Anti-Nectin-4 antibody, antibody-drug conjugate, method for producing the same, and use

Abstract

The present invention relates to an anti-Nectin-4 antibody, an antibody-drug conjugate, and methods for producing and using the same that are effective in treating and / or preventing Nectin-4-positive tumors, including cancers such as breast cancer and bladder cancer.

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals, and more particularly to antibodies and complexes against the Nectin-4 protein, and to the use of such antibodies and complexes in the treatment of tumors. [Background technology]

[0002] Nectin-4 (Nectin Cell Adhesion Molecule 4), also known as PVRL4 (Poliovirus Receptor like 4), is a type I membrane protein belonging to the family of cell adhesion molecules that are specifically expressed in the embryo and placenta. This family consists of four members (Nectin-1 to Nectin-4). 2+ Nectin-1 to Nectin-3 are transmembrane cell adhesion molecules of independent immunoglobulins. They are widely expressed in normal adult tissues. Nectin-4 protein, on the other hand, is undetectable in healthy adult tissues, including breast tissue, but is overexpressed in tissues of various cancers (breast cancer, lung cancer, ovarian cancer, etc.), with overexpression observed in 62% of triple-negative breast cancers.

[0003] Studies have shown that Nectin-4 overexpression promotes intratumoral angiogenesis and tumor growth in cancer development. Furthermore, the PI3K / AKT signaling pathway is involved in promoting Nectin-4-mediated cancer cell proliferation. In addition, Nectin-4 overexpression is associated with poor prognosis in lung, breast, and ovarian cancers. Nectin-4 interacts with the Afadin protein (actin filament-binding protein) to form a complex, ultimately increasing cell survival and preventing apoptosis via the PI3K-AKT signaling pathway. High Nectin-4 expression is a risk factor for lymph node metastasis in papillary thyroid carcinoma (PTC) patients. Nectin-4 depletion effectively inhibits proliferation and invasion in two PTC cell lines (i.e., TPC1 and KTC-1 human thyroid cancer cell lines) and induces apoptosis in vitro. Nectin-4 overexpression in human esophageal cancer tissue is closely associated with tumor size, tumor invasion depth, and poor patient prognosis. Intervention of Nectin-4 expression in esophageal cancer cell lines shows that increased Nectin-4 expression significantly promotes cell viability, migration, invasion, and tumorigenesis.

[0004] Bladder cancer is a malignant tumor that develops in the bladder mucosa, and is mostly transitional cell carcinoma. It most commonly occurs in the lateral and posterior walls of the bladder, followed by the triangular region and upper region, which can occur multicentrically. Bladder cancer is the most common tumor of the urinary system and is more common in men. According to data published by the National Cancer Center of China in 2019, bladder cancer ranked among the top seven cancers with an incidence rate of 8.83 per 100,000 people, and has shown a significant upward trend in recent years. There are differences in the incidence of bladder cancer by region, race, and sex, and it occurs in all age groups, with a high incidence rate of 50-70 years old, and the incidence rate increases with age.

[0005] Bladder cancer is clinically classified as non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) or metastatic bladder cancer. Urothelial (transitional cell) carcinoma is the most common type of bladder cancer, accounting for approximately 90% of all cases. Metastatic bladder cancer generally has a poor prognosis, with a 5-year survival rate of only 15% for patients with phase IV bladder cancer. For NMIBC and MIBC, transurethral resection of bladder tumor (TURBT) is the standard treatment, while local bladder perfusion chemotherapy followed by immunotherapy (using bacillus Calmette-Guerin) is adjuvant therapy. Over the past several decades, chemotherapy has been the preferred treatment for metastatic cancer, most commonly using platinum-containing anticancer drugs.

[0006] Breast cancer is currently the most common cancer in women worldwide, with an annual incidence rate of 0.2% to 8% globally. Approximately 1.4 million women are diagnosed with breast cancer each year, and around 500,000 die from the disease. Breast cancer is the leading cause of death for women aged 40-55. Since the late 1970s, breast cancer has been the leading cause of malignant tumors in women worldwide. According to the American Association for Cancer Research, there are approximately 200,000 new cases of breast cancer annually in the United States, with an incidence rate of 1.16 per 100,000. While China has historically had a low incidence of breast cancer, the incidence rate has increased significantly in recent years. In major Chinese cities, the incidence rate has increased by 37% and the mortality rate by 38.9% over the past decade. Shanghai, Beijing, Tianjin, and coastal areas, in particular, have become high-incidence areas for breast cancer, leading the way in the incidence of malignant tumors in women.

[0007] Currently, international anti-tumor drugs are primarily targeted monoclonal antibodies, which constitute the largest sub-industry in the biomedical field. According to some data, in 2016, monoclonal antibody drugs accounted for 43% of the total biopharmaceutical market. While monoclonal antibodies possess characteristics such as high target specificity and low side effects, their therapeutic effects are relatively limited when used alone. Therefore, the majority of monoclonal antibody drugs are used in combination with chemotherapy, and the current primary pathway for enhancing the therapeutic effect of monoclonal antibodies is antibody-drug conjugates. Antibody-drug conjugates are a novel type of anti-cancer biopharmaceutical and consist of three parts: an antibody, a drug, and a linker that connects both. When a monoclonal antibody is bound to a drug, the antibody-drug conjugate utilizes the targeting of the monoclonal antibody to specifically recognize receptors on the surface of cancer cells, bind to the receptors, enter the cell, and release the drug via intracellular proteases, inhibiting cancer cell proliferation and ultimately leading to cell death. Antibody-drug conjugation technology integrates small molecule drugs with biological proteins, combining the advantages of both to significantly improve drug efficacy, reduce drug side effects, and create next-generation therapeutic products.

[0008] Currently, in projects targeting Nectin-4 both domestically and internationally, there are no chemical drugs, and only six biological drugs. Of these, the only Nectin-4-targeting antibody-drug conjugate (ADC) drug on the market is a Nectin-4-targeting ADC jointly developed by Agensys Japan (a subsidiary of Astellas Pharma) and Seattle Genetics in the United States. It received accelerated approval from the FDA on December 19, 2019, and its trade name is Padcev. Its indication is locally advanced or metastatic urothelial carcinoma. Relevant clinical data showed an ORR of 44%, a CR of 12%, and a median DOR of 7.6 months. Among patients treated with Padcev, 46% experienced severe adverse reactions, the most common of which (≧3%) were urinary tract infection (6%), cellulitis (5%), hyperthermic neutropenia (4%), diarrhea (4%), sepsis (3%), acute kidney injury (3%), dyspnea (3%), and rash (3%). Fatal adverse reactions occurred at an incidence of 3.2%, including acute respiratory failure (0.8%), aspiration pneumonia (0.8%), heart disease (0.8%), and sepsis (0.8%).

[0009] Thus, given the relatively serious adverse reactions associated with Padcev, and based on the current state of research on Nectin-4 targets, patients still have unsatisfied treatment needs regarding related antibody-drug conjugates, and there is a strong desire for antitumor targeted drugs that are highly effective, have few side effects, and are reasonably priced, thus benefiting cancer patients.

[0010] The development of ADCs is an extremely complex task, requiring precise target selection, drug optimization and improvement, appropriate linker selection, and enhancement of the efficacy and safety of ADC drugs, which are the challenges of ADC drug development. In ADC drug design, based on a comprehensive consideration of the antibody, linker, and drug as a whole, the oncological indications and targets of drug action are clarified, the mechanism of action of the ADC drug is thoroughly and comprehensively studied, and ultimately, effective killing of tumor cells by the ADC drug is achieved while simultaneously improving the quality of life of patients.

[0011] Furthermore, one of the keys to the success of ADCs is selecting antibodies suitable for delivering drugs to tumor cells while maintaining drug activity. Antibodies used in the preparation of ADCs must not only specifically bind to antigen-positive cells in tumors, but also possess the ability for the antigen-antibody conjugate to facilitate internal distribution of the ADC. Antibody screening must consider at least three factors: specific binding, internal distribution, and post-distribution antibody localization. Moreover, antibody affinity does not have a clear correlation with the rate of internal distribution (Laurent Ducry, Antibody-Drug Conjugates, Scientific Press, pp. 36-37). Therefore, antibodies that function as highly accurate "localization tools" must be able to specifically bind to target cells and effectively mediate "endocytosis" and localization; these are important physical properties for the specificity and toxicity of antibody-drug conjugates.

[0012] Based on the shortcomings of the prior art, the inventors focused on the problem that the binding between linkers of conventional drugs and small molecule drugs is unstable, and developed NH2 -(CH 2 -CH 2 -O) 3 -CH 2 -C(=O)Using Val-Cit as a linker, a human antibody is conjugated to a small molecule drug (e.g., MMAE) via an enzyme-mediated site crosslinking method. The resulting ADC drug exhibits extremely potent agonistic activity against Nectin-4-overexpressing cancer cells, particularly tumors such as breast cancer and bladder cancer. Specifically, firstly, endocytosis experiments showed that the anti-Nectin-4 antibody-drug conjugate obtained by the present invention exhibited a better endocytosis effect compared to the control Padcev, simultaneously achieving specific binding, highly efficient internalization, and accurate cell localization, thereby improving the specificity of the antibody-drug conjugate and reducing its toxicity. Secondly, in vivo experiments showed that intravenous administration of the antibody-drug conjugate to nude mice with Nectin-4-positive xenograft tumors inhibited tumor growth, and a significant therapeutic effect was observed even with a low single intravenous dose of 1 mg / kg. Its overall therapeutic effect was significantly superior to that of the control Padcev. Finally, in toxicity experiments with monkeys, Padcev showed severe side effects at a second dose of 6 mg / kg, proving to be lethal. In contrast, the antibody-drug conjugate obtained by the present invention showed no substantial adverse reactions even with two high doses of 9 mg / kg, exhibiting a wider therapeutic window and better drug formation properties. The antibody-drug conjugate provided by the present invention achieved unexpected technical benefits. [Overview of the project] [Problems that the invention aims to solve]

[0013] The present invention provides antibodies, functional fragments (e.g., antigen-binding fragments), and antibody-drug conjugates (ADCs) that bind to the Nectin-4 protein and / or Nectin-4 protein polypeptide fragments.

[0014] In one embodiment, the present invention provides an antibody or antibody functional fragment comprising a heavy chain and a light chain that specifically binds to Nectin-4, among which, (i) The heavy chain contains three CDR regions, and the amino acid sequence of at least one CDR region has the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or has an amino acid sequence having at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; and / or (ii) The light chain contains three CDR regions, and the amino acid sequence of at least one CDR region has the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or has an amino acid sequence having at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0015] In some specific embodiments, the antibody comprises a heavy chain and a light chain, (i) The heavy chain contains three CDR regions, which are the CDR1 region, the CDR2 region, and the CDR3 region respectively, and the CDR1 region, the CDR2 region, and the CDR3 region have the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively; and / or (ii) The light chain contains three CDR regions, which are the CDR1 region, the CDR2 region, and the CDR3 region respectively, and the CDR1 region, the CDR2 region, and the CDR3 region have the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively.

[0016] In some specific embodiments, the antibody has a sequence of the heavy chain variable region that is the amino acid sequence shown in SEQ ID NO: 9, or a sequence having at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; and the sequence of the light chain variable region is selected from the amino acid sequences shown in SEQ ID NO: 10 - SEQ ID NO: 12 or sequences having at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0017] In some specific embodiments, the antibody or its functional fragment of the present invention is isolated.

[0018] In some specific embodiments, the antibody of the present invention is a monoclonal antibody.

[0019] In some specific embodiments, the antibody or its functional fragment of the present invention is a human antibody, preferably a fully human antibody.

[0020] In some specific embodiments, the antibody of the present invention is a bispecific antibody.

[0021] In some specific embodiments, the antibody or its functional fragment of the present invention has ADCC activity.

[0022] In some specific embodiments, the antibody or its functional fragment of the present invention has CDC activity.

[0023] In some specific embodiments, the antibody or its functional fragment of the present invention specifically binds to Nectin-4 without substantially binding to Nectin-1, Nectin-2, or Nectin-3.

[0024] In some specific embodiments, the antibody or its functional fragment of the present invention includes bispecific antibodies, Fab fragments, Fab' fragments, F(ab)'2, scFv, dsFv, and single domain antibodies. The scFv protein is a fusion protein in which the variable region of the light chain and the variable region of the heavy chain of an immunoglobulin are linked by a linker. The dsFv has cysteine introduced at specific sites in the conserved framework regions of VH and VL, thereby stabilizing the structure of dsFv by a disulfide bond.

[0025] In some specific embodiments, the antibody or its functional fragment of the present invention is IgM, IgD, IgG, IgA, or IgE. IgG antibodies have four subtypes, IgG1, IgG2, IgG3, and IgG4, and preferably have IgG1 antibodies.

[0026] In some specific embodiments, the heavy chain constant region sequence of the antibody is shown as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 16).

[0027] In some specific embodiments, the constant region sequence of the antibody light chain is shown as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 17).

[0028] In certain specific embodiments, the antibody or functional fragment thereof of the present invention is useful for the treatment or prevention of cancer that overexpresses Nectin-4.

[0029] In some embodiments, an antibody or functional fragment having the ability to bind to Nectin-4 binds to a native epitope of Nectin-4 present on the surface of living cells. In some embodiments, an antibody or functional fragment having the ability to bind to Nectin-4 binds to the extracellular domain of Nectin-4. In some embodiments, an antibody or functional fragment having the ability to bind to Nectin-4 binds to the first extracellular region of Nectin-4. In some embodiments, an antibody or functional fragment having the ability to bind to Nectin-4 binds to the 1-147aa domain of Nectin-4. In some embodiments, an antibody or functional fragment having the ability to bind to Nectin-4 binds to the 32-142aa domain of Nectin-4.

[0030] In another embodiment, the present invention provides isolated polynucleotides encoding the antibody of the present invention.

[0031] In yet another embodiment, the present invention provides a combination of isolated polynucleotides comprising a polynucleotide encoding the light chain of the antibody or functional fragment thereof and a polynucleotide encoding the heavy chain of the antibody or functional fragment thereof.

[0032] In another embodiment, the present invention provides an expression vector comprising a polynucleotide or a combination of polynucleotides of the present invention, wherein a polynucleotide is linked to an expressible regulatory sequence of the polypeptide encoded by it in a host cell or cell-free expression system.

[0033] In some embodiments of the present invention, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell may be Escherichia coli, Bacillus subtilis, Streptomyces, or Aspergillus chimaericus. Examples of eukaryotic host cells include fungi such as Pasteurella, Saccharomyces cerevisiae, fission yeast, and Xylaria, insect cells such as Meadow Slime, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the host cell of the present invention is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0034] In another embodiment, the present invention provides an antibody-drug conjugate comprising an antibody or functional fragment thereof conjugated to one or more drugs, preferably the drugs being cytotoxic drugs (e.g., antimetabolites, antitumor antibiotics, alkaloids), immunostimulants, or radioisotopes. More preferably the drugs are selected from the group consisting of auristatin derivatives, maytansinoids (e.g., ansamitocin or mertansine), dolastatin and its derivatives), camptothecin analogs, DNA topoisomerase I inhibitors and their derivatives, and most preferably the drugs are selected from MMAE (Monomethyl auristatin E) and MMAF (Monomethyl auristatin F).

[0035] In some embodiments, an antibody or functional fragment thereof capable of binding to Nectin-4 is covalently bound to the drug moiety via a linker. In some embodiments, the junction is a cleavable junction. In some embodiments, the junction is cleavable under intracellular conditions. In some embodiments, the junction is hydrolyzable at a pH less than 5.5. In some embodiments, the junction is cleavable by intracellular proteases. In some embodiments, the junction is tissue protease-cleavable. In some embodiments, the junction comprises a dipeptide or tetrapeptide. In some embodiments, the dipeptide is valine (Val)-citrulline (Cit). In some embodiments, the antibody is bound to the linker via the sulfhydryl group of the antibody's cysteine. In some embodiments, the antibody is bound to the linker via the amino group of the antibody (particularly the amino group of a glutamine residue).

[0036] In some specific embodiments, the antibody-drug conjugate of the present invention has the general formula Ab-(LU)n, where Ab represents a monoclonal antibody targeting Nectin-4, and L represents NH2-( CH 2 -CH 2 -O )m -CH 2 -C(=O) -Val-Cit, NH2-( CH 2 -CH 2 -O )m -CH 2 -C(=O) -A linker selected from Val-Cit-pABC, mc-Val-Cit-pABC, Val-Cit-pABC, Val-Cit-pAB, or Val-Cit, where U is a drug selected from DM1, DM4, MMAE, MMAF, DXD, and SN38, and m is (CH 2 -CH 2 -O)n represents the number of , an integer from 1 to 8, preferably 2, 3, 4, 5, 6, 7, 8, more preferably 3, 4, 5, 6, and most preferably 3, and n represents the drug-antibody binding ratio DAR, an integer from 1 to 8, preferably 2, 4, 6, 8, more preferably 2, 4, and most preferably 2. In some specific embodiments, n is a decimal from 1 to 8, where mc-Val-Cit-pABC is a collective term for maleimidocaproyl-valine-citrulline-p-aminocarbamate and Val-Cit-pAB is a collective term for valine-citrulline-p-aminobenzyloxycarbonyl.

[0037] In some specific embodiments, the present invention relates to an antibody-drug conjugate (ADC) that specifically binds to Nectin-4, wherein the antibody or a functional fragment thereof comprises a heavy chain variable region and a light chain variable region, the sequence of the heavy chain variable region being the amino acid sequence shown in SEQ ID NO: 9, and the sequence of the light chain variable region being an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 10-12.

[0038] In some specific embodiments, the sequence of the heavy chain variable region of the antibody or its functional fragment is the amino acid sequence shown in SEQ ID NO: 9, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence of the light chain variable region is selected from the amino acid sequences shown in SEQ ID NOs: 10-12, or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0039] In some specific embodiments, the antibody is a monoclonal antibody.

[0040] In some specific embodiments, the antibody is a human antibody.

[0041] In some specific embodiments, the present invention relates to an isolated polynucleotide encoding the light chain and / or the heavy chain of the antibody or functional fragment.

[0042] In some specific embodiments, the present invention relates to an expression vector comprising a polynucleotide, wherein the polynucleotide is linked to an expressible regulatory sequence of the polypeptide encoded by it in a host cell or cell-free expression system.

[0043] In some specific embodiments, the present invention relates to a host cell containing the expression vector.

[0044] In another embodiment, the present invention provides a pharmaceutical formulation comprising the antibody-drug conjugate of the present invention and a pharmaceutically acceptable diluent, carrier, or excipient.

[0045] In another embodiment, the present invention provides a medical preparation comprising the antibody-drug conjugate of the present invention. In some embodiments, the medical preparation exists in the form of a kit comprising a container for housing the antibody-drug conjugate. In some embodiments, the medical preparation further includes printed instructions for using the product in a method of treating or preventing cancer (particularly cancer expressing Nectin-4).

[0046] In another embodiment, the present invention provides an antibody-drug conjugate for the effective treatment and / or prevention of tumors associated with cells expressing Nectin-4.

[0047] In another embodiment, the present invention provides the use of an antiproliferative agent and the antibody-drug conjugate in the manufacture of a pharmaceutical product for the treatment of tumors.

[0048] In another embodiment, the present invention provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate and antiproliferative agent.

[0049] In some specific embodiments, the antiproliferative agent may be an antibody, an antibody-drug conjugate, or a fusion protein.

[0050] In another embodiment, the present invention relates to a method for treating a tumor in a subject, comprising administering the antibody-drug conjugate, the pharmaceutical composition, or the pharmaceutical product to the subject.

[0051] In some embodiments, the present invention relates to a method for treating a tumor in a subject, comprising administering an effective amount of an antibody-drug conjugate, a pharmaceutical composition, or a pharmaceutical product, along with radiation to the subject.

[0052] In some embodiments, the present invention relates to a method for treating a tumor in a subject, comprising administering an effective amount of an antibody-drug conjugate or the pharmaceutical composition or the pharmaceutical product and an antiproliferative agent to the subject.

[0053] In some embodiments, the present invention relates to the aforementioned antibodies, antibody-drug conjugates, and pharmaceutical compositions for use in the treatment of cancer, preferably Nectin-4 positive tumors.

[0054] In some embodiments, the tumor (including cancer) includes, but is not limited to, hematological malignancies or solid tumors.

[0055] In some embodiments, the tumor (including cancer) is a hematological malignancy, preferably a lymphoma or leukemia, and includes, but is not limited to, myeloma, B-cell lymphoma, mantle cell lymphoma, non-Hodgkin B-cell lymphoma, non-Hodgkin T-cell lymphoma, cutaneous lymphoma, anaplastic large cell lymphoma, multiple myeloma, inactive non-Hodgkin lymphoma, plasmacytoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, and follicular lymphoma. In some embodiments, the hematological malignancy is recurrent or refractory.

[0056] In some embodiments, the tumor (including cancer) is a solid tumor and includes, but is not limited to, tumors of the respiratory system, gastrointestinal tract, urinary tract, male organ tumors, female organ tumors, skin cancer, endothelial cell tumors, brain tumors, nervous system tumors, and endocrine organ tumors.

[0057] In some embodiments, the respiratory tumors include, but are not limited to, lung cancer, nasopharyngeal cancer, and laryngeal cancer.

[0058] In some embodiments, the gastrointestinal tumors include, but are not limited to, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, and bile duct cancer.

[0059] In some embodiments, the urological tumors include, but are not limited to, kidney cancer, renal pelvis and ureteral cancer, bladder cancer, and urethral cancer.

[0060] In some embodiments, the male organ tumors include, but are not limited to, penile cancer, prostate cancer, or testicular cancer.

[0061] In some embodiments, the female genital tumors include, but are not limited to, breast cancer, vulvar cancer, vaginal cancer, cervical cancer, uterine cancer, or ovarian cancer.

[0062] In some embodiments, the neurological tumors include, but are not limited to, astrogliomas, oligodendrogliomas, ependymomas, medulloblastomas, and meningiomas.

[0063] In some embodiments, the brain tumor includes, but is not limited to, glioblastoma, neurocytoma, germoid mesenchymal tumor, stromal tumor, epithelial tumor, teratoma, and pineal tumor.

[0064] In some embodiments, the skin cancer includes, but is not limited to, cutaneous melanoma or non-melanoma skin cancer.

[0065] In some embodiments, the tumor is a solid tumor and includes, but is not limited to, bladder cancer, brain tumor, breast cancer, cervical cancer, thoracic tumor, endometrial cancer, esophageal squamous cell carcinoma, stomach cancer, head tumor, pancreatic cancer, bile duct cancer, colorectal cancer, eye cancer, head and neck squamous cell carcinoma, urothelial carcinoma, kidney cancer, liver cancer, lymph node cancer, lung cancer, oral cancer, cervical tumor, ovarian cancer, prostate cancer, testicular cancer, laryngeal and uterine cancer, melanoma, salivary gland cancer, fibrosarcoma, soft tissue sarcoma, and osteosarcoma. In some embodiments, the cancers described above are recurrent or refractory.

[0066] In some embodiments, the breast cancers include ductal carcinoma, lobular carcinoma, myelin carcinoma, glioma, tubular carcinoma, inflammatory breast cancer, and triple-negative breast cancer (TNBC).

[0067] In some embodiments, the ovarian cancer includes adenocarcinoma in the ovary and epithelial ovarian tumors such as adenocarcinoma that migrates from the ovary into the abdominal cavity.

[0068] In some embodiments, the leukemia includes acute myeloid leukemia (AML), acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, myelodysplasia, myeloproliferative disorders, NK cell leukemia (e.g., blastic plasmacytoid dendritic cell neoplasm), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and myelodysplastic syndromes (MDS).

[0069] In some embodiments, the pancreatic cancer is acinar cell adenocarcinoma of the pancreas, ductal cell adenocarcinoma of the pancreas, or phase IV pancreatic cancer.

[0070] In some embodiments, the lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and in some embodiments, the non-small cell lung cancer (NSCLC) includes, but is not limited to, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. In some embodiments, the lung cancer is recurrent, and in some embodiments, the lung cancer is recurrent squamous cell lung cancer or stage IV (advanced) squamous cell lung cancer.

[0071] In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC). [Brief explanation of the drawing]

[0072] [Figure 1] A schematic diagram of the Nectin-4 protein structure is shown. [Figure 2] A schematic diagram of the Nectin-4-ADC (SWY2001-Ab1-LND1002) structure is shown. In the formula, LND1002 represents the linker + drug molecule portion, and the circled portion represents the amide bond between the linker and antibody in L&D. The Nectin-4 ADC shown in this diagram is a site-directed antibody-drug conjugate, in which one anti-Nectin-4 monoclonal antibody is bound to one molecule of the MMAE derivative at the amino acid (Kabat) at position Q295 of each heavy chain via a linker (NH2-(CH2-CH2-O)3-CH2-C(=O)-Val-Cit) in each molecule. The bond between the antibody and the linker is a stable amide bond (hetero-peptide bond), and the average drug-antibody binding ratio (DAR) was 2.0. [Figure 3] A schematic diagram of the Nectin-4-ADC(SWY2001-Ab1-VC MMAE) structure is shown. [Figure 4] The pcDNA3.1 map is shown. [Figure 5] The DSC map for the human antibody SWY2001-Ab1 is shown. [Figure 6] This shows the modification rate identification map for Nectin-4 ADC (SWY2001-Ab1-LND1002). [Figure 7A] This shows the map for SWY2001-Ab1-LND1002 with confirmed DAR values. [Figure 7B] This shows the map of the determined DAR values ​​for SWY2001-Ab1-VC-MMAE. [Figure 8] This shows the endocytosis effect of SWY2001-Ab1-LND1002 in SK-BR-3 cells. [Figure 9] This shows the endocytosis effect of SWY2001-Ab1-LND1002 in T47D cells. [Figure 10] This shows in vivo tumor inhibition experiments of SWY2001-Ab1-LND1002 against mouse PC3-Nectin-4 stabilized strains. Note: m pk = mg / kg [Figure 11] This paper shows in vivo tumor inhibition experiments of SWY2001-Ab1-LND1002 against mouse MDA-MB-468 tumors. [Figure 12] This paper demonstrates in vivo tumor inhibition by SWY2001-Ab1-LND1002 against mouse HT-1376 tumors. [Modes for carrying out the invention]

[0073] Definition: Unless otherwise defined, all scientific terms used herein have the same meaning as understood by those skilled in the art. For definitions and terminology in this art, those skilled in the art can specifically refer to the Current Protocols in Molecular Biology (Ausubel). Amino acid residue abbreviations are three-letter and / or one-letter standard codes used in this art to represent one of the 20 common L-amino acids.

[0074] In this invention, numerical ranges and parameter approximations are described over a wide range, but all numerical values ​​shown in the specific examples are described as accurately as possible. However, any numerical value inherently contains a certain degree of error due to the standard deviation in each measurement. Furthermore, it should be understood that all ranges disclosed herein encompass all and any subranges contained within them. For example, when described herein, the range "1-10" encompasses all and any subranges contained between the minimum value of 1 and the maximum value of 10 (including the endpoints), that is, all subranges starting with a minimum value of 1 or a value greater than 1, e.g., 1-6.1, and all subranges ending with a maximum value of 10 or a value less than 10, e.g., 5.5-10. In addition, it should be understood that all references described as "incorporated herein" are incorporated in their entirety.

[0075] As used herein, the terms “pharmaceutical composition,” “combination drug,” and “drug combination” may be used interchangeably and mean a combination of at least one drug and optionally a pharmaceutically acceptable carrier or adjuvant to achieve a certain particular purpose. In some embodiments, a pharmaceutical composition includes combinations separated at the temporal and / or spatial level, insofar as they work together to achieve the objectives of the present invention. For example, the components contained in a pharmaceutical composition (e.g., antibodies, nucleic acid molecules, nucleic acid molecule combinations, and / or complexes) may be administered to a subject whole or separately. When the components contained in a pharmaceutical composition are administered to a subject separately, the components may be administered simultaneously or sequentially. Preferably, pharmaceutically acceptable carriers include water, buffered aqueous solutions, isotonic saline solutions, e.g., PBS (phosphate buffer solution), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol, or polyalkylene glycols, e.g., poly(propylene glycol), triglycerides, etc. The type of pharmaceutically acceptable carrier used depends particularly on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition according to the present invention may also contain humectants, emulsifiers, or buffering materials as additives.

[0076] The pharmaceutical composition, vaccine, or pharmaceutical preparation according to the present invention can be administered by any suitable route, such as oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, or intravenous administration.

[0077] As used herein, “therapeutically effective dose” or “effective dose” refers to a dose sufficient to demonstrate benefit to the recipient. The actual dose, rate, and duration of administration will vary depending on the condition and severity level of the recipient. The decision regarding treatment (e.g., the determination of dosage) is ultimately the responsibility of the general practitioner and other physicians, and the decision will be based on the factors described above. Typically, the decision will be made considering the disease being treated, the individual patient’s condition, the site of delivery, the method of administration, and any other factors known to the physician.

[0078] As used herein, the term "subject" means a mammal, for example, a human, but may also mean other animals, for example, wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.), or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

[0079] The term "antibody" refers to intact antibodies and their functional fragments. A "full-length antibody" refers to a protein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains one heavy chain variable region (abbreviated as VH) and one heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains one light chain variable region (abbreviated as VL) and one light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into various highly variable regions called complementarity-determining regions (CDRs), and may contain various more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding domains for interaction with the antigen. The constant region of the antibody may be mediated by 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. Chimeric or human antibodies are also included in the antibodies of the present invention. The CDR coding pattern of the present invention is IMGT.

[0080] Conventional genetic engineering techniques can be used to synthesize the genes for the light and heavy chains of antibodies; for example, the method disclosed by Chen Jianjun et al. (Chen Jianjun et al., Cellular and Molecular Immunology, 1997, No. 3) can be referenced.

[0081] The term "human antibody" refers to an antibody containing a CDR region derived from a human antibody, the rest of which is derived from one (or more) types of human antibodies. Furthermore, several residues in the framework region (FR) segment can be modified for the purpose of maintaining binding affinity. Human antibodies or fragments thereof according to the present invention can be prepared by techniques known to those skilled in the art.

[0082] The term "chimeric antibody" refers to an antibody in which the sequence of the variable region originates from one species, while the sequence of the constant region originates from another species; for example, the sequence of the variable region originates from a mouse antibody, while the sequence of the constant region originates from a human antibody. Chimeric antibodies or fragments thereof according to the present invention can be prepared by using genetic recombination technology. For example, a chimeric antibody can be produced by cloning recombinant DNA containing a promoter and sequences encoding the variable region of a non-human (particularly mouse) monoclonal antibody according to the present invention and sequences encoding the constant region of a human antibody. The chimeric antibody of the present invention encoded by this recombinant gene is, for example, a mouse-human chimera, which has specificity determined by the variable region derived from mouse DNA and isotype determined by the constant region derived from human DNA.

[0083] The term "monoclonal antibody" refers to an antibody molecular preparation having a single molecular structure. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0084] The term "bispecific antibody" refers to an antibody that can bind to two different antigens or antigenic epitopes, and includes a light chain and heavy chain of an antibody that can specifically bind to a first antigen, and a light chain and heavy chain of an antibody that can specifically bind to a second antigen.

[0085] As used herein, the term “functional fragment” more specifically means an antibody fragment, such as Fv, scFv (sc indicates a single chain), Fab, F(ab')2, Fab', scFv-Fc fragment, or bispecific antibody (diabody), or any fragment whose half-life can be increased by chemical modification or by incorporation into a liposome. Such chemical modification includes, for example, the addition of poly(alkylene) glycol, such as polyethylene glycol ("polyethylene glycolated") (referred to as polyethylene glycolated fragments of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG, where "PEG" indicates polyethylene glycol), and such fragment has Nectin-4 binding activity. Preferably, the functional fragment consists of or contains a partial sequence derived from the heavy chain or light chain variable chain of the antibody from which it is derived, the partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived. This functional fragment contains at least five amino acids from the antibody sequence from which it is derived, preferably 10, 15, 25, 50, and 100 consecutive amino acids.

[0086] Typically, for the purpose of preparing monoclonal antibodies or their functional fragments, particularly mouse-derived monoclonal antibodies or their functional fragments, techniques described in detail in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988), or techniques for preparation from hybridoma cells, as described by Kohler and Milstein (Nature, 256:495-497, 1975), may be referenced.

[0087] The terms “antibody-drug conjugate” (ADC) or “complex,” as used herein, generally refer to an antibody or its antigen-binding fragment linked to another agonist, such as a chemotherapeutic agent, toxin, immunotherapy agent, or imaging probe. The linkage may be covalent or non-covalent, such as through electrostatic interaction. Various linkers known in the art and described herein can be used to form antibody-drug conjugates. In addition, antibody-drug conjugates can be provided in the form of fusion proteins that can be expressed from polynucleotides encoding an immunoconjugate. As used herein, “fusion protein” refers to a protein produced by joining two or more genes or gene fragments that originally encoded separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.

[0088] In a "complex," two or more compounds are linked together. In certain embodiments, at least some of the properties of each compound are maintained in the complex. Linking can be achieved by covalent or non-covalent bonds. Preferably, the compounds of the complex are linked by covalent bonds. Different compounds of a complex may be directly bonded to each other via one or more covalent bonds between the atoms of the compounds. Alternatively, the compounds may be bonded to each other via a chemical moiety such as a linker molecule, where the linker is covalently linked to the atoms of the compounds. If the complex consists of more than two compounds, such compounds may be linked in a chain conformation, for example, one attached to the next compound, or several compounds each attached to one central compound.

[0089] The cytotoxic drugs described in this specification refer particularly to substances that inhibit or block the expression activity and cell functions of cells and / or bring about cell destruction. Examples include auristatin derivatives (e.g., MMAE, MMAF), aureobasidin (chlortetracycline), maytansinoid and its derivatives (DM0, DM1, DM2, DM3, DM4, etc.), Ricin, combrestatin, ansamitocin, calicheamicin, duocarmycin, dolastatin and its derivatives, DNA topoisomerase inhibitors and their derivatives (e.g., Etoposide, teniposide, Dxd, SN38), Amanitin, cc1065 and its analogs, Mitomycin C, Camptothecin (CPT) and its analogs, Vincristine, Vinblastine, colchicine, mitoxantrone, Actinomycins, diphtheria toxin, Pseudomonas exotoxin A, Abrin, Gelonin, Micronomicin, etc., but are not limited thereto.

[0090] The term "immunopotentiator" refers to substances that can activate non-specific immunity and enhance the body's immune response, such as TLR agonists and STING agonists.

[0091] Radioisotopes include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 or Bi 213 , P 32 , Pb212 This includes, but is not limited to, Lu.

[0092] The term "linker" refers to a structural element of a compound that links one structural element of that compound to one or more other structural elements of the same compound. A linker may be a non-cleavable linker. Suitable non-cleavable linkers include, but are not limited to, NH2-RX, NH2NH-RX, and NH2-ORX (wherein R is an alkyl or polyethylene glycol group (also known as PEG), and X is the active moiety). A polyethylene glycol group or PEG group has the general formula -(CH2CH2O) n The formula has (wherein n is an integer of at least 1). In some embodiments, n is one of 2, 4, 6, 8, 10, 12, 16, 20, or 24.

[0093] The linkers that can be cut are Lys-Phe-X, Lys-Val-Cit-PABC-X, and NH2-(CH2CH2O). n -Val-Cit-pABC-X and NH2-(CH2CH2O)n-(Val-Cit-PABC-X)2 (wherein X is the active moiety and n is an integer of at least 1, e.g., 2, 4, 6, 8, 10, 12, 16, 20, or 24), respectively. PABC stands for p-aminobenzyloxycarbonyl.

[0094] just As an example, the linker is NH2-( CH 2 -CH 2 -O )m -CH 2 -C(=O) -Val-Cit, NH2-( CH 2 -CH 2 -O )m -CH 2 -C(=O) -Val-Cit-pABC, mc-Val-Cit-pABC, Val-Cit-pABC, or Val-Cit may be selected, and m is (CH 2 -CH 2 -O)It represents a number, and is an integer from 1 to 8, i.e., 1, 2, 3, 4, 5, 6, 7, 8.

[0095] The term "endogenous glutamine" refers to a glutamine residue conserved at position 295 of the heavy chain (Q295) in full-length human isotype IgG antibodies, and this glutamine residue is close to the N-glycosylation site (N297). [Examples]

[0096] Apparatus and experimental materials: [Table 1]

[0097] Example 1: Antibody production and humanization process Nectin-4 mouse antibodies were prepared according to WO2022051591A2. The basic procedure was as follows: Animals (BALB / c mice) were immunized by intraperitoneal injection with human Nectin-4-His fusion protein containing an adjuvant (Freund's adjuvant). Additional immunizations were induced every two weeks until an appropriate titer was achieved. Blood samples were collected from the animals after each additional immunization, and titers were detected by ELISA and FACS. Four days after the final immunization, the spleens of animals with an appropriate titer were collected, and single-cell suspensions were prepared. These cells were fused with SP2 / 0 mouse myeloma cells using electrofusion. The fused cells were resuspended in a medium containing the hybridoma cell selectors thymidine, hypoxanthine, and aminopterin (HAT), seeded in 96-well plates, and cultured.

[0098] After 7-10 days of culture, the culture supernatant was collected, and clones bound to human Nectin-4 protein were detected by ELISA or FACS to confirm whether they bound to mouse Nectin-4 protein. Hybridoma cells were cultured after adding fresh HAT-containing medium. After 2 days, the culture supernatant obtained from the first screening was collected, and the functional activity of the antibodies was measured. Subsequently, the selected positive clones were further subcloned. After successful subcloning, these antibodies were purified using conventional antibody purification methods, and the variable regions of the antibodies were sequenced for some of the desired clones.

[0099] Nectin-4 mouse antibodies obtained after immunization and screening in animals were subjected to humanization. The humanization process was entrusted to GenScript, yielding three human antibodies: SWY2001-Ab1, SWY2001-Ab2, and SWY2001-Ab3. Their sequences are shown in Table 2, and the humanization process was as follows.

[0100] 1.1 Chimeric Antibody Expression Purification Intact heavy and light chain plasmids were constructed by inserting the variable region of a chimeric antibody, modified based on a mouse antibody in a constant region selected from human IgG1, into a pcDNA3.4(IgG1,kappa) vector. The plasmids were transfected into HEK293 cells, and the supernatant was collected and purified using protein A magnetic beads to obtain full-length antibodies. The solution was replaced with PBS by dialyzing and desalting. Antibody concentration and purity were detected by OD280 and SDS-PAGE gel electrophoresis, respectively. Next, their affinity was detected by surface plasmon resonance (SPR) technology.

[0101] 1.2 Detection of Chimeric Antibody Binding Activity and Affinity Affinity detection of chimeric antibodies is primarily determined by the Biacore instrument using surface plasmon resonance (SPR) technology. Fc fragments were captured using a protein A chip and bound to the antibody, with the antigen used as the mobile phase. The corresponding binding constant (ka) and dissociation constant (pk) were determined from the obtained detection data using the instrument's software fit, and the corresponding equilibrium constant (KD) was calculated.

[0102] 1.3 Design of Reverse Mutations in Human Antibodies Reverse mutations and combinations were performed at multiple amino acid sites in the nuclear regions of the light chain and heavy chain structures of human antibodies. The corresponding light chain genes were synthesized using GenScript®.

[0103] 1.4 Ranking of production and affinity of reverse mutation human antibodies After obtaining heavy and light chain plasmids, the corresponding light and heavy chain combinations were paired and transfected in a 4 mL system for expression. After obtaining the expression supernatant, affinity was detected using SPR technology, and the antibodies in the supernatant were captured by capturing antibody Fc fragments using a protein A chip and immobilized on a sensor chip. The analyte antigen was used as the mobile phase. Surface regeneration was performed before injecting another antibody supernatant, and this process was repeated until all antibodies were analyzed. Experimental data were fitted using a 1:1 interaction model with Biacore analysis software. Antibody binding and dissociation rates were obtained, and antibody affinities were ordered by the dissociation rate constant (pk). From the sequence results, the top three affinity clones were selected as candidate antibodies, and three human antibodies named SWY2001-Ab1, SWY2001-Ab2, and SWY2001-Ab3 were obtained using the above procedure.

[0104] 1.5 Construction, expression, and affinity assay of candidate antibodies The genes of three candidate antibody strains were inserted into the vector pcDNA3.4 (IgG1, kappa) to construct complete heavy-chain and light-chain antibody plasmids. Next, Expi293F cells were simultaneously transfected with the light-chain and heavy-chain plasmids. After collecting the supernatant, it was purified using protein A magnetic beads to obtain full-length antibodies. The solution was replaced with PBS by dialyzing and desalting, and the antibody concentration and purity were detected by OD280 and SDS-PAGE gel electrophoresis, respectively. Subsequently, its affinity was detected by surface plasmon resonance (SPR) technology.

[0105] [Table 2-1] [Table 2-2]

[0106] Example 2: Construction of an expression vector and expression of a human antibody The heavy and light chain DNA sequences of the Nectin-4 human antibody were synthesized by Biointron Tazhou, and the expression vector pcDNA3.1 was provided by Biointron Tazhou (see Figure 4). The basic procedure was as follows:

[0107] Heavy chain design and synthesis: The artificially synthesized heavy chain was named Nectin-4-HC. A HindIII endonuclease moiety was introduced at the 5' end, and an EcoRI endonuclease moiety at the 3' end. Furthermore, a Kozak sequence and a signal peptide sequence (19 amino acids) MELGLCWVFLVAILEGVQC (SEQ ID NO: 14) were introduced after the HindIII endonuclease moiety at the 5' end. The heavy chain expression cassette was designed as follows. HindIII-Kozak sequence-signal peptide-Nectin-4-HC-stop codon-EcoRI

[0108] Light chain design and synthesis: The artificially synthesized light chain was named Nectin-4-LC. During synthesis, a HindIII endonuclease moiety was introduced to the 5' end of the light chain and an EcoRI endonuclease moiety to the 3' end. A Kozak sequence and a signal peptide sequence (22 amino acids): MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 15) were then introduced after the HindIII endonuclease moiety at the 5' end. The light chain expression cassette was designed as follows. HindIII-Kozak sequence-signal peptide-Nectin-4-LC-stop codon-EcoRI

[0109] 2) Construction of recombinant plasmids The PCR amplification product Nectin-4-HC and the plasmid vector pcDNA3.1 were cleaved with the HindIII / EcoRI double enzyme and ligated, and then positive clones were screened with the Amp+ resistance marker to confirm that the correct recombinant heavy chain expression vector was obtained. The PCR amplification product Nectin-4-HC and the plasmid vector pcDNA3.1 were double-cleaved with HindIII / EcoRI, ligated, and positive clones were screened with the Amp+ resistance marker to obtain the correct recombinant light chain expression vector.

[0110] In this experiment, human antibody expression was induced by transient transfection of HEK293 cells. HEK293 cells were placed in a 5% CO2 constant temperature shaking bed and cultured at 37°C and 120 rpm. The cells were cultured at a density of 2.0 × 10⁶. 6Cells were cultured until the cell count reached 100 cells / mL, and antibody heavy chain and light chain plasmids were added at a rate of 0.5 mg HC and 0.5 mg LC per liter of cells. First, KPM (transfection buffer) and sterile plasmids were mixed, and then a separate centrifuge tube containing KPM and TA-293 transfection reagent was removed. The transfection reagent was slowly added to the KPM mixture containing the plasmid and gently mixed to prepare the plasmid-vector complex. After standing for 10 minutes, the plasmid-vector complex was added to the cells. After 24 hours, a cell protein expression enhancer and transient transfection nutrients were added, and cells were harvested and purified on day 6 post-transfection.

[0111] Capillary isoelectric focusing (cIEF) method: 4.3M urea, 3M urea-cIEF gum solution, and cIEF MIX solution were prepared. The sample was diluted to 5 mg / mL, 234 μL of cIEF MIX solution and 10 μL of the sample were mixed, and vortexing was performed thoroughly. 200 μL of the mixture was transferred to an intubation tube and detected.

[0112] Monoclonal antibody size variant measurement (CE-SDS) method: Non-reducing sample preparation: 100 μg of sample solution was taken, 95 μL of SDS sample buffer was added, and then 5 μL of 250 mM iodoacetamide solution was added and mixed. A reference solution was prepared by the same method. Reducing sample preparation: 100 μg of sample solution was taken, 95 μL of SDS sample buffer was added, and then 5 μL of 2-mercaptoethanol was added and mixed. The mixed sample was incubated at 70±2°C for 10 minutes, cooled to room temperature, and then centrifuged at 6000 rpm for 1 minute. The supernatant was collected in 80 μL portions into sample tubes and immediately analyzed. The physicochemical properties were as follows:

[0113] [Table 3]

[0114] Thus, it was revealed that the human antibodies obtained by the present invention exhibit high expression levels. While the transient expression level of typical antibodies is 50-100 mg / L, all three human antibodies of the present invention exceeded 100 mg / L, and the SWY2001-Ab1 expression level reached 200 mg / L. Purity was examined using various methods. SEC-HPLC purity was >99%, and reduced CD-SDS purity was >98%, meeting the requirements for further experiments.

[0115] Example 3: Detection of Nectin-4 human antibody DSC In this experiment, the stability of Nectin-4 human antibody was measured using differential scanning calorimeter. The experimental parameters are shown in the table below, the antibody concentration was 1 mg / mL, and the sample volume was 325 μL. The experimental results are shown in Figure 5. The specific DSC parameters were set as follows.

[0116] [Table 4]

[0117] The results shown in Figure 5 indicate that the antibodies obtained by this invention exhibit excellent stability.

[0118] Example 4: Affinity and species cross-detection of Nectin-4 human antibodies In this experiment, the affinity of human Nectin-4 antibodies to the human Nectin-4 protein was detected using Fortebio. After incubating the Nectin-4 protein with an anti-Nectin-4 antibody sample, the affinity of the sample to human Nectin-4 was analyzed by detecting the signal value using Fortebio. As shown in Table 5, the experimental results demonstrated that the affinity of the three human antibodies to the human Nectin-4 protein hardly decreased even after humanization was complete. The chimeric antibodies were obtained by denaturing mouse antibodies, and the constant region was selected from human IgG1.

[0119] [Table 5]

[0120] The affinity of human Nectin-4 antibodies to Nectin-4 proteins of different species was determined using ELISA. Different species of Nectin-4 proteins (human Nectin-4, cynomolgus monkey Nectin-4, rat Nectin-4, and mouse Nectin-4) were incubated with Nectin-4 human antibody samples at different concentrations. These were then incubated with IgG-bound secondary antibodies (Goat anti-Human IgG(H+L) Cross-Adsorbed Secondary Antibody), and the affinity of the samples to different species of Nectin-4 proteins was analyzed by measuring the signal values ​​at different concentrations using ELISA. The experimental results are shown in Table 6. The results indicated that human Nectin-4 antibodies exhibited good affinity for human, rat, and cynomolgus monkey Nectin-4 proteins.

[0121] [Table 6]

[0122] Example 5: Preparation of Nectin-4 antibody-drug conjugate 1. Enzymatic binding of SWY2001-Ab1-LND1002 LND1002 (1 g dissolved in 5 mL DMSO, structure shown in Figure 2), 10× reaction buffer, anti-Nectin-4 antibody, mTgase (transglutaminase, used to catalyze the transglutamine reaction at receptor glutamine adjacent to the N-glycosylation site, and can specifically catalyze the transglutamine reaction at Kabat number Q295), and 20% H2O were added sequentially to an EP tube, sealed and mixed, and reacted at 30°C for less than 72 hours. The reaction was stopped when the binding rate reached 95%, and immediately purified. The reaction conditions were as follows:

[0123] [Table 7]

[0124] Experimental results confirmed that the transglutaminase catalytic site of the antibody of the present invention is located near Q295 of the N-glycosylation site.

[0125] The transglutaminase sequence used was as follows (SEQ ID NO: 13): DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNED ARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDPEAFRPDRGTGLVDMSRDRNIIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKSWNTAPDKVTQGWP

[0126] 2. Chemical bonding of SWY2001-Ab1-VC-MMAE The antibody was replaced with PBS buffer containing 5 mM EDTA pH = 6.0 to adjust the antibody concentration to 10 mg / mL. Three molar equivalents of TCEP reducing agent and 1 / 50 volume of 1M dipotassium phosphate were added according to the molar concentration of the antibody (SWY2001-Ab1). The mixture was heated in a 37°C water bath for 1 hour. Then, six molar equivalents of MC-VC-pABC-MMAE were added, followed by 80 μL of DMSO per 1 mL of volume. The mixture was reacted at room temperature for 30 minutes. Twelve molar equivalents of L-cysteine ​​were added, and the reaction was stopped after 20 minutes to form the final product. The antibody was linked to the linker-drug via sulfhydryl groups in the interchain cysteine, and its structure is shown in Figure 3.

[0127] Example 6: Physicochemical analysis and identification of SWY2001-Ab1-ADC 1. Identification of the enzymatic modification rate of SWY2001-Ab1-LND1002 Experimental procedure: 1) Sample preparation: The SWY2001-Ab1-LND100272h modified solution was treated with 50 mM ammonium acetate, 20 mM DTT, and 55 mM Tris-HCl buffer at 30°C ± 2°C for 30 minutes, and then centrifuged at 12000 rpm for 5 minutes. 2) Sample addition: 10 μL (30 μg) of the supernatant was added to a liquid chromatograph (Waters Xbridge C4, 3.5 μm, 4.6 mm × 250 mm). 3) Elution: Mobile phase A was a 0.1% TFA aqueous solution, and mobile phase B was a 0.1% acetonitrile solution. At 0, 5, 8, 15, 20, 22, 25, and 30 minutes, mobile phase A was adjusted so that the ratio of mobile phase A to B was 9:1, 7:3, 6.5:3.5, 6:4, 5.5:4.5, 5:5, 1:9, and 9:1, respectively. The flow rate was controlled to 0.8 mL / min, the column temperature was set to 60°C, and the detection wavelength was set to 280 / 254 nm.

[0128] As shown in Figure 6, the experimental results for SWY2001-Ab1-LND1002 showed a modification rate of 96.32% after 72 hours of reaction.

[0129] 2. SWY2001-Ab1-ADCDAR value detection Experimental procedure: Referring to high-performance liquid chromatography described in Part 4, Item 0512 of the Chinese Pharmacopoeia 2020, the drug-antibody binding ratio (DAR) of this product was measured using reverse-phase chromatography (RP-HPLC).

[0130] Laboratory equipment: High-performance liquid chromatography system Agilent 1260 Column: PLRP-S1000A, 5um, 50*2.1mm Mobile phase: Mobile phase A: 0.1% (v / v) TFA aqueous solution Mobile phase B: 0.1% (v / v) TFA acetonitrile solution

[0131] The detection method is as follows: Flow rate 0.25mL / min Detection wavelength: 280 nm Injection volume 10μL Column temperature 80°C

[0132] The gradient elution procedure was as follows: [Table 8]

[0133] The experimental results were analyzed using area normalization: The calculation results were as follows:

number

[0134] The experimental results are shown in Figure 7 (Figures 7A and 7B) and Table 8. [Table 9]

[0135] Example 7: Endocytosis of SWY2001-Ab1-LND1002 Experimental procedure: SK-BR-3 cells and T47D cells (human breast cancer cells) were collected and suspended in culture medium. The target cells were gently blown several times until a single-cell suspension was obtained, and cell viability and cell number were determined by trypan blue staining. The cell density was set to 1 × 10⁻⁶. 5 Adjust to cells / mL, transfer to a confocal 96-well cell culture dish at 100 μL / well, and fill with 1 × 10⁶ cells per well. 4 Cells were seeded at a specific number of cells, and ADC labeled with Zenon® pHrodo® iFL was added to a 96-well plate at a final concentration of 2 μg / mL. The cells were then incubated continuously for 24 hours at 37°C under 5% CO2 incubator conditions. All images were observed and captured using a laser confocal microscope with a 20X objective lens.

[0136] As shown in Figures 8 and 9, the experimental results showed that SWY2001-Ab1-LND1002 underwent endocytosis within cells, localized to lysosomes in an acidic environment, and exhibited a much higher endocytosis rate than the drug PADCEV. Generally, for antibody-drug conjugates, the faster the endocytosis, the greater the drug's ability to enter tumor cells, enabling better toxin release and allowing the drug to exert its effects more rapidly. Therefore, it was found that the ability of the antibody-drug conjugate obtained in this invention to penetrate tumor cells is significantly superior to that of PADCEV.

[0137] Example 8: Inhibitory effect of SWY2001-Ab1-LND1002 on different extracellular growth processes. Experimental procedure: Target cells were harvested and resuspended as a single-cell suspension, and cell viability and cell number were measured by trypan blue staining. Cell density was set to 1 × 10⁻⁶. 5 The solution was adjusted to cells / mL and added 100 μL / well to each of the 96-well black flat-bottom cell culture plates. Diluted samples were added to each of the 96-well black flat-bottom cell culture plates already seeded with cells, 20 μL per well, and incubated in a cell incubator (37°C, 5% CO2) for 66 ± 3 hours. Resazurin solution (0.03%) was added at 20 μL per well, and the mixture was reacted at 37°C for 3-4 hours. Fluorescence values ​​were read at 550 nm / 610 nm using a microplate reader, and plotted using Prism or similar plotting software. The half-inhibition concentration IC of the reference standard and the sample was then measured. 50 It was fitted to the IC. The output parameter C is IC 50 And there was a unit called ng / mL.

[0138] As shown in Table 9, experimental results demonstrated that SWY2001-Ab1-LND1002 inhibited the growth of 293T-Nectin-4, SK-BR-3, and PC3-Nectin-4 cancer cells in vitro. For 293T-Nectin-4 stabilized transformants, SWY2001-Ab1-LND1002 showed slightly better results than PADECV based on in vitro killer data. For SK-BR-3, SWY2001-Ab1-LND1002 was four times more effective than PADCEV based on in vitro assay data. For PC3-Nectin-4, SWY2001-Ab1-LND1002 showed results comparable to PADCEV based on in vitro assay data.

[0139] [Table 10]

[0140] Example 9: In vivo efficacy of SWY2001-Ab1-LND1002 1. In vivo efficacy experiments of prostate cancer inhibitors In this study, female NUNU mice of appropriate age were inoculated with cells stably transformed with PC3-Nectin-4 (Nectin-4 expression was high in NUNU mouse madel transplanted with human prostate cancer). The tumor volume was approximately 190 mm². 3 If the tumors grew to D19 (after dissemination), 42 animals with good tumor growth were selected and divided equally into 6 groups of 7 mice each, according to tumor volume (D0): a solvent control group, a Nectin4-mab 1 mg / kg group, a PADCEV 1 mg / kg group, a SWY2001-Ab1-LND1002 0.5 mg / kg group, a 1 mg / kg group, and a 2 mg / kg group. Tumor growth was dynamically observed by weighing the mice after administration, recording the data, and measuring the tumor diameter at various time points after administration. The experiment was terminated on d17 (17 days after administration), and after asphyxiating the mice with carbon dioxide, the tumors were removed and weighed.

[0141] Under these experimental conditions (see Figure 10), SWY2001-Ab1-LND1002 dose-dependently inhibited tumor growth (P<0.05) and showed significantly better tumor inhibition rates than PADCEV at a 1 mg / kg dose (58.3% vs. 39.0%). The tumor weight reduction rates for the Nectin4-mab 1 mg / kg, PADCEV 1 mg / kg, and SWY2001-Ab1-LND1002 0.5 mg / kg, 1 mg / kg, and 2 mg / kg groups were 28.2%, 39.0%, 22.1%, 58.3%, and 79.4%, respectively.

[0142] 2. In vivo efficacy experiments of drugs inhibiting breast cancer In this experiment, nude mice were inoculated with MDA-MB-468 cells to construct a human breast cancer MDA-MB-468 nude mouse transplantation model. The tumor volume was approximately 100 mm². 3 When the tumors had grown to a certain size (D25 after dissemination), 16 animals with good tumor growth were selected and divided into three groups according to tumor volume (D0): a solvent control group of 8 animals and the remaining 4 animals in the experimental group. Each group received intravenous administration of 0.9% sodium chloride injection (0.9% INJ NS (physiological saline), solvent control group), 3 mg / kg of the drug PADCEV group, and 3 mg / kg of the drug SWY2001-Ab1-LND1002 group (single dose). The body weight of the mice was weighed after administration, the data was recorded, and tumor growth was dynamically observed by measuring the tumor diameter at various time points after administration. The experiment was terminated on D29, and after asphyxiating the mice with carbon dioxide, the tumors were removed and weighed.

[0143] The experimental results are shown in Figure 11. Under these test conditions, the inhibition rates were 72.3% in the PADCEV 3 mg / kg group and 66.3% in the SWY2001-Ab1-LND1002 3 mg / kg group, respectively. Compared to the solvent control group, tumor growth was significantly inhibited in both groups (P<0.001).

[0144] 3. In vivo efficacy experiments of drugs inhibiting bladder cancer In this study, female NUNU mice of appropriate age were given 5 × 10⁶ mice per mouse. 6HT-1376 cells were inoculated into individual cells. The tumor volume was approximately 100 mm². 3 If the tumors grew to D21 (after seeding), 12 animals with good tumor growth were selected and divided into three groups according to tumor volume (D0): A: 4 animals in the solvent control group, administered 0.9% sodium chloride injection (0.9% INJ NS (physiological saline), solvent control group); B: 4 animals in the PADCEV experimental group, administered PADCEV 3 mg / kg (single dose); C: 4 animals in the SWY2001-Ab1-LND1002 experimental group, administered 3 mg / kg (single dose). The body weight of the mice was weighed after administration, the data was recorded, and tumor growth was dynamically observed by measuring the diameter of the tumor at various time points after administration. The experiment was terminated at D22, the mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed.

[0145] The experimental results are shown in Figure 12. Under these experimental conditions, the tumor suppression rates were 42.1% in the PADCEV 3 mg / kg group and 49.4% in the SWY2001-Ab1-LND1002 3 mg / kg group, respectively. The Nectin-4-ADC 3 mg / kg group (single dose) significantly suppressed tumor growth compared to the solvent control group. The SWY2001-Ab1-LND1002 3 mg / kg group showed a remarkably improved tumor inhibitory effect compared to the positive control PADCEV 3 mg / kg (single dose) group.

[0146] Example 10: Safety evaluation study of SWY2001-Ab1-ADC 10.1 Comparative assay of the effect of SWY2001-Ab1-VC-MMAE on chemical binding In this study, six male cynomolgus monkeys of appropriate age were selected, and their toxic reactions were observed after intravenous injection of the control and the sample, SWY2001-Ab1-ADC. As shown in the table below, the sample SWY2001-Ab1-LND1002 (DAR2) and the control SWY2001-Ab1-VC-MMAE (DAR4) were administered intravenously once a week for two weeks. Continuous observation was performed for 7 days after the second administration, with general observations twice a day and detailed observations once a day. Body weight changes were observed 1 day before administration, and 7 and 14 days after administration, and hematological and biochemical indices were measured.

[0147] [Table 11]

[0148] The experimental results are shown in the table below. [Table 12]

[0149] As is clear from the experimental results above, overall, clinical observations showed that the control product was more severe than the sample in terms of skin toxicity and ocular toxicity at the same dose. Specifically, the sample SWY2001-Ab1-LND1002 showed only slight ocular toxicity (redness around the eyes) in clinical observations in the high-dose group (9 mg / kg), whereas the control group not only showed symptoms of ocular toxicity in the low-dose group of 6 mg / kg, but also exhibited serious toxic reactions such as ulcers on the left hind leg, mild swelling from the knee joint to the ankle joint, discoloration around the eyes, and desquamation of both forelimbs, as well as a serious adverse reaction that resulted in the death of an animal after the third dose. The sample prepared according to the present invention showed no abnormalities in any animal even when adjusted to 12 mg / kg (a much larger dose than the control dose of 6 mg / kg) after the third dose, demonstrating significant superiority over the control product in terms of safety.

[0150] 10.2 Safety Experiments in Cynomolgus Monkeys Regarding PADCEV (Commercial Product) In the study, 10 cynomolgus monkeys, half male and half female, were randomly divided into 5 groups of 2 monkeys each. Group 1 was administered 6 mg / kg of the commercially available control (PADCEV®), while Groups 2-5 were administered the sample SWY2001-Ab1-LND1002 by intravenous infusion at a dose volume of 10 mL / kg and an infusion rate of 0.5 mL / kg / min. The dose, concentration, frequency, and duration administered to the animals in each group are shown in the table below.

[0151] [Table 13]

[0152] At the same dose (6 mg / kg), the sample exhibited the same abnormalities as the commercial control, including skin abnormalities, leukocyte and classification counts, red blood cell-related indicators (RBC, HGB, HCT), AST, ALT, PLT, and FIB, as well as corneal histopathological abnormalities. However, the toxicological characterization was substantially consistent with that of the commercial control. The commercial control showed abnormalities earlier than the sample (D7 vs. D14), and the expression of skin toxicity was more severe (desquamation vs. ulceration).

[0153] According to FDA data on the commercial control, in a 4-week long-term toxicity study in which the commercial control was administered in duplicate at doses of 1 mg / kg, 3 mg / kg, and 6 mg / kg per week, three animals died early in the study (D11), exhibiting major toxicity including skin damage, myelotoxicity, and mild hepatotoxicity. To assess severe toxicity characteristics, all animals in the 6 mg / kg group were discontinued after the second dose (D8).

[0154] This revealed that samples at the same dose posed a lower risk of toxicity compared to the commercially available control product PADCEV®.

[0155] In summary, the experimental results show that the antibody-drug conjugate obtained by the present invention exhibits significantly superior overall efficacy compared to PADCEV in endocytosis, in vitro tumor cell inhibition experiments, and in vivo tumor inhibition drug efficacy experiments (prostate cancer, breast cancer, and bladder cancer). In particular, safety evaluation experiments revealed that the antibody-drug conjugate obtained by the present invention has a wide therapeutic window, fewer side effects, and no significant adverse reactions such as skin toxicity or ocular toxicity.

[0156] Example 11: Stability experiment of SWY2001-Ab1-LND1002 Plasma stability experiment In the experiment, the stability of SWY2001-Ab1-LND1002 and MMAE in SD rat, cynomolgus monkey, and human plasma was investigated using an in vitro plasma incubation method at 37°C. PBST was used as a negative control group to examine the overall reliability of the test system. Incubation samples were collected from each incubation group at 0, 24, 48 (D2), 72 (D3), 96 (D4), 168 (D7), and 336 (D14). The concentrations of MMAE in various plasmas and PBSTs were measured using LC-MS / MS. Table 12 shows the average percentage of MMAE production after incubation of SWY2001-Ab1-LND1002 in various plasmas and PBSTs.

[0157] [Table 14]

[0158] The experimental results showed that, within the measured concentration range, after 336 hours of incubation in human, cynomolgus monkey, and rat plasma, the three concentrations of antibody-drug conjugates produced small molecule MMAEs at approximately 1% of the theoretical amount, confirming their good stability.

[0159] The above description is merely illustrative and does not limit the combination of features necessary to carry out the present invention. Furthermore, the title provided is not intended to limit the diverse embodiments of this application. For example, the terms “comprising,” “including,” and “including” are not intended to be limiting. Moreover, unless otherwise specified, the plural form is included where there is no count word modifier, and “or” and “or” mean “and / or.” Unless otherwise specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0160] All disclosures and patents referenced herein are incorporated herein by reference. Many modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of this application. Although this application is described by certain preferred embodiments, it should be understood that the application for which protection is claimed should not be unduly limited to these particular embodiments. In fact, several of those variations of the described embodiments for carrying out this application, which will be apparent to those skilled in the art, are intended to be included in the appended claims.

Claims

1. An antibody-drug conjugate (ADC) that specifically binds to Nectin-4, wherein the antibody or its functional fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR1 containing the amino acid sequence shown in SEQ ID NO: 1, CDR2 containing the amino acid sequence shown in SEQ ID NO: 2, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 3, the light chain variable region comprising CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, the drug is bound to the antibody or its functional fragment by a linker, the linker being NH₂-(CH₂-CH₂-O)m-CH₂-C(=O)-Val-Cit, NH₂-(CH₂-CH₂-O)m-CH₂ The antibody-drug conjugate is selected from -C(=O)-Val-Cit-pABC, mc-Val-Cit-pABC, mc-Val-Cit, Val-Cit-pABC, Val-Cit-pAB, and Val-Cit, where m is an integer from 1 to 8.

2. The antibody-drug conjugate according to claim 1, wherein the sequence of the heavy chain variable region of the antibody or its functional fragment is the amino acid sequence shown in SEQ ID NO: 9, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence of the light chain variable region is selected from the amino acid sequences shown in SEQ ID NOs: 10-12, or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

3. The antibody-drug conjugate according to claim 1, wherein the antibody is a monoclonal antibody.

4. The antibody-drug conjugate according to claim 1, wherein the antibody is a humanized antibody.

5. Encoding the light chain and / or the heavy chain of the antibody or functional fragment described in claim 2; or Encoding the antibody or functional fragment thereof according to claim 2, Isolated polynucleotides.

6. An expression vector comprising a polynucleotide according to claim 5, wherein the polynucleotide is linked to a regulatory sequence that enables the expression of the polypeptide encoded by it in a host cell or cell-free expression system.

7. A host cell comprising the expression vector described in claim 6.

8. The complex according to Claim 1, wherein the drug is selected from cytotoxic drugs, immunostimulants and radioisotopes, preferably the drug is selected from auristatin derivatives, mytansinoid derivatives, camptothecin analogs, DNA topoisomerase I inhibitors and their derivatives, most preferably the drug is selected from MMAE, MMAF, DM1, DM4, DXD and SN38 and their derivatives.

9. The linker binds to the antibody or its functional fragment via a sulfhydryl group or an amino group, and the linker is mc-Val-Cit-pABC, mc-Val-Cit, NH m , 2 -(CH 2 -CH 2 -O) m -CH 2 -C(=O)-Val-Cit, and NH 2 -(CH 2 -CH 2 -O) m -CH 2 -C(=O)-Val-Cit-pABC, and is selected from the group consisting of, where m is an integer of 1-8. The conjugate according to claim 8.

10. Ab-(LU) n The complex according to claim 8, having the structure, wherein Ab represents an antibody or a functional fragment thereof, L represents a linker, U represents a drug, and n is an integer or decimal number from 1 to 8.

11. The complex according to claim 8, wherein the antibody or functional fragment thereof is a monoclonal antibody or a bispecific antibody, preferably a humanized antibody.

12. The complex according to claim 8, wherein the antibody or functional fragment thereof is an IgG type, preferably an IgG1 antibody.

13. A pharmaceutical composition comprising the complex described in claim 8 and a pharmaceutically acceptable carrier.

14. Use of the complex according to any one of claims 8-12 or the pharmaceutical composition according to claim 13 in the manufacture of a pharmaceutical for the treatment or prevention of tumors.

15. The use according to claim 14, wherein the tumor is a Nectin-4 positive tumor, preferably a Nectin-4 positive solid tumor, preferably selected from the group consisting of prostate cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, bladder cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer, particularly preferably prostate cancer, breast cancer, bladder cancer, lung cancer, or ovarian cancer, and most preferably prostate cancer, breast cancer, or bladder cancer.

16. A pharmaceutical product comprising the complex described in claim 8.

17. The pharmaceutical product according to claim 16, which exists in the form of a kit comprising a container for containing the complex according to any one of claims 8-12.

18. Use of a combination of the complex and an antiproliferative agent according to any one of claims 8-12 in the manufacture of an antitumor drug.

19. A pharmaceutical composition comprising the complex according to any one of claims 8-12 and an antiproliferative agent.

20. An agent for treating a tumor in a target, comprising an effective amount of an antibody-drug conjugate according to any one of claims 8-12, a pharmaceutical composition according to claim 13, or a pharmaceutical product according to claim 16.

21. An agent for use in combination with radiotherapy for treating a tumor in a target, comprising an effective amount of an antibody-drug conjugate according to any one of claims 8-12, a pharmaceutical composition according to claim 13, or a pharmaceutical preparation according to claim 16.

22. An agent for treating a tumor in a target, comprising an effective amount of an antibody-drug conjugate according to any of claims 8-12, a pharmaceutical composition according to claim 13, or a pharmaceutical formulation according to claim 16 and an antiproliferative agent.

23. A method for producing a complex, comprising the step of linking an antibody or a functional fragment thereof according to any one of claims 1 to 4 to a drug via a linker in the presence of transglutaminase, wherein the linker is conjugated at position Q295 of the endogenous receptor glutamine residue of the antibody.

24. The drug is selected from auristatin derivatives, mytansin alkaloid derivatives, camptothecin analogs, DNA topoisomerase I inhibitors and their derivatives, preferably from MMAE, MMAF, DM1, DM4, DXD and SN38 and their derivatives, and the linker is mc-Val-Cit-pABC, mc-Val-Cit, NH 2 -(CH 2 -CH 2 -O) m -CH 2 -C(=O)-Val-Cit and NH 2 -(CH 2 -CH 2 -O) m -CH 2 The method according to claim 23, wherein a selection is made from -C(=O)-Val-Cit-pABC, and m is an integer from 1 to 8.

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