Compound and antibody-conjugated drug containing same

Abstract

The invention belongs to the field of biological medicine, and relates to a compound and an antibody coupling medicine containing the compound. The invention further relates to a pharmaceutical composition containing the antibody coupling medicine and application of the pharmaceutical composition. Specifically, the antibody coupling medicine is an antibody coupling medicine for resisting HER3 (Human Epidermal Growth Factor Receptor 3). The antibody coupling drug provided by the invention has good stability and biological activity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] The present invention belongs to the field of biomedicine, 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 connection technology [J]. Chinese Journal of New Drugs and Clinical Practice, 2022, 41(12): 10.).

[0005] Antibody drug conjugate (ADC) is an emerging macromolecular targeted drug that combines the powerful killing effect of traditional small molecule chemotherapy with the tumor targeting of antibody drugs. ADC consists of three main parts: antibodies responsible for selectively recognizing antigens on the surface of cancer cells, payloads responsible for killing cancer cells, and linkers that connect antibodies and payloads. After ADC drugs enter the blood circulation, their antibody part specifically binds to antigens on the surface of tumor cells, and the complex formed by the binding enters the cell through receptor-mediated endocytosis (which can be divided into four types: clathrin-mediated endocytosis, cave-like invagination, macropinocytosis, and clathrin-and caveolin-independent endocytosis). Cleavable linkers are sensitive to environmental factors in tumor cells and will be cleaved by specific pH environments, proteases, or certain chemicals; ADC drugs carrying non-cleavable linkers are digested by lysosomes, thereby releasing the drug. The small molecules of some ADC drugs can penetrate the cell membrane to 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] The accumulation of payload in tumor is limited, so it is necessary to kill tumor at sub-nanomolar concentration. In addition, it needs to have good stability to maintain structural integrity and activity in the circulatory system and lysosomes. Common payloads mainly include tubulin inhibitors and DNA damaging agents. Tubulin inhibitors kill tumor cells and inhibit the rapid proliferation of tumor cells by inhibiting the generation and aggregation of tubulin. Tubulin inhibitors commonly used in ADC construction include maytansines, auristatins and tubulysins. DNA damaging agents are divided into three categories due to different mechanisms of action: DNA double-strand breakers, DNA intercalators and DNA alkylating agents. DNA is critical 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 indole chlorobenzazepines (e.g. pyrrolo[2,1-c][1,4]benzodiazepine, PBD), dukamycin, camptothecin (CPT) and its derivatives (e.g. 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 parts: an extracellular ligand binding region, an α-helical transmembrane region, and an intracellular tyrosine kinase region. HER3 is abnormally overexpressed in a variety of invasive 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 can bind to its ligand neuregulin (NRG). Although it lacks intrinsic tyrosine kinase activity, HER3 can form heterodimers with HER2 and induce cross-linking phosphorylation of highly conserved intracellular kinase residues, that is, one receptor phosphorylates specific tyrosine residues on another receptor, thereby recruiting and activating downstream proteins, directly activating the PI3K / AKT pathway, causing a signal cascade reaction, and promoting the occurrence and development of tumor cells.

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

[0009] After in-depth research and creative work, the inventors have prepared a compound that can serve as a linker and an effective load, and further prepared an antibody-drug conjugate. The prepared antibody-drug conjugate has good stability and / or biological activity. The following invention is provided:

[0010] 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,

[0011]

[0012]

[0013] in,

[0014] n is 2, 3, 4, 5 or 6;

[0015] The compound of formula III, n alanines, and the compound of formula IV are independently connected directly (eg, connected by a chemical bond) or connected by a chemical group.

[0016] The 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, etc.

[0017] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof has a structural formula as shown in the following Formula I:

[0018]

[0019] in,

[0020] A represents alanine (alanine residue);

[0021] m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0022] n is 2, 3, 4, 5 or 6.

[0023] In some embodiments of the present invention, the compound or pharmaceutically acceptable salt thereof, wherein the compound is as shown in the following formula II,

[0024]

[0025] 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,

[0026] Wherein, the linker and the effective load are the compounds described in any one of the present invention or their pharmaceutically acceptable salts;

[0027] Preferably, the linker is linked to the antibody or antigen-binding fragment thereof via one or more thioether bonds;

[0028] Preferably, the linker forms a thioether bond with the sulfur atom at the disulfide bond position of the antibody hinge portion. 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.

[0029] In some embodiments of the present invention, the antibody-drug conjugate is an IgG antibody, preferably an IgG1 antibody.

[0030] In some embodiments of the present invention, the antibody-drug conjugate, wherein the antibody is an anti-HER3 antibody.

[0031] In some embodiments of the present invention, the antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region.

[0032] in,

[0033] 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;

[0034] 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.

[0035] In some embodiments of the present invention, in the antibody-drug conjugate, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9.

[0036] In some embodiments of the present invention, in the antibody-drug conjugate, the heavy chain constant region of the antibody is Ig gamma-1chain C region or Ig gamma-4chain C region; and the light chain constant region is Ig kappachain C region.

[0037] 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.

[0038] In some embodiments of the present invention, in the antibody-drug conjugate, the average number of linkers-payloads conjugated to each antibody molecule is 1-8, 2-8, 3-8, 4-8, 5-8, 6-8 or 7-8.

[0039] 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.

[0040] 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 Ig kappa chain C region.

[0041] In some embodiments of the present invention, the antibody-drug conjugate has ADCC activity.

[0042] In some embodiments of the present invention, the antibody-drug conjugate, wherein:

[0043] "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 50 "The same or substantially the same. The meaning of "substantially the same" here can be, for example:

[0044] The antibody-drug conjugate binds to its targeted antigen 50 " and "the antibody or antigen-binding fragment thereof contained in the antibody-drug conjugate binds to the same antigen 50” within ±10%, within ±15%, within ±20%, within ±25%, within ±30%, within ±35%, within ±40% or within ±45%.

[0045] The antibody-drug conjugate of the present invention can be used to treat or prevent tumors;

[0046] Preferably, the tumor is a HER3-positive tumor;

[0047] 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;

[0048] Preferably, the lung cancer is non-small cell lung cancer (NSCLC);

[0049] Preferably, the colon cancer is metastatic colon cancer.

[0050] The antibody-drug conjugate can be prepared by reacting the 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 by forming a thioether bond in the disulfide bond portion present in the hinge portion of the antibody.

[0051] Another aspect of the present invention relates to a pharmaceutical composition comprising the antibody-drug conjugate described in any one of the present invention and one or more pharmaceutically acceptable excipients.

[0052] In another aspect, the present invention relates to use of the antibody-drug conjugate described in any one of the present invention in the preparation of a drug for treating or preventing tumors;

[0053] Preferably, the tumor is a HER3-positive tumor;

[0054] 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;

[0055] Preferably, the lung cancer is non-small cell lung cancer (NSCLC);

[0056] Preferably, the colon cancer is metastatic colon cancer.

[0057] 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 described in any one of the present invention to a subject in need;

[0058] Preferably, the tumor is a HER3-positive tumor;

[0059] 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;

[0060] Preferably, the lung cancer is non-small cell lung cancer (NSCLC);

[0061] Preferably, the colon cancer is metastatic colon cancer.

[0062] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0063] 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.

[0064] As used herein, the term "no ADCC activity" means that there is no ADCC activity, ADCC activity cannot be detected, or the 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).

[0065] As used herein, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined 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 immunoglobulins 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 subdivided into regions with high variability, called complementary determining regions (CDRs), interspersed with more conservative 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, respectively. The assignment of amino acids to various regions or domains follows Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the IMGT numbering system definition, 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 acid research, 2009; 38 (suppl_1): D301-D307.

[0066] The variable regions of the light chain and the heavy chain determine the binding of the antigen; the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs), wherein the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3. In the present invention, CDRs are defined by 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 acid research, 2009; 38 (suppl_1): D301-D307.

[0067] 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 an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0068] 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 natural mutations that may occur spontaneously. 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, which usually recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique 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 recombinant DNA technology (see, for example, US Patent 4,816,567).

[0069] 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 may 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 receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by 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 of 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).

[0070] As used herein, the term "single chain antibody (single chain fragment variable, 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. Wherein 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., Birdet al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883). Such scFv molecules may have a general structure: NH2-VL-linked fragment-VH-COOH or NH2-VH-linked fragment-VL-COOH. Suitable prior art linkers are composed 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 useful 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.

[0071] As used herein, the term "antibody portion" refers to the antibody portion in the antibody-drug conjugate, which is connected to the intermediate linker portion through a specific functional group in certain specific embodiments, and the antibody portion can specifically bind to the antigen.

[0072] 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, which 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 connected in the form of hydrazone bonds, disulfide bonds, and polypeptides. Non-cleavable linkers do not have built-in chemical bonds that can trigger cleavage, and antibodies need to be converted into amino acids through a proteolytic mechanism in cancer cells, thereby releasing cytotoxic drugs with linkers and amino acid fragments, mainly connected in the form of thioethers.

[0073] As used herein, the term "payload" refers to the cytotoxic molecule in the antibody-drug conjugate that is primarily responsible for executing the cell-killing function. It is connected to the intermediate linker portion through chemical synthesis, enters the tumor cell through internalization, and releases the cytotoxic 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.

[0074] As used herein, the term "DAR (drug-to-antibody ratio)" refers to the average number of small molecule drugs coupled to each antibody molecule, which is one of the important quality attributes of ADC. According to the chemical properties of the linker, small molecule cytotoxin and the coupling method (lysine coupling, cysteine ​​coupling, glycosyl coupling, etc.), commonly used characterization analysis methods include: UV-visible spectrophotometry, hydrophobic chromatography, reverse phase chromatography and mass spectrometry (MS).

[0075] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated 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.

[0076] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. 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, papillomaviruses (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. In addition, the vector may also contain a replication initiation site.

[0077] 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.

[0078] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. 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 M or less affinity (KD) binds to the antigen.

[0079] 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. Generally, antibodies bind to the antigen 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 M or less binds to the antigen. KD can be determined using methods known to those skilled in the art, such as using a Fortebio molecular interaction instrument.

[0080] 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. And 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.

[0081] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0082] 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 gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0083] Advantageous Effects of the Invention

[0084] The present invention achieves one or more of the following technical effects:

[0085] (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.

[0086] (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.

[0087] (3) The anti-HER3 antibody-drug conjugate KA0013 of the present invention can effectively bind to HER3.

[0088] (4) Antibody-drug conjugates prepared using the compounds of the present invention, such as the anti-HER3 antibody-drug conjugate KA0013, have internalization activity.

[0089] (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.

[0090] (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.

[0091] (7) Antibody-drug conjugates prepared using the compounds of the present invention, such as HER3 antibody-drug conjugate KA0013, have ADCC activity.

[0092] (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.

[0093] (9) Antibody-drug conjugates prepared using the compounds of the present invention, such as HER3 antibody-drug conjugate KA0013, have good safety.

[0094] (10) Antibody-drug conjugates prepared using the compounds of the present invention, such as HER3 antibody-drug conjugate KA0013, have a bystander killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 : Results of the binding activity test of the anti-HER3 antibody-drug conjugate KA0013 to the surface HER3 of SK-BR-3 cells expressing HER3 on the cell surface.

[0096] Figure 2 : Results of internalization activity detection of anti-HER3 antibody-drug conjugate KA0013 in MDA-MB-453 cells expressing HER3 on the cell surface.

[0097] Figure 3 : Results of stability test on nonspecific killing caused by cleavage of linker-payload portion of anti-HER3 antibody-drug conjugate KA0013.

[0098] Figure 4 : Plasma stability test results of anti-HER3 antibody-drug conjugates KA0013 and KA0011.

[0099] Figure 5 : In vitro detection of the linker-payload efficiency of cathepsin B cleavage of KA0013 and KA0011.

[0100] Figure 6 : The results of the indirect ELISA method to determine the binding activity of anti-HER3 antibody-drug conjugates KA0013 and KA0011 with the antigen human HER3-his.

[0101] Figure 7 : Detection results of kinetic characteristic parameters of anti-HER3 antibody-drug conjugate KA0013.

[0102] Figure 8 : Detection results of kinetic characteristic parameters of anti-HER3 antibody-drug conjugate KA0011.

[0103] Fig. 9 :Specific cell killing activity test results of anti-HER3 antibody-drug conjugates.

[0104] Fig.10 : ADCC activity test results of anti-HER3 antibody-drug conjugates.

[0105] Fig.11 : In vivo efficacy results of anti-HER3 antibody-drug conjugates on HCC827 human lung cancer cell transplanted tumor model in CB-17SCID mice.

[0106] Fig.12 : Effects of anti-HER3 antibody-drug conjugates on body weight in the HCC827 human lung cancer cell transplantation tumor model in CB-17SCID mice. DETAILED DESCRIPTION

[0107] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0108] 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 refer to Chinese patent document CN106163559 B.

[0109] In the following experimental examples of the present invention, the isotype control antibodies used, i.e., 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 anti-hen egg lysosome (HEL), and the variable region sequences of these antibodies are 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 the constant region fragments thereof 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 are all prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0110] Preparation Example 1: Preparation of anti-HER3 antibody Patritumab

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

[0112] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQP PGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSV TAADTAVYYCARDKWTWYFDLWGRGTLVTVSS(SEQ ID NO:7)

[0113] Nucleic acid sequence encoding the heavy chain variable region of Patritumab:

[0114] CAGGTGCAGCTGCAGCAGTGGGGCGCCGGACTGCTGAAGCCATCCGAGACCCTGTCTCTGACATGTGCCGTGTACGGCGGCTCCTTTTCTGGCTACTATTGGAGCTGGATCAGGCAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCGAGATCAACCACAGCGGCTCCACCAACTATAATCCCTCTCTGAAGAGCCGGGTGACCATCAGCGTGGAGACATCCAAGAATCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGCGCCCGGGACAAGTGGACCTGGTACTTTGATCTGTGGGGCAGAGGCACCCTGGTGACAGTGTCTAGC(SEQ ID NO:8)

[0115] Amino acid sequence of the variable region of the Patritumab light chain:

[0116] DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSTPRTFGQGTKVEIK(SEQ ID NO:9)

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

[0118] GACATCGAGATGACCCAGTCCCCTGATTCTCTGGCCGTGAGCCTGGGAGAGAGGGCAACAATCAACTGTAGAAGCTCCCAGTCCGTGCTGTACTCTAGCTCCAACCGGAATTACCTGGCCTGGTATCAGCAGAATCCAGGCCAGCCCCCTAAGCTGCTGATCTATTGGGCCAGCACCAGGGAGTCCGGAGTGCCAGACCGCTTCTCCGGCTCTGGCAGCGGCACAGACTTCACCCTGACAATCTCTAGCCTGCAGGCCGAGGACGTGGCCGTGTACTATTGCCAGCAGTACTATAGCACCCCCAGGACATTTGGCCAGGGCACCAAGGTGGAGATCAAG(SEQ ID NO:10)

[0119] The heavy chain constant region of Patritumab uses the Ig gamma-1 chain C region; the light chain constant region uses the Igkappa chain C region.

[0120] Amino acid sequence of the heavy chain constant region of Patritumab:

[0121] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:11)

[0122] Nucleic acid sequence of the heavy chain constant region of Patritumab:

[0123] GCCTCTACAAAGGGGCCCAGCGTGTTTCCTCTGGCCCCTAGCAGCAAGTCTACAAGCGGAGGAACAGCAGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCAGAGCCCGTGACCGTGTCTTGGAACTCAGGAGCCCTGACCAGCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCAGCGGACTGTATAGCCTGAGCAGCGTGGTGACCGTGCCTTCTTCTAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGGGTGGAGCCCAAGTCTTGCGACAAGACCCACACTTGTCCTCCTTGTCCAGCCCCAGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCTCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAATAAGGCCCTGCCAGCCCCTATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTTCTCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATTGCCGTCGAGTGGGAGTCTAACGGCCAGCCCGAGAACAACTACAAGACCACACCTCCAGTGCTGGATAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAAAGCCGCTGGCAGCAGGGCAACGTGTTTTCTTGCAGCGTGATGCACGAAGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCTCCAGGCAAG(SEQ IDNO:12)

[0124] Amino acid sequence of the light chain constant region of Patritumab:

[0125] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:13)

[0126] Nucleic acid sequence of the light chain constant region of Patritumab:

[0127] CGTACGGTGGCAGCCCCATCTGTCTTCATTTTTCCCCCTAGTGACGAGCAGCTGAAATCCGGAACAGCCTCTGTGGTCTGTCTGCTGAACAATTTCTACCCTCGCGAAGCCAAGGTGCAGTGGAAAGTCGATAACGCTCTGCAGAGTGGCAATTCACAGGAG AGCGTGACTGAACAGGACTCCAAGGATTCTACCTATAGTCTGAGTCCCACTCTGACCCTGTCCAAAGCAGATTACGAAAAGCACAAAGTGTATGCCTGTGAGGTCACCCACCAGGGGCTGAGTTCTCCAGTCACCAAATCCTTCAACAGAGGCGAATGT(SEQ ID NO:14)

[0128] The heavy chain cDNA and light chain cDNA of Patritumab were cloned into the pcDNA3.1 vector to obtain the recombinant expression plasmid of the antibody Patritumab. The recombinant plasmid was transfected into 293F cells. The 293F cell culture fluid was purified and then tested.

[0129] The anti-HER3 monoclonal antibody Patritumab was produced.

[0130] According to the IMGT numbering system, the six CDRs of the anti-HER3 monoclonal antibody Patritumab are as follows:

[0131] HCDR1: GGSFSGYY (SEQ ID NO: 1)

[0132] HCDR2: INHSGST (SEQ ID NO: 2)

[0133] HCDR3:ARDKWTWYFDL(SEQ ID NO:3)

[0134] LCDR1:QSVLYSSSNRNY(SEQ ID NO:4)

[0135] LCDR2: WAS (SEQ ID NO: 5)

[0136] LCDR3: QQYYSTPRT (SEQ ID NO: 6).

[0137] Preparation Example 2: Preparation of Mc-AAA-Dxd (KA0012)

[0138] 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.

[0139] Mc-AAA-Dxd is named KA0012 in the present invention, and its structural formula is shown in the following formula II:

[0140]

[0141] Wherein Mc is a maleimide-N-group, and its structure is shown in the following formula III:

[0142]

[0143] Wherein the structural formula of -(NH-DX) is shown in the following formula IV:

[0144]

[0145] The synthesis steps of KA0012 are as follows: Step 1 to Step 10.

[0146] Step 1: Preparation of intermediate HM-1315_6B_2

[0147]

[0148] The raw material HM-1315_6B_1 (450.0g, 1.44mol, 1eq) was suspended in DCM (dichloromethane) (4.5L, 10V), the temperature was controlled at 0-5°C, HOSU (182.9g, 1.59mol, 1.1eq) and EDCI (304.8g, 1.59mol, 1.1eq) were added in sequence, and the reaction solution was dissolved until clear after the addition. The reaction was reacted at (10-20°C) for 3 hours. Half-saturated brine (1.0L) was added to the reaction solution, stirred, and then allowed to stand for separation. The organic phase was washed once with half-saturated brine (1.0L), and then washed twice with saturated brine (1.0L×2), dried over anhydrous sodium sulfate, and concentrated to obtain 579.0g of white solid, with a yield of 98%.

[0149] 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.

[0150] Step 2: Preparation of intermediate HM-1315_1

[0151]

[0152] The raw material HM-1315_6B_2 (579.0g, 1.42mol, 1.0eq) was dissolved in acetonitrile (2895mL, 5v), and glycine (117.2g, 1.56mol, 1.1eq) and water (2895mL, 5v) were added. The temperature was lowered to -5~0℃, and DIPEA (N,N-diisopropylethylamine) (275.3g, 2.13mol, 1.5eq) was slowly added. After the addition, the reaction was reacted at 20~25℃ for 3 hours. Ethyl acetate (1.0L) was added to the reaction solution to dilute it, and the pH was adjusted to 3~4 with dilute hydrochloric acid (0.5N). Ethyl acetate (500mL) was added to extract the liquid, and the aqueous phase was extracted twice with ethyl acetate (1.0L×2). The organic phases were combined, washed once with saturated brine (1.5L), 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%.

[0153] 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.

[0154] Step 3: Preparation of intermediate HM-1315_2

[0155]

[0156] The raw material HM-1315_1 (474.0g, 1.29mol, 1.0eq) was dissolved in ultra-dry DMF (2370mL, 5v), and under nitrogen protection, lead tetraacetate (1143.9g, 2.58mol, 2.0eq), anhydrous copper acetate (234.3g, 1.29mol, 1.0eq) and acetic acid (170.5g, 2.84mol, 2.2eq) were added. After the addition, the reaction temperature rose to about 50°C. The reaction solution was placed in an oil bath at 50-55°C for 0.5 hours. The reaction solution was cooled, ethyl acetate (2370mL, 5v) was added thereto, and then ice water (2370mL, 5v) was added, and 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 a white solid product with a yield of 74%.

[0157] 1H NMR (400MHz, DMSO) δ8.90 (s, 1H), 7.89 (d, J = 7.5Hz, 2H), 7.73 (t, J = 6.6Hz, 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.

[0158] Step 4: Preparation of intermediate HM-1315_4

[0159]

[0160]

[0161] The raw material HM-1315_2 (326.0 g, 0.85 mol, 1.0 eq) was dissolved in THF (3260.0 mL, 10 v), and HM-1315_3 (211.9 g, 1.27 mol, 1.5 eq) was added. After the addition, the reaction temperature was lowered to 0-10 ° C, and an aqueous solution of lithium hydroxide (24.4 g, 1.02 mol, 1.2 eq) (163.0 mL, 0.5 v) was slowly added. After the addition, the temperature was naturally raised to room temperature (15-25 ° C) and the reaction was carried out for 1 hour. Ethyl acetate (1.5 L) and water (1.5 L) were added to the reaction solution, and the liquids 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), dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column chromatography. The product was eluted with petroleum ether / ethyl acetate = 1 / 1. The product was collected, concentrated and dried with an oil pump to obtain 311.8 g of a white solid product with a yield of 74%.

[0162] 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.

[0163] Step 5: Preparation of intermediate HM-1315_7

[0164]

[0165]

[0166] 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 solution was cooled to -5-0°C, and PPTS (4-methylbenzenesulfonic acid pyridine) (30.86g, 122.82mmol, 1.0eq), EDCI (23.54g, 122.82mmol, 1.0eq), HOBT (1-hydroxybenzotriazole) (16.59g, 122.82mmol, 1.0eq) and HM-1315_6 (42.27g, 110.54mmol, 0.9eq) were added to the reaction solution in sequence. After the addition, the mixture was reacted at -5-0°C for 16 hours. After adding n-hexane (1200mL, 20v) to the reaction solution, the mixture was allowed to stand for separation, and the DMAc layer was washed four times with n-hexane (1.2L×4), and then poured into water (1800mL, 30v). A large amount of white solid precipitated. The mixture was filtered with a Buchner funnel, and the filter cake was dried to obtain a white solid crude product. Add acetonitrile (300.0 mL, 5 v) to the reaction bottle, add the white crude product obtained in the previous step, beat for 1 hour, filter, and pump the filter cake dry to obtain 56.2 g of white solid product with 80% yield.

[0167] 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.

[0168] Step 6: Preparation of intermediate HM-1315_8

[0169]

[0170]

[0171] 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), cooled to 0-10 ° C, added with 10% palladium carbon (0.39 g, 0.15 w / w), replaced with hydrogen three times, and reacted at 0-10 ° C for 16 hours. The palladium carbon was filtered off, and the filter cake was rinsed with acetonitrile / water (10 mL / 10 mL). 2-methyltetrahydrofuran (50 mL) was added to the filtrate for extraction, and the organic phase was washed once with water (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain 2.0 g of a white solid crude product. The crude product (2.0 g, 3.70 mmol, 1.0 eq) was suspended in dichloromethane (20.0 mL, 10 v), and DBU (0.56 g, 3.70 mmol, 1.0 eq) was slowly added dropwise at 20-25 ° C. After the addition, the reaction solution was partially dissolved and reacted at 20-25 ° C for 16 hours, and a white solid precipitated. Methyl tert-butyl ether (40.0 mL, 20 v) was added to the reaction solution, and more white solid precipitated. Filter, transfer the filter cake to a single-mouth bottle, add acetonitrile (20.0 mL), beat for 2 hours, filter, and use an oil pump to dry the filter cake to obtain 0.5 g of a white solid product with a yield of 38%.

[0172] 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.

[0173] Step 7: Preparation of intermediate HM-1315_9

[0174]

[0175] 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), HM-297D_9 (290 mg, 0.94 mmol, 1.0 eq) was added, cooled to 0-10 ° C, DIPEA (121 mg, 0.94 mmol, 1.0 eq) was added, and the reaction was carried out at 0-10 ° C for 16 hours to prepare and separate the product (290 mg, 60% yield) as a white solid.

[0176] 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.

[0177] Step 8: Preparation of intermediate HM-582_9

[0178]

[0179] 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. 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%).

[0180] 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.

[0181] Step 9: Preparation of intermediate HM-582_10

[0182]

[0183] Compound HM-582_9 (30 g) was suspended in water (600 mL), and methanesulfonic acid (300 mL) was slowly added. The solid dissolved and exothermic phenomenon occurred. After nitrogen replacement, the mixture was heated to 112°C for 7 h, cooled to room temperature, filtered, and the filter cake was washed with water (100 mL). The filtrate was diluted with ethanol (4 L), and the solid precipitated. The mixture was stirred at room temperature for 20 min, filtered, and dried. The crude product was suspended in ethanol / water = 4:1 (1 L), heated to reflux for 2 h, cooled to room temperature, filtered, and the solid was washed with a small amount of ethanol and dried. After freeze-drying, isotecan mesylate (16.7 g, 50%) was obtained.

[0184] 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.

[0185] Step 10: Preparation of HM-1315_10 (KA0012)

[0186]

[0187] The raw material HM-1315_9 (150 mg, 0.29 mmol, 1.0 eq) was dissolved in DMAc (3.0 mL, 20 v), HM-582_10 (156 mg, 0.29 mmol, 1.0 eq) was added, cooled to 0-10 ° C, 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, and the reaction was carried out at 0-10 ° C for 16 hours. LCMS showed that the raw material was completely converted, and the product (135 mg, 50% yield) was prepared and isolated as a light yellow solid.

[0188] 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,2H),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.

[0189] Preparation Example 3: Preparation of anti-HER3 antibody-drug conjugate KA0013

[0190] The structures of KA0013 and KA0011 are shown in Table 1 below.

[0191] Table 1

[0192] name structure KA0013 Patritumab-Mc-AAA-Dxd KA0011 Patritumab-Mc-GGFG-Dxd

[0193] 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 generate an antibody-drug conjugate. Specifically, the compound reacts with an antibody having a thiol group to produce an antibody-drug conjugate.

[0194] 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 Traut's reagent with amino groups of antibodies; reacting N-succinimidyl S-acetylthioalkanoate with amino groups of antibodies, followed by reaction with hydroxylamine; reacting with N-succinimidyl 3-(pyridyldithio) propionate, followed by reaction with a reducing agent; reacting antibodies with reducing agents such as dithiothreitol, 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce disulfide bonds in the hinge portion of antibodies to form thiol groups, but are not limited thereto.

[0195] Specifically, 0.3 to 10 molar equivalents of TCEP per disulfide bond in the hinge of the antibody is 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 in the hinge of the antibody are partially or completely reduced. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA).

[0196] It may be at a concentration of 1 mM to 20 mM. Examples of usable buffers include sodium phosphate, sodium borate or sodium acetate solutions. Specifically, an antibody having a partially or completely reduced thiol group may be obtained by reacting the antibody with TCEP at 4°C-37°C for 1 to 4 hours.

[0197] Meanwhile, by performing a reaction for adding a thiol group to the drug-linker moiety, the drug-linker moiety can be coupled via a thioether bond.

[0198] By using 2 to 20 molar equivalents of the compound for each antibody having a sulfhydryl group, an antibody-drug conjugate in which 2 to 8 drug molecules are coupled to each antibody can be prepared. Specifically, it is sufficient to add a solution in which the compound is dissolved in a buffer containing an antibody having a sulfhydryl group to react. Here, examples of buffers that can be used include sodium acetate solution, sodium phosphate and sodium borate. The pH of the reaction is 5 to 9, and the reaction is more preferably carried out at about 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).

[0199] The reaction can be carried out by adding a 1 to 20% v / v organic solvent solution in which the compound is dissolved 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.

[0200] After conjugation, the produced antibody-drug conjugates can be subjected to buffer exchange, antibody concentration, and average number of drug conjugates per antibody molecule can be measured to identify the antibody-drug conjugates.

[0201] Common operation A: Measurement of antibody concentration

[0202] The antibody concentration was measured using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At this time, the absorption coefficient at 280 nm can be estimated by the amino acid sequence of the antibody using a known calculation method (Protein Science, 1995, Vol. 4, 2411-2423), and a different absorption coefficient at 280 nm for each antibody (1.3 mL mg-1 cm-1 to 1.8 mL mg-1 cm-1) was used. In the case of KA0013, an absorption coefficient at 280 nm of 1.768 mL mg-1 cm-1 was used as an estimated value based on its amino acid sequence.

[0203] Common procedure B: Buffer exchange of antibody-drug conjugates

[0204] The antibody-drug conjugate was ultrafiltrated and concentrated using an ultrafiltration membrane package (Millipore P3C030C00, 30 kDa) according to the manufacturer's method, and the liquid was replaced 10 times.

[0205] Common procedure C: Measurement of the average number of drug conjugates per antibody molecule in an antibody-drug conjugate

[0206] The average number of drug conjugates per antibody molecule in an antibody-drug conjugate was measured by LC-MS:

[0207] The sample was diluted with water to 2 mg / mL, 50 μL of the diluted sample was taken, 1 μL of 0.5 M TCEP was added, and the reaction was carried out at room temperature for 30 min, and then the sample was prepared for injection. The chromatographic column was MabPac RP (2.1×50 mm); the mobile phase was mobile phase A-0.1% formic acid aqueous solution; the mobile phase B-0.1% formic acid acetonitrile solution; the injection volume was 2 μg; the column temperature was 80°C; and the detection wavelength was 280 nm.

[0208] The liquid phase gradient is shown in Table 2 below.

[0209] Table 2

[0210]

[0211] The DAR value was calculated based on the signal value of each component.

[0212]

[0213] In the above formula, LCn represents the signal value of the light chain coupled to n linkers-payloads, and HCn represents the signal value of the heavy chain coupled to n linkers-payloads.

[0214] Specific coupling examples are as follows.

[0215] Antibody reduction: Partritumab was diluted to about 10 mg / mL using phosphate buffer PB / EDTA (15 mM Na2HPO4, 5 mM NaH2PO4, 5 mM EDTA-2Na, pH 7.0). The antibody solution (100 mL) was added to a beaker at room temperature, followed by addition of 10 mM TCEP aqueous solution (0.68 mL; 10.0 equivalents per antibody molecule), and stirred with a magnetic stirrer, and incubated at room temperature for 3 hours to reduce the disulfide bonds of the antibody hinge.

[0216] Coupling of antibody and drug linker: Slowly add 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 antibody.

[0217] Purification: Ultrafiltration and concentration were performed using an ultrafiltration membrane package to replace the buffer with a formulation buffer while removing the unconjugated drug linker and other low molecular weight reagents, and the solution was then concentrated. The purified solution was sterile filtered to obtain a solution containing the antibody-drug conjugate KA0013.

[0218] The antibody concentration was measured using common procedure A: 23.64 mg / mL, and the average number of drug conjugations (n) per antibody molecule in KA0013 was measured using common procedure C: 7.87.

[0219] Preparation Example 4: Preparation of control drug

[0220] KA0010: Mc-GGFG-Dxd, a control linker-payload.

[0221] The structural formula of reference KA0010 is shown in the following formula V:

[0222]

[0223]

[0224] KA0011: Patritumab-Mc-GGFG-Dxd.

[0225] IgG-KA0010: IgG-Mc-GGFG-Dxd, abbreviated as IgG-Dxd.

[0226] IgG-KA0012: IgG-Mc-AAA-Dxd.

[0227] The preparation methods of KA0010 and KA0011 refer to the relevant description in Chinese patent document CN 106163559B.

[0228] Both IgG-KA0010 and IgG-KA0012 were prepared by a common coupling method for thiol coupling. The specific preparation method can refer to the preparation method of KA0013 mentioned above, and can also refer to the relevant description in Chinese patent document CN 106163559 B.

[0229] Example 1: FACS detection of the binding of anti-HER3 antibody-drug conjugate KA0013 to HER3 on the surface of SK-BR-3 cells Synthetic activity

[0230] 1. Experimental Drugs

[0231] KA0013,

[0232] HER3 target monoclonal antibody Patritumab (prepared with reference to Preparation Example 1).

[0233] KA0011.

[0234] 2. Experimental Methods

[0235] SK-BR-3 cells (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were routinely collected, washed once with an appropriate amount of PBS, counted and the 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 gradient diluted experimental drugs (900nM, 300nM, 100nM, 33.3nM, 11.1nM, 3.7nM, 1.23nM, 0.41nM, 0.041nM, 0.0041nM according to 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 (SouthernBiotech, 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 the washing twice; add 200μL 1% PBSA, resuspend the cell pellet and then detect on the instrument.

[0236] 3. Experimental results

[0237] The results are as follows Figure 1 shown.

[0238] The results showed that under the same experimental conditions, KA0013, KA0011, and HER3-targeting monoclonal antibody Patritumab bound to SK-BR-3 cells and EC 50 They are 15.36nM, 12.89nM and 8.481nM respectively.

[0239] The results showed that under the same experimental conditions, KA0013, KA0011 and Patritumab all had the activity of effectively binding to SK-BR-3 cells expressing HER3.

[0240] Example 2: FACS detection of the internalization activity of anti-HER3 antibody-drug conjugate KA0013 on MDA-MB-453 cells

[0241] 1. Experimental Drugs

[0242] KA0013,

[0243] IgG-Dxd,

[0244] KA0012,

[0245] KA0011.

[0246] 2. Experimental Methods

[0247] MDA-MB-453 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60386) were collected, counted and the cell viability was determined; 1×10 5Add the cell suspension to a 96-well low-adsorption plate, centrifuge at 750×g for 5 minutes, and discard the supernatant; add 100 μL of experimental drugs (working concentration is 100 nM) respectively, and design an isotype control (IgG-Dxd, working concentration is 100 nM) at the same time, mix well, and incubate in a 37°C incubator for 1 h, 3 h, and 5 h. After the 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 patritumab (working concentration was 100 nM) was added, and the cells were 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-AlexaFluor 647 (Southern Biotech, Cat. 9040-31) was added, the cell pellet was resuspended, and the cells were incubated on ice and protected from light 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 1% PBSA was added, the cell pellet was resuspended, and the cells were tested on the machine.

[0248] 3. Experimental results

[0249] The results are as follows Figure 2 shown.

[0250] The results showed that under the same experimental conditions, compared with the isotype control IgG-Dxd (i.e., IgG-Mc-GGFG-Dxd, produced by Zhongshan Kangfang Biotechnology Co., Ltd.), KA0013 and KA0011 could be internalized in MDA-MB-453, and the endocytic activity of the two was significantly different from that of IgG-Dxd.

[0251] Example 3: Non-specific killing activity measurement of the linker-payload portion of the anti-HER3 antibody-drug conjugate Certainly

[0252] 1. Experimental Drugs

[0253] IgG-KA0010,

[0254] IgG-KA0012.

[0255] 2. Experimental Methods

[0256] 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.

[0257] After collecting human venous blood, the sodium heparin anticoagulation tube was gently mixed upside down and inverted, centrifuged at 3500 rpm for 10 minutes at room temperature, and the plasma layer was taken for the experiment. MDA-MB-453 cells were routinely collected, counted and determined for cell viability, and the cell density was adjusted to 2.5×10 4 / mL, add the cell suspension to a 96-well black bottom plate at 100μL / well, and incubate overnight in a 37°C, 5% CO2 incubator; dilute the antibody with DMEM medium (5% human plasma), add 100μL of gradient diluted experimental drugs (100nM, 50nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM, 0.05nM, 0.01nM according to the working concentration of the antibody), and design a blank control group, with 3 replicates for each group; incubate in a 37°C, 5% CO2 incubator for 6 days. After the incubation, remove the 96-well plate, centrifuge at 350×g for 5 minutes, discard 135μL of supernatant, add 65μL CellTiter-Glo (promega, Cat.: G8461), incubate at room temperature for 20 minutes, and detect the fluorescence value in a multi-label microplate reader.

[0258] 3. Experimental results

[0259] The results are as follows Figure 3 shown.

[0260] 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.

[0261] Example 4: Plasma stability test of anti-HER3 antibody-drug conjugate KA0013

[0262] 1. Experimental Drugs

[0263] KA0013,

[0264] KA0011.

[0265] 2. Experimental Methods

[0266] After collecting human venous blood, gently mix the sodium heparin anticoagulation tube upside down and invert it, centrifuge it at 3500rpm for 10 minutes at room temperature, and the supernatant is plasma. Dilute the experimental drug with human plasma (working concentration is 100μg / mL), place it in a 37℃, 5% CO2 incubator for 0h, 24h, 48h, 72h, 96h, 139h, and after the incubation, take out 150μL of the sample and freeze it in liquid nitrogen. After all the samples are collected, the changes in free Dxd in plasma are detected by mass spectrometry.

[0267] Mass spectrometry sample preparation: Take an appropriate amount of the above sample, mix with acetonitrile in a ratio of 1:1 to precipitate protein, centrifuge at 12000rpm for 3min, and take 10μL of the supernatant for mass spectrometry detection. Chromatographic column: MabPac RP 2.1×50mm 4μm; mobile phase A: 0.1% formic acid water; mobile phase B: 0.1% formic acid acetonitrile; column temperature: 30℃;

[0268] The gradient is shown in Table 3 below.

[0269] Table 3

[0270]

[0271]

[0272] 3. Experimental results

[0273] The results are as follows Figure 4 shown.

[0274] 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.

[0275] Example 5: In vitro detection of cathepsin B cleavage efficiency of different linkers-payloads

[0276] 1. Experimental samples:

[0277] KA0010,

[0278] KA0012.

[0279] 2. Experimental Methods

[0280] PBS (20-fold solution) (Sinobio, Cat.: B548117) was diluted 20 times 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 was 3 nM) was mixed with an equal volume of linker-payload (working concentration was 10 μM), and incubated in a 37°C, 5% CO2 incubator for 0 hours, 0.5 hours, 1 hour, 2 hours, and 4 hours; Halt was added after the incubation. TM The reaction was terminated by using Protease Inhibitor Cocktail, EDTA-Free (30X) (Thermo Fisher, Cat.: 87785).

[0281] The enzymatically hydrolyzed samples were directly loaded for mass spectrometry detection. The chromatographic column: MabPac RP 2.1×50mm 4μm; mobile phase A: 0.1% formic acid water; mobile phase B: 0.1% formic acid acetonitrile; sample volume: 10μM sample injection 1μL; column temperature: 30℃;

[0282] The gradient is shown in Table 4 below.

[0283] Table 4

[0284]

[0285]

[0286] 3. Experimental results

[0287] The results are as follows Figure 5 shown.

[0288] 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.

[0289] Example 6: ELISA method to detect the binding activity of anti-HER3 antibody-drug conjugate KA0013 with antigen

[0290] The ELISA plate was coated with human HER3-his and incubated. After blocking, the antibodies to be tested were added respectively. 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, TMB (Neogen, 308177) was used for color development. After color development was terminated, the absorbance at a wavelength of 450 nm was detected in an ELISA instrument. The data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0291] The results are as follows Figure 6 And as shown in Table 5.

[0292] Table 5: Binding activity of KA0013 and KA0011 to the antigen human HER3-his

[0293]

[0294]

[0295] 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.

[0296] Example 7: Determination of Kinetic Parameters of Anti-HER3 Antibody-Drug Conjugates

[0297] 1. Experimental samples

[0298] KA0013

[0299] KA0011

[0300] 2. Experimental Methods

[0301] The dilution buffer of the experimental sample was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 1 μg / ml huHER3-his-Biotin was fixed on the SA sensor with a fixed height of 1 nm. The sensor was balanced in the buffer for 60 s. The huHER3-his-Biotin fixed on the sensor was bound to each antibody with an antibody concentration of 0.2469-20 nM (three-fold dilution) for 120 s. The antibody was dissociated in the buffer for 300 s. The sample plate vibration rate was 1000 rpm, the detection temperature was 30°C, and the frequency was 5.0 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constant. The data acquisition software was Fortebio DataAcquisition 12.0, and the data analysis software was Fortebio Data Analysis 12.0.

[0302] 3. Experimental results

[0303] The results are shown in Table 6 and Figures 7 and 8 shown.

[0304] Table 6: Kinetic parameters for binding of anti-HER3 antibody-drug conjugates to human HER3-his-biotin

[0305] sample KD(M) kon(1 / Ms) SE(kon) kdis(1 / s) SE(kdis) Rmax(nm) KA0013 8.08E-11 1.49E+06 4.63E+03 1.21E-04 2.77E-06 0.116-0.243 KA0011 5.52E-11 1.40E+06 6.33E+03 7.70E-05 3.99E-06 0.128-0.197

[0306] KD is the affinity constant; kon is the antigen-antibody binding rate; kdis is the antigen-antibody dissociation rate; KD = kdis / kon.

[0307] The results showed that the anti-HER3 antibody-drug conjugates KA0013 and KA0011 had good affinity to the antigen human HER3-his-biotin.

[0308] Example 8: Determination of specific cell killing activity of anti-HER3 antibody-drug conjugates

[0309] 1. Experimental Drugs

[0310] KA0013

[0311] KA0011

[0312] IgG-KA0012

[0313] 2. Experimental Methods

[0314] Collect NCI-H1781 cells (Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60116), centrifuge at 1000x g for 5 minutes, remove the supernatant, resuspend, count and viability, dilute the cells, add NCI-H1781 (2500 / 100μL) to each well of a black-bottom 96-well plate. Place in a 37℃5% CO2 incubator overnight; dilute the experimental drug with culture medium (10% human AB serum), add 100μL of experimental drug to each well according to the experimental design, and design a blank control, with 3 replicates per group. Place in a 37℃5% CO2 incubator for 6 days; after incubation, centrifuge the 96-well plate at 350x g for 5 minutes, discard 135μL of supernatant; add 60μL CellTiter-Glo (promega, Cat.: G8461) to each well, incubate at room temperature for 15 minutes, and detect the fluorescence value with a multi-label microplate reader.

[0315] 3. Experimental results

[0316] The results are as follows Fig. 9 The results showed that both anti-HER3 antibody-drug conjugates KA0013 and KA0011 could specifically kill NCI-H1781 cells.

[0317] Example 9: Study on ADCC activity of anti-HER3 antibody-drug conjugates

[0318] 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 min, and the supernatant was discarded. The cell pellet was resuspended in assay medium (RPMI-1640 containing 1% FBS), washed twice by centrifugation, and the number of cells was counted. The cell density was adjusted with assay medium, and the target cell suspension was inoculated into a 96-well plate at 50 μL / well (each well contained 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 antibody working concentration) 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 5PBMC, 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 100μL of cell supernatant and transfer to a new 96-well plate. According to the instructions of the Cytotoxicity Detection Kit (Roche, catalog number: 11644793001), add 100μL 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 490 nm -OD 650 nm The ADCC activity (expressed as ADCC%) was calculated based on the measured OD value of each group.

[0319] 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%

[0320] The results are as follows Fig.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.

[0321] Example 10: In vivo efficacy testing of anti-HER3 antibody-drug conjugates

[0322] To detect the anti-HER3 antibody-drug conjugate in vivo tumor inhibition activity, HCC827 human lung cancer cells (purchased from Guangzhou Jinio Biotechnology Co., Ltd.) were first inoculated subcutaneously above the right back thigh of 5-8 week-old female CB-17SCID mice (purchased from Guangdong Nanmo Biotechnology Co., Ltd.). The day of grouping was defined as D0. The administration method was intravenous injection (iv), once a week, for a total of 3 times. 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.

[0323] Table 7: Dosing regimen of anti-HER3 antibody-drug conjugates in the treatment of HCC827 human lung cancer cell transplant tumor model in CB-17SCID mice

[0324]

[0325] The results are as follows Fig.11 As shown. The results showed that compared with the isotype control antibody, the anti-HER3 antibody-drug conjugate KA0013 can effectively inhibit the growth of mouse tumors. In addition, Fig.12 As shown, the tumor-bearing mice had good tolerance to the test drug KA0013, and each group had no effect on the body weight of tumor-bearing mice.

[0326] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached 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-1chain C region or Ig gamma-4chain C region; the light chain constant region is Ig kappachain 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.

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