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WO2023025243A1 - 抗Nectin-4抗体、药物缀合物及其制备方法和用途 - Google Patents

抗Nectin-4抗体、药物缀合物及其制备方法和用途 Download PDF

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WO2023025243A1
WO2023025243A1 PCT/CN2022/114793 CN2022114793W WO2023025243A1 WO 2023025243 A1 WO2023025243 A1 WO 2023025243A1 CN 2022114793 W CN2022114793 W CN 2022114793W WO 2023025243 A1 WO2023025243 A1 WO 2023025243A1
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antibody
cancer
nectin
drug
functional fragment
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PCT/CN2022/114793
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English (en)
French (fr)
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孙召朋
沈明月
淡墨
袁灿
王艳翠
武玉芬
惠希武
刘伯宁
姚兵
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石药集团巨石生物制药有限公司
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Priority to JP2024536344A priority Critical patent/JP2024532598A/ja
Priority to EP22860589.5A priority patent/EP4393951A1/en
Priority to CN202280057818.XA priority patent/CN117897407A/zh
Priority to AU2022335573A priority patent/AU2022335573A1/en
Priority to US18/685,971 priority patent/US20240366778A1/en
Priority to CA3230122A priority patent/CA3230122A1/en
Priority to KR1020247008450A priority patent/KR20240136924A/ko
Publication of WO2023025243A1 publication Critical patent/WO2023025243A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, in particular to an anti-Nectin-4 protein antibody and its conjugate, and the use of the antibody and the conjugate in treating tumors.
  • Nectin-4 (Nectin Cell Adhesion Molecule 4), also known as PVRL4 (Poliovirus Receptor like 4), is a type I membrane protein belonging to the cell adhesion factor (Nectin) family One of the members, specifically expressed in embryos and placenta; this family is a group of Ca2+-independent immunoglobulin-like transmembrane cell adhesion molecules, consisting of four members (Nectin-1 to Nectin-4).
  • Nectin-1 to Nectin-3 are ubiquitously expressed in normal adult tissues.
  • Nectin-4 protein cannot be detected in adult healthy tissues (including breast tissue), but it is overexpressed in various cancers (breast cancer, lung cancer, ovarian cancer, etc.), and overexpression is found in 62% of triple-negative breast cancers .
  • Nectin-4 in cancer progression can promote intratumoral angiogenesis and promote tumor growth.
  • the PI3K/AKT signaling pathway is involved in the promotion of Nectin-4-mediated cancer cell proliferation.
  • overexpression of Nectin-4 is associated with poor prognosis in lung, breast and ovarian cancers.
  • Nectin-4 interacts with Afadin protein (actin filament-binding protein) to form a complex, and ultimately improves cell survival and prevents cell apoptosis through the PI3K-AKT signaling pathway; high expression of Nectin-4 is a key factor in papillary thyroid carcinoma (PTC ) risk factors for lymph node metastasis; Nectin-4 depletion can effectively inhibit the proliferation and invasion of two PTC cell lines (ie TPC1, KTC-1 human thyroid cancer cell lines) in vitro and induce apoptosis; Nectin in human esophageal cancer tissue The overexpression of -4 is closely related to tumor size, tumor invasion depth and poor prognosis of patients. Intervention of Nectin-4 expression in esophageal cancer cell lines showed that increased Nectin-4 expression can significantly promote cell viability, migration, invasion and tumor formation.
  • Afadin protein actin filament-binding protein
  • Bladder cancer refers to malignant tumors occurring on the bladder mucosa, most of which are transitional epithelial cell carcinomas. It is most common in the side wall and back wall of the bladder, followed by the trigone and top, and its occurrence can be multicentric. Bladder cancer is the most common tumor of the urinary system, more common in men. According to the data released by the National Cancer Center in 2019, bladder cancer ranks seventh among male cancers, with an incidence rate of 8.83/100,000, and the upward trend has been obvious in recent years. There are differences in the incidence of bladder cancer in regions, races, and genders, and it can occur In all age groups, the highest incidence age is 50-70 years old, and the incidence rate gradually increases with age.
  • NMIBC non-muscle-invasive
  • MIBC muscle-invasive
  • TURBT transurethral resection of bladder tumor
  • Breast cancer is still the female tumor with the highest incidence rate in the world.
  • the annual incidence of breast cancer in the world increases by 0.2% to 8%.
  • About 1.4 million people in the world are diagnosed with breast cancer every year, and about 50 Ten thousand people die from the disease, and breast cancer is the first cause of death among women aged 40 to 55. Since the late 1970s, the incidence of breast cancer has been ranked first in female malignant tumors worldwide.
  • According to figures released by the American Cancer Society there are about 200,000 new cases of breast cancer in the United States every year, with an incidence rate of 116/100,000. Although my country is a low-incidence area for female breast cancer, the incidence of breast cancer has increased significantly in recent years.
  • monoclonal antibodies are the largest sub-sector in the field of biopharmaceuticals.
  • monoclonal antibody drugs accounted for 43% of the total biopharmaceutical market share; although monoclonal antibody therapy has the characteristics of high target specificity and low side effects, its curative effect is relatively limited when used alone. Therefore, most monoclonal antibody drugs are used in combination with chemotherapy, and the main way to improve the curative effect of monoclonal antibodies is antibody drug conjugates.
  • Antibody-drug conjugates belong to a new class of anti-cancer biomissile drugs, which are composed of three parts: antibody, drug and linker connecting them.
  • the antibody-drug conjugate uses the targeting of the monoclonal antibody to specifically recognize the receptor on the surface of the cancer cell, bind to the receptor, and then enter the interior of the cell, using the cell
  • the proteases in the body release drugs to prevent cancer cells from multiplying and killing cancer cells.
  • Antibody-drug conjugation technology integrates small-molecule drugs and biological proteins, combines the strengths of both, greatly enhances drug efficacy, and reduces toxic and side effects, becoming a new generation of therapeutic products.
  • Nectin-4-targeting projects there are no chemical drugs for Nectin-4-targeting projects at home and abroad, and there are only 6 biological drugs.
  • ADCs Nectin-4-targeted antibody-drug conjugates
  • the product name is Padcev, and the indications are: locally advanced or metastatic Urothelial carcinoma.
  • ORR is 44%, CR is 12%, and median DOR is 7.6 months; in patients receiving Padcev treatment, 46% of serious adverse reactions occurred; the most common serious adverse reactions ( ⁇ 3 %) were urinary tract infection (6%), cellulitis (5%), febrile neutropenia (4%), diarrhea (4%), sepsis (3%), acute kidney injury (3% ), dyspnea (3%), and rash (3%); fatal adverse reactions occurred in 3.2% of patients: including acute respiratory failure (0.8%), aspiration pneumonia (0.8%), cardiac disease (0.8%) ) and sepsis (0.8%).
  • ADC drugs needs to be based on a comprehensive consideration of antibodies, linkers and drugs as an organic whole, to clarify tumor indications and drug targets, and to fully and thoroughly study the mechanism of action of ADC drugs, so as to finally realize the effect of ADC drugs on tumor cells. effective killing, while improving the quality of life of patients.
  • ADC antibody-drug conjugates
  • the antibody used to prepare the ADC must have certain characteristics. It not only needs to specifically bind to the antigen-positive cells in the tumor, but also the antigen-antibody complex needs to be able to mediate the internalization of the ADC; screening antibodies need to consider at least the following three factors : specific binding, internalization, and cellular localization of antibodies after internalization; and, there is no clear link between antibody affinity and internalization speed (Laurent Ducry, Antibody Drug Conjugates, Science Press, pp. 36-37 ). Therefore, as a highly precise "positioning device", antibodies can specifically bind to target cells and effectively mediate "endocytosis" and their localization, which is crucial to the specificity and toxicity of antibody-drug conjugates;
  • the inventors aimed at the problem of unstable conjugation of existing drug linkers and small molecule drugs, using NH 2 -PEG 3 -Val-Cit as the linker, and using enzyme-mediated site-specific coupling to human
  • the derived antibody is coupled with a small molecule drug (such as MMAE) through a linker, and the obtained ADC drug has a strong killing effect on cancer cells with high expression of Nectin-4, especially breast cancer, bladder cancer and other tumors; specifically, first , according to the results of endocytosis experiments, the anti-Nectin-4 antibody drug conjugate obtained in the present invention has a better endocytosis effect than the control drug Padcev, and can simultaneously achieve specific binding, efficient internalization, and precise cell localization, improving The specificity of antibody-drug conjugates is reduced while reducing its toxicity; secondly, in vivo experiments show that: in nude mice with Nectin-4-positive xenograft tumors, intravenous administration of antibody-
  • the antibody drug conjugate obtained in the present invention basically has no adverse reactions when administered twice at a high dose of 9 mg/kg, has a wider therapeutic window period, and has better druggability.
  • the antibody provided by the application Drug conjugates achieve unexpected technical results.
  • the present invention provides antibodies, functional fragments (such as antigen-binding fragments) and antibody-drug conjugates (ADCs) that bind to Nectin-4 protein and/or Nectin-4 protein polypeptide fragments.
  • ADCs antibody-drug conjugates
  • the invention provides an antibody or a functional fragment thereof capable of specifically binding Nectin-4, said antibody comprising a heavy chain and a light chain, wherein
  • the heavy chain comprises three CDR regions, wherein the amino acid sequence of at least one of the CDR regions has the amino acid sequence shown in SEQ ID NO.1, 2 or 3 or at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and/or
  • the light chain comprises three CDR regions, wherein the amino acid sequence of at least one of the CDR regions has the amino acid sequence shown in SEQ ID NO.4, 5 or 6 or at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the antibody comprises a heavy chain and a light chain, wherein
  • the heavy chain comprises three CDR regions, which are respectively CDR1 region, CDR2 region and CDR3 region, and the CDR1 region, CDR2 region and CDR3 region have amino acids shown in SEQ ID NO.1, 2 and 3 respectively sequence; and/or
  • the light chain comprises three CDR regions, which are respectively CDR1 region, CDR2 region and CDR3 region, and the CDR1 region, CDR2 region and CDR3 region have amino acids shown in SEQ ID NO.4, 5 and 6 respectively sequence.
  • the heavy chain variable region sequence of the antibody is the amino acid sequence shown in SEQ ID NO.9 or at least 80% (preferably 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity sequence; the light chain variable region sequence is selected from the amino acid sequence shown in SEQ ID NO.10-12 or at least 80% (preferably 85%, 90%, 95%) thereof , 96%, 97%, 98% or 99%) sequence identity.
  • antibodies of the invention or functional fragments thereof are isolated.
  • antibodies of the invention are monoclonal antibodies.
  • the antibodies or functional fragments thereof of the present invention are humanized antibodies, preferably fully humanized antibodies.
  • antibodies of the invention are bispecific antibodies.
  • an antibody of the invention or a functional fragment thereof has ADCC activity.
  • an antibody of the invention or a functional fragment thereof has CDC activity.
  • an antibody of the invention or a functional fragment thereof specifically binds Nectin-4, but does not substantially bind Nectin-1, Nectin-2 or Nectin-3.
  • the antibodies of the present invention or functional fragments thereof include: double antibodies, Fab fragments, Fab' fragments, F(ab)'2, scFv, dsFv, and single domain antibodies;
  • the scFv protein is an immunoglobulin A fusion protein in which the light chain variable region and the heavy chain variable region of the protein are combined through a linker; in the dsFv, cysteine is introduced at a specific site in the conserved framework region of VH and VL, thereby introducing a disulfide bond to stabilize Structure of dsFv.
  • the antibody or functional fragment thereof of the present invention is IgM, IgD, IgG, IgA or IgE, wherein IgG antibodies have four subtypes: IgG1, IgG2, IgG3, IgG4, preferably IgG1 antibodies.
  • the heavy chain constant region sequence of the antibody is:
  • the light chain constant region sequence of the antibody is:
  • the antibodies of the invention or functional fragments thereof are useful in the treatment or prevention of cancer, wherein the cancer overexpresses Nectin-4.
  • the antibody or functional fragment thereof having the ability to bind Nectin-4 binds a native epitope of Nectin-4 present on the surface of a living cell. In some embodiments, the antibody or functional fragment thereof having the ability to bind nectin-4 binds to the extracellular domain of nectin-4.
  • the antibody having the ability to bind to Nectin-4 or a functional fragment thereof binds to the first extracellular region of Nectin-4; in some embodiments, the antibody or functional fragment thereof having the ability to bind to Nectin-4 The functional fragment binds to the 1-147aa domain of Nectin-4; in some embodiments, an antibody or a functional fragment thereof capable of binding to Nectin-4 binds to the 32-142aa domain of Nectin-4;
  • the invention provides an isolated polynucleotide encoding an antibody of the invention.
  • the invention provides a combination of isolated polynucleotides comprising a polynucleotide encoding a light chain of an antibody of the invention or a functional fragment thereof and a heavy chain encoding an antibody of the invention or a functional fragment thereof of polynucleotides.
  • the present invention provides an expression vector comprising the polynucleotide of the present invention or a combination of the polynucleotides of the present invention, the polynucleotide and the polypeptide that allow it to be expressed in a host cell or Regulatory sequences expressed in a cell-free expression system are operably linked.
  • 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 Proteus mirabilis, etc.
  • the eukaryotic host cell can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, fission yeast, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, such as BHK cells, CHO cells, COS cells, myeloma cells and other mammalian cells.
  • the host cells of the present invention are preferably mammalian cells, more preferably BHK cells, CHO cells, NSO cells or COS cells.
  • the present invention provides an antibody drug conjugate comprising the antibody of the present invention or a functional fragment thereof conjugated to one or more drugs, preferably the drug is a cytotoxic drug (such as anti-metabolites, anti-tumor antibiotics, alkaloids), immune enhancers or radioactive isotopes, more preferably the drug is selected from auristatin derivatives, maytansine alkaloid derivatives (such as ansamitocin ( Ansamitocin) or Maytansine (Mertansine)), dolastatin (dolastatin) and derivatives thereof), camptothecin analogs, DNA topoisomerase I inhibitors and derivatives thereof; most preferably the drug is selected from From MMAE (Monomethyl auristatin E) and MMAF (Monomethyl auristatin F).
  • a cytotoxic drug such as anti-metabolites, anti-tumor antibiotics, alkaloids
  • immune enhancers or radioactive isotopes more preferably the drug is selected from
  • the antibody or functional fragment thereof capable of binding to Nectin-4 is covalently linked to the drug moiety via a linker.
  • the linker is a cleavable linker.
  • linkers are cleavable under intracellular conditions.
  • the linker is hydrolyzable at a pH of less than 5.5.
  • linkers are cleavable by intracellular proteases.
  • the linker is a cathepsin cleavable linker.
  • the linker comprises a dipeptide, a tetrapeptide.
  • the dipeptide is valine (Val)-citrulline (Cit); in some embodiments, the antibody is linked to a linker via a cysteine sulfhydryl group of the antibody. In one embodiment, the antibody is linked to the linker via an amino group of said antibody, particularly an amino group of a glutamine residue.
  • the antibody drug conjugate of the present invention has the following general formula Ab-(LU)n, wherein Ab represents a monoclonal antibody targeting Nectin-4, L is a linker selected from NH 2 -( PEG)m-Val-Cit, NH 2 -(PEG)m-Val-Cit-pABC, mc-Val-Cit-pABC, Val-Cit-pABC, Val-Cit-pAB or Val-Cit, U is a drug, Selected from DM1, DM4, MMAE, MMAF, DXD and SN38; and m represents the number of PEG, which is an integer from 1 to 8, preferably 2, 3, 4, 5, 6, 7, 8, more preferably 3, 4, 5, 6, most preferably 3; n represents the drug antibody ratio DAR, which is an integer from 1 to 8, preferably 2, 4, 6, 8, more preferably 2, 4, and most preferably 2.
  • Ab represents a monoclonal antibody targeting Nectin-4
  • L is a linker selected from NH 2 -
  • n is a decimal number from 1 to 8; wherein, the full name of mc-Val-Cit-pABC is: maleimidocaproyl-valine-citrulline-p-aminocarbamate; the full name of Val-Cit-pAB is: valine-citrulline -p-aminobenzoxyloxycarbonyl.
  • the present invention relates to an antibody drug conjugate (ADC) capable of specifically binding Nectin-4, wherein the antibody or a functional fragment thereof comprises a heavy chain variable region and a light chain variable region. region, the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO.9; the light chain variable region sequence is selected from the amino acid sequence shown in SEQ ID NO.10-12.
  • ADC antibody drug conjugate
  • the heavy chain variable region sequence of the antibody or its functional fragment is the amino acid sequence shown in SEQ ID NO.9 or at least 80%, 85%, 90%, 95% , 96%, 97%, 98% or 99% identity sequence; the light chain variable region sequence is selected from the amino acid sequence shown in SEQ ID NO.10-12 or at least 80%, 85%, 90%, 95% thereof %, 96%, 97%, 98%, or 99% identical sequences.
  • the antibody is a monoclonal antibody.
  • the antibody is a humanized antibody.
  • the invention relates to an isolated polynucleotide encoding the light chain of said antibody or functional fragment thereof and/or the heavy chain of said antibody or functional fragment thereof; or Its encoding is based on said antibody or a functional fragment thereof.
  • the present invention relates to an expression vector comprising said polynucleotide in effect with regulatory sequences allowing expression of a polypeptide encoded thereby in a host cell or in a cell-free expression system connect.
  • the present invention relates to a host cell comprising said expression vector.
  • the present invention provides a pharmaceutical formulation comprising an antibody drug conjugate of the present invention, and a pharmaceutically acceptable diluent, carrier or excipient.
  • the invention provides a medical preparation comprising an antibody drug conjugate of the invention.
  • the medical article is in the form of a kit comprising a container comprising an antibody drug conjugate.
  • the medical article further comprises printed instructions for using the article in a method of treating or preventing cancer, particularly a Nectin-4 expressing cancer.
  • the present invention provides antibody drug conjugates for effective treatment and/or prevention of tumors associated with Nectin-4 expressing cells.
  • the present invention provides the use of the aforementioned antibody drug conjugate and anti-proliferative agent in the preparation of a drug for treating tumors.
  • the present invention provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate and an antiproliferative agent.
  • the anti-proliferative agent can also be an antibody, an antibody drug conjugate, or a fusion protein.
  • the present invention also relates to a method for treating tumors in a subject, the method comprising administering the antibody drug conjugate or the pharmaceutical composition or the medical product to the subject.
  • the present invention also relates to a method of treating a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody drug conjugate or the pharmaceutical composition or the medical products and radiation.
  • the present invention also relates to a method of treating a tumor in a subject, the method comprising administering to the subject an effective amount of the antibody drug conjugate or the pharmaceutical composition or the medical products and antiproliferative agents.
  • the present invention also relates to the aforementioned antibody, antibody drug conjugate and pharmaceutical composition, which are used for treating cancer, preferably Nectin-4 positive tumors.
  • the tumors (including cancers) described above include, but are not limited to, hematological tumors or solid tumors.
  • the tumors (including cancers) described above are hematological tumors, preferably lymphomas or leukemias, including but not limited to myeloma, B-cell lymphoma, mantle cell lymphoma, non-Hodgkin B- Lymphoma, non-Hodgkin's lymphoma, T-cell lymphoma, cutaneous lymphoma, anaplastic large cell lymphoma, multiple myeloma, indolent non-Hodgkin's lymphoma, plasmacytoma, chronic lymphocytic leukemia, small Lymphoblastic lymphoma, follicular lymphoma.
  • the hematological neoplasm is relapsed or refractory.
  • the tumors (including cancers) described above are solid tumors, including but not limited to: Respiratory system tumors, digestive tract tumors, urinary system tumors, male organ tumors, female organ tumors, skin cancer, endothelial cell Tumors, brain tumors, nervous system tumors, endocrine organ tumors.
  • the respiratory tumors include, but are not limited to, lung and nasopharyngeal and laryngeal cancers.
  • the gastrointestinal tumors include but not limited to: esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer.
  • the tumor of the urinary system includes, but is not limited to: kidney cancer, ureteral carcinoma, bladder cancer, urethral cancer.
  • the male organ tumor includes, but is not limited to, penile cancer, prostate cancer, or testicular cancer.
  • the female organ tumor includes, but is not limited to, breast cancer, vulvar cancer, vaginal cancer, cervical cancer, uterine body cancer, or ovarian cancer.
  • the nervous system tumor includes, but is not limited to: astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, meningioma.
  • the brain tumor includes, but is not limited to, glioblastoma, neuroblastoma, germinal stromal tumor, mesenchymal tumor, epithelial tumor, teratoma, pineal tumor.
  • the skin cancer includes, but is not limited to, cutaneous melanoma or non-melanoma skin cancer.
  • the tumor is a solid tumor, including but not limited to bladder cancer, brain cancer, breast cancer, cervical cancer, breast cancer, endometrial cancer, esophageal squamous cell carcinoma, gastric cancer, head tumor, pancreatic cancer, Cholangiocarcinoma, colorectal cancer, eye cancer, head and neck squamous cell carcinoma, urothelial carcinoma, kidney cancer, liver cancer, lymph node cancer, lung cancer, oral cavity cancer, neck cancer, ovarian cancer, prostate cancer, testicular cancer, laryngeal cancer and uterine cancer , melanoma, salivary gland carcinoma, fibrosarcoma, soft tissue sarcoma, and osteosarcoma.
  • the above cancer is relapsed or refractory.
  • the breast cancer comprises: ductal carcinoma, lobular carcinoma, medullary carcinoma, colloid carcinoma, tubular carcinoma, inflammatory breast cancer, triple negative breast cancer (TNBC).
  • TNBC triple negative breast cancer
  • the ovarian cancer includes: epithelial ovarian tumors, such as adenocarcinoma in the ovary and adenocarcinoma that migrates from the ovary into the abdominal cavity.
  • epithelial ovarian tumors such as adenocarcinoma in the ovary and adenocarcinoma that migrates from the ovary into the abdominal cavity.
  • the leukemia includes: acute myeloid leukemia (AML), acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, myelodysplasia, myeloproliferative disorder, NK cell leukemia (eg, blastic plasmacytoid dendritic cell neoplasm), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma ( MM) and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • AML acute lymphoblastic leukemia
  • chronic lymphocytic leukemia chronic myelogenous leukemia
  • hairy cell leukemia myelodysplasia
  • myeloproliferative disorder eukemia (eg, blastic plasmacytoid dendritic cell
  • the pancreatic cancer is acinar cell adenocarcinoma of the pancreas, ductal cell adenocarcinoma of the pancreas, or stage IV pancreatic cancer.
  • the lung cancer includes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC); in some embodiments, non-small cell lung cancer (NSCLC) includes but is not limited to: squamous cell carcinoma, adenocarcinoma , large cell carcinoma; in some embodiments, the lung cancer is recurrent; in some embodiments, the lung cancer is recurrent squamous cell lung cancer or (advanced) stage IV squamous cell lung cancer.
  • NSCLC non-small cell lung cancer
  • NSCLC small cell lung cancer
  • the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).
  • FIG. 1 Schematic diagram of the structure of Nectin-4 protein
  • FIG. 1 Schematic diagram of the structure of Nectin-4-ADC (SWY2001-Ab1-LND1002);
  • LND1002 represents the linker (Linker) + drug molecule (Drug) part, and the circled part indicates that the linker in L&D is connected with the amide bond of the antibody.
  • the Nectin-4ADC shown in this figure is a site-specific antibody-drug conjugate, and each molecule consists of an anti-Nectin-4 monoclonal antibody at the Q295 amino acid (Kabat) of each heavy chain through a linker (NH 2 -PEG 3 -Val-Cit) coupled with one molecule of MMAE derivative, the antibody and the linker are connected by a stable amide bond (isopeptide bond), and the average ratio (DAR) of the drug molecule to the antibody is 2.0;
  • FIG. 3 Schematic diagram of the structure of Nectin-4-ADC (SWY2001-Ab1-VC MMAE);
  • Figure 5 DSC spectrum of humanized antibody SWY2001-Ab1;
  • Figure 7A SWY2001-Ab1-LND1002 DAR value identification map
  • Figure 7B SWY2001-Ab1-VC-MMAE DAR value identification map
  • Figure 8 Endocytosis effect experiment of SWY2001-Ab1-LND1002 in SK-BR-3 cells
  • Figure 9 Endocytosis effect experiment of SWY2001-Ab1-LND1002 in T47D cells
  • Figure 10 In vivo inhibitory effect experiment of SWY2001-Ab1-LND1002 on mouse PC3-Nectin-4 stably transfected tumor;
  • Figure 11 In vivo inhibitory effect experiment of SWY2001-Ab1-LND1002 on mouse MDA-MB-468 tumor;
  • Figure 12 In vivo inhibitory effect experiment of SWY2001-Ab1-LND1002 on mouse HT-1376 tumor;
  • the terms “pharmaceutical composition”, “combination drug” and “pharmaceutical combination” are used interchangeably to mean at least one drug combined together for a specific purpose and optionally a pharmaceutically acceptable carrier or Combination of accessories.
  • the pharmaceutical composition includes temporally and/or spatially separated combinations as long as they can act together to achieve the purpose of the present invention.
  • the components contained in the pharmaceutical composition eg, antibodies, nucleic acid molecules, nucleic acid molecule combinations and/or conjugates according to the present invention
  • the ingredients contained in the pharmaceutical composition may be administered to the subject simultaneously or sequentially.
  • the pharmaceutically acceptable carrier is water, buffered aqueous solution, isotonic saline solution such as PBS (phosphate buffer saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, carbonic acid Magnesium, 0.3% glycerin, hyaluronic acid, alcohol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.
  • compositions, vaccine or pharmaceutical formulation according to the invention may be administered by any suitable route, for example may be administered orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
  • terapéuticaally effective amount refers to a dose sufficient to demonstrate a benefit to the subject to which it is administered.
  • the actual amount administered, as well as the rate and time course of administration, will depend upon the individual condition and severity of the individual being treated.
  • the prescribing of treatment e.g. decisions on dosage etc.
  • the prescribing of treatment is ultimately the responsibility of and relies on the general practitioner and other medical practitioners to make decisions, usually taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration and what is known to the physician. other known factors.
  • the term "subject” refers to mammals, such as humans, but may also be other animals, such as wild animals (such as herons, storks, cranes, etc.), domestic animals (such as ducks, geese, etc.) or experimental animals (such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).
  • wild animals such as herons, storks, cranes, etc.
  • domestic animals such as ducks, geese, etc.
  • experimental animals such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.
  • antibody refers to whole antibodies and functional fragments thereof
  • Full-length antibody refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region.
  • the light chain constant region comprises one domain, CL.
  • the VH and VL regions can be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • These variable regions of the heavy and light chains contain the binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.
  • Chimeric or humanized antibodies are also included in the antibodies according to the present invention, and the CDRs of the antibodies of the present invention are coded as IMGT.
  • Antibody light and heavy chain genes can be synthesized using conventional genetic engineering techniques, for example, refer to the method disclosed by Chen Jianjun et al. (Chen Jianjun et al, Journal of Cellular and Molecular Immunology, 1997, No. 3).
  • humanized antibody refers to an antibody, which may comprise a CDR region derived from a human antibody, and other parts of the antibody molecule derived from one (or several) human antibodies. Furthermore, some residues of the framework (referred to as FR) segments may be modified in order to preserve binding affinity; humanized antibodies or fragments thereof according to the invention may be prepared by techniques known to those skilled in the art.
  • chimeric antibody refers to an antibody in which the variable region sequences are from one species and the constant region sequences are from another species, eg, an antibody in which the variable region sequences are from a mouse antibody and the constant region sequences are from a human antibody.
  • the chimeric antibody or fragment thereof according to the present invention can be prepared by using gene recombination technology.
  • said chimeric antibodies can be produced by cloning recombinant DNA comprising a promoter and sequences encoding the variable regions of non-human, especially murine monoclonal antibodies according to the invention, and sequences encoding the constant regions of human antibodies .
  • the chimeric antibody of the present invention encoded by such a recombinant gene will be, for example, a mouse-human chimera whose specificity is determined by a variable region derived from murine DNA and whose isotype is determined by a variable region derived from human DNA. to determine the constant region.
  • monoclonal antibody refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.
  • bispecific antibody is capable of binding to two antigens or epitopes, respectively, and includes the light chain and heavy chain of an antibody that specifically binds a first antigen, and the light chain and heavy chain of an antibody that specifically binds a second antigen. chain.
  • the term "functional fragment” as used herein especially refers to antibody fragments such as Fv, scFv (sc refers to single chain), Fab, F(ab') 2 , Fab', scFv-Fc fragments or diabodies, or Any fragment that should be able to increase the half-life by chemical modification such as the addition of poly(alkylene) glycols such as polyethylene glycol (“pegylation, PEGylation”) or by incorporation into liposomes ) (a pegylated fragment known as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG” is polyethylene glycol), the The fragment has nectin-4 binding activity.
  • poly(alkylene) glycols such as polyethylene glycol (“pegylation, PEGylation") or by incorporation into liposomes )
  • pegylated fragment known as Fv-PEG, scF
  • the functional fragment will consist of or comprise a partial sequence of the variable chain of the heavy or light chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived.
  • Fragments will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which they are derived.
  • antibody drug conjugate or "conjugate” as used herein generally refers to the combination of an antibody or antigen-binding fragment thereof with another agent (such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, etc.) connections.
  • the attachment may be a covalent bond or a non-covalent interaction, for example by electrostatic force.
  • linkers known in the art and described herein can be employed.
  • antibody drug conjugates can be provided as fusion proteins that can be expressed from polynucleotides encoding the immunoconjugates.
  • fusion protein means a protein produced by the joining of two or more genes or gene fragments that originally encoded separate proteins, including peptides and polypeptides. Translation of the fusion genes produces a single protein with functional properties from each original protein.
  • conjugate two or more compounds are linked together. In certain embodiments, at least some properties from each compound are retained in the conjugate. Linkage can be achieved by covalent or non-covalent bonds.
  • the compounds of the conjugate are linked by covalent bonds.
  • the different compounds of the conjugate may be directly bound to each other by one or more covalent bonds between the atoms of the compounds.
  • compounds may be bound to each other by chemical moieties such as linker molecules, where linkers are covalently attached to atoms of the compounds. If the conjugate consists of more than two compounds, these compounds may be linked, for example, in a chain conformation, with one compound linked to the next, or several compounds each linked to a central compound.
  • cytotoxic drugs described herein specifically refer to substances that inhibit or prevent cell expression activity, cell function, and/or cause cell destruction.
  • examples include, but are not limited to: Auristatin derivatives (e.g. MMAE, MMAF), chlortetracycline, maytansinoids and their derivatives (DMO, DM1, DM2, DM3, DM4, etc.) , Ricin, Combrestatin, Ansamitocin, Calicheamicin, Duocarmycin, Dolastatin and its derivatives Drugs, DNA topoisomerase inhibitors and their derivatives (for example: etoposide Etoposide, teniposide teniposide, Dxd, SN38), amanitin (Amanitin), cc1065 and its analogs, mitomycin C (Mitomycin C), camptothecin (CPT) and its analogues, vincristine, vinblastine, colchicine, mitoxantrone, actinomycetes Actin
  • immune enhancer refers to substances that can activate non-specific immunity and enhance the body's immune response, such as TLR agonists and STING agonists.
  • Radioactive isotopes include, but are not limited to: At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 or Bi 213 , p 32 , Pb 212 , Lu.
  • linker refers to a structural element of a compound that enables the linking of two compounds through one structural element of said compound to one or more other structural elements of said same compound.
  • the linker may be a non-cleavable linker.
  • Suitable non-cleavable linkers include, but are not limited to: NH2 -RX, NH2NH -RX, and NH2 - ORX, where R is an alkyl group or a polyethylene glycol group (also known as PEG), where X is a reactive part.
  • a polyethylene glycol group or a PEG group may have the general formula -( CH2CH2O ) n , where n is an integer of at least one. In some embodiments, n is any of 2, 4, 6, 8, 10, 12, 16, 20, or 24;
  • Cleavable linkers include, but are not limited to , Lys-Phe-X, Lys-Val-Cit-PABC-X, NH2-( CH2CH2O ) n -Val-Cit-pABC-X, and NH2- ( CH2CH2O )n-(Val-Cit-PABC-X) 2 , wherein X is an active moiety, and n is at least 1 (such as any of 2, 4, 6, 8, 10, 12, 16, 20 or 24) integer.
  • PABC refers to p-aminobenzyloxycarbonyl (p-aminobenzyloxycarbonyl);
  • the linker may be selected from: NH2- (PEG)m-Val-Cit, NH2- (PEG)m-Val-Cit-pABC, mc-Val-Cit-pABC, Val-Cit-pABC or Val-Cit, m represents the number of PEGs, which is an integer from 1 to 8, that is, 1, 2, 3, 4, 5, 6, 7, 8;
  • endogenous glutamine means that full-length IgG antibodies of the human isotype contain a conserved glutamine residue (Q295) at position 295 of their heavy chain, which is located in close proximity to the N - Glycosylation site (N297).
  • Nectin-4 mouse antibody For the preparation of Nectin-4 mouse antibody, please refer to the patent application WO2022051591A2. The brief steps are as follows:
  • mice Animals (BALB/c mice) were immunized with human Nectin-4-His fusion protein containing adjuvant (Freund's adjuvant) by intraperitoneal injection. Animals were given booster immunizations every 2 weeks until appropriate titers were induced. After each booster immunization, blood was collected from the animals, and the titer was detected by ELISA and FACS; 4 days after the last immunization, the spleen of the animal with a suitable titer was obtained, and a single cell suspension was prepared. Cells were fused with SP2/0 mouse myeloma cells using electrofusion. The fused cells were resuspended in a medium containing hybridoma selective agents thymidine, hypoxanthine and aminopterin (HAT), and then seeded into 96-well plates for culture.
  • HAT hybridoma selective agents
  • the culture supernatant was collected, and the clones capable of binding to human Nectin-4 protein were screened out by ELISA or FACS, and whether they could bind to mouse Nectin-4 protein was verified.
  • Hybridoma cells were continued in culture after addition of fresh medium containing HAT. After 2 days, the culture supernatant of the positive clones selected in the first round of screening was collected, and then the functional activity of the antibody was tested. Afterwards, the selected positive clones were further subcloned. After the subcloning is successful, these antibodies are purified by conventional antibody purification methods, and at the same time, antibody variable region sequencing is performed on some clones that meet the requirements.
  • the Nectin-4 mouse antibody obtained after animal immunization and screening was humanized (commissioned GenScript to complete the humanization work and obtained three humanized antibodies, SWY2001-Ab1, SWY2001-Ab2, and SWY2001-Ab3, with specific sequences See Table 2; humanization steps are as follows:
  • variable region sequences of chimeric antibodies (the chimeric antibodies are modified on the basis of murine antibodies, and the constant region is selected from human IgG1) into the vector pcDNA3.4 (IgG1, kappa) by gene synthesis , to construct complete heavy and light chain plasmids.
  • the plasmid was then transfected into HEK293 cells, and the supernatant was collected and purified using protein A magnetic beads to obtain the full-length antibody.
  • the solution was replaced with PBS by desalting by dialysis, and the concentration and purity of the antibody were detected by OD280 and SDS-PAGE gel electrophoresis, respectively. Then its affinity was detected by surface plasmon resonance (Surface Plasmon Resonance, SPR) technique.
  • the detection of chimeric antibody affinity is mainly determined by Biacore instrument using surface plasmon resonance (Surface Plasmon Resonance, SPR) technology.
  • Detection method use the protein A chip to couple the antibody by capturing the Fc fragment of the antibody, and use the antigen as the mobile phase for detection.
  • the obtained detection data is fitted by the software that comes with the instrument to determine the corresponding association constant (ka) and dissociation constant ( pk) and calculate the corresponding equilibrium constant (KD).
  • the on-dissociation rate of the antibody was obtained, and the antibody affinity was ranked by the dissociation rate constant (pk). According to the sorting results, the top 3 clones with the highest affinity were selected as candidate antibodies, and three humanized antibodies were obtained through the above steps, which were named: SWY2001-Ab1, SWY2001-Ab2 and SWY2001-Ab3.
  • the three candidate antibody genes were respectively inserted into the vector pcDNA3.4 (IgG1, kappa) to construct complete heavy chain and light chain antibody plasmids.
  • the light and heavy chain plasmids were co-transfected into Expi293F cells, and the supernatant was collected and purified with protein A magnetic beads to obtain the full-length antibody.
  • the solution was replaced with PBS by desalting by dialysis, and the concentration and purity of the antibody were detected by OD280 and SDS-PAGE gel electrophoresis, respectively. Then its affinity was detected by surface plasmon resonance (Surface Plasmon Resonance, SPR) technique.
  • the synthetic heavy chain was named Nectin-4-HC.
  • the 5' end introduces the HindIII endonuclease site, the 3' end introduces the EcoRI endonuclease site, and introduces the Kozak sequence behind the 5' end HindIII endonuclease site, and the signal peptide sequence (19 amino acids) MELGLCWVFLVAILEGVQC( SEQ ID NO.14).
  • the expression cassette for the heavy chain was designed as:
  • the synthetic light chain was named Nectin-4-LC.
  • a HindIII endonuclease site was introduced at the 5' end of the light chain
  • an EcoRI endonuclease site was introduced at the 3' end
  • a Kozak sequence and a signal peptide sequence 22 amino acids: MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO. 15).
  • the expression cassette for the light chain was designed as:
  • the PCR amplification product Nectin-4-HC and the plasmid vector pcDNA3.1 were subjected to HindIII/EcoRI double enzyme digestion and ligation transformation, and the positive clones were screened by the Amp+ resistance marker to confirm that the recombinant heavy chain expression vector was constructed correctly;
  • the amplification product Nectin-4-LC and the plasmid vector pcDNA3.1 were subjected to HindIII/EcoRI double enzyme digestion and ligated transformation, and the positive clones were screened by the Amp+ resistance marker to obtain the correctly constructed recombinant light chain expression vector.
  • HEK293 cells were transiently transfected to express humanized antibodies.
  • HEK293 cells were cultured in a 5% CO2 constant temperature shaker at 37° C. and 120 rpm constant temperature shaking. Cells were cultured to a density of 2.0 ⁇ 10 6 cells/mL, and antibody heavy chain and light chain plasmids were added at a ratio of 0.5 mg HC and 0.5 mg LC per liter of cells.
  • Capillary isoelectric focusing (cIEF) method prepare 4.3M urea, 3M urea-cIEF gel solution, and cIEF MIX solution, dilute the test sample to 5 mg/mL, mix 234 ⁇ L cIEF MIX solution with 10 ⁇ L sample, vortex fully, take 200 ⁇ L of the mixture was transferred to the inner tube for detection.
  • cIEF Capillary isoelectric focusing
  • CE-SDS Monoclonal antibody molecular size variant determination
  • non-reducing sample preparation take 100 ⁇ g of the test solution, add SDS sample buffer to 95 ⁇ L, then add 5 ⁇ L 250mM iodoacetamide solution and mix well. Prepare the reference solution in the same way.
  • Reduction sample preparation Take 100 ⁇ g of the test solution, add SDS sample buffer to 95 ⁇ L, then add 5 ⁇ L 2-mercaptoethanol and mix well. Incubate the mixed samples at 70 ⁇ 2°C for 10 min, cool to room temperature, centrifuge at 6000 rpm for 1 min, pipette 80 ⁇ L of the supernatant into the sample tubes, and load the samples immediately for analysis.
  • the physical and chemical properties test data are as follows:
  • the expression level of the humanized antibody obtained in the present invention is relatively high, the transient expression level of common antibody is 50-100 mg/L, the three humanized antibodies of the present invention are all greater than 100 mg/L, and the expression level of SWY2001-Ab1 Reached 200mg/L.
  • multiple methods were used to detect the purity, and the SEC-HPLC purity was >99%, and the reduced CD-SDS purity was >98%, all of which met the requirements of further experiments.
  • Nectin-4 humanized antibody to Nectin-4 protein of different species was tested by ELISA.
  • Nectin-4 proteins of different species human Nectin-4, cynomolgus monkey Nectin-4, rat Nectin-4, mouse Nectin-4 were incubated with different concentrations of Nectin-4 humanized antibody samples, and then combined with IgG secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody) was incubated, and the affinity of the sample to different species of Nectin-4 proteins was analyzed by ELISA to detect signal values at different concentrations.
  • the experimental results are shown in Table 6. The results showed that the Nectin-4 humanized antibody had good affinity to human, rat, and cynomolgus monkey Nectin-4 proteins.
  • transglutaminase catalytic site of the antibody of the present application is near the Q295 position of N-glycosylation.
  • the transglutaminase sequence used is as follows (SEQ ID NO: 13):
  • this product uses reverse phase chromatography (RP-HPLC) to determine the drug-antibody coupling ratio DAR value.
  • Mobile Phase Mobile Phase A: 0.1% (v/v) TFA in water
  • the detection method is as follows:
  • SK-BR-3 cells and T47D cells human breast cancer cells
  • resuspend the cells in culture medium gently pipette and resuspend the target cells several times to a single-cell suspension, and use trypan blue staining to identify cell viability and cell Count; adjust the cell density to 1 ⁇ 10 5 cells/mL
  • Zenon TM pHrodo TM iFL ADC was added to the 96-well plate with a final concentration of 2 ⁇ g/mL, and then placed in a 37°C, 5% CO 2 incubator for continuous culture for up to 24 hours. All images were observed and captured by a laser confocal microscope with a 20X objective lens.
  • the experimental results show that SWY2001-Ab1-LND1002 was endocytosed in cells and localized in lysosomes in an acidic environment, and its endocytosis rate was much higher than that of the drug PADCEV.
  • the endocytosis rate is fast, the ability of the drug to enter tumor cells is strong, the toxin can be released better, and the drug has a faster onset of action. Therefore, the ability of the antibody drug conjugate obtained in this application to enter tumor cells is significantly better than that of PADCEV.
  • Example 8 Inhibitory effect of SWY2001-Ab1-LND1002 on the growth of different cells in vitro
  • SWY2001-Ab1-LND1002 can inhibit the growth of 293T-Nectin-4, SK-BR-3, and PC3-Nectin-4 cancer cells in vitro.
  • the in vitro killing data of 293T-Nectin-4 stable transfection showed that SWY2001-Ab1-LND1002 was slightly better than PADECV;
  • the in vitro killing data of SK-BR-3 showed that SWY2001-Ab1-LND1002 was 4 times that of PADCEV;
  • the in vitro killing data of PC3-Nectin-4 showed no significant difference between SWY2001-Ab1-LND1002 and PADCEV.
  • mice After the administration, the mice were weighed, the data was recorded, and the growth of the tumor was dynamically observed by measuring the diameter of the tumor at different times after the administration. On d17 (the 17th day after administration), the test was over, and after the mice were asphyxiated with carbon dioxide, the tumors were peeled off and weighed.
  • SWY2001-Ab1-LND1002 can inhibit tumor growth in a dose-dependent manner (P ⁇ 0.05). %vs 39.0%); Nectin4-mab 1mg/kg, PADCEV 1mg/kg, SWY2001-Ab1-LND10020.5, 1 and 2mg/kg tumor weight inhibition rates were 28.2%, 39.0%, 22.1%, 58.3% , 79.4%.
  • nude mice were inoculated with MDA-MB-468 cells to construct a human breast cancer MDA-MB- 468 nude mouse xenograft tumor model.
  • the 16 animals were divided into 3 groups (D0) according to the tumor volume, 8 in the solvent control group, and 4 in the remaining experimental groups. They were given 0.9% sodium chloride injection (0.9% INJ NS (normal saline) intravenously, respectively. solvent control group), drugs PADCEV, SWY2001-Ab1-LND1002 3mg/kg (single administration). After the administration, the mice were weighed, the data was recorded, and the growth of the tumor was dynamically observed by measuring the diameter of the tumor at different times after the administration. At the end of the D29 experiment, after the mice were asphyxiated with carbon dioxide, the tumors were peeled off and weighed.
  • A 4 rats in the solvent control group, given 0.9% sodium chloride injection (0.9% INJ NS (normal saline), solvent control group); B: 4 rats in the PADCEV experimental group, PADCEV 3mg/kg (single administration); C: 4 mice in the SWY2001-Ab1-LND1002 experimental group were given 3mg/kg (single administration); after administration, the mice were weighed, and the data was recorded. By measuring the diameter of the tumor at different times after administration, dynamic observation Tumor growth. At the end of the D22 experiment, after the mice were asphyxiated with carbon dioxide, the tumors were peeled off and weighed.
  • the test product has basically the same toxicity characteristics, including skin abnormalities, white blood cell and differential count, erythroid related indicators (RBC, HGB, HCT), AST, ALT, PLT, and FIB were abnormal, and corneal histopathology was abnormal.
  • the abnormal symptoms of the commercially available reference substance appeared earlier than that of the test substance (D7 vs D14), and the skin toxicity symptoms were more severe (dandruff vs ulceration).
  • the overall effect of the antibody drug conjugate obtained in this application is significantly better than that of PADCEV in endocytosis, in vitro tumor suppressor cell experiments, and in vivo tumor suppressor efficacy experiments (prostate cancer, breast cancer and bladder cancer);
  • the preliminary safety evaluation test shows that the antibody drug conjugate obtained in this application has a wider therapeutic window, fewer side effects, and no obvious adverse reactions such as skin toxicity and ocular toxicity.

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Abstract

一种抗Nectin-4抗体、药物缀合物及其制备方法和用途，其能够有效用于治疗和/或预防Nectin-4阳性肿瘤，包括以乳腺癌和膀胱癌为代表的癌症。

Description

抗Nectin-4抗体、药物缀合物及其制备方法和用途 技术领域
本发明涉及生物医药领域，尤其涉及一种抗Nectin-4蛋白的抗体及其缀合物，以及上述抗体和缀合物在治疗肿瘤方面的用途。
背景技术
Nectin-4(Nectin Cell Adhesion Molecule 4)，也被称为PVRL4(脊髓灰质炎病毒受体样4，Poliovirus Receptor like 4)，是一种I型膜蛋白，隶属于细胞粘附因子(Nectin)家族成员之一，在胚胎和胎盘中特异性表达；该家族是一组不依赖Ca2+的免疫球蛋白样跨膜细胞粘附分子，由四个成员组成(Nectin-1至Nectin-4)。Nectin-1至Nectin-3在正常的成人组织中广泛表达。Nectin-4蛋白无法在成人健康组织(包括乳腺组织)中检测到，但在多种癌症(乳腺癌、肺癌、卵巢癌等)组织中过表达，在62％的三阴性乳腺癌中发现过表达。
研究表明：在癌症进展中Nectin-4的过表达可促进肿瘤内血管生成并促进肿瘤生长。此外，PI3K/AKT信号通路参与促进Nectin-4介导的癌细胞增殖。此外，Nectin-4的过表达与肺癌，乳腺癌和卵巢癌的不良预后相关。Nectin-4与Afadin蛋白(actin filament-binding protein)相互作用形成复合物，并通过PI3K-AKT信号通路最终提高细胞存活率并防止细胞凋亡；Nectin-4的高表达是甲状腺乳头状癌(PTC)患者淋巴结转移的危险因素；Nectin-4耗竭能够有效抑制体外两个PTC细胞系(即TPC1、KTC-1人甲状腺癌细胞系)的增殖和侵袭并诱导细胞凋亡；人食道癌组织中Nectin-4的过度表达与肿瘤的大小，肿瘤的浸润深度和患者的预后不良密切相关。食道癌细胞系中Nectin-4表达的干预表明，增加的Nectin-4表达可以显着促进细胞活力，迁移，侵袭和肿瘤形成。
膀胱癌是指发生在膀胱黏膜上的恶性肿瘤，多数为移行上皮细胞癌。在膀胱侧壁及后壁最多，其次为三角区和顶部，其发生可为多中心。膀胱癌是泌尿系统最常见的肿瘤，在男性中更为常见。根据2019年国家癌症中心公布的数据，膀胱癌位列男性癌症第七位，发病率为8.83/10万，且近年来上升趋势较为明显，膀胱癌发病存在地区、种族及性别的差异，可发生于各年龄段，高发年龄50～70岁，且随着年龄增加，发病率也逐渐增加。
膀胱癌在临床上归类为非肌层浸润肿瘤(NMIBC)和肌层浸润肿瘤(MIBC)或转移性肿瘤。尿路上皮(移行上皮)癌是膀胱癌的常见类型，约占所有病例的90％。转移性 膀胱癌一般都伴随一个不佳的预后，IV期膀胱癌患者5年生存率仅为15％。对于NMIBC和MIBC，经尿道膀胱肿瘤切除术(TURBT)为标准疗法，局部膀胱灌注化疗以及后续的免疫治疗(应用法芽孢杆菌卡介苗BCG)为辅助疗法；在过去数十年里，对于转移性癌症，化疗早已成为癌症治疗的首选疗法，最常用的是含铂的相关疗法。
乳腺癌目前依旧是全世界发生率最高的女性肿瘤，全世界每年乳腺癌的发病率以0.2％～8％的幅度上升，每年全世界约有140万人被诊断为乳腺癌，而大约有50万人死于该病，乳腺癌是40～55岁女性的第一位死亡原因。自20世纪70年代末开始，乳腺癌的发病在全球范围内一直位居女性恶性肿瘤的首位。根据美国癌症学会公布的数字，美国每年约有20万乳腺癌新发病例，发病率为116/10万，我国虽属女性乳腺癌的低发地区，但近年来乳腺癌的发病率明显增高。中国主要城市10年来乳腺癌发病率增长了37％，死亡率增长了38.9％。尤其以上海、北京、天津及沿海地区为我国乳腺癌的高发地区，已占女性恶性肿瘤发病率的首位。
目前，国际上抗肿瘤用药以靶向药物单克隆抗体为主，单抗是生物制药领域最大的子行业。数据表明：2016年单克隆体抗类药物占据整个生物药市场份额的43％；虽然单克隆抗体治疗因具有靶点特异性高、副作用低等特点，但是单独使用，其疗效比较有限。因此，大多数单克隆抗体药物都与化疗联合使用，目前提高单克隆抗体疗效的主要途径是抗体药物缀合物。抗体药物缀合物属于一类新型抗癌生物导弹药物，是由三部分组成的：抗体，药物与连接两者的接头。将单克隆抗体与药物偶联后，抗体-药物缀合物利用单克隆抗体的靶向性，特异性地识别癌细胞表面的受体，并与受体结合，然后进入到细胞内部，利用细胞内的蛋白酶释放药物阻止癌细胞繁殖与杀灭癌细胞。抗体药物偶联技术使小分子药物与生物蛋白融为一体，兼具二者之长，极大增强了药效，并减少毒副作用，成为新一代治疗产品。
目前国内外靶向Nectin-4的项目没有化学药物，只有6款生物药，其中以Nectin-4为靶点的抗体药物缀合物(ADC)仅有一款上市药物，即为日本Agensys公司(安斯泰来旗下公司)和美国Seattle Genetics公司合作开发的一款靶向Nectin-4的ADC药物，2019年12月19日被FDA加速批准，商品名为Padcev，适应症为：局部晚期或转移性尿路上皮癌。相关临床数据表明：ORR为44％，CR为12％，中位DOR为7.6个月；在接受Padcev治疗的患者中，有46％发生了严重的不良反应；最常见的严重不良反应(≥3％)是尿路感染(6％)，蜂窝织炎(5％)，高热性中性粒细胞减少症(4％)，腹泻(4％)，败血症(3％)，急性肾损伤(3％)，呼吸困难(3％)和皮疹(3％)；致命的不良反应发生在3.2％的患者中：包括急性呼吸衰竭(0.8％)，误吸性肺炎(0.8％)，心脏疾病(0.8％)和败血症(0.8％)。
由此可见，Padcev的不良反应比较严重，基于Nectin-4靶点的研究现状，患者针对相关抗体药物缀合物仍在存在未被满足的治疗需求，急需高效、低副作用、价格合理的能够惠及广大肿瘤患者的抗肿瘤靶向药品。
ADC的研发是一项非常复杂的工作，正确的选择靶点、对药物进行优化和改进、选择合适的接头提升ADC药物的疗效和安全性，均是ADC药物研发的难点。ADC药物设计需要基于对抗体、接头和药物作为一个有机整体进行全面的考量，明确肿瘤适应症和药物作用靶点，并充分、透彻地研究ADC药物的作用机制，从而最终实现ADC药物对肿瘤细胞的有效杀伤，同时提高患者的生活质量。
需要强调的是，ADC成功的关键之一在于选择合适的抗体，在保持药物活性的条件下将药物输送到肿瘤细胞。用于制备ADC的抗体要有一定的特性，它不仅需要特异性结合至肿瘤中的抗原阳性细胞，而且抗原-抗体复合物需要能够介导ADC的内化；筛选抗体需要至少考虑以下三个因素：特异性结合、内化以及内化后抗体的细胞定位；并且，抗体的亲和力与内化速度并无明确的联系(Laurent Ducry著，抗体药物偶联物，科学出版社，第36-37页)。因此，抗体作为精确度极高的“定位装置”，能够特异性结合目标细胞以及有效介导“内吞”及其定位，对抗体药物缀合物的特异性和毒性至关重要；
基于现有技术的缺陷，发明人针对现有药物的接头和小分子药物缀合不稳定的问题，采用NH 2-PEG 3-Val-Cit作为接头，采用酶介导的定点偶联方式将人源化抗体通过接头与小分子药物(例如：MMAE)偶联，获得的ADC药物对Nectin-4高表达癌症细胞尤其是乳腺癌、膀胱癌等肿瘤具有极强的杀伤作用；具体而言，首先，根据胞吞实验结果，本发明获得的抗Nectin-4的抗体药物缀合物相对于对照药Padcev具有更好的胞吞效果，能够同时实现特异性结合、高效内化以及精准细胞定位，提高了抗体药物缀合物的特异性的同时降低其毒性；其次，体内实验表明：在具有Nectin-4阳性异种移植肿瘤的裸鼠中静脉内施用抗体药物缀合物导致肿瘤生长抑制，在低至1mg/kg的单剂量静脉内施用下即可观察到显著的治疗效果；其总体治疗效果显著优于对照药Padcev；最后，Padcev在猴子毒理实验中6mg/kg第二次给药即出现严重的不良反应并致死。相比之下，本发明获得的抗体药物缀合物在9mg/kg高剂量两次给药基本上无不良反应，治疗窗口期更宽，成药性更好，综合来看，本申请提供的抗体药物缀合物取得了预料不到的技术效果。
发明内容
本发明提供结合于Nectin-4蛋白和/或Nectin-4蛋白多肽片段的抗体、功能性片 段(例如抗原结合片段)及抗体药物缀合物(ADC)。
在一个方面，本发明提供了能够特异性结合Nectin-4的抗体或其功能性片段，所述抗体包含重链和轻链，其中
(i)所述重链包含三个CDR区，其中至少一个所述CDR区的氨基酸序列具有如SEQ ID NO.1、2或3所示的氨基酸序列或者与其具有至少80％(优选85％、90％、95％、96％、97％、98％或者99％)序列同一性的序列；和/或
(ii)所述轻链包含三个CDR区，其中至少一个所述CDR区的氨基酸序列具有如SEQ ID NO.4、5或6所示的氨基酸序列或者与其具有至少80％(优选85％、90％、95％、96％、97％、98％或者99％)序列同一性的序列。
在一些具体的实施方案中，所述抗体包含重链和轻链，其中
(i)所述重链包含三个CDR区，分别为CDR1区、CDR2区和CDR3区，所述CDR1区、CDR2区和CDR3区分别具有如SEQ ID NO.1、2和3所示的氨基酸序列；和/或
(ii)所述轻链包含三个CDR区，分别为CDR1区、CDR2区和CDR3区，所述CDR1区、CDR2区和CDR3区分别具有如SEQ ID NO.4、5和6所示的氨基酸序列。
在一些具体的实施方案中，所述抗体的重链可变区序列为如SEQ ID NO.9所示的氨基酸序列或者与其具有至少80％(优选85％、90％、95％、96％、97％、98％或者99％)序列同一性的序列；轻链可变区序列选自如SEQ ID NO.10-12所示的氨基酸序列或者与其至少80％(优选85％、90％、95％、96％、97％、98％或者99％)序列同一性的序列。
在一些具体实施方案中，本发明的抗体或其功能性片段是分离的。
在一些具体实施方案中，本发明的抗体是单克隆抗体。
在一些具体实施方案中，本发明的抗体或其功能性片段是人源化抗体，优选全人源化抗体。
在一些具体实施方案中，本发明的抗体是双特异性抗体。
在一些具体实施方案中，本发明的抗体或其功能性片段具有ADCC活性。
在一些具体实施方案中，本发明的抗体或其功能性片段具有CDC活性。
在一些具体实施方案中，本发明的抗体或其功能性片段特异性地结合Nectin-4，而基本上不结合Nectin-1、Nectin-2或Nectin-3。
在一些具体实施方案中，本发明的抗体或其功能性片段包括：双抗、Fab片段、Fab’片段、F(ab)’2、scFv、dsFv以及单域抗体；所述scFv蛋白是免疫球蛋白的轻链可变区和重链可变区通过接头结合的融合蛋白；所述dsFv中，在VH与VL的保守 框架区的特定位点引入半胱氨酸，由此引入二硫键稳定dsFv的结构。
在一些具体实施方案中，本发明的抗体或其功能性片段是IgM、IgD、IgG、IgA或IgE，其中，IgG抗体有4种亚型：IgG1、IgG2、IgG3、IgG4，优选IgG1抗体。
在一些具体实施方案中，所述抗体的重链恒定区序列为：
Figure PCTCN2022114793-appb-000001
在一些具体实施方案中，所述抗体的轻链恒定区序列为：
Figure PCTCN2022114793-appb-000002
在一些具体实施方案中，本发明的抗体或其功能性片段可用于治疗或预防癌症，其中所述癌症过表达Nectin-4。
在一些实施方案中，具有与Nectin-4结合之能力的抗体或其功能性片段与存在于活细胞表面上的Nectin-4的天然表位结合。在一些实施方案中，具有与Nectin-4结合之能力的抗体或其功能性片段与Nectin-4的细胞外结构域结合。在一些实施方案中，具有与Nectin-4结合之能力的抗体或其功能性片段与Nectin-4的第一胞外区结合；在一些实施方案，具有与Nectin-4结合之能力的抗体或其功能性片段与Nectin-4的1-147aa结构域结合；在一些实施方案，具有与Nectin-4结合之能力的抗体或其功能性片段与Nectin-4的32-142aa结构域结合；
在另一个方面，本发明提供了编码本发明抗体的分离的多核苷酸。
在又一个方面，本发明提供了分离的多核苷酸的组合，所述组合包括编码本发明抗体或其功能性片段之轻链的多核苷酸和编码本发明抗体或其功能性片段之重链的多核苷酸。
在另一个方面，本发明提供了表达载体，其包含本发明所述的多核苷酸或者本发明所述的多核苷酸的组合，所述多核苷酸与允许其所编码的多肽在宿主细胞或无细胞表达系统中表达的调节序列有效连接。
在本发明的一些实施方案中，所述宿主细胞可以为原核宿主细胞、真核宿主细 胞或噬菌体。所述原核宿主细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。所述真核宿主细胞，可以为如巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，如草地粘虫等昆虫细胞，如烟草等植物细胞，如BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施方案中，本发明所述宿主细胞优选为哺乳动物细胞，更优选为BHK细胞、CHO细胞、NSO细胞或COS细胞。
在另一个方面，本发明提供了一种抗体药物缀合物，其包含与一个或更多个药物偶联的本发明所述的抗体或其功能性片段，优选地所述药物为细胞毒性药物(例如抗代谢药、抗肿瘤抗生素、生物碱)、免疫增强剂或放射性同位素，更优选地所述药物选自奥瑞斯他汀衍生物、美登素生物碱衍生物(例如安丝菌素(Ansamitocin)或者美登素(Mertansine))、海兔毒素肽(dolastatin)及其衍生物)、喜树碱类似物、DNA拓扑异构酶I抑制剂及其衍生物；最优选地所述药物选自MMAE(Monomethyl auristatin E)和MMAF(Monomethyl auristatin F)。
在一些实施方案中，具有与Nectin-4结合能力的抗体或其功能性片段通过接头与药物部分共价连接。在一些实施方案中，接头是可切割接头。在一些实施方案中，接头在细胞内条件下可被切割。在一个实施方案中，接头在小于5.5的pH下可水解。在一些实施方案中，接头可被细胞内蛋白酶切割。在一个实施方案中，接头是组织蛋白酶可切割接头。在一些实施方案中，接头包含二肽、四肽。在一些实施方案中，二肽是缬氨酸(Val)-瓜氨酸(Cit)；在一些实施方案中，抗体通过所述抗体的半胱氨酸巯基与接头连接。在一个实施方案中，抗体通过所述抗体的氨基团(特别是谷氨酰胺残基的氨基团)与接头连接。
在一些具体实施方案中，本发明所述抗体药物缀合物具有以下通式Ab-(L-U)n，其中Ab表示靶向Nectin-4的单克隆抗体，L为接头，选自NH 2-(PEG)m-Val-Cit、NH 2-(PEG)m-Val-Cit-pABC、mc-Val-Cit-pABC、Val-Cit-pABC、Val-Cit-pAB或Val-Cit，U为药物，选自DM1、DM4、MMAE、MMAF、DXD和SN38；以及m表示PEG的个数，为1到8的整数，优选为2、3、4、5、6、7、8，更优选为3、4、5、6，最优选3；n表示药物抗体比率DAR，为1到8的整数，优选为2、4、6、8，更优选为2、4，最优选为2。在一些具体的实施方案中，n为1到8的小数；其中，mc-Val-Cit-pABC全称为：maleimidocaproyl-valine-citrulline-p-aminocarbamate；Val-Cit-pAB的全称为：valine-citrulline-p-aminobenzyloxycarbonyl。
在一些具体的实施方案中，本发明涉及一种能够特异性结合Nectin-4的抗体药物缀合物(ADC)，其中所述抗体或其功能性片段包含重链可变区和轻链可变区，所述重链可变区序列为如SEQ ID NO.9所示的氨基酸序列；所述轻链可变区序列选自如 SEQ ID NO.10-12所示的氨基酸序列。
在一些具体的实施方案中，所述抗体或其功能性片段的重链可变区序列为如SEQ ID NO.9所示的氨基酸序列或者与其具有至少80％、85％、90％、95％、96％、97％、98％或者99％同一性的序列；轻链可变区序列选自如SEQ ID NO.10-12所示的氨基酸序列或者与其至少80％、85％、90％、95％、96％、97％、98％或者99％同一性的序列。
在一些具体的实施方案中，所述抗体为单克隆抗体。
在一些具体的实施方案中，所述抗体为人源化抗体。
在一些具体的实施方案中，本发明涉及一种病分离的多核苷酸，其编码所述抗体或其功能性片段之轻链的和/或所述抗体或其功能性片段之重链；或者其编码根据所述抗体或其功能性片段。
在一些具体的实施方案中，本发明涉及一种表达载体，其包含所述多核苷酸，所述多核苷酸与允许其所编码的多肽在宿主细胞或无细胞表达系统中表达的调节序列有效连接。
在一些具体的实施方案中，本发明涉及一种宿主细胞，其包含所述的表达载体。
在另一个方面，本发明提供了药物制剂，其包含本发明的抗体药物缀合物，以及可药用稀释剂、载体或赋形剂。
在另一个方面，本发明提供了医用制品(medical preparation)，其包含本发明的抗体药物缀合物。在一些实施方案中，医用制品以药盒的形式存在，所述药盒包含含有抗体药物缀合物的容器。在一些实施方案中，医用制品还包含用于在治疗或预防癌症(特别是表达Nectin-4的癌症)的方法中使用所述制品的打印说明书(printed instructions)。
在另一方面，本发明提供了用于有效地治疗和/或预防与表达Nectin-4细胞相关的肿瘤的抗体药物缀合物。
在另一方面，本发明提供了前述的抗体药物缀合物与抗增生剂在制备治疗肿瘤的药物中的用途。
在另一方面，本发明提供了一种药物组合物，其包括前述的抗体药物缀合物和抗增生剂。
在某些具体实施方案中，所述抗增生剂也可以是抗体、抗体药物缀合物、融合蛋白。
另一方面，本发明还涉及一种在对象中治疗肿瘤的方法，所述方法包括向所述对象给予所述的抗体药物缀合物或所述的药物组合物或所述的医用制品。
在一些实施方案中，本发明还涉及一种在对象中治疗肿瘤的方法，所述方法包括向所述对象给予有效量的所述的抗体药物缀合物或所述的药物组合物或所述的医用制品和辐射。
在一些实施方案中，本发明还涉及一种在对象中治疗肿瘤的方法，所述方法包括向所述对象给予有效量的所述的抗体药物缀合物或所述的药物组合物或所述的医用制品和抗增生剂。
另一方面，本发明还涉及前述的抗体、抗体药物缀合物和药物组合物，其用于治疗癌症，优选Nectin-4阳性肿瘤。
在一些实施方案中，上文所述的肿瘤(包括癌症)包括但不限于血液瘤或实体瘤。
在一些实施方案中，上文所述的肿瘤(包括癌症)是血液瘤，优选淋巴瘤或白血病，包括但不限于骨髓瘤、B-细胞淋巴瘤、套细胞淋巴瘤、非霍奇金B-细胞淋巴瘤、非霍奇金淋巴瘤T-细胞淋巴瘤、皮肤淋巴瘤、间变性大细胞淋巴瘤、多发性骨髓瘤、惰性非霍奇金淋巴瘤、浆细胞瘤、慢性淋巴细胞白血病、小淋巴细胞淋巴瘤、滤泡性淋巴瘤。在一些实施方案中，血液瘤是复发或难治性的。
在一些实施方案中，上文所述的肿瘤(包括癌症)是实体瘤，包括但不限于：呼吸系统肿瘤、消化道肿瘤、泌尿系统肿瘤、男性器官肿瘤、女性器官肿瘤、皮肤癌、内皮细胞肿瘤、脑部肿瘤、神经系统肿瘤、内分泌器官肿瘤。
在一些实施方案中，所述呼吸系统肿瘤包括但不限于：肺癌和鼻咽癌以及喉癌。
在一些实施方案中，所述消化道肿瘤包括但不限于：食管癌、胃癌、大肠癌、肝癌、胰腺癌、胆管癌。
在一些实施方案中，所述泌尿系统肿瘤包括但不限于：肾癌、肾盂输尿管癌、膀胱癌、尿道癌。
在一些实施方案中，所述男性器官肿瘤包括但不限于阴茎癌、前列腺癌或睾丸癌。
在一些实施方案中，所述女性器官肿瘤包括但不限于乳腺癌、外阴癌、阴道癌、宫颈癌、子宫体癌或卵巢癌。
在一些实施方案中，所述神经系统肿瘤包括但不限于：星形胶质细胞瘤、少突胶质细胞瘤、室管膜瘤、髓母细胞瘤、脑膜瘤。
在一些实施方案中，所述脑部肿瘤包括但不限于：胶质细胞瘤、神经细胞瘤、类胚叶间质肿瘤、间质类肿瘤、上皮类肿瘤、畸胎瘤、松果体瘤。
在一些实施方案中，所述皮肤癌包括但不限于皮肤黑色素瘤或非黑色素瘤皮肤 癌。
在一些实施方案中，所述肿瘤是实体瘤，包括但不限于膀胱癌、脑癌、乳腺癌、宫颈癌、胸部肿瘤、子宫内膜癌、食管鳞癌、胃癌、头部肿瘤、胰腺癌、胆管癌、结直肠癌、眼癌、头颈鳞癌、尿路上皮癌、肾癌、肝癌、淋巴结癌、肺癌、口腔癌、颈部肿瘤、卵巢癌、前列腺癌、睾丸癌、喉癌和子宫癌、黑色素瘤、唾液腺癌、纤维肉瘤、软组织肉瘤和骨肉瘤。在一些实施方案中，以上癌症复发或难治性的。
在一些实施方案中，所述乳腺癌包括：导管癌、小叶癌、髓性癌、胶质性癌、管状癌、炎性乳腺癌、三阴性乳腺癌(TNBC)。
在一些实施方案中，所述卵巢癌包括：上皮卵巢肿瘤，如卵巢中的腺癌和从卵巢迁移至腹腔中的腺癌。
在一些实施方案中，所述白血病包括：急性髓性白血病(AML)、急性淋巴细胞白血病、慢性淋巴细胞白血病、慢性髓性白血病、毛细胞白血病、脊髓发育不良、骨髓增生性障碍、NK细胞白血病(例如，母细胞性浆细胞样树突状细胞肿瘤)、急性骨髓性白血病(AML)、慢性骨髓性白血病(CML)、肥大细胞增多症、慢性淋巴细胞白血病(CLL)、多发性骨髓瘤(MM)和骨髓增生异常综合征(MDS)。
在一些实施方案中，所述胰腺癌是胰腺的腺泡细胞腺癌，胰腺的导管细胞腺癌或IV期胰腺癌。
在一些实施方案中，所述肺癌包括非小细胞肺癌(NSCLC)和小细胞肺癌(SCLC)；在一些实施方案中，非小细胞肺癌(NSCLC)包括但不限于：鳞状细胞癌、腺癌、大细胞癌；在一些实施方案中，肺癌是复发性的；在一些实施方案中，肺癌是复发性鳞状细胞肺癌或(晚期)IV期鳞状细胞肺癌。
在一些实施方案中，所述前列腺癌是转移性去势抗性前列腺癌(mCRPC)。
附图说明
图1：Nectin-4蛋白结构示意图；
图2：Nectin-4-ADC(SWY2001-Ab1-LND1002)结构示意图；
其中LND1002表示接头(Linker)+药物分子(Drug)的部分，圆圈标记部分表示L&D中的接头与抗体的酰胺键连接。该图所展示的Nectin-4ADC是位点特异性的抗体药物缀合物，每个分子由1个抗Nectin-4单克隆抗体在每条重链的Q295位氨基酸(Kabat)通过接头(NH 2-PEG 3-Val-Cit)偶联1分子的MMAE衍生物，抗体与接头的连接方式为稳定的酰胺键(异肽键)，药物分子与抗体的平均比值(DAR)为2.0；
图3：Nectin-4-ADC(SWY2001-Ab1-VC MMAE)结构示意图；
图4：pcDNA3.1图谱；
图5：人源化抗体SWY2001-Ab1 DSC图谱；
图6：Nectin-4ADC(SWY2001-Ab1-LND1002)修饰率鉴定图谱；
图7A：SWY2001-Ab1-LND1002DAR值鉴定图谱；
图7B：SWY2001-Ab1-VC-MMAE DAR值鉴定图谱；
图8：SWY2001-Ab1-LND1002在SK-BR-3细胞内吞效果实验；
图9：SWY2001-Ab1-LND1002在T47D细胞内吞效果实验；
图10：SWY2001-Ab1-LND1002对小鼠PC3-Nectin-4稳转株肿瘤体内抑制作用实验；
注：mpk＝mg/kg
图11：SWY2001-Ab1-LND1002对小鼠MDA-MB-468肿瘤体内抑制作用实验；
图12：SWY2001-Ab1-LND1002对小鼠HT-1376肿瘤体内抑制作用实验；
具体实施方式
定义：
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
尽管本发明的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
本文使用的术语“药物组合物”、“组合药物”和“药物组合”可互换地使用，其表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述药物组合物中所含的成分(例如根据本发明的抗体、核酸分子、核酸分子组合和/或缀合物)可以以整体施用于对象，或 者分开施用于对象。当所述药物组合物中所含的成分分开地施用于对象时，所述成分可以同时或依次施用于对象。优选地，所述可药用载体是水、缓冲水溶液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
根据本发明的药物组合物、疫苗或者药物制剂可通过任何适宜的途径施用，例如可口服、鼻、皮内、皮下、肌内或静脉内施用。
本文使用的“治疗有效量”或“有效量”是指足以显示其对于所施用对象益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
本文所使用的术语“对象”是指哺乳动物，如人类，但也可以是其它动物，如野生动物(如苍鹭、鹳、鹤等)，家畜(如鸭、鹅等)或实验动物(如猩猩、猴子、大鼠、小鼠、兔子、豚鼠、土拨鼠、地松鼠等)。
术语“抗体”系指完整抗体和其功能性片段“全长抗体”系指包含通过二硫键而互连的至少两条重(H)链和两条轻(L)链的蛋白。每条重链包含一重链可变区(缩写为VH)和一重链恒定区。该重链恒定区包含三个域(domain)，CH1、CH2和CH3。每条轻链包含一轻链可变区(缩写为VL)和一轻链恒定区。该轻链恒定区包含一个域，CL。VH和VL区域还可再细分为具有高可变性的多个区，被称为互补决定区(CDR)，其间散布有更为保守的被称为框架区(FR)的多个区域。每个VH和VL均由三个CDR和四个FR构成，按照以下顺序从氨基端至羧基端排布：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的这些可变区包含与抗原相互作用的结合域。抗体的恒定区可介导免疫球蛋白与宿主的组织或因子结合，包括免疫系统的各种细胞(如效应细胞)和经典补体系统的第一成分(Clq)。嵌合或人源化抗体也涵盖在根据本发明的抗体中，本发明抗体的CDR编码方式为IMGT。
抗体轻重链基因的合成可以采用常规的基因工程技术方法，例如，可以参考：陈建军等(陈建军et al，细胞与分子免疫学杂志，1997年，第3期)公开的方法。
术语“人源化抗体”是指一种抗体，其可以包含来源于人源抗体的CDR区、并且该抗体分子的其他部分来源于一种(或几种)人抗体。而且，为了保留结合亲和力，可 以修饰骨架(称为FR)区段的一些残基；通过本领域技术人员已知的技术可以制备根据本发明的人源化抗体或其片段。
术语“嵌合抗体”系指以下抗体，其中的可变区序列来自一物种而恒定区序列来自另一物种，例如可变区序列来自小鼠抗体而恒定区序列来自人抗体的抗体。通过使用基因重组技术可以制备根据本发明的嵌合抗体或其片段。例如，所述嵌合抗体可以通过克隆重组DNA来生产，所述重组DNA包含启动子和编码根据本发明的非人尤其是鼠单克隆抗体可变区的序列、以及编码人抗体恒定区的序列。由这种重组基因编码的本发明嵌合抗体将是，例如，鼠-人嵌合体，该抗体的特异性由来源于鼠DNA的可变区确定，并且其同种型由来源于人DNA的恒定区来确定。
术语“单克隆抗体”系指具有单一分子组成的抗体分子的制备物。单克隆抗体组合物显示出对于特定表位的单一结合特异性和亲和性。
术语“双特异性抗体”能够分别和两种抗原或抗原表位结合，其包括特异性结合第一抗原的抗体的轻链和重链，以及特异性结合第二抗原的抗体的轻链和重链。
本文所使用的术语“功能性片段”尤其是指抗体片段如Fv、scFv(sc指单链)、Fab、F(ab’) 2、Fab’、scFv-Fc片段或者双抗体(diabody)、或者通过化学修饰或通过掺入脂质体中应能够增加半寿期的任何片段，所述化学修饰例如添加聚(亚烷基)二醇如聚乙二醇(“聚乙二醇化，PEG化”)(被称为Fv-PEG、scFv-PEG、Fab-PEG、F(ab’)2-PEG或Fab’-PEG的聚乙二醇化片段)(“PEG”为聚乙二醇)，所述片段具有Nectin-4结合活性。优选地，所述功能片段将由其来源抗体的重链或轻链可变链的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，这种功能片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。
通常，为了制备单克隆抗体或其功能片段，尤其是鼠源的单克隆抗体或其功能片段，可以参考尤其描述在手册“Antibodies”中的技术(Harlow and Lane，Antibodies：A Laboratory Manual，Cold Spring Harbor Laboratory，Cold Spring Harbor NY，pp.726，1988)或者参考Kohler和Milstein描述的从杂交瘤细胞制备的技术(Nature，256：495-497，1975)。
本文中使用的术语“抗体药物缀合物(ADC)”或“缀合物”通常是指抗体或其抗原结合片段与另一种试剂(诸如化学治疗剂、毒素、免疫治疗剂、成像探针等)的连接。所述连接可以是共价键或非共价相互作用，例如通过静电力。为了形成抗体药物缀合物，可以采用本领域已知的和本文所述的各种接头。另外，抗体药物缀合物可以以融合蛋白的形式提供，所述融合蛋白可以从编码免疫缀合物的多核苷酸表 达。本文中使用的“融合蛋白”表示通过两个或更多个最初编码单独蛋白(包括肽和多肽)的基因或基因片段的连接而产生的蛋白质。融合基因的翻译产生具有来自每个原始蛋白的功能特性的单个蛋白。
在“缀合物”中，两个或更多个化合物连接在一起。在某些实施方案中，来自每种化合物的至少一些性质保留在缀合物中。可以通过共价或非共价键实现连接。优选地，缀合物的化合物通过共价键连接。缀合物的不同化合物可以通过化合物的原子之间的一个或多个共价键彼此直接结合。可替换地，化合物可以通过化学部分诸如接头分子彼此结合，其中接头共价连接到化合物的原子上。如果缀合物由超过两种化合物组成，则这些化合物可以例如以链构象连接，一种化合物连接至下一种化合物，或者数种化合物各自连接至一种中心化合物。
本文所述的细胞毒性药物具体是指抑制或阻止细胞的表达活性、细胞功能，和/或导致细胞破坏的物质。示例包括但不限于：奥瑞斯他汀衍生物(例如MMAE、MMAF)、金霉素(chlortetracycline)、美登素生物碱(maytansinoid)及其衍生物(DM0、DM1、DM2、DM3、DM4等)、蓖麻毒蛋白(Ricin)、考布他汀(combrestatin)、安丝菌素(Ansamitocin)、卡奇霉素(calicheamicin)、倍癌霉素(Duocarmycin)、海兔毒素肽(dolastatin)及其衍生物、DNA拓扑异构酶抑制剂及其衍生物(例如：依托泊苷Etoposide、替尼泊苷teniposide、Dxd、SN38)、鹅膏毒素(Amanitin)、cc1065及其类似物、丝裂霉素C(Mitomycin C)、喜树碱(Camptothecin，CPT)及其类似物、长春新碱(Vincristine)、长春花碱(Vinblastine)、秋水仙碱(colchicine)、米托蒽醌(mitoxantrone)、放线菌素(Actinomycins)、白喉毒素(diphtheria toxin)、假单胞菌外毒素A(Pseudomonas exotoxin)、相思豆毒蛋白(Abrin)、白树毒素(Gelonin)、小诺霉素(Micronomicin)等；
术语“免疫增强剂”是指能够激活非特异性免疫，增强机体免疫应答的物质，例如：TLR激动剂、STING激动剂。
放射性同位素包括但不限于：At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212或Bi 213、p 32、Pb 212、Lu。
术语“接头”是指这样一种化合物结构元件，其可通过所述化合物的一个结构元件与所述相同化合物的一个或多个其他结构元件实现两种化合物的连接。所述接头可以是不可切割接头(non-cleavable linker)。合适的不可切割接头包括但不限于：NH 2-R-X、NH 2NH-R-X和NH 2-O-R-X，其中R是烷基或聚乙二醇基团(也被称为PEG)，其中X是活性部分。聚乙二醇基团或PEG基团可以具有-(CH 2CH 2O) n的通式，其中n是至少1的整数。在一些实施方案中，n是2、4、6、8、10、12、16、20或24中 的任一个；
可切割接头包括但不限于Lys-Phe-X、Lys-Val-Cit-PABC-X、NH2-(CH 2CH 2O) n-Val-Cit-pABC-X和NH2-(CH 2CH 2O)n-(Val-Cit-PABC-X) 2，其中X是活性部分，且n是至少1(诸如2、4、6、8、10、12、16、20或24中的任一个)的整数。PABC是指对氨基苄氧基羰基(p-aminobenzyloxycarbonyl)；
仅为举例说明，接头可以选自：NH 2-(PEG)m-Val-Cit、NH 2-(PEG)m-Val-Cit-pABC、mc-Val-Cit-pABC、Val-Cit-pABC或Val-Cit，m表示PEG的个数、为1到8的整数，即为1、2、3、4、5、6、7、8；
术语“内源性谷氨酰胺”是指：人同种型的全长IgG抗体在其重链的295位含有一个保守的谷氨酰胺残基(Q295)，该谷氨酰胺残基紧密靠近N-糖基化位点(N297)。
设备与实验材料：
表1：设备与实验材料
仪器设备 生产厂家 货号
Human Nectin-4 Protein，His Tag ACRO NE4-H52H3
Cynomolgus Nectin-4 Protein，His Tag ACRO NE4-C52H4
Mouse Nectin-4 Protein，His Tag ACRO NE4-M52H3
Recombinant Rat Nectin-4 His-tag Protein，CF R&D systems 9997-N4-050
SK-BR-3 ATCC HTB-30 TM
293T-Nectin-4 康源博创 定制
PC3-Nectin-4 康源博创 定制
HT1376 赛百慷 iCell-h077
实施例1：抗体的获得以及人源化改造
Nectin-4鼠源抗体制备相关内容参考专利申请WO2022051591A2，简要步骤如下：
用含有佐剂(弗氏佐剂)的人Nectin-4-His融合蛋白通过腹腔注射的方式对动物(BALB/c小鼠)进行免疫。每2周对动物进行加强免疫持续诱导直至产生合适效价。每次加强免疫后对动物进行采血，通过ELISA和FACS检测效价；最后一次免疫后4天，获取合适效价动物的脾脏，并制备单细胞悬液。使用电融合将细胞与SP2/0小鼠骨髓瘤细胞进行融合。将融合的细胞重悬于含杂交瘤细胞选择剂胸腺核苷嘧啶，次黄嘌呤和氨基喋呤(HAT)的培养基中，然后接种到96孔板中进行培养。
孵育7-10天之后，收集培养上清，通过ELISA或者FACS的方法检测筛选出能够结合人Nectin-4蛋白的克隆，并验证是否结合小鼠Nectin-4蛋白。杂交瘤细胞在添加新鲜含有HAT的培养基之后继续培养。2天后收集第一轮筛选出来的阳性克隆培养上清再进行抗体功能活性检测。之后，对挑选出的阳性克隆进行进一步的亚克隆。亚克隆成功后，用常规的抗体纯化方法对这些抗体进行纯化，同时对部分符合要求的克隆进行抗体可变区测序。
经动物免疫及筛选后获得的Nectin-4鼠源抗体，进行人源化改造(委托金斯瑞完成人源化工作得到SWY2001-Ab1、SWY2001-Ab2、SWY2001-Ab3三个人源化抗体，具体序列见表2；人源化步骤如下：
1.1嵌合抗体表达纯化
通过基因合成将嵌合抗体(所述嵌合抗体是在鼠源抗体的基础上改造而成，恒定区选自human IgG1)的可变区序列分别插入到载体pcDNA3.4(IgG1，kappa)中，构建完整的重链及轻链质粒。再把质粒转染至HEK293细胞，收集上清后采用protein A磁珠纯化得到全长抗体。通过透析脱盐将溶液置换为PBS，抗体的浓度和纯度分别通过OD280和SDS-PAGE凝胶电泳检测。然后通过表面等离子共振(Surface Plasmon Resonance，SPR)技术检测其亲和力。
1.2嵌合抗体结合活性及亲和力检测
嵌合抗体亲和力检测主要通过Biacore仪器运用表面等离子共振(Surface Plasmon Resonance，SPR)技术来测定。检测方法：使用protein A芯片通过捕获抗体Fc片段来偶联结合抗体，以抗原作为流动相来检测，获得的检测数据通过仪器自带的软件拟合测定相应的结合常数(ka)和解离常数(pk)及计算对应平衡常数(KD)。
1.3人源化抗体回复突变的设计
对人源化抗体的轻、重链结构内核区的多个氨基酸位点进行回复突变及组合。相应的轻重链基因由金斯瑞生物科技有限公司(GenScript)合成。
1.4回复突变人源化抗体的生产及亲和力排序
获得重链和轻链的质粒后，将对应轻重链组合配对并进行4mL体系的转染表达。获取表达上清后，运用SPR技术对其上清进行亲和力检测和排序：使用protein A芯片通过捕获抗体Fc片段来捕获上清中抗体，将抗体固定在传感芯片上。分析物抗原用作流动相。在注射另一株抗体上清之前进行表面再生，重复该过程直至分析完所有抗体。通过使用Biacore分析软件，运用1∶1相互作用模型拟合实验数据。获得抗体的结合解离速率，通过解离速率常数(pk)对抗体亲和力进行排序。根据排序结果，选择亲和力前3高的克隆作为候选抗体，通过上述步骤获得三个人源化抗体， 分别命名为：SWY2001-Ab1、SWY2001-Ab2以及SWY2001-Ab3。
1.5候选抗体的构建、表达及亲和力测定验证
将3株候选抗体基因分别插入到载体pcDNA3.4(IgG1，kappa)中，构建完整的重链及轻链抗体质粒。再把轻重链质粒共转染至Expi293F细胞，收集上清后采用protein A磁珠纯化得到全长抗体。通过透析脱盐将溶液置换为PBS，抗体的浓度和纯度分别通过OD280和SDS-PAGE凝胶电泳检测。然后通过表面等离子共振(Surface Plasmon Resonance，SPR)技术检测其亲和力。
表2：本申请涉及的相关序列
Figure PCTCN2022114793-appb-000003
Figure PCTCN2022114793-appb-000004
Figure PCTCN2022114793-appb-000005
实施例2：表达载体的构建及人源化抗体的表达
Nectin-4人源化抗体重、轻链DNA序列由泰州百英合成，表达载体pcDNA3.1载体由泰州百英提供(参见附图4)。简要步骤如下：
重链的设计与合成：
人工合成的重链命名为Nectin-4-HC。5’端引入HindIII内切酶位点，3’端引入EcoRI内切酶位点，同时在5’端HindIII内切酶位点后面引入了Kozak序列，以及信号肽序列(19个氨基酸)MELGLCWVFLVAILEGVQC(SEQ ID NO.14)。重链的表达框设计为：
HindIII-Kozak序列-信号肽-Nectin-4-HC-终止密码子-EcoRI
轻链的设计与合成：
人工合成的轻链命名为Nectin-4-LC。合成过程中，在轻链5’端引入HindIII内切酶位点，3’端引入EcoRI内切酶位点，同时在5’端HindIII内切酶位点后面引入Kozak序列，以及信号肽序列(22个氨基酸)：MDMRVPAQLLGLLLLWFPGSRC(SEQ ID NO.15)。轻链的表达框设计为：
HindIII-Kozak序列-信号肽-Nectin-4-LC-终止密码子-EcoRI
2)重组质粒的构建
对PCR扩增产物Nectin-4-HC和质粒载体pcDNA3.1进行HindIII/EcoRI双酶切和连接转化，通过Amp+抗性标记筛选阳性克隆，确认获得构建正确的重组重链表达载体；对PCR扩增产物Nectin-4-LC和质粒载体pcDNA3.1进行HindIII/EcoRI双酶切和连接转化，通过Amp+抗性标记筛选阳性克隆，获得构建正确的重组轻链表达载体。
本实验采用瞬转HEK293细胞的方式进行人源化抗体的表达。将HEK293细胞置于5％的C02恒温摇床中，37℃、120rpm恒温震荡培养。将细胞培养至密度在2.0×10 6个/mL，按照每升细胞加入0.5mg HC、0.5mg LC的比例加入抗体重链和轻链 质粒。首先将KPM(转染缓冲液)与无菌质粒混匀，然后另取一支离心管，将KPM与TA-293转染试剂混匀，将转染试剂缓慢加入有质粒的KPM混合物中，轻轻混匀制备成质粒-载体复合物，静置10min后将质粒-载体复合物加入细胞中；24h后加入细胞蛋白表达增强剂和瞬时传染营养添加剂，转染后第6天收获细胞，进行纯化。
毛细管等电聚焦(cIEF)方法：配制4.3M尿素、3M尿素-cIEF胶溶液、cIEF MIX溶液，将供试品稀释至5mg/mL，取234μL cIEF MIX溶液与10μL样品混合，充分涡旋，取200μL混合液转移至内插管中进行检测。
单抗分子大小变异体测定(CE-SDS)方法：非还原样品制备：取100μg供试品溶液，加入SDS样品缓冲液至95μL，再加入5μL 250mM碘乙酰胺溶液混匀。同法制备参比品溶液。还原样品制备：取100μg供试品溶液，加入SDS样品缓冲液至95μL，再加入5μL 2-巯基乙醇混匀。将混合好的样品在70±2℃孵育10min，冷却至室温后，6000rpm离心1min，分别吸取80μL上清至样品管中，立即进行上样分析。理化性质检测数据如下：
表3：Nectin-4人源化抗体表达及理化性质检测
Figure PCTCN2022114793-appb-000006
由此可见，本发明获得的人源化抗体的表达量较高，普通抗体瞬转表达量在50-100mg/L，本发明三个人源化抗体均大于100mg/L，且SWY2001-Ab1表达量达到了200mg/L。同时采用了多重方法检测纯度，其SEC-HPLC纯度＞99％，还原CD-SDS纯度＞98％，均满足进一步实验的要求。
实施例3：Nectin-4人源化抗体DSC检测
本实验采用差示扫描量热仪检测Nectin-4人源化抗体的稳定性，实验参数如下表，抗体浓度为1mg/mL，上样量325μL。实验结果如图5所示。具体DSC参数设置如下：
表4：DSC参数设置
Figure PCTCN2022114793-appb-000007
从图5结果可以看出，本申请获得的抗体稳定性很好。
实施例4：Nectin-4人源化抗体的亲和力以及种属交叉检测
本实验采用Fortebio检测Nectin-4人源化抗体与人Nectin-4蛋白的亲和力。Nectin-4蛋白与抗Nectin-4抗体样品孵育后，通过Fortebio检测信号值，分析样品对人Nectin-4的亲和力。实验结果如表5所示，显示3个人源化抗体对人Nectin-4蛋白的亲和力在完成人源化后基本没有降低。所述嵌合抗体是在鼠源抗体的基础上改造而成，恒定区选自human IgG1。
表5：抗Nectin-4抗体对人Nectin-4蛋白的亲和力
Figure PCTCN2022114793-appb-000008
采用ELISA进行Nectin-4人源化抗体与不同种属Nectin-4蛋白的亲和力。不同种属的Nectin-4蛋白(人Nectin-4、食蟹猴Nectin-4、大鼠Nectin-4、小鼠Nectin-4)与不同浓度的Nectin-4人源化抗体样品孵育后，用结合IgG的二抗(Goat anti-Human IgG(H+L)Cross-Adsorbed Secondary Antibody)孵育，通过ELISA检测不同浓度下的信号值分析样品对不同种属Nectin-4蛋白的亲和力。实验结果如表6所示。结果显示Nectin-4人源化抗体对人、大鼠、食蟹猴Nectin-4蛋白都具有良好亲和力。
表6：Nectin-4人源化抗体种属交叉反应性
Figure PCTCN2022114793-appb-000009
实施例5：Nectin-4抗体药物缀合物的制备
1.SWY2001-Ab1-LND1002酶法偶联：
分别将LND1002(按1g∶5mL DMSO溶解，结构如图2所示)、10×反应缓冲液、抗Nectin-4抗体、mTgase(转谷氨酰胺酶，用于催化靠近N-糖基化位点的受体谷氨酰胺处的转谷氨酰胺反应，可特异性催化Kabat编号Q295处的转谷氨酰胺反应)、20％H 2O按照顺序加入EP管中，密封混匀后置于30℃，反应时间不超过72h。当偶联率95％时，结束反应，立即纯化。反应条件如下：
表7：SWY2001-Ab1-LND1002偶联反应体系
Figure PCTCN2022114793-appb-000010
根据实验结果确认：本申请的抗体的转谷氨酰胺酶催化位点在靠近N-糖基化的Q295位置。
使用的转谷氨酰胺酶序列如下(SEQ ID NO：13)：
Figure PCTCN2022114793-appb-000011
2、SWY2001-Ab1-VC-MMAE化学偶联：
将抗体置换到含有5mM EDTA pH＝6.0的PBS缓冲液中，使抗体浓度为10mg/mL，按照抗体(SWY2001-Ab1)的物质的量浓度加入3摩尔当量的TCEP还原剂，1/50体积的1M磷酸氢二钾，37℃水浴加热1h；然后加入6摩尔当量的MC-VC-pABC-MMAE，每1mL体积加入80μLDMSO，室温反应30min；加入12摩尔当量的L-半胱氨酸，20min后停止反应，形成终产物，抗体通过其链间半胱氨酸上的巯基与接头-药物相连，结构如图3所示。
实施例6：SWY2001-Ab1-ADC理化性质分析鉴定
1、酶法偶联SWY2001-Ab1-LND1002修饰率鉴定
实验步骤：
1)样品处理：将SWY2001-Ab1-LND100272h修饰液用50mM醋酸铵，20mM DTT，
55mM Tris-HCl缓冲液处理于30℃±2℃反应30min，12000rpm离心5min。
2)上样：取10μL(30μg)上清液体上色谱柱(waters xbridge C4，3.5μm，4.6mm*250mm)
3)洗脱：流动相A液为0.1％TFA水溶液，流动相B液为0.1％乙腈溶液，分别于第0、5、8、15、20、22、25、30min调整流动相A液：B液的比例为9∶1、7∶3、6.5∶3.5、6∶4、5.5∶4.5、5∶5、1∶9、9∶1洗脱，控制流速为0.8mL/min，柱温为60℃，检测波长为280/254nm。
实验结果如图6所示，显示SWY2001-Ab1-LND1002反应72h修饰率为96.32％。
2、SWY2001-Ab1-ADC DAR值检测
实验步骤：
参照《中国药典》2020年版四部0512高效液相色谱法，本品采用反相色谱法(RP-HPLC)测定药物抗体偶联比DAR值。
实验仪器：高效液相色谱仪Agilent 1260
色谱柱：PLRP-S 1000A，5um，50*2.1mm
流动相：流动相A：0.1％(v/v)TFA水溶液
流动相B：0.1％(v/v)TFA乙腈溶液
检测方法如下：
流速          0.25mL/min
检测波长      280nm
进样量        10μL
柱温箱温度    80℃
梯度洗脱程序如下：
时间(min) 流动相A液(％) 流动相B液(％)
0 73 27
3 73 27
8 65 35
25 57 43
26 5 95
31 5 95
31.5 73 27
40 73 27
采用面积归一化法，对实验结果进行分析：
计算结果如下：
DAR平均＝DAR0％×0+DAR1％×1+DAR2％×2+DAR3％×3
实验结果如图7(图7A和图7B)、表8所示。
表8：SWY2001-Ab1-ADC DAR值
Figure PCTCN2022114793-appb-000012
实施例7：SWY2001-Ab1-LND1002的内吞作用
实验步骤：
收集SK-BR-3细胞、T47D细胞(人乳腺癌细胞)，并使用培养基重悬细胞；轻柔吹打重悬靶细胞数次至单细胞悬液，使用台盼蓝染色法鉴定细胞活力和细胞计数；调整细胞密度为1×10 5cells/mL；每孔100μL接种于共聚焦96孔板细胞培养皿中，每孔的细胞接种数为1×10 4个，将标记Zenon TM pHrodo TMiFL的ADC加入到96孔板中，其终浓度为2μg/mL，然后置于37℃、5％CO 2培养箱条件下连续培养至24小时。所有图像均经激光共聚焦显微镜20X物镜观察并摄取图像。
实验结果如图8、图9所示，SWY2001-Ab1-LND1002在细胞中发生了内吞，并且定位于酸性环境的溶酶体，且其内吞速率远高于药物PADCEV。针对抗体药物缀合物来说，一般而言，内吞速率快，药物进入肿瘤细胞的能力强，能够更好的释放毒素，药物起效更快。因此，本申请获得的抗体药物缀合物进入肿瘤细胞的能力显著优于PADCEV。
实施例8：SWY2001-Ab1-LND1002对不同细胞体外生长的抑制作用
实验步骤：
收集靶细胞重悬为单细胞悬液，台盼蓝染色法鉴定细胞活力和细胞计数；调整细胞密度为1×10 5cells/mL；每孔100μL加至96孔黑色平底细胞培养板；将稀释好的供试品每孔20μL加至已接种细胞的96孔黑色平底细胞培养板；置于细胞培养箱(37℃，5％CO 2)孵育66±3hr；加入刃天青钠溶液(0.03％)，每孔20μL；37℃作用3-4h，酶标仪550nm/610nm读取荧光值，利用Prism或同类作图软件作图，拟合出参考标准品和样品的半抑制浓度IC50。输出参数C为IC 50，单位为ng/mL。
实验结果如表9所示，显示SWY2001-Ab1-LND1002在体外均能抑制293T-Nectin-4、SK-BR-3、PC3-Nectin-4癌细胞的生长。293T-Nectin-4稳转株体外杀伤数据显示SWY2001-Ab1-LND1002略优于PADECV；SK-BR-3体外杀伤数据显示SWY2001-Ab1-LND1002是PADCEV的4倍；PC3-Nectin-4体外杀伤数据显示SWY2001-Ab1-LND1002与PADCEV没有明显差异。
表9：SWY2001-Ab1-LND1002对几种癌细胞的体外抑制作用
Figure PCTCN2022114793-appb-000013
实施例9：SWY2001-Ab1-LND1002的体内药效
1.体内抑制前列腺癌药效实验
本试验选用适龄雌性NUNU小鼠接种PC3-Nectin-4稳转株细胞(人前列腺癌裸小鼠移植瘤模型，高表达Nectin-4)，待肿瘤体积长至约190mm 3(接种后d19)时，挑选肿瘤生长良好的42只动物，将动物按肿瘤体积均衡分为6组(D0)，溶剂对照组，Nectin4-mab 1mg/kg组，PADCEV 1mg/kg组，SWY2001-Ab1-LND10020.5，1和2mg/kg组，每组7只。给药后称量小鼠体重，记录数据，通过测量给药后不同时间的肿瘤瘤径，动态观察肿瘤的生长情况。d17(给药后第17天)试验结束，用二氧化碳窒息小鼠后，剥瘤称量瘤重。
本实验条件下(参见图10)，SWY2001-Ab1-LND1002可剂量依赖性的抑制肿瘤生长(P＜0.05)，1mg/kg剂量条件下，SWY2001-Ab1-LND1002抑瘤率显著优于PADCEV(58.3％vs 39.0％)；Nectin4-mab 1mg/kg，PADCEV 1mg/kg，SWY2001-Ab1-LND10020.5，1和2mg/kg组的瘤重抑制率分别为28.2％，39.0％，22.1％，58.3％，79.4％。
2.体内抑制乳腺癌药效实验
本实验选用裸小鼠接种MDA-MB-468细胞，构建人乳腺癌MDA-MB-468裸小鼠移植瘤模型，待肿瘤体积长至约100mm 3(接种后d25)时，挑选肿瘤生长良好的16只动物，将动物按肿瘤体积均衡分为3组(D0)，溶剂对照组8只，其余实验组4只，分别静脉给予0.9％氯化钠注射液(0.9％INJ NS(生理盐水)，溶剂对照组)，药物PADCEV、SWY2001-Ab1-LND1002 3mg/kg(单次给药)。给药后称量小鼠体重，记录数据，通过测量给药后不同时间的肿瘤瘤径，动态观察肿瘤的生长情况。D29试验结束，用二氧化碳窒息小鼠后，剥瘤称量瘤重。
实验结果如图11所示，本试验条件下，PADCEV 3mg/kg组、SWY2001-Ab1-LND1002 3mg/kg组抑制率分别为72.3％，66.3％。与溶剂对照组相比，均能显著抑制肿瘤的生长(P＜0.001)。
3.体内抑制膀胱癌药效实验
本实验选用适龄雌性NUNU小鼠接种HT-1376细胞，每只接种5×10 6个细胞，待肿瘤体积长至约100mm 3(接种后d21)时，挑选肿瘤生长良好的12只动物，将动物按肿瘤体积均衡分为3组(D0)。A：溶剂对照组4只，给予0.9％氯化钠注射液(0.9％INJ NS(生理盐水)，溶剂对照组)；B：PADCEV实验组4只，PADCEV 3mg/kg(单次给药)；C：SWY2001-Ab1-LND1002实验组4只，给与3mg/kg(单次给药)； 给药后称量小鼠体重，记录数据，通过测量给药后不同时间的肿瘤瘤径，动态观察肿瘤的生长情况。D22试验结束，用二氧化碳窒息小鼠后，剥瘤称量瘤重。
实验结果如图12所示，本实验条件下，PADCEV 3mg/kg、SWY2001-Ab1-LND1002 3mg/kg抑瘤率分别为42.1％、49.4％。与溶剂对照组相比，Nectin-4-ADC 3mg/kg(单次给药)组均能显著抑制肿瘤的生长。与阳性对照PADCEV 3mg/kg(单次给药)组相比，SWY2001-Ab1-LND1002 3mg/kg组对肿瘤的抑制作用显著提高。
实施例10：SWY2001-Ab1-ADC安全性评价研究
10.1相对于化学法偶联SWY2001-Ab1-VC-MMAE的效果对比试验
本试验选用适龄雄性食蟹猴6只，通过静脉注射对照品和供试品SWY2001-Ab1-ADC，观察毒性反应。供试品SWY2001-Ab1-LND1002(DAR2)，对照品SWY2001-Ab1-VC-MMAE(DAR4)，剂量设计如下表所示，每周静脉注射1次，连续注射2周，第二次给药后连续观察7天，每天2次一般观察，每天1次详细观察。在给药前1D、给药后7D、14D观察体重变化，并进行血液学和血生化指标检测。
表10：毒性实验设计方案
Figure PCTCN2022114793-appb-000014
实验结果如下表所示：
表11：毒性实验结果
Figure PCTCN2022114793-appb-000015
Figure PCTCN2022114793-appb-000016
上述实验结果表明：总体而言，根据临床观察同剂量下对照品皮肤毒性、眼部毒性比供试品严重；具体来看，供试品SWY2001-Ab1-LND1002在高剂量组(9mg/kg)的临床观察，仅见轻微的眼毒性(双眼眼周发红)，相比之下，对照组在6mg/kg低剂量组不仅显示出眼毒性的症状，还表现出：左后爪破溃，左后肢膝关节以下至踝关节轻度肿胀，双眼眼周变色，双前肢脱屑等严重毒性反应，甚至在第3次给药后出现动物死亡的严重不良反应，相比之下，本申请制备的供试品即使在第3次给药调整为12mg/kg(远高于6mg/kg的对照品施用剂量)，两只动物均未见异常，安全性显著优于对照品。
10.2相对于PADCEV(市售)的食蟹猴安全性实验
试验使用食蟹猴10只，雌雄各半，随机分为5组，每组2只。第1组给予6mg/kg市售对照品
Figure PCTCN2022114793-appb-000017
第2～5组给予供试品SWY2001-Ab1-LND1002，静脉输注给药，给药容量为10mL/kg，输注速度为0.5mL/kg/min。各组动物给药剂量、浓 度、频次和周期见下表：
表12：毒性实验设计方案
Figure PCTCN2022114793-appb-000018
a：自D15开始，给药剂量调整为18mg/kg
相同剂量(6mg/kg)下，供试品与市售对照品相比，二者毒性表征基本一致，包括皮肤异常，白细胞及分类计数、红系相关指标(RBC、HGB、HCT)，AST、ALT、PLT和FIB异常，角膜组织病理学异常。市售对照品异常症状出现比供试品更早(D7vs D14)，皮肤毒性表征更严重(皮屑vs破溃)。
市售对照品FDA资料显示：食蟹猴重复给药4周长毒试验中，每周给予1、3和6mg/kg的市售对照品，3只动物在试验早期出现死亡(D11)，主要毒性包括皮肤损伤、骨髓毒性和轻度肝脏毒性，由于严重毒性表征，6mg/kg所有动物在第2次给药后(D8)均停止给药。
由此可见，相同剂量的供试品与市售对照品
Figure PCTCN2022114793-appb-000019
相比，毒性风险更小。
综合上述实验结果可知：本申请获得的抗体药物缀合物在胞吞、体外抑瘤细胞实验、体内抑瘤药效实验(前列腺癌、乳腺癌以及膀胱癌)中，总体效果显著优于PADCEV；特别是在安全性评价初步试验表明：本申请获得的抗体药物缀合物治疗窗口更宽，副作用更小，未见明显的皮肤毒性、眼部毒性等不良反应。
实施例11：SWY2001-Ab1-LND1002稳定性实验
血浆稳定性实验
试验采用37℃体外血浆孵育的方法对SWY2001-Ab1-LND1002和MMAE在SD大鼠、食蟹猴和人血浆中的稳定性进行测定。另外，选用PBST作为阴性对照组，用于考察整个试验系统的可靠性。其中，各孵育组分别于0h、24h、48h(D2)、72h(D3)、96h(D4)、168h(D7)和336h(D14)采集孵育样本。采用LC-MS/MS方法测定各种属血浆和PBST中MMAE的浓度。SWY2001-Ab1-LND1002在各种属血浆及PBST中 孵育后MMAE平均生成百分率结果见表12：
表13各种属血浆中MMAE平均生成百分率(％)
Figure PCTCN2022114793-appb-000020
实验结果表明：测试浓度范围内，三个浓度的抗体药物缀合物在人、猴、大鼠血浆孵育336h后，小分子MMAE的生成量约占其理论量的1％左右，证实其具有良好的稳定性。
以上描述地仅是优选实施方案，其只作为示例而不限制实施本发明所必需特征的组合。所提供的标题并不意指限制本发明的多种实施方案。术语例如“包含”、“含”和“包括”不意在限制。此外，除非另有说明，没有数词修饰时包括复数形式，以及“或”、“或者”意指“和/或”。除非本文另有定义，本文使用的所有技术和科学术语的意思与本领域技术人员通常理解的相同。
本申请中提及的所有公开物和专利通过引用方式并入本文。不脱离本发明的范围和精神，本发明的所描述的方法和组合物的多种修饰和变体对于本领域技术人员是显而易见的。虽然通过具体的优选实施方式描述了本发明，但是应该理解所要求保护的本发明不应该被不适当地局限于这些具体实施方式。事实上，那些对于相关领域技术人员而言显而易见的用于实施本发明的所描述的模式的多种变体意在包括在随附的权利要求的范围内。

Claims (24)

  1. 一种能够特异性结合Nectin-4的抗体药物缀合物(ADC)，其中所述抗体或其功能性片段包含重链可变区和轻链可变区，所述重链可变区序列为如SEQ ID NO.9所示的氨基酸序列；所述轻链可变区序列选自如SEQ ID NO.10-12所示的氨基酸序列。
  2. 根据权利要求1所述的抗体药物缀合物，其抗体或其功能性片段的重链可变区序列为如SEQ ID NO.9所示的氨基酸序列或者与其具有至少80％、85％、90％、95％、96％、97％、98％或者99％同一性的序列；轻链可变区序列选自如SEQ ID NO.10-12所示的氨基酸序列或者与其至少80％、85％、90％、95％、96％、97％、98％或者99％同一性的序列。
  3. 根据权利要求1-或2所述的抗体药物缀合物，所述抗体为单克隆抗体。
  4. 根据权利要求1-3任一项所述的抗体药物缀合物，所述抗体为人源化抗体。
  5. 分离的多核苷酸，其编码根据权利要求1-4中任一项所述抗体或其功能性片段之轻链的和/或根据权利要求1-4中任一项所述抗体或其功能性片段之重链；或者其编码根据权利要求1-4中任一项所述抗体或其功能性片段。
  6. 表达载体，其包含根据权利要求5的多核苷酸，所述多核苷酸与允许其所编码的多肽在宿主细胞或无细胞表达系统中表达的调节序列有效连接。
  7. 宿主细胞，其包含权利要求6所述的表达载体。
  8. 缀合物，其包含与一个或更多个药物偶联的抗体或其功能性片段，所述药物选自细胞毒性药物、免疫增强剂和放射性同位素，优选地，所述药物选自奥瑞斯他汀衍生物、美登素生物碱衍生物、喜树碱类似物、DNA拓扑异构酶I抑制剂及其衍生物，最优选地所述药物选自MMAE、MMAF、DM1、DM4、DXD和SN38及其衍生物，并且，所述抗体为权利要求1-4任一项所述的抗体或其功能性片段。
  9. 根据权利要求8的缀合物，其中所述药物与所述抗体或其功能性片段通过接头偶联，所述接头与抗体或其功能性片段通过巯基或氨基连接，所述接头选自mc-Val-Cit-pABC、mc-Val-Cit、NH 2-(PEG) m-Val-Cit和NH 2-(PEG) m-Val-Cit-pABC，m为1至8的整数。
  10. 根据权利要求8的缀合物，其具有Ab-(L-U) n的结构，其中Ab表示抗体或其功能性片段，L表示接头，U表示药物，n为1至8的整数或小数。
  11. 权利要求8-10任一项所述的缀合物，所述抗体或其功能性片段为单克隆抗体或双特异性抗体，优选为人源化抗体，最优选全人源抗体。
  12. 权利要求8-11任一项所述的缀合物，所述抗体或其功能性片段是IgG类型，优选IgG1抗体。
  13. 药物组合物，其包含权利要求8至12中任一项的缀合物，以及可药用载体。
  14. 权利要求8至12中任一项的缀合物或权利要求13的药物组合物在制备用于治疗或预防肿瘤的药物中的用途。
  15. 根据权利要求14的用途，其中所述肿瘤为Nectin-4阳性肿瘤，优选Nectin-4阳性实体瘤，优选选自前列腺癌、胃癌、食管癌、胰腺癌、乳腺癌、膀胱癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌和胆囊癌，特别优选的所述肿瘤是前列腺癌、乳腺癌、膀胱癌、肺癌、卵巢癌；最优选前列腺癌、乳腺癌或膀胱癌。
  16. 医用制品，其包含权利要求8至14中任一项所述的抗体药物缀合物。
  17. 权利要求16所述的医用制品，其以药盒的形式存在，所述药盒包含含有权利要求9-13任一项所述抗体-药物缀合物的容器。
  18. 含有权利要求8-12任一项所述的缀合物与抗增生剂在制备抗肿瘤药物中的用途。
  19. 一种药物组合物，包括权利要求8-12任一项所述的缀合物和抗增生剂。
  20. 一种在对象中治疗肿瘤的方法，所述方法包括向所述对象给予有效量的如权利要求8-12中任一项所述的抗体药物缀合物或权利要求13所述的药物组合物或权利要求16所述的医用制品。
  21. 一种在对象中治疗肿瘤的方法，所述方法包括向所述对象给予有效量的如权利要求8-12中任一项所述的抗体药物缀合物或权利要求13所述的药物组合物或权利要求16所述的医用制品和辐射。
  22. 一种在对象中治疗肿瘤的方法，所述方法包括向所述对象给予有效量的如权利要求8-12中任一项所述的抗体药物缀合物或权利要求13所述的药物组合物或权利要求16所述的医用制品和抗增生剂。
  23. 一种制备缀合物的方法，包括：在转谷氨酰胺酶存在的情况下，使权利要求1-4任一项所述抗体或其功能性片段与药物通过接头连接，其中所述接头与抗体上的内源性受体谷氨酰胺残基Q295缀合。
  24. 权利要求23所述方法，所述药物选自：奥瑞斯他汀衍生物、美登素生物碱衍生物、喜树碱类似物、DNA拓扑异构酶I抑制剂及其衍生物，优选地所述药物选自MMAE、MMAF、DM1、DM4、DXD和SN38及其衍生物；所述接头选自mc-Val-Cit-pABC、mc-Val-Cit、NH 2-(PEG) m-Val-Cit和NH 2-(PEG) m-Val-Cit-pABC，m为1至8的整数。
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