Antibody-drug conjugate binding to nectin-2 and use thereof

Abstract

The present invention relates to an antibody-drug conjugate binding to Nectin-2 and use thereof, in which a mouse monoclonal antibody (m12G1 clone) and a chimeric anti-Nectin-2 antibody (chimeric 12G1; c12G1), which are capable of specifically targeting Nectin-2, have been prepared. The c12G1 antibody specifically bound to the C2 domain of human Nectin-2 with high affinity, but did not bind to mouse Nectin-2. Subsequently, an antibody-drug conjugate comprising a c12G1 antibody conjugated to DM1 was prepared, and as a result of examining the cytotoxic effect thereof on cancer cells in vitro and in vivo, c12G1-DM1 induced cell cycle arrest in the mitotic stage of Nectin-2-positive ovarian cancer cells, but not in Nectin-2-negative cancer cells. c12G1-DM1 induced approximately 100-fold cytotoxicity at IC50 in the range of 0.1-7.4 nM in ovarian cancer cells as compared to normal IgG-DM1, and c12G1-DM1 exhibited approximately 91% tumor growth inhibition in a mouse xenograft model transplanted with OV-90 cells. These results suggest that c12G1-DM1 can be effectively used as a potential therapeutic agent for Nectin-2-positive ovarian cancer.

Description

Antibody drug conjugate binding to nectin-2 and uses thereof

[0001] The present invention relates to antibody drug conjugates (ADCs) that bind to Nectin-2 and uses thereof.

[0002] Ovarian cancer is the second most common malignancy of the female reproductive system, causing an estimated 210,000 deaths worldwide in 2020. Because more than 70% of cases are diagnosed at an advanced stage, the five-year survival rate for ovarian cancer patients is only 47%. Ovarian cancer is classified into epithelial, sex cord stromal, germ cell, and mixed types based on cell origin. Approximately 90% of patients are diagnosed with epithelial ovarian cancer (EOC), and its histological subtypes include serous, endometrioid, clear cell, mucinous, malignant Brenner tumor, and mixed histology, with more than 60% of cases classified as serous. High-grade serous carcinomas arise from well-differentiated low-grade serous carcinomas. Recent advances in molecular characterization have allowed EOC to be further classified into two distinct groups: type I and type II. Type I EOC primarily arises from endometriosis or serous borderline fallopian tube involvement and is characterized by genomic alterations involving KRAS, BRAF, PTEN, PI3K3CA, CTNNB1, and ARID1A. In contrast, type II EOC originates from precursor lesions in the fallopian tube epithelium, most often harboring TP53 mutations.

[0003] Treatment for ovarian cancer typically involves surgery and chemotherapy. The integration of targeted therapies, including PARP inhibitors and bevacizumab, has significantly impacted disease control in EOC. Despite these advances, most women diagnosed with advanced EOC ultimately develop platinum-resistant relapse. Recently, targeted therapies targeting vascular endothelial growth factor and folate receptor alpha have shown remarkable progress in improving progression-free survival. Nevertheless, there remains an unmet medical need to develop other effective therapies for the treatment of patients with ovarian cancer.

[0004] Nectin-2 is Ca 2+ Nectin-2 is a cell adhesion molecule that is independent of CD34 and has a structure similar to immunoglobulin. It functions at adherens junctions and recent studies have shown that it is overexpressed in various cancer cells, making it a potential target for cancer treatment. In particular, nectin-2 is a CD34-dependent cell adhesion molecule. + It is known to be highly expressed in hematopoietic and endothelial cells, tumor cells, and immune cells. For example, comparing the expression levels of nectin-2 in various normal and cancerous tissues reveals that high levels are observed in cancerous tissues, particularly breast, ovarian, pancreatic, and prostate cancers. Furthermore, nectin-2 has been reported to be overexpressed in mature dendritic cells, a causative factor in autoimmune diseases, suggesting its potential as a therapeutic agent for autoimmune diseases.

[0005] Nectin-2 mRNA and protein expression is upregulated in various cancer tissues, including breast, ovarian, and prostate cancer, compared to adjacent non-tumor tissues. It is overexpressed in ovarian tumor biopsies, regardless of histological subtype, grade, or distant metastasis. Approximately 48% of ovarian cancer patients were found to be nectin-2 positive. Surprisingly, nectin-2 gene expression was found to be higher in tumors with lymph node metastases compared to lymph node-negative tumors. Recent studies have reported that nectin-2 antibodies that reduce antibody-dependent cellular phagocytosis could be developed as therapeutics for ovarian cancer. Therefore, antibody-drug conjugates that bind to nectin-2 have the potential to be developed as therapeutics for ovarian cancer.

[0006] An object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to Nectin-2; and an antibody drug conjugate (ADC) to which a drug is bound.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer in which Nectin-2 is overexpressed, comprising the antibody drug conjugate as an active ingredient.

[0008] In order to achieve the above object, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6; and an antibody-drug conjugate to which a drug is bound.

[0009] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer in which Nectin-2 is overexpressed, comprising the antibody drug conjugate as an active ingredient.

[0010] The present invention relates to an antibody drug conjugate binding to Nectin-2 and its use. A mouse monoclonal antibody (m12G1 clone) and a chimeric anti-Nectin-2 antibody (chimeric 12G1: c12G1) capable of specifically targeting Nectin-2 were produced. Epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization analysis were performed to characterize the chimeric 12G1 antibody. The c12G1 antibody has a high affinity (K D = 2.90 × 10 -10 M) specifically bound to the C2 domain of human nectin-2, but not to mouse nectin-2. Subsequently, an antibody-drug conjugate containing c12G1 antibody conjugated to DM1 was prepared, and its cytotoxic effect on cancer cells was examined in vitro and in vivo. As a result, c12G1-DM1 induced cell cycle arrest at the mitotic phase of nectin-2-positive ovarian cancer cells, but not in nectin-2-negative cancer cells. c12G1-DM1 had an IC ranging from 0.1 nM to 7.4 nM compared to normal IgG-DM1 in ovarian cancer cells. 50 c12G1-DM1 induced ~100-fold cytotoxicity, and showed ~91% tumor growth inhibition in a mouse xenograft model implanted with OV-90 cells. These results suggest that c12G1-DM1 can be useful as a potential therapeutic agent for nectin-2-positive ovarian cancer.

[0011] Figure 1 shows the results of the confirmation of mouse monoclonal Nectin-2 antibody.

[0012] Figure 2 shows the results of multiple production and purification of mouse monoclonal Nectin-2 antibodies.

[0013] Figure 3 shows the results of binding affinity measurement.

[0014] Figure 4 is V H and V L Shows the amplification results of the area.

[0015] Figure 5 shows V in 12G1 antibody H and V L It shows the results of amino acid and nucleic acid sequence analysis.

[0016] Figure 6 shows the optimization results for FACS analysis.

[0017] Figure 7 shows the results of immunoprecipitation analysis.

[0018] Figure 8 shows the expression level of Nectin-2 in HEK-293 cells.

[0019] Figure 9 shows the results that the 12G1 antibody specifically recognizes Nectin-2 on the cell surface.

[0020] Figure 10 shows the results of FACS analysis of Nectin-2 expression in various cancer cell lines.

[0021] Figure 11 shows the results of qRT-PCR analysis of Nectin-2 expression in various cancer cell lines.

[0022] Figure 12 shows the results of characterization of chimeric 12G1 (c12G1) antibody.

[0023] Figure 13 shows the results of confirming the Nectin-2 binding domain of c12G1.

[0024] Figure 14 shows the results showing that c12G1 antibody is internalized into ovarian cancer cells.

[0025] Figure 15 shows the results of the characteristic analysis of c12G1-DM1.

[0026] Figure 16 shows the results showing that c12G1-DM1 exhibits anti-tumor activity both in vitro and in vivo.

[0027] Figure 17 shows the results of the characteristic analysis of c12G1-MMAE.

[0028] Figure 18 shows the anti-tumor activity results of c12G1-MMAE.

[0029] The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6; and antibody drug conjugates (ADCs) to which a drug is bound.

[0030] Preferably, the heavy chain variable region may include an amino acid sequence represented by SEQ ID NO: 7, and the light chain variable region may include an amino acid sequence represented by SEQ ID NO: 8, but is not limited thereto.

[0031] Preferably, the antibody may be a mouse antibody or a chimeric antibody, but is not limited thereto.

[0032] Preferably, the antibody may comprise a kappa chain or a constant region derived from human IgG1, but is not limited thereto.

[0033] Preferably, the drug may be an anticancer agent, and more preferably, the anticancer agent is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, Cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine,Clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin It may be at least one selected from the group consisting of E (monomethyl auristatin E, MMAE) and monomethyl auristatin F (monomethyl auristatin F), but is not limited thereto.

[0034] Preferably, the antibody or antigen-binding fragment thereof can be linked to a drug via a linker, more preferably, the linker can be, but is not limited to, 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB) or N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB).

[0035] In the present invention, the term "antibody drug conjugate (ADC)" requires that the anticancer drug be stably bound to the antibody before it is delivered to the target cancer cell. Once delivered to the target, the drug must be released from the antibody and induce target cell death. To achieve this, the drug must be stably bound to the antibody and, upon release from the target cell, possess sufficient cytotoxicity to induce target cell death.

[0036] Meanwhile, the antibody may be bound to a drug via a linker. The linker serves as a linking site between the antibody of the present invention and the drug, enabling the drug to be released from the antibody in the intracellular environment. Reflecting the long half-life of the antibody, the antibody should be stable in systemic circulation, and the binding of the linker to the drug should not affect the stability and pharmacokinetics of the antibody. The linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, like a peptide linker, it can be cleaved by an intracellular peptidase or protease enzyme, such as a lysosomal or endosomal protease. In the case of a non-cleavable linker, for example, a thioether linker, the drug can be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0037] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically reacts with a specific antigen, and examples thereof may include monoclonal antibodies, polyclonal antibodies, full-length antibodies, and antibody fragments. In addition, the term "antibody" may include bivalent or dual-specific molecules (e.g., bispecific antibodies), diabodies, triabodies, or tetrabodies.

[0038] As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibodies exhibit single binding affinity and binding affinity for a specific epitope, unlike polyclonal antibodies that can bind to multiple epitopes. As used herein, the term "full-length antibody" refers to a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types. IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.

[0039] In the present invention, the term "chimeric antibody" is an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and is an antibody with a greatly improved immune response compared to a mouse antibody.

[0040] In the present invention, the term "heavy chain" may include both a full-length heavy chain and fragments thereof, which include a variable region VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant regions CH1, CH2, and CH3. In addition, the term "light chain" in the present invention may include both a full-length light chain and fragments thereof, which include a variable region VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region CL.

[0041] In the present invention, the terms "fragment," "antibody fragment," and "antigen-binding fragment" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.

[0042] The antibodies or antigen-binding fragments thereof of the present invention may include not only the sequences of the antibodies described herein, but also biological equivalents thereof, to the extent that they can exhibit the ability to specifically bind to Nectin-2. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on this, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0043] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer in which Nectin-2 is overexpressed, comprising the antibody drug conjugate as an active ingredient.

[0044] Preferably, the cancer may be, but is not limited to, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, esophageal cancer, gallbladder cancer, or acute myeloid leukemia.

[0045]

[0046] The pharmaceutical composition of the present invention can be manufactured using pharmaceutically suitable and physiologically acceptable auxiliary agents in addition to the active ingredient, and the auxiliary agents can be solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and can be used as a mixture of saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

[0047] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.

[0048] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0049]

[0050] <Experimental Example>

[0051] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0052]

[0053] 1. Cell culture and cell lines

[0054] In the present invention, human embryonic kidney cells, HEK-293, breast cancer cell lines, MCF-7, pancreatic cancer cell lines, PANC-1, AsPC-1, and BxPC-3, and ovarian cancer cell lines, Caov-3, ES-2, OVCAR-3, OV-90, SK-OV-3, TOV-112D, and UWB1.289 were used. HEK-293, MCF-7, PANC-1, and Caov-3 were cultured in Dulbecco's high glucose modified Eagle's medium (DMEM, Hyclone, Logan, USA). AsPC-1, BxPC-3, SK-OV-3, OVCAR-3, and UWB1.289 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone, Logan, USA), and ES-2 was cultured in McCoy's 5A medium (Hyclone, Logan, USA). OV-90 and TOV-112D were cultured in a 1:1 mixture of MCDB-105 medium (Sigma-Aldrich, St. Louis, USA) and Medium 199 (M199, Hyclone, Logan, USA). All cell culture media were supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, USA) and 1% penicillin / streptomycin (Hyclone, Logan, USA), except that 15% FBS and 1% penicillin / streptomycin were added to a mixture of MCDB-105 medium and Media 199 medium. Detailed information on cell lines is described in Table 1. Cells were cultured at 37°C in a humidified 5% CO2 incubator.

[0055] CellDescriptionTissue typeCulture mediaHEK-293Embryonic adrenalprecursor cellEmbryonic kidneyDMEM, 10% FBSMCF-7AdenocarcinomaBreast cancerDMEM, 10% FBSAsPC-1AdenocarcinomaPancreatic cancerRPMI 1640, 10% FBSBxPC-3AdenocarcinomaRPMI 1640, 10% FBSPANC-1Endothelioid carcinomaDMEM, 10% FBSCaov-3AdenocarcinomaOvarian cancerDMEM, 10% FBSES-2Clear cell carcinomaMcCoy's 5A, 10% FBSSK-OV-3AdenocarcinomaDMEM, 10% FBSOVCAR-3AdenocarcinomaRPMI 1640, 10% FBSOV-90AdenocarcinomaM199, MCDB105, 15% FBSSW626AdenocarcinomaLeibovitz' L-15, 10% FBSTOV-112DEndometrioid carcinomaM199, MCDB105, 15% FBSUWB1.289Ovarian carcinomaRPMI 1640, 10% FBS

[0056]

[0057] 2. 항체 제작

[0058] To generate monoclonal antibodies against nectin-2, recombinant nectin-2 protein (50 μg, Sino biological Inc., Beijing, China) together with complete Freund's adjuvant (Sigma-Aldrich, St. Louis, USA) was injected subcutaneously into 6-week-old female Balb / c mice. Subsequent immunizations were repeated once at one-week intervals with an equal volume of immunogen emulsified in incomplete Freund's adjuvant (Sigma-Aldrich, St. Louis, USA). After the second injection, high-titer antibodies in the serum were measured using an indirect ELISA. For hybridoma construction, spleen cells were harvested 3 days after the final booster injection and fused with SP2 / 0 cells using 50% polyethylene glycol (PEG, Sigma-Aldrich, St. Louis, USA). Fused hybridomas were screened in hypoxanthine, aminopterin, and thymidine (HAT) medium containing HAT supplement (GIBCO, Grand Island, USA) in DMEM medium. Selected hybridomas were measured by indirect ELISA using cell culture supernatants. To amplify antibodies, hybridomas were diluted in calcium- and magnesium-free Dulbecco's phosphate-buffered saline (DPBS, Hyclone, Logan, USA) one week after inoculation of pristane (Sigma-Aldrich, St. Louis, USA) into Balb / c mice and injected intraperitoneally into 10-week-old Balb / c mice (5 × 10 6Two weeks later, ascites were harvested from immunized mice and centrifuged at 842 × g for 15 min to obtain the supernatant. Immunization-induced ascites was confirmed by indirect ELISA, Western blot, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Monoclonal antibodies were purified from ascites using protein A / G plus beads (Santa Cruz Biotechnology, Dallas, USA). The antibody isotype was confirmed using Beadlyte-Mouse Immunoglobulin Isotyping Kit (Upstate Co., Temecula, USA).

[0059] The amino acid sequence of the variable domain of the 12G1 clone was determined by nucleic acid sequencing, and the variable domain of the mouse 12G1 antibody was grafted onto human IgG1 to generate a chimeric 12G1 (c12G1) antibody. c12G1 was subcloned into the pCHO 1.0 vector (Thermo Fisher Scientific, Waltham, MA, USA), and the recombinant plasmid was transiently transfected into CHO-S cells (Thermo Fisher Scientific) using OptiPRO™ SFM and FreeStyle™ MAX Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The antibody was purified using Protein A Sepharose and SP Sepharose columns (Invitrogen, Waltham, MA, USA).

[0060]

[0061] 3. Indirect ELISA

[0062] A 96-well plate (SPL Life Sciences, Pocheon, Korea) was coated with recombinant nectin-2 protein (100 ng / well) diluted in phosphate-buffered saline (PBS) and incubated overnight at 4°C. The plate was then washed three times with PBS, blocked with PBS containing 1% bovine serum albumin (BSA; Bovogen Biologicals, East Keilor, Australia) for 1 h, and washed three times. Serum, hybridoma culture supernatant, ascites, and purified antibodies were used as primary antibodies, added to each well with various diluents, and incubated for 1 h at room temperature (RT) to screen for specificity for nectin-2 protein. The plate was then washed three times with PBST containing 0.05% Tween-20 (Sigma-Aldrich, St. Louis, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:10,000, Thermo Fisher Scientific, Waltham, USA) was reacted in each well containing blocking buffer (100 μl / well) for 1 h at RT. The plates were washed three times with PBST, and 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich, St. Louis, USA) peroxidase substrate (100 μl / well) was added and incubated at RT for 15 min without light exposure. The reaction was stopped by adding 50 μl of H2SO4 for 5 min at RT. Optical density (OD) was measured at a wavelength of 450 nm using a Perkinelmer Victor X3 (Perkin Elmer, Waltham, USA).

[0063]

[0064] 4. RNA extraction and cDNA synthesis

[0065] TRIzol reagent (Ambion ® Total RNA was isolated from HEK-293 or hybridoma cells using a 100 μM oligosaccharide (100 μM) ... Subsequently, 10 mM dNTP, 200 U / μl M-MLV reverse transcriptase (Beamsbio, Seongnam, Korea), and 5X reaction buffer (50 mM Tris-HCl [pH 7.6], 1 mM dithiothreitol (DTT), 0.1% Nonidet P-40, 0.1 mM ethylenediaminetetraacetica acid (EDTA), 150 mM NaCl, and 50% glycerol) were added to the tube. Polymerase chain reaction (PCR) conditions for cDNA synthesis consisted of pre-reaction at 37°C for 5 min, synthesis at 37°C for 60 min, reverse transcriptase inactivation at 72°C for 5 min, and storage at 4°C until use. PCR analysis was performed with MultiGene OptiMax (Labnet, NJ, USA). cDNA was diluted 1:2 with RNase-free water and then used for reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR).

[0066]

[0067] 5. Cloning of variable regions in monoclonal antibodies

[0068] The synthesized cDNA was amplified with 5 U / μl GS-TaqDNA polymerase (Beamsbio, Seongnam, Korea), 10 mM dNTP, 10 μM specific primer (Table 2), and 10X GS-Taq reaction buffer (200 mM Tris-HCl [pH 8.8], 100 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 1.0% Triton X-100, and 50% glycerol). Thermal cycling of the heavy chain in the antibody was performed under the following conditions: Pre-denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 1 minute, 5 cycles per temperature from 61°C to 58°C with 1 minute of binding per cycle (exceptionally, MHV7 or 11 primers bind from 57°C to 53°C) and 25 cycles of elongation at 72°C for 1 minute, final elongation at 72°C for 7 minutes and storage at 4°C until use. In addition, the amplification conditions of the light chain in the antibody have a binding temperature of 12 cycles per temperature from 51°C to 52°C (in the case of MKV 10 primers, 5 cycles per temperature from 62°C to 54°C). The products were confirmed by EDTA-stained 1% agarose gel. Positive PCR products were identified by TOPcloner TM Cloning was performed using the TA kit (Enzynomics, Daejeon, Korea) according to the manufacturer's instructions. The plasmids were transformed into DH5α (Enzynomics, Daejeon, Korea), and positive clones were subjected to Fast DNA-spin TM(iNtRON Biotechnology, Seongnam, Korea) and confirmed by EtBr-stained 0.8% agarose gel. The sequence of the purified plasmid was screened using the analysis service of Enzynomics Co., Ltd.

[0069] PrimersSequencesHeavy ChainV H ForwardMHV15'-ATGAAATGCAGCTGGGGCATSTTCTTC-3'(SEQ ID NO: 9)MHV25'-ATGGGATGGAGCTRTATCATSYTCTT-3'(SEQ ID NO: 10)MHV35'-ATGAAGWTGTGGTTAACTGGGTTTTT-3'(SEQ ID NO: 11)MHV45'-ATGRACTTTGGGYTCAGCTTGRTTT-3'(SEQ ID NO: 12)MHV55'-ATGGACTCCAGGCTCAATTTAGTTTTCCTT-3'(SEQ ID NO: 13)MHV65'-ATGGCTGTCYTRGSGCTRCTCTTCTGC-3'(SEQ ID NO: 14)MHV75'-ATGGRATGGAGCKGGRTCTTTMTCTT-3'(SEQ ID NO: 15)MHV85'-ATGAGAGTGCTGATTCTTTTGTG-3'(SEQ ID NO: 16) MHV95'-ATGGMTTGGGTGTGGAMCTTGCTATTCCTG-3' (SEQ ID NO: 17) MHV105'-ATGGGCAGACTTACATTCTCATTCCTG-3' (SEQ ID NO: 18) MHV115'-ATGGATTTTGGGCTGATTTTTTTTATTG-3' (SEQ ID NO: 19) MHV125'-ATGATGGTGTTAAGTCTTCTGTACCTG-3' (SEQ ID NO: 20) V H ReverseCκI3'-GGACAGGGATCCAGAGTTCCA-5'(SEQ ID NO: 21)Light ChainV LForwardMKV15'-ATGAAGTTGCCTGTTAGGCTGTTGTGTCTC-3'(SEQ ID NO: 22)MKV25'-ATGGAGWCAGACACATCCTGYTATGGGTG-3'(SEQ ID NO: 23)MKV35'-ATGAGTGTGCTCACTCACTCCTGGSGTTG-3'(SEQ ID NO: 24)MKV45'-ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG-3'(SEQ ID NO: 25)MKV55'-ATGGATTTWCAGGTGCAGATTWTCAGCTTC-3'(SEQ ID NO: 26)MKV65'-ATGAGGTKCYYTGYTSAGYTYCTGRGG-3'(SEQ ID NO: 27)MKV75'-ATGGGCWTCAAGATGGAGTCACAKWYYCWGG-3'(SEQ ID NO: 28) MKV8 5'-ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG-3' (SEQ ID NO: 29) MKV9 5'-ATGGTRTCCWCASCTCAGTTCCTTG-3' (SEQ ID NO: 30) MKV10 5'-ATGTATATATGTTTGTTGTCTATTTCT-3' (SEQ ID NO: 31) MKV11 5'-ATGGAAGCCCCAGCTCAGCTTCTCTTCC-3' (SEQ ID NO: 32) V L ReverseCκI3'-TGAGGCACCTCCAGATGTTAA-5' (SEQ ID NO: 33)

[0070]

[0071] 6. Surface plasmon resonance analysis

[0072] Surface plasmon resonance (SPR) analysis was performed using an SR7500DC (Reichert Technologies, Depew, USA). Recombinant nectin-2 (1 μg, Sinobio, Beijing, China) dissolved in 20 mM sodium acetate buffer (pH 4.5) was immobilized on gold chips (PEG chips, Reichert Technologies, Depew, USA) according to the manufacturer's instructions. For binding affinity analysis, the flow rate of 12G1 antibody at various concentrations dissolved in 1X PBS (pH 7.4) was 30 μl / min. K D The numbers are the equilibrium dissociation constants, and the association constants (K a ) and dissociation constant (K d ) was calculated using Scrubber 2 software (Reichert Technologies, Depew, USA).

[0073]

[0074] 7. FACS analysis

[0075] To measure the expression of nectin-2 on the cell surface, various cell lines were harvested when the confluence reached 70-80% using enzyme-free cell dissociation buffer (Merck-Millipore, Darmstadt, Germany) for 5-7 min at 37°C. Cells were washed with ice-cold wash buffer containing 1% BSA in modified DPBS. The cells were centrifuged at 200 × g for 3 min at 4°C without calcium and magnesium. 12G1 and normal mouse IgG (Santa Cruz Biotechnology, Dallas, USA) antibodies were reacted in ice-cold wash buffer using a shaker at 4°C for 1 h. Normal mouse IgG served as an isotype control. After the reaction, the cells were washed twice with ice-cold wash buffer. Afterwards, secondary antibody, mouse fluorescein isothiocyanate (FITC)-conjugated Alexa 488 (Thermo Fisher Scientific, Waltham, USA), was reacted and stirred at 4°C in ice-cold washing buffer. Cells were washed twice with ice-cold washing buffer, and single cell populations were identified. Data were acquired with CyFlow cube 6 (Sysmex Partec GmbH, Gφrlitz, Germany) and analyzed using FCS express 4 flow research edition (De Novo Software™, Los Angeles, USA).

[0076]

[0077] 8. Immunoprecipitation analysis and Western blot

[0078] HEK-293 cells were harvested at 70–80% confluency and homogenized in cold radioimmunoprecipitation assay buffer (RIPA) lysis buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM β-glycerophosphate, 1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, St. Louis, USA), and 2 mM 2-mercaptoethanol, protease inhibitor cocktail (PIC, Calbiochem, Darmstadt, Germany), and 100 mM PMSF. Resuspended cells were incubated on ice for 20 minutes and centrifuged at 25,000 ×g for 20 minutes at 4°C.

[0079] For IP analysis, 1 μg of normal mouse IgG or 12G1 antibody as a control was reacted with whole cell lysate (200 μg) in the presence of A / G plus agarose beads (Santa Cruz Biotechnology, Dallas, USA), and the mixture was stirred at 4°C for 2 h. The beads were precipitated by centrifugation at 842 × g for 1 min at 4°C and washed three times with RIPA lysis buffer. The precipitated beads were resuspended in 2X SDS-PAGE sample buffer and boiled for 7 min. The supernatant was applied to a 7% SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, MA, USA).

[0080] For Western blotting, the membrane was blocked with tris-based saline (TBS, 20 mM Tris-HCl [pH 7.6], 150 mM HCl) containing 5% skim milk and 0.2% Tween-20 for 1 h at RT. Subsequently, the membrane was incubated overnight at 4°C with anti-rabbit nectin-2 antibody (1:5000, Abcam, Cambridge, UK) or anti-mouse purified nectin-2 IgG antibody such as 12G1 (diluted from 1:1000 to 1:10,000), and ascites diluted 1:100 in a primary antibody solution dissolved in TBS containing 1% BSA and 0.2% Tween-20. After the reaction, the membrane was washed three times for 5 min each with TBST. The secondary antibodies used in the present invention were HRP-conjugated anti-mouse IgG (1:20,000, Thermo Scientific, Waltham, USA) and HRP-conjugated anti-rabbit IgG (1:3,000, Thermo Scientific, Waltham, USA), and the membrane was reacted with TBST containing 1% skim milk at RT for 1 hour. The membrane was washed three times with TBST, and specific signals were detected using enhanced chemiluminescence (ECL) solution (Santa Cruz Biotechnology, Dallas, USA).

[0081]

[0082] 9. RNA interference

[0083] For the knockdown experiment of Nectin-2, the following Nectin-2 siRNA sequences were used. #1 sense; 5'-UGACCUGGCUCAGAGUCAUAGCCAA-3' (SEQ. 34) and antisense; 5'-UUGGCUAUGACUCUGAGCCAGGUCA-3' (SEQ. 35), #2 sense; 5'-CCUGAUACCUGUGACCCUCUCUGUA-3' (SEQ. 36) and antisense; 5'-UACAGAGAGGGUCACAGGUAUCAGG-3' (SEQ. 37), #3 sense; 5'-CACCUUCGUCUGCACAGUCACCAAU-3' (SEQ. 38) and antisense; 5'-AUUGGUGACUGUGCAGACGAAGGUG-3' (SEQ. 39). Nectin-2 siRNAs were synthesized as sense and antisense oligomers (Bioneer, Daejeon, Korea), and negative siRNA was purchased from Bioneer. HEK-293 cells (2 × 10 5 (cells / well) were seeded in 6-well plates (Thermo Fisher Scientific, Waltham, USA) and cultured for 12–18 h. Subsequently, 40 nM siRNA was transfected into HEK-293 cells for 72 h using K2 reagent (Biontex Laboratories GmbH, München, Germany) according to the manufacturer's instructions. Knockdown efficiency was compared with control siRNA using RT-PCR or qRT-PCR.

[0084]

[0085] 10. RT-PCR and qRT-PCR

[0086] Target genes were amplified by RT-PCR and qRT-PCR. RT-PCR was performed on an AccuPower PCR PreMix (Bioneer, Daejeon, Korea) using cDNA as a template and 10 μM specific primers (Table 3). The amplification conditions were as follows: pre-denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, followed by a final extension cycle at 72°C for 10 min, and storage at 4°C until use. The PCR products were analyzed for bands on a 3% agarose gel stained with ethidium bromide. qRT-PCR was performed using a reaction mixture consisting of cDNA, 10 μM primers, and TOPreal qPCR 2X PreMIX with SYBR Green (Enzynomics, Daejeon, Korea) on a StepOne Real-Time PCR System (Applied Biosystems, Foster City, USA) for gene expression analysis. Thermal cycling conditions consisted of a single denaturation at 94°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, and a final cycle of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. In each experiment, the expression level of nectin-2 was expressed as the C of GAPDH under the given experimental conditions. t Normalized to numerical values ​​and expressed as a numerical value of fold change (RQ, 2 -△△Ct ) was calculated. GAPDH was used as a loading control.

[0087] GenesPrimer SequencesGAPDHF: 5'-GGGTGTGAACCATGAGAAGTATGAC-3'(SEQ ID NO: 40)R: 5'-GTCCTTCCACGATACCAAAGTTGTC-3'(SEQ ID NO: 41)Nectin-2F: 5'-CCAGAAGGTCACGTTCAGCC-3'(SEQ ID NO: 42)R: 5'-CAGTCCAGGGATGAGAGCCA-3'(SEQ ID NO: 43)Nectin-2αF: 5'-CGAAAGCTCAGGTGTTGGGA-3'(SEQ ID NO: 44)R: 5'-AGGGACTACTGGTGTCCAGA-3'(SEQ ID NO: 45)Nectin-2δF: 5'-GTGGCTCCACTATGACCCCT-3'(SEQ ID NO: 46)R: 5'-CTGGGAGAGGAGTCCTTGGG-3' (SEQ ID NO: 47)

[0088]

[0089] 11. ELISA

[0090] Human recombinant nectin-2 protein was diluted to 20 ng / 100 μL / well in 1× PBS (lab-produced) and coated onto a 96-well Immuno Clear Standard Module (Thermo Fisher Scientific) overnight at 4°C. After blocking with 300 μL / well of blocking buffer (PBS containing 5% BSA) for 2 h at RT, the plates were reacted with the following primary antibodies (100 μL / well): mouse anti-12G1 or anti-c12G1 diluted in PBS containing 0.1% Tween-20 and 1% BSA for 1 h and 30 min at RT. The plates were washed four times with PBST and reacted with secondary antibodies (100 μL / well) for 1 h at RT. The secondary antibodies were HRP-conjugated anti-mouse IgG (1:1500 dilution, Thermo Fisher Scientific) and anti-human IgG (1:1500 dilution, Thermo Fisher Scientific) diluted in PBST. The plates were then washed four times with PBST, and the samples were reacted with 100 μL / well of 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) for 3 minutes. The reaction was terminated with 1 N H2SO4 (50 μL / well), and then incubated with SPECTROstar 　 Absorbance measurements were performed at a wavelength of 450 nm using a Nano microplate reader (BMG Labtech, Ortenberg, Germany).

[0091] To determine whether c12G1 could interfere with the binding of nectin-2 to PVRIG, a competitive ELISA was performed. Human recombinant PVRIG protein (100 ng / 100 μL / well; Sino Biological Inc.) was coated onto ELISA plates, and each well was blocked as described above. Human recombinant nectin-2 protein (100 ng) was incubated with or without 12G1 or hIgG isotypes at RT for 1 h. After blocking, the plates were incubated with the pre-reaction mixture (100 μL / well) at RT for 1 h 30 min. The plates were washed four times with PBST and then incubated with 100 μL / well of rabbit polyclonal anti-nectin-2 antibody (1:500, Abcam) at RT for 1 h. HRP-conjugated anti-rabbit IgG (100 μL / well, 1:1500 dilution, Thermo Fisher Scientific) was added to each well for 1 h at RT. The plate was then washed four times with PBST, and the HRP signal was measured.

[0092]

[0093] 12. Effector Function Analysis

[0094] For ADCC assay, OV-90 cells used as target cells (T) were harvested using cell dissociation buffer and stained with 10 μM Hoechst 33,342 (Invitrogen) for 20 min. The stained cells were washed three times with DPBS and seeded at 5 × 10 per well on a 96-well black cell culture microplate (Greiner Bio-One, Kremsmünster, Austria). 3Cells were seeded at a density of 100 μg / ml. The cells were then treated with the indicated concentrations of c12G1 and incubated for 30 minutes. During the incubation, human peripheral blood mononuclear cells (PBMCs; Cellular Technology Ltd., Cleveland, OH, USA) used as effector cells (E) were lysed in DMEM containing 10% FBS with low glucose (HyClone) and washed three times with DPBS. The viability of PBMCs was greater than 90% when measured using a hemacytometer. PBMCs were incubated with target cells (E / T ratio, 30:1) depending on the presence or absence of c12G1. OV-90 cells were incubated at 37°C for 6 hours and counted using a Celigo Imaging Cytometer (Nexelom Bioscience, Lawrence, MA, USA).

[0095] For CDC analysis, OV-90 cells (5 × 10 3 / well) were seeded onto a 96-well black cell culture microplate (Greiner Bio-One) and incubated overnight. c12G1 was then added to the plate, and the cells were incubated for 30 minutes. Next, 20% (v / v) human serum complement (Quidel, San Diego, CA, USA) was added to the plate, and the cells were incubated at 37°C for 6 hours. The cells were stained with 10 μM Hoechst 33342 for 20 minutes and counted using a Celigo Imaging Cytometer.

[0096]

[0097] 13. Internalization Analysis

[0098] Cells were blocked with Human BD Fc Block™ (BD Biosciences, San Diego, CA, USA) in DPBS containing 5% BSA for 10 min at RT. Then, c12G1 (1 μg / mL) diluted in cold serum-free medium containing 2% BSA and 75 μg / mL cycloheximide was added to the cells, and the cells were incubated on ice for 1 h.

[0099] After washing twice, cells were resuspended in cold or prewarmed serum-free medium containing 2% BSA and 75 μg / mL cycloheximide and seeded in 60-mm dishes. Each sample was incubated for 1 or 3 h at 4°C or 37°C. The cells were harvested and stained with fluorescein isothiocyanate-conjugated anti-human IgG (0.3 μg / mL; Thermo Fisher Scientific) diluted in wash buffer (cold DPBS containing 2% BSA) on ice for 1 h. After washing three times, the fluorescence signal was detected using a CyFlow Cube 6 and analyzed using FCS Express™ 6 flow cytometry software. Additional internalization analysis was performed using pH-sensitive Zenon™ dye. OV-90 cells, SK-OV-3 cells, or Caov-3 cells (1 × 10 5Cells) were seeded in 48-well plates and incubated at 37°C for 1 h. Afterwards, the cells were treated with c12G1 antibody / Zenon™ dye mixture (10:100 nM) for 5 min at RT and incubated for an additional 15 h at 37°C. Cells were harvested using 0.25% Trypsin / 1 mM EDTA (Welgene Inc., Gyeongsan, Korea) and centrifuged at 1000× g for 3 min at 4°C. Cells were washed with cold DPBS and resuspended in cold DPBS containing 0.1% BSA. Fluorescent signals were detected using CytoFLEX (Beckman Coulter Inc., Brea, CA, USA) and analyzed using CytExpert software (Beckman Coulter Inc.).

[0100]

[0101] 14. Production of ADCs

[0102] Antibodies were dialyzed against BupH™ PBS Packs (Thermo Fisher Scientific) and conjugated with smcc-DM1 (MedChemExpress, Monmouth Junction, NJ, USA) at a molar ratio of 1:20 for 1.5 h at RT. The mixture was centrifuged at 21,000 × g for 20 min at 20°C to remove aggregates. ADCs were purified using a Sephadex G-25 PD-10 desalting column (GE Healthcare, Chicago, IL, USA) and stored in 10 mM sodium succinate, 6% (v / v) sucrose, and 0.05% (v / v) Tween-20, pH 5.0. ADCs were further analyzed using SDS-PAGE and UV spectroscopy. DAR ratio analysis was performed as previously described.

[0103]

[0104] 15. Cell cycle and in vitro cytotoxicity analysis

[0105] Cells were seeded in 96-well black cell culture microplates (3-8 × 10 for adherent cells 3 cells / well density) or 6-well plates (for suspension cells, 1 × 10 5 Cells were seeded at a density of 10 cells / well and incubated overnight in a humidified 5% CO2-containing chamber. Afterwards, the cells were incubated with 1 μg / mL of antibodies or ADCs for 24 or 48 h. The cells were fixed with 70% ice-cold ethanol for 30 min at 4°C and washed twice with cold DPBS. In addition, the cells were stained with propidium iodide solution (50 μg / mL, containing 0.1 mg / mL RNase and 0.05% Triton™ X-100) for 1 h at 37°C. The cells were then documented using a Celigo Imaging Cytometer and analyzed using FCS Express™ 6 flow cytometry software.

[0106] For cytotoxicity assays, cells were seeded in 96-well black cell culture microplates (2-5 × 10 3 Cells were seeded at 10 μg / well (cells / well). Afterwards, cells were treated with antibodies or ADCs at concentrations ranging from 0 to 400 μg / mL, and the plates were further cultured for 3 to 4 days depending on the cell growth rate. When cell confluency reached 80–90%, cells were stained with 10 μM Hoechst 33342 or 1 μg / mL Calcein AM (Invitrogen) for 20 minutes and counted using a Celigo Imaging Cytometer. IC 50 The figures were calculated using Prism 5 (GraphPad Software Inc., San Diego, CA, USA).

[0107]

[0108] 16. In vivo experiments

[0109] Animal studies were approved by the Institutional Animal Care and Use Committee of Ajou University (IACUC Approval Number: 2021-0019). The experiments complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. All experiments were performed in accordance with relevant guidelines and regulations. To evaluate the toxicity of c12G1-DM1, C57BL / 6 mice (Koatech, Pyeongtaek, Korea) were intravenously administered vehicle or 20 mg / kg of c12G1-DM1, and body weight changes were observed every 3 days for 15 days.

[0110] For xenograft analysis, OV-90 (5 × 10 6 / 100 μL), SK-OV-3 (5 × 10 6 / 100 μL) and Caov-3 (5 × 10 6 / 100 μL) cells in an equal volume of Matrigel 　 (Corning, Corning, NY, USA) and injected subcutaneously into 5-week-old female CB-17 severe combined immunodeficiency mice (Orient Bio Inc., Seongnam, Korea). The average tumor volume was 100-200 mm 3 Upon reaching the target dose, mice were randomized into treatment groups for efficacy studies. Vehicle, c12G1, or ADCs diluted in DPBS were administered intravenously to mice once a week for three times at the indicated concentrations. Tumor size was measured using a Vernier caliper, and tumor volume was calculated as follows:

[0111] Tumor volume = (4 / 3) × π × (length / 2) × (width / 2) × (depth / 2)

[0112] Tumor growth inhibition rate was calculated as follows:

[0113] Tumor growth inhibition rate =

[0114] [1 - (Treatment group RTV) / (Control group RTV) × 100 (%)]

[0115] RTV = (tumor volume on measurement date) / (tumor volume on day 0)

[0116] Here, RTV is the relative tumor volume. At the end of the experiment, mice were euthanized using CO2.

[0117]

[0118] 17. Statistical Analysis

[0119] Statistical analysis was performed using Prism 5. Data are expressed as mean ± standard error of the mean (SEM) or ± standard deviation (SD), and were analyzed using one-way ANOVA and unpaired Student's test to determine statistical significance. A p < 0.05 was considered statistically significant.

[0120]

[0121] <Example 1> Production of monoclonal Nectin-2 antibody

[0122] To produce a mouse monoclonal antibody, recombinant mature human nectin-2 was injected into Balb / c mice. Nectin-2 protein with a His-tag (340 amino acids) was purchased. The protein purity was confirmed to be greater than 95%. The nectin-2 protein was determined to have a molecular weight of 48 kDa by reducing SDS-PAGE. This mass was slightly higher than the predicted molecular weight of 36.2 kDa due to glycosylation.

[0123] Mouse sera were harvested before and after immunization with nectin-2 protein and assessed for the presence of nonspecific (Figure 1A) and specific antibodies (Figure 1B) using indirect ELISA. The OD values ​​of pre-immunized sera were 0.069, 0.064, 0.06, and 0.071 at a 1:100 dilution, indicating that the pre-immunized sera did not exhibit any nonspecific signals (Figure 1A). These results indicate that the antigen injection was adequate. Even before the final injection, mouse sera were harvested and the presence of specific antibodies was confirmed using indirect ELISA. The immunized sera exhibited an OD value greater than 1.0 even at a 1:10,000 dilution (Figure 1B). Because high antibody titers were confirmed to be formed, spleens were harvested after the final administration, and splenocytes were fused with SP2 / 0 mouse myeloma cells to generate hybridomas. The fused cells were selected in HAT medium, and finally, a hybridoma clone, 12G1, which showed high antibody titer when tested by indirect ELISA was selected. The hybridoma clone was subcloned through limiting dilution and then screened to harvest single colonies. The cell culture supernatant obtained from the expanded cells of the clone was verified by indirect ELISA and Western blotting (Figures 1C and 1D). The OD value of each cell culture supernatant was ≥1.0 (Figure 1C). This result indicates the presence of a high titer of antibody. To confirm that the clone, 12G1, binds to the nectin-2 protein, Western blotting was performed. The antibodies were able to detect more than 50 ng of the nectin-2 protein (Figure 1D).

[0124] The 12G1 monoclonal antibody secreting anti-Nectin-2 antibodies was amplified by hybridoma injection into the ascites of 10-week-old Balb / c mice. Ascites were harvested two weeks after hybridoma injection. The ascites produced by the injection tested positive for the antigen by indirect ELISA, Western blot, and SDS-PAGE analyses (Figures 2A, 2B, and 2C). The presence of the antibody was confirmed in the ascites using SDS-PAGE (Figure 2A). The OD value of the 12G1 ascites was 1.18 even at a 1:100,000 dilution (Figure 2B). Western blot analysis also demonstrated that 12G1 was sensitive enough to recognize at least 20 ng of nectin-2 protein (Figure 2C). The antibody isotype was identified as an IgG1 subclass with a kappa chain (Table 4). The IgG antibody was purified using protein A / G plus beads, and the clone concentration was determined. The purified antibody was analyzed by indirect ELISA for various dilution factors, and the changes in OD values ​​were similar to the ELISA results (Fig. 2D). Analysis of SDS-PAGE gels stained with Coomassie blue R-250 revealed that the 12G1 antibody was pure, with heavy (~50 kDa) and light (~25 kDa) chains detected (Fig. 2E).

[0125] To measure the binding affinity between nectin-2 and its antibody 12G1, SPR analysis was performed (Fig. 3). K D The numerical value is the coupling constant (K a ) and dissociation constant (K d ) was calculated. The 12G1 antibody was 499 pM (Fig. 3, lower panel).

[0126] Additionally, to identify the antigen-binding site, total RNA of the hybridomas was extracted and synthesized to make cDNA, which was amplified using PCR with primers specific for the variable region of the antibody. The product size was similar to that of the heavy chain variable region (V) in the 12G1 antibody. H) was approximately 500 bp, and the light chain variable region (V L ) was approximately 450 bp (Fig. 4). Since complementarity-determining regions (CDRs) contribute to diverse antigen specificities, the PCR products were cloned into a TA vector, and the ligated plasmids were analyzed to find the CDRs within the variable region in each antibody through sequence analysis (Fig. 5). V H All PCR products were one from each clone (Fig. 4, left). The 12G1 antibody obtained positive products using the MHV6 primers. In the 12G1 antibody, V L There were three PCR products (MKV2, MKV3, and MKV6 primers). In the case of the PCR product using the MKV2 primer, it was the result of a nonspecific reaction because the MKV2 primer amplified the pseudogene in the hybridoma, not the pseudogene in the antibody (Fig. 4, right). Amplification using the MKV3 and MKV6 primers showed the same sequence analysis results in the 12G1 antibody, respectively (Fig. 5 and Table 5).

[0127] CloneO.D. 450nmIgG1IgG2aIgG2bIgG3IgAIgMKappaLambda12G10.5830.050.0430.0440.0440.0420.1440.048

[0128] OD values ​​were measured at a wavelength of 450 nm.

[0129]

[0130] Sequence Information Amino Acid Sequence Heavy Chain CDR1 SEQ ID NO: 1 CTVSGFSLSRYGVHCDR2 SEQ ID NO: 2 VIWRGGSTDYNAAFMS CDR3 SEQ ID NO: 3 KRDNDGALDY Light Chain CDR1 SEQ ID NO: 4 RASENIIY SYLA CDR2 SEQ ID NO: 5 NAKTLAECDR3 SEQ ID NO: 6 QHHYGPPYT

[0131]

[0132] <Example 2> Characterization of Nectin-2 mouse monoclonal antibody

[0133] The potential applicability of the developed Nectin-2 mouse monoclonal antibody was confirmed using FACS and IP analysis. HEK-293 cells were used for FACS analysis, and antibody concentrations were optimized to determine whether cells reacted or not under fixed conditions. When cells were fixed with 4% paraformaldehyde, they did not react with the 12G1 antibody. Therefore, all cells were tested using live cells.

[0134] To optimize FITC-conjugated secondary antibodies for FACS analysis, we confirmed that 4 μg of FITC exhibited higher non-specific background staining compared to 2 μg of FITC-conjugated secondary antibody (Fig. 6A, middle and right). Furthermore, 1 μg of FITC-conjugated secondary antibody exhibited lower non-specific background staining, but showed a decrease in positive staining. Therefore, 2 μg of FITC-conjugated secondary antibody was used.

[0135] To optimize the primary antibody for FACS analysis, normal mouse IgG antibody was tested at various concentrations (0.05–10 μg) as an isotype control (Fig. 6B). Non-specific staining of normal mouse IgG for the primary antibody increased in a dose-dependent manner. However, when normal mouse IgG was used at 0.05 μg, nectin-2 positive staining also decreased along with the non-specific signal, making this concentration inapplicable to nectin-2 staining. Therefore, 0.1 μg of primary antibody was selected for FACS analysis. FACS analysis of the nectin-2 antibody, 12G1, was performed under optimized experimental conditions, and the results showed positive staining with the 12G1 antibody (Fig. 6C). Therefore, the 12G1 antibody was subjected to further characterization.

[0136] For IP analysis, proteins were extracted from HEK-293 cells. Protein extracts (200 μg) were reacted with 1 μg of normal mouse IgG or 12G1 antibody in the presence of protein A / G plus beads. The mixture was incubated at 4°C for 2 h with vortexing. The eluted proteins were loaded onto 7% SDS-PAGE and blotted with anti-rabbit nectin-2 antibody as the primary antibody (Fig. 7). In the endogenous IP with anti-rabbit nectin-2 antibody, precipitation with 12G1 antibody was detected at ~75 kDa. Furthermore, no precipitation with normal mouse IgG antibody was detected using anti-rabbit nectin-2 antibody, indicating that nectin-2 precipitation was dependent on the nectin-2 antibody (Fig. 7, upper panel). Additionally, the IgG heavy chain of each sample was detected by staining the gel with Coomassie blue dye or by HRP-conjugated anti-mouse IgG (Figure 7, middle panel). These results support the use of the 12G1 antibody in IP analysis.

[0137]

[0138] <Example 3> Expression of Nectin-2 in cell lines

[0139] To confirm nectin-2 expression in HEK-293, RT-PCR and qRT-PCR analyses were performed to amplify GAPDH, nectin-2, nectin-2α, and nectin-2δ. GAPDH was used as an endogenous control. Nectin-2 amplified the common region (895-995 bp) of its variants. The sizes of the amplified products of GAPDH, nectin-2, nectin-2α, and nectin-2δ by RT-PCR were 122, 102, 56, and 139 bp, respectively (Fig. 8A). These results indicated that HEK-293 expressed both nectin-2α and δ, and that the expression level of nectin-2δ was higher than that of nectin-2α (Figs. 8A, 8B, and 8C). To further confirm antibody specificity, we designed specific siRNAs to knockdown nectin-2 expression, targeting a common region within the extracellular domain of nectin-2 (Figure 8D). Each siRNA was then transfected into HEK-293 cells, and nectin-2 knockdown was confirmed using RT-PCR and qRT-PCR (Figures 8E and 8F). The results indicate that the expression of both nectin-2α and δ was downregulated, and all siRNA transfections resulted in significant knockdown, with an efficiency of approximately 50% (Figures 8E and 8F).

[0140] HEK-293 cells transfected with Nectin-2 by mixing siRNAs (#1 + #2 + #3) or negative control siRNAs were evaluated by FACS with the 12G1 antibody (Fig. 9). The mean fluorescence intensity (MFI) of cells transfected with control siRNA after staining with the 12G1 antibody was 138 (Fig. 9, left bar in the lower panel), compared to 66.6 in cells transfected with the mixed nectin-2 siRNA (Fig. 9, right bar in the lower panel). When nectin-2 siRNA was transfected into HEK-293 cells, the degree of staining by 12G1 was also reduced, as was the dramatic decrease in the stained nectin-2 entities. These results demonstrate that the 12G1 antibody specifically binds to the nectin-2 protein.

[0141] Based on FACS analysis, the expression levels of high-expressing nectin-2 cells in various cancer cell lines, including breast, ovarian, and pancreatic cancer cell lines, were verified using the 12G1 antibody (Figure 10). Nectin-2 expression was highest in OV-90 cells, while it was lowest in Caov-3 cells. These results support the 12G1 antibody's potential for biochemical analysis.

[0142] In FACS analysis, nectin-2 expression levels represent protein levels on the cell surface. To confirm the correlation between mRNA and protein levels, qRT-PCR was performed to determine how much nectin-2 mRNAs were transcribed (Fig. 11). A correlation between mRNA-protein expression levels in OV-90 and Caov-3 cells was observed between FACS and qRT-PCR analyses. As a result, OV-90 cells expressed high levels of nectin-2, whereas Caov-3 cells showed low expression levels. However, other cell types showed discrepancies in expression levels, and there was no difference in the expression ratio of nectin-2 across various cells (Fig. 11).

[0143]

[0144] <Example 4> Characterization of chimeric 12G1 (c12G1) antibody

[0145] The present inventors grafted the variable domain of the m12G1 antibody onto human IgG1 to produce a chimeric 12G1 antibody (c12G1). FACS analysis showed that the binding of the c12G1 antibody to ovarian cancer cell lines was similar to that of m12G1 (Fig. 12A). In addition, additional knockdown experiments confirmed that the c12G1 antibody specifically binds to Nectin-2 (Fig. 12B). In addition, SPR analysis showed that the human nectin-2 protein (K D = 2.90 × 10 -10 There was no difference in binding affinity for M) (Fig. 12C).

[0146] Recently, it was discovered that the nectin-2 protein, which is overexpressed in cancer cells, functions as an immune checkpoint by binding to the nectin-2 protein receptor (PVRIG) on NK and cytotoxic T cells. Cytokine secretion and cytotoxic activity of immune cells were inhibited when the V domain of nectin-2 bound to PVRIG. To determine whether the c12G1 antibody could be used as an immune cell activator by inhibiting the interaction between nectin-2 and PVRIG, we performed a competitive enzyme-linked immunosorbent assay (ELISA). As shown in Figure 13A, the c12G1 antibody did not interfere with the binding of nectin-2 to PVRIG, supporting that the c12G1 antibody binds to a site other than the V domain of nectin-2. To identify the binding domain of the c12G1 antibody, we constructed several defective mutants of human nectin-2 (Figure 13B). Immunoprecipitation analysis after transfection of wild-type (WT), Δ1, or Δ2 mutants showed that c12G1 antibody could bind to WT and Δ1 mutant, but not to Δ2 mutant, indicating that c12G1 antibody binds to the first C2 domain of nectin-2 (Fig. 13C). Furthermore, since nectin-2 protein may be structurally different from endogenous nectin-2 protein in case of artificial overexpression, we examined whether c12G1 antibody could bind to endogenous nectin-2 protein. Immunoprecipitation analysis using HEK293 cell lysates showed that c12G1 antibody could bind to endogenous nectin-2 protein (Fig. 13D).

[0147] Furthermore, the therapeutic efficacy of naked antibodies in oncology is determined by their ability to bind antigen while simultaneously mediating effector functions, including complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In vitro effector function assays using the OV-90 cell line revealed that the CDC and ADCC activities of the c12G1 antibody did not exceed a concentration of 20 μg / mL. These results indicate that the naked c12G1 antibody cannot be expected to exhibit anticancer effects through its effector functions, suggesting that its application as an immunotherapy for cancer treatment is challenging.

[0148]

[0149] <Example 5> c12G1 ADC exhibiting anti-tumor activity in vitro and in vivo

[0150] Although Nectin-2 is highly overexpressed in the OV-90 cell line, the c12G1 antibody cannot be used as an anti-tumor agent in its own form. Therefore, ADCs using the c12G1 antibody may be a reasonable option for the effective treatment of ovarian cancer. To confirm its applicability as an ADC, we investigated the internalization efficiency of the c12G1 antibody in ovarian cancer cells. FACS analysis showed that the internalization efficiency of the c12G1 antibody was 54.9% (OV-90), 59.1% (SK-OV-3), and 57.9% (Caov-3) (Figure 14A). The pH within lysosomes is known to be slightly acidic. Therefore, we further analyzed the transport of the c12G1 antibody / Nectin-2 complex into lysosomes using anti-Fc FAB conjugated with Zenon™, a pH-sensitive fluorescent dye. As shown in Figure 14B, the internalization efficiency of the c12G1 antibody was 81.4% in OV-90 cells, 45% in SK-OV-3 cells, and 28% in Caov-3 cells. Taken together, these results support the possibility that the c12G1 antibody can be used as an efficient carrier for the specific delivery of toxins for the treatment of ovarian cancer. Subsequently, we constructed ADCs using smcc-DM1, which consists of a non-cleavable linker and a microtubule inhibitor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that conjugation of smcc-DM1 to the c12G1 antibody induced a slight size change (Figure 15A). Furthermore, since DM1 and antibodies absorb UV light at 252 nm and 280 nm, respectively, the present inventors compared and analyzed the absorbance of the naked antibody (c12G1) and ADC (c12G1-DM1) at 252 nm and 280 nm. Conjugation of DM1 and c12G1 increased the absorbance at 252 nm (Fig. 15B). The drug-to-antibody ratio (DAR) was measured as previously described and was found to be approximately 5.07.Since the target-binding affinity of ADCs can be reduced by conformational changes resulting from conjugation of the N-hydroxysuccinimide ester of the smcc linker to the primary amine of lysine in the antibody, we compared the binding affinities of the 12G1 antibody and 12G1 ADC. ELISA and FACS analyses revealed that the binding affinities of the c12G1 antibody and c12G1 ADC to Nectin-2 were similar (Figures 15C and 15D). These results indicate that conjugation of smcc-DM1 to the c12G1 antibody did not affect the binding affinity to Nectin-2.

[0151] DM1 induces cell cycle arrest in the G2 / M phase by inhibiting microtubule assembly and promoting apoptosis in actively dividing cells. Cell cycle analysis revealed that c12G1-DM1 inhibits cell division in the G2 / M phase, leading to an increase in apoptotic cells in the sub-G1 population (Fig. 16A). However, c12G1-DM1 did not affect the cell cycle of Daudi, a nectin-2-negative cell line. Subsequently, we analyzed the cytotoxic activity of c12G1-DM1 in ovarian cancer cell lines. c12G1-DM1 exhibited in vitro cytotoxic activity in OV-90, SK-OV-3, and Caov-3 cells, and the half maximal inhibitory concentration (IC 50) values ​​ranged from 100 picomolar to nanomolar. The in vitro cytotoxic activity of c12G1-DM1 against nectin-2-positive cell lines was 5-300 times higher than that against nectin-2-negative cell lines (Fig. 16B). Next, the in vivo efficacy of c12G1-DM1 was investigated using mouse models xenografted with OV-90, SK-OV-3, and Caov-3 cells. In the OV-90 cell line, naked c12G1 antibody and IgG-DM1 did not inhibit tumor growth, whereas c12G1-DM1 inhibited tumor growth in a dose-dependent manner (Fig. 16C). Additionally, c12G1-DM1 at 3 mg / kg and 5 mg / kg induced complete tumor remission for approximately 25 days (Fig. 16C). Furthermore, except for one mouse in the 5 mg / kg treatment group that did not respond to the administration from the early stage of the treatment itself for unknown reasons, c12G1-DM1 did not exhibit any antitumor activity in SK-OV-3 cells, whereas c12G1-DM1 inhibited tumor growth in a dose-dependent manner in Caov-3 cells (Fig. 16C). There was no change in the body weight of ADC-treated mice, and a single toxicity study of 20 mg / kg c12G1-DM1 using normal C57BL / 6 mice did not affect body weight changes. Taken together, these results support that ADCs targeting nectin-2 may be useful for the treatment of not only ovarian cancer but also other nectin-2-positive cancers, including breast, colon, and lung cancers.

[0152] Furthermore, we compared c12G1-smcc-DM1 and c12G1-vc-PAB-MMAE (Figs. 17 and 18). DM1 and MMAE, microtubule inhibitors, exhibited similar cytotoxicity between nanomolar and tens of nanomolar concentrations. In assays using ovarian cancer cell lines, smcc-DM1 and vc-MMAE exhibited similar activities. Although the DAR and target-binding affinities of c12G1-smcc-DM1 and c12G1-vc-PAB-MMAE were similar (5.07 vs. 4.79), and smcc-DM1 did not exhibit a bystander effect, the non-cleavable linker payload, smcc-DM1, exhibited significantly higher cytotoxic activity than the cathepsin B-cleavable linker payload, vc-PAB-MMAE. Proteases, including cathepsin B, are highly expressed and secreted in EOC, promoting metastasis through degradation of the extracellular matrix. While the non-cleavable linker payload, smcc-DM1, is released upon antibody degradation by various proteases in lysosomes, the cleavable linker can be cleaved in the extracellular environment by secreted cathepsin B, potentially releasing the MMAE in c12G1-vc-PAB-MMAE into the extracellular environment before it is internalized by cancer cells, potentially reducing its therapeutic efficacy. If the in vitro activity of the non-cleavable linker in ADCs demonstrates effective therapeutic efficacy, this could directly translate into in vivo efficacy, as the cleaved peptides of the antibody could promote immune cell activation near cancer cells. However, further investigation is required to confirm this. Additionally, amine coupling of c12G1 antibody with smcc-DM1 showed a uniform band pattern in both non-reducing and reducing SDS-PAGE, whereas coupling of free thiol groups within the antibody with mc-vc-PAB-MMAE showed a uniform band pattern observed in reducing SDS-PAGE but a non-uniform band pattern in non-reducing SDS-PAGE.The above results support that free thiol groups still exist in the antibody after conjugation with mc-vc-PAB-MMAE, which allows separation into heavy and light chains in non-reducing SDS-PAGE and structural instability in solution.

[0153]

[0154] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6; and an antibody drug conjugate (ADC) to which a drug is bound.

2. An antibody drug conjugate according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence represented by SEQ ID NO:

8.

3. An antibody drug conjugate according to claim 1, characterized in that the antibody is a mouse antibody or a chimeric antibody.

4. An antibody drug conjugate according to claim 3, characterized in that the antibody comprises a kappa chain or a constant region derived from human IgG1.

5. An antibody drug conjugate according to claim 1, characterized in that the drug is an anticancer agent.

6. In paragraph 5, the anticancer agent is daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, Carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine,Cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E, An antibody drug conjugate characterized by at least one selected from the group consisting of MMAE and monomethyl auristatin F.

7. An antibody-drug conjugate according to claim 1, characterized in that the antibody or antigen-binding fragment thereof is bound to a drug via a linker.

8. An antibody drug conjugate according to claim 7, wherein the linker is 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB), or N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

9. A pharmaceutical composition for preventing or treating cancer in which Nectin-2 is overexpressed, comprising the antibody-drug conjugate of any one of claims 1 to 8 as an active ingredient.

10. A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer in paragraph 10 is breast cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, esophageal cancer, gallbladder cancer, or acute myeloid leukemia.

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