54-1.40(m,4H),1.28-1.10(m,11H).LCMS[M+23]:534.3.

[0154] Step 8: Preparation of intermediate HM-582_9

[0155] Compound HM-582_7 (10 g, 39.98 mmol, 1.0 eq), compound HM-582_8 (10 g, 38.01 mmol, 0.95 eq), and pyridinium p-toluenesulfonate (6 g, 23.88 mmol, 0.60 eq) were added to a three-necked flask, and toluene (500 mL) was added. The mixture was reacted at 130-140°C for 48 hours to separate water. The reaction solution was cooled, filtered, washed with methyl tert-butyl ether (100 mL), and the solid was collected and dried to obtain the product (18.4 g, 96.5%).

[0156] 1 H NMR(DMSO)δ:8.43-8.55(m,1H),7.80(d,J=10.9Hz,1H),7.31(d,J=3.8Hz,1H),6.55(brs,1H),5.52-5.57(m,1H),5.42(s,2H), 5.16-5.20(m,2H),3.14-3.18(m,2H),2.39(s,3H),2.11-2.16(m,2H),1.80-1.92(m,5H),0.86-0.89(m,3H).LCMS[M+1]478.2.

[0157] Step 9: Preparation of intermediate HM-582_10

[0158] Compound HM-582_9 (30 g) was suspended in water (600 mL), and methanesulfonic acid (300 mL) was slowly added. The solid dissolved and an exotherm occurred. After nitrogen displacement, the reaction mixture was heated to 112°C for 7 h, cooled to room temperature, filtered, and the filter cake washed with water (100 mL). The filtrate was diluted with ethanol (4 L), and a solid precipitated. The mixture was stirred at room temperature for 20 min, filtered, and drained. The crude product was suspended in ethanol / water (4:1) (1 L), heated at reflux for 2 h, cooled to room temperature, filtered, and the solid washed with a small amount of ethanol and drained. After lyophilization, isotecan mesylate (16.7 g, 50%) was obtained.

[0159] 1 H NMR(DMSO)δ8.41-8.50(m,3H),7.86(d,J=10.8Hz,1H),7.33(s,1H),5.66-5.74(m,1H),5.37-5.44(m,2H),5.10(s,1H),3.27-3.32( m,1H),3.08-3.17(m,1H),2.41(s,3H),2.31(s,3H),2.17-2.24(m,1H),1.80-1.95(m,2H),0.88(t,J=7.3Hz,3H).LCMS[M+1]436.1.

[0160] Step 10: Preparation of HM-1315_10 (KA0012)

[0161] The starting material HM-1315_9 (150 mg, 0.29 mmol, 1.0 eq) was dissolved in DMAc (3.0 mL, 20 v), and HM-582_10 (156 mg, 0.29 mmol, 1.0 eq) was added. The mixture was cooled to 0-10°C, and HATU (2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (121 mg, 0.32 mmol, 1.0 eq) was added. 2,4,6-Trimethylpyridine (70 mg, 0.58 mmol, 2.0 eq) was added dropwise. The mixture was reacted at 0-10°C for 16 hours. LCMS showed complete conversion of the starting material, and the product (135 mg, 50% yield) was obtained from the preparative isolation as a light yellow solid.

[0162] 1H NMR (400MHz, DMSO) δ8.64(t,J=6.6Hz,1H),8.47(d,J=8.8Hz,1H),7.95(dd,J=9. 3,7.3Hz,2H),7.87(d,J=7.2Hz,1H),7.77(d,J=11.0Hz,1H),7.30(s,1H),6.99( s,2H),6.51(s,1H),5.60(d,J=8.0Hz,1H),5.42(s,2H),5.18(s,2H),4.61(dt,J =10.1,3.3Hz,2H),4.17(dt,J=14.1,7.1Hz,3H),3.98(s,2H),3.38(s,1H),3.16 (dd,J=14.1,6.4Hz,2H),2.38(s,3H),2.18(d,J=5.4Hz,2H),2.07(t,J=7.3Hz,2H),1.85(dd,J=15.0,7.4Hz,2 H), 1.51-1.39 (m, 4H), 1.17 (dt, J=14.5, 7.4Hz, 11H), 0.86 (t, J=7.3Hz, 3H). LCMS [M+l]: 929.3, [M+23]: 952.3.

[0163] Preparation Example 3: Preparation of anti-HER3 antibody-drug conjugate KA0013

[0164] The structures of KA0013 and KA0011 are shown in Table 1 below.

[0165] Table 1

[0166] An antibody-drug conjugate is obtained by forming a thioether bond at a disulfide bond site present in the hinge portion of an anti-HER3 antibody, wherein the anti-HER3 antibody is treated under reducing conditions and then reacted with a compound (such as Formula II) to produce the antibody-drug conjugate. Specifically, the antibody-drug conjugate can be produced by reacting the compound with an antibody having a sulfhydryl group.

[0167] Antibodies having thiol groups can be obtained using methods known in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). Examples include: reacting an antibody amino group with Traut's reagent; reacting an antibody amino group with N-succinimidyl S-acetylthioalkanoate, followed by reaction with hydroxylamine; reacting an antibody with N-succinimidyl 3-(pyridyldithio)propionate, followed by reaction with a reducing agent; and reacting an antibody with a reducing agent such as dithiothreitol, 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce disulfide bonds in the antibody hinge region to form thiol groups, but are not limited thereto.

[0168] Specifically, 0.3 to 10 molar equivalents of TCEP per disulfide bond within the antibody hinge region are used as a reducing agent and reacted with the antibody in a buffer containing a chelating agent to obtain an antibody in which the disulfide bonds within the antibody hinge region are partially or completely reduced. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA).

[0169] It can be used at a concentration of 1 mM to 20 mM. Examples of usable buffers include sodium phosphate, sodium borate, or sodium acetate solutions. Specifically, by reacting the antibody with TCEP at 4°C-37°C for 1 to 4 hours, an antibody having partially or completely reduced thiol groups can be obtained.

[0170] Meanwhile, by performing a reaction to add a thiol group to the drug-linker moiety, the drug-linker moiety can be coupled via a thioether bond.

[0171] By using 2 to 20 molar equivalents of the compound per antibody having a sulfhydryl group, an antibody-drug conjugate in which 2 to 8 drug molecules are coupled per antibody can be produced. Specifically, it is sufficient to add a solution in which the compound is dissolved to a buffer containing an antibody having a sulfhydryl group to carry out the reaction. Examples of buffers that can be used here include sodium acetate solution, sodium phosphate, and sodium borate. The pH of the reaction is 5 to 9, and more preferably the reaction is carried out at around pH 7. Examples of solvents for dissolving compound (1) include organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyridone (NMP).

[0172] The reaction can be carried out by adding a 1 to 20% v / v organic solvent solution of the compound to a buffer solution containing an antibody having a thiol group. The reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours.

[0173] After conjugation, the produced antibody-drug conjugates can be subjected to buffer exchange, and the antibody concentration and the average number of drug conjugates per antibody molecule can be measured to identify the antibody-drug conjugates.

[0174] Common operation A: Measurement of antibody concentration

[0175] Antibody concentration was measured using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the manufacturer's method. The absorption coefficient at 280 nm was estimated from the amino acid sequence of the antibody using a known calculation method (Protein Science, 1995, Vol. 4, 2411-2423). A different absorption coefficient at 280 nm was used for each antibody (1.3 mL mg cm-1 to 1.8 mL mg cm-1). In the case of KA0013, an absorption coefficient at 280 nm of 1.768 mL mg cm-1 was used as an estimated value based on its amino acid sequence.

[0176] Common Procedure B: Buffer Exchange for Antibody-Drug Conjugates

[0177] The antibody-drug conjugate was ultrafiltrated and concentrated using an ultrafiltration membrane (Millipore P3C030C00, 30 kDa) according to the manufacturer's instructions, and the solution was replaced 10 times.

[0178] Common procedure C: Determination of the average number of drug conjugates per antibody molecule in an antibody-drug conjugate

[0179] The average number of drug conjugates per antibody molecule in an antibody-drug conjugate was measured using liquid chromatography-mass spectrometry (LC-MS / MS).

[0180] The sample was diluted with water to 2 mg / mL. 50 μL of the diluted sample was added to 1 μL of 0.5 M TCEP. The reaction was allowed to react at room temperature for 30 minutes before injection. The chromatographic column was MabPac RP (2.1 × 50 mm); the mobile phases were: mobile phase A - 0.1% formic acid in water; mobile phase B - 0.1% formic acid in acetonitrile; the injection volume was 2 μg; the column temperature was 80°C; and the detection wavelength was 280 nm.

[0181] The liquid phase gradient is shown in Table 2 below.

[0182] Table 2

[0183] The DAR value was calculated based on the signal value of each component.

[0184] In the above formula, LCn represents the signal value of the light chain coupled with n linker-payloads, and HCn represents the signal value of the heavy chain coupled with n linker-payloads.

[0185] Specific coupling examples are as follows.

[0186] Antibody Reduction: Partritumab was diluted to approximately 10 mg / mL using phosphate buffered saline (PB / EDTA) (15 mM Na2HPO4, 5 mM NaH2PO4, 5 mM EDTA-2Na, pH 7.0). This antibody solution (100 mL) was added to a beaker at room temperature, followed by the addition of 10 mM TCEP aqueous solution (0.68 mL; 10.0 equivalents per antibody molecule). The mixture was stirred using a magnetic stirrer and incubated at room temperature for 3 hours to reduce the disulfide bonds in the antibody hinge region.

[0187] Conjugation of the antibody and the drug linker: Slowly add a DMSO solution containing 10 mM KA0012 (0.748 mL; 11.0 equivalents per antibody molecule) and stir at room temperature for 1 hour to couple the drug linker and the antibody.

[0188] Purification: Ultrafiltration and concentration are performed using an ultrafiltration membrane to replace the buffer with formulation buffer while removing unconjugated drug linker and other low molecular weight reagents. The resulting purified solution is sterile filtered to obtain a solution containing the antibody-drug conjugate KA0013.

[0189] The antibody concentration was measured using common procedure A: 23.64 mg / mL, and the average number of drug conjugates (n) per antibody molecule in KA0013 was measured using common procedure C: 7.87.

[0190] Preparation Example 4: Preparation of control drug

[0191] KA0010: Mc-GGFG-Dxd, a control linker-payload.

[0192] The structural formula of the control KA0010 is shown in the following formula V:

[0193] KA0011: Patritumab-Mc-GGFG-Dxd.

[0194] IgG-KA0010:IgG-Mc-GGFG-Dxd, abbreviated as IgG-Dxd.

[0195] IgG-KA0012: IgG-Mc-AAA-Dxd.

[0196] The preparation methods of KA0010 and KA0011 refer to the relevant description in Chinese patent document CN 106163559 B.

[0197] IgG-KA0010 and IgG-KA0012 were both prepared using a common thiol coupling method. The specific preparation method refers to the preparation method of KA0013 described above, and can also refer to the relevant description in Chinese patent document CN 106163559 B.

[0198] Example 1: FACS detection of the binding activity of anti-HER3 antibody-drug conjugate KA0013 to HER3 on the surface of SK-BR-3 cells

[0199] 1. Experimental Drugs

[0200] KA0013,

[0201] HER3 target monoclonal antibody Patritumab (prepared with reference to Preparation Example 1).

[0202] KA0011.

[0203] 2. Experimental Methods

[0204] SK-BR-3 cells (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were routinely collected, washed once with appropriate amount of PBS, counted and cell viability was determined; 3×10 5Add the cell suspension to a 96-well plate, centrifuge at 1000×g for 5 minutes, and discard the supernatant; add 100 μL of serially diluted experimental drugs (900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM based on the antibody working concentration), resuspend the cell pellet, and incubate on ice for 40 minutes; add 1% PBSA (PBS + 1% BSA), centrifuge at 1000×g for 5 minutes, discard the supernatant, and repeat washing twice; add 100 μL of 300-fold diluted fluorescent antibody Mouse Anti-Human IgG Fc-Alexa Fluor 647 (Southern Biotech, Cat.9040-31), resuspend the cell pellet, and incubate on ice for 30 minutes in the dark; add 1% PBSA, centrifuge at 1000×g for 5 minutes, discard the supernatant, and repeat washing twice; add 200 μL 1% PBSA, resuspend the cell pellet and detect on the instrument.

[0205] 3. Experimental Results

[0206] The results are shown in Figure 1.

[0207] The results showed that under the same experimental conditions, KA0013, KA0011, and HER3-targeting monoclonal antibody Patritumab could bind to SK-BR-3 cells and EC 50 They are 15.36nM, 12.89nM and 8.481nM respectively.

[0208] The results showed that under the same experimental conditions, KA0013, KA0011 and Patritumab all had the activity of effectively binding to HER3-expressing SK-BR-3 cells.

[0209] Example 2: FACS detection of the internalization activity of anti-HER3 antibody-drug conjugate KA0013 in MDA-MB-453 cells

[0210] 1. Experimental drugs

[0211] KA0013,

[0212] IgG-Dxd,

[0213] KA0012,

[0214] KA0011.

[0215] 2. Experimental Methods

[0216] MDA-MB-453 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60386) were collected, counted, and cell viability was determined; 1×10 5 Add the cell suspension to a 96-well low-adhesion plate, centrifuge at 750 × g for 5 minutes, and discard the supernatant; add 100 μL of experimental drug (working concentration is 100 nM) and an isotype control (IgG-Dxd, working concentration is 100 nM) respectively, mix well, and incubate in a 37°C incubator for 1 hour, 3 hours, and 5 hours. After incubation, 1% PBSA (PBS + 1% BSA) was added, centrifuged at 750 × g for 5 minutes, the supernatant was discarded, and the cells were washed twice with 1% PBSA; 100 μL of patritumab (working concentration of 100 nM) was added and incubated on ice for 30 minutes; 1% PBSA (PBS + 1% BSA) was added, centrifuged at 750 × g for 5 minutes, the supernatant was discarded, and the cells were washed twice with 1% PBSA; 100 μL of 300-fold diluted fluorescent antibody Mouse Anti-Human IgG Fc-Alexa Fluor 647 (Southern Biotech, Cat. 9040-31) was added, the cell pellet was resuspended, and the cells were incubated on ice in the dark for 30 minutes; 1% PBSA (PBS + 1% BSA) was added, centrifuged at 1000 × g for 5 minutes, the supernatant was discarded, and the cells were washed twice again; 200 μL of 1% PBSA was added, the cell pellet was resuspended, and the cells were detected by flow cytometry.

[0217] 3. Experimental Results

[0218] The results are shown in Figure 2.

[0219] The results showed that under the same experimental conditions, KA0013 and KA0011 could be internalized in MDA-MB-453 compared with the isotype control IgG-Dxd (i.e., IgG-Mc-GGFG-Dxd, produced by Zhongshan Kangfang Biotechnology Co., Ltd.), and the endocytic activity of the two was significantly different from that of IgG-Dxd.

[0220] Example 3: Determination of nonspecific killing activity of the linker-payload portion of the anti-HER3 antibody-drug conjugate

[0221] 1. Experimental Drugs

[0222] IgG-KA0010,

[0223] IgG-KA0012.

[0224] 2. Experimental Methods

[0225] The nonspecific killing activity of the linker-payload part Mc-AAA-Dxd (named KA0012) of the anti-HER3 antibody-drug conjugate KA0013 and the linker-payload part Mc-GGFG-Dxd (named KA0010) of the control KA0011 were tested.

[0226] After collecting human venous blood, gently mix the sodium heparin anticoagulation tube by inverting it upside down and centrifuging it at 3500 rpm for 10 minutes at room temperature. The plasma layer was collected for use in the experiment. MDA-MB-453 cells were routinely collected, counted, and cell viability was determined. The cell density was adjusted to 2.5×10 4 Cells were plated at a concentration of 100 μL / well of the cell suspension at 5% CO2 per well in a 96-well black-bottom plate and incubated overnight at 37°C in a 5% CO2 incubator. Antibodies were diluted in DMEM (5% human plasma) and 100 μL of serially diluted experimental drugs (100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, and 0.01 nM, respectively, were added. A blank control group was also added, with triplicate wells per group. The plates were incubated at 37°C in a 5% CO2 incubator for 6 days. After incubation, the 96-well plate was removed and centrifuged at 350 × g for 5 minutes. 135 μL of supernatant was discarded, and 65 μL of CellTiter-Glo (Promega, Cat. G8461) was added. The plates were incubated at room temperature for 20 minutes, and fluorescence was measured using a multi-label microplate reader.

[0227] 3. Experimental Results

[0228] The results are shown in Figure 3.

[0229] The results showed that under the same experimental conditions, compared with the isotype control (IgG-KA0010, produced by Zhongshan Kangfang Biological Co., Ltd.), the nonspecific killing of IgG-KA0012 was weaker, indicating that Mc-AAA-Dxd was more stable than Mc-GGFG-Dxd.

[0230] Example 4: Plasma stability test of anti-HER3 antibody-drug conjugate KA0013

[0231] 1. Experimental Drugs

[0232] KA0013,

[0233] KA0011.

[0234] 2. Experimental Methods

[0235] After collecting human venous blood, gently mix the tube by inverting it upside down and centrifuging it at 3500 rpm for 10 minutes at room temperature. The supernatant is plasma. Experimental drugs were diluted in human plasma (working concentration: 100 μg / mL) and incubated in a 37°C, 5% CO2 incubator for 0 h, 24 h, 48 h, 72 h, 96 h, and 139 h. After the incubation period, 150 μL of the sample was removed and quickly frozen in liquid nitrogen. After all samples were collected, changes in free Dxd in plasma were detected by mass spectrometry.

[0236] Mass spectrometry sample preparation: Take an appropriate amount of the above sample and mix it with acetonitrile in a 1:1 ratio to precipitate the protein. Centrifuge at 12,000 rpm for 3 min. Collect 10 μL of the supernatant for mass spectrometry analysis. Column: MabPac RP 2.1×50 mm 4 μm; Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Column temperature: 30°C.

[0237] The gradient is shown in Table 3 below.

[0238] Table 3

[0239] 3. Experimental Results

[0240] The results are shown in Figure 4.

[0241] The results showed that under the same experimental conditions, the amount of free Dxd in the plasma of the KA0013 group was lower than that of the KA0011 group, indicating that the stability of KA0013 in plasma was better than that of KA0011.

[0242] Example 5: In vitro detection of cathepsin B cleavage efficiency of different linkers-payloads

[0243] 1. Experimental samples:

[0244] KA0010,

[0245] KA0012.

[0246] 2. Experimental Methods

[0247] PBS (20x solution) (Sinobio, Cat.: B548117) was diluted 20-fold to obtain PBS working solution, and cathepsin B (CTSB) (Sinobio, Cat.: 10483-H08H) and linker-payload were diluted with PBS working solution; 150 μL CTSB (working concentration of 3 nM) was mixed with an equal volume of linker-payload (working concentration of 10 μM), and incubated in a 37°C, 5% CO2 incubator for 0 h, 0.5 h, 1 h, 2 h, and 4 h; after the incubation, Halt TMThe reaction was terminated with Protease Inhibitor Cocktail, EDTA-Free (30X) (Thermofisher, Cat.: 87785).

[0248] The enzymatically digested sample was directly loaded onto the mass spectrometer for detection. The chromatographic column was MabPac RP 2.1×50mm4μm; mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile; sample volume was 1μL for 10μM sample; column temperature was 30°C;

[0249] The gradient is shown in Table 4 below.

[0250] Table 4

[0251] 3. Experimental Results

[0252] The results are shown in Figure 5.

[0253] The results showed that under the same experimental conditions, both KA0012 (Mc-AAA-Dxd) and KA0010 (Mc-GGFG-Dxd) could be effectively cleaved by CSTB and could effectively release the payload, with the same efficiency.

[0254] Example 6: ELISA method to detect the binding activity of anti-HER3 antibody-drug conjugate KA0013 with antigen

[0255] Human HER3-his was coated on an ELISA plate and incubated. After blocking, the test antibodies were added. After incubation and washing, goat anti-human IgG Fc, HRP (purchased from Jackson ImmunoResearch Inc., catalog number: 109-035-098) was added. After incubation and washing, the plate was developed with TMB (Neogen, 308177). After color development, absorbance at 450 nm was measured in a microplate reader. Data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0256] The results are shown in Figure 6 and Table 5.

[0257] Table 5: Binding activity of KA0013 and KA0011 to the antigen human HER3-his

[0258] The results showed that the anti-HER3 antibody-drug conjugates KA0013, KA0011 and Patritumab could effectively bind to the antigen human HER3-his, and the binding efficiency was dose-dependent.

[0259] Example 7: Determination of Kinetic Parameters of Anti-HER3 Antibody-Drug Conjugates

[0260] 1. Experimental samples

[0261] KA0013

[0262] KA0011

[0263] 2. Experimental Methods

[0264] The dilution buffer for the experimental samples was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 1 μg / ml huHER3-his-Biotin was immobilized on the SA sensor at a fixed height of 1 nm. The sensor was equilibrated in the buffer for 60 seconds. The huHER3-his-Biotin immobilized on the sensor bound to each antibody at an antibody concentration of 0.2469-20 nM (three-fold dilution) for 120 seconds, and the antibody dissociated in the buffer for 300 seconds. The sample plate was vibrated at a rate of 1000 rpm, the detection temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model fit to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 12.0, and the data analysis software was Fortebio Data Analysis 12.0.

[0265] 3. Experimental Results

[0266] The results are shown in Table 6 and Figures 7 and 8.

[0267] Table 6: Kinetic parameters for binding of anti-HER3 antibody-drug conjugates to human HER3-his-biotin

[0268] KD is the affinity constant; kon is the antigen-antibody association rate; kdis is the antigen-antibody dissociation rate; KD = kdis / kon.

[0269] The results showed that the anti-HER3 antibody-drug conjugates KA0013 and KA0011 both had good affinity to the antigen human HER3-his-biotin.

[0270] Example 8: Determination of specific cell killing activity of anti-HER3 antibody-drug conjugates

[0271] 1. Experimental drugs

[0272] KA0013

[0273] KA0011

[0274] IgG-KA0012

[0275] 2. Experimental Methods

[0276] NCI-H1781 cells (Nanjing Kebai Biotechnology Co., Ltd., Catalog No. CBP60116) were harvested and centrifuged at 1000 x g for 5 minutes. The supernatant was removed, the cells were resuspended, counted, and viability was determined. The cells were diluted and added to each well of a black-bottomed 96-well plate (2500 cells / 100 μL). The plates were incubated overnight at 37°C in a 5% CO2 incubator. The experimental drugs were diluted in culture medium (10% human AB serum) and 100 μL of the experimental drug was added to each well according to the experimental design. A blank control was also included, with triplicate wells per group. The plates were incubated at 37°C in a 5% CO2 incubator for 6 days. After incubation, the 96-well plate was centrifuged at 350 x g for 5 minutes, and 135 μL of the supernatant was discarded. 60 μL of CellTiter-Glo (Promega, Cat. G8461) was added to each well. The plates were incubated at room temperature for 15 minutes, and fluorescence was measured using a multi-label microplate reader.

[0277] 3. Experimental Results

[0278] The results are shown in Figure 9. The results showed that both anti-HER3 antibody-drug conjugates KA0013 and KA0011 could specifically kill NCI-H1781 cells.

[0279] Example 9: Study on ADCC activity of anti-HER3 antibody-drug conjugates

[0280] MDA-MB-453 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60386) and PBMC (provided by Zhongshan Blood Station) in the logarithmic growth phase were collected and centrifuged at 170×g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in assay medium (RPMI-1640 containing 1% FBS), washed twice by centrifugation, and the cell number was counted. The cell density was adjusted with assay medium, and the target cell suspension was seeded into a 96-well plate at 50 μL / well (each well containing approximately 3×10 4 Target cells). Then add the drug in gradient dilution (5 μg / mL, 15 μg / mL, 45 μg / mL according to the working concentration of the antibody) and pre-incubate at room temperature for 1 hour. After pre-incubation, add PBMC suspension to the sample wells at 100 μL / well (each well contains about 9×10 5PBMCs, target ratio of 30:1), mix thoroughly, and incubate in a 37°C, 5% CO2 incubator for 4 hours. After incubation, remove the 96-well plate, centrifuge at 250×g for 5 minutes, carefully aspirate 100 μL of cell supernatant and transfer it to a new 96-well plate. According to the instructions of the Cytotoxicity Detection Kit (Roche, Catalog No.: 11644793001), add 100 μL of freshly prepared reaction solution to each well and incubate at room temperature in the dark for 30 minutes. Measure the OD value at 490nm and 650nm respectively. The OD value of each group = OD 490nm -OD 650nm The ADCC activity (expressed as ADCC%) was calculated based on the measured OD values ​​of each group.

[0281] ADCC% = (OD of experimental group - mean OD of target cell spontaneous group) / (mean OD of positive control - mean OD of target cell spontaneous group) × 100%

[0282] The results are shown in Figure 10. The results showed that in the MDA-MB-453 target cell system, KA0011 had no ADCC activity against MDA-MB-453 cells, while KA0013 had ADCC activity against MDA-MB-453 cells.

[0283] Example 10: In vivo efficacy testing of anti-HER3 antibody-drug conjugates

[0284] To test the in vivo anti-tumor activity of anti-HER3 antibody-drug conjugates, HCC827 human lung cancer cells (purchased from Guangzhou Genio Biotechnology Co., Ltd.) were first inoculated subcutaneously into the right dorsal upper thigh of 5-8-week-old female CB-17 SCID mice (purchased from Guangdong Nanmo Biotechnology Co., Ltd.). The day of grouping was defined as day 0. The drug was administered by intravenous (iv) injection once a week for a total of 3 doses. The modeling and specific administration methods are shown in Table 7. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.

[0285] Table 7: Dosing regimen of anti-HER3 antibody-drug conjugates for the treatment of HCC827 human lung cancer cell xenograft tumor models in CB-17 SCID mice

[0286] The results are shown in Figure 11. The results demonstrate that the anti-HER3 antibody-drug conjugate KA0013 effectively inhibited tumor growth in mice compared to an isotype control antibody. Furthermore, as shown in Figure 12, the tumor-bearing mice tolerated the test drug KA0013 well, with no effect on the body weight of the mice in either group.

[0287] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein: The compound comprises: a compound of formula III, n alanines, and a compound of formula IV connected in sequence, in, n is 2, 3, 4, 5 or 6; The compound of formula III, n alanines and the compound of formula IV are independently connected directly or through chemical groups.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, whose structural formula is shown in the following formula I, in, A represents alanine; m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; n is 2, 3, 4, 5 or 6.

3. The compound or pharmaceutically acceptable salt according to any one of claims 1 to 2, wherein: The compound is shown in the following formula II, 4. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof, a linker and a payload, wherein: The linker and the effective load are the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3; Preferably, the linker is linked to the antibody or antigen-binding fragment thereof via one or more thioether bonds; Preferably, the linker forms a thioether bond with a sulfur atom at the disulfide bond position of the antibody hinge portion.

5. The antibody-drug conjugate according to claim 4, wherein: The antibody is an IgG antibody, preferably an IgG1 antibody.

6. The antibody-drug conjugate according to any one of claims 4 to 5, wherein: The antibody is an anti-HER3 antibody.

7. The antibody-drug conjugate according to any one of claims 4 to 6, wherein: The antibody comprises a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3; The light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:

6.

8. The antibody-drug conjugate according to claim 7, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

9. The antibody-drug conjugate according to any one of claims 4 to 8, wherein: The heavy chain constant region of the antibody is Ig gamma-1 chain C region or Ig gamma-4 chain C region; the light chain constant region is Ig kappa chain C region.

10. The antibody-drug conjugate according to any one of claims 4 to 9, wherein: The amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO:11; and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

13.

11. The antibody-drug conjugate according to any one of claims 4 to 10, wherein: The average number of linker-payloads coupled to each antibody molecule was 1-8.

12. The antibody-drug conjugate according to any one of claims 4 to 11, which has ADCC activity.

13. The antibody-drug conjugate according to any one of claims 4 to 12, wherein: The antibody-drug conjugate binds to its targeted antigen 50 , and the antibody or antigen-binding fragment thereof contained in the antibody-conjugated drug binds to the same antigen 50 The same or substantially the same. 14 . A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 4 to 13 and one or more pharmaceutically acceptable excipients.

15. Use of the antibody-drug conjugate according to any one of claims 4 to 13 in the preparation of a drug for treating or preventing tumors; Preferably, the tumor is a HER3-positive tumor; Preferably, the tumor is selected from one or more of lung cancer, clear cell sarcoma, skin cancer, kidney cancer, urothelial carcinoma, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer and penile cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the colon cancer is metastatic colon cancer.

16. The antibody-drug conjugate according to any one of claims 4 to 13, which is used for treating or preventing tumors; Preferably, the tumor is a HER3-positive tumor; Preferably, the tumor is selected from one or more of lung cancer, clear cell sarcoma, skin cancer, kidney cancer, urothelial carcinoma, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer and penile cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the colon cancer is metastatic colon cancer.

17. A method for treating or preventing tumors, comprising the step of administering to a subject in need thereof an effective amount of the antibody-drug conjugate according to any one of claims 4 to 13; Preferably, the tumor is a HER3-positive tumor; Preferably, the tumor is selected from one or more of lung cancer, clear cell sarcoma, skin cancer, kidney cancer, urothelial carcinoma, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, colon cancer, rectal cancer, uterine cancer, salivary gland cancer, vulvar cancer, thyroid cancer, anal cancer and penile cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the colon cancer is metastatic colon cancer.

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