Antibody specifically binding to nectin-2 and use thereof

Abstract

The present invention relates to an antibody specifically binding to Nectin-2 and uses thereof. A mouse anti-Nectin-2 antibody capable of specifically targeting Nectin-2 was discovered and analyzed for characteristics, and a chimeric anti-Nectin-2 antibody (chimeric 12G1; c12G1) was produced. In addition, in order to verify the specificity of mouse 12G1 antibody, siRNA for Nectin-2 was constructed and transduced into HEK-293 cells, and the expression level of Nectin -2 was confirmed through reverse transcription polymerase chain reaction, real-time polymerase chain reaction, and flow cytometry with fluorescent labeling. Furthermore, epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization assay were performed for characterization of the chimeric 12G1 antibody. Accordingly, the antibody developed in the present invention has specific reactivity to Nectin-2 and thus presents potential as an antibody therapeutic agent for cancer and autoimmune diseases.

Description

Antibodies that specifically bind to nectin-2 and uses thereof

[0001] The present invention relates to an antibody that specifically binds to Nectin-2 and uses thereof.

[0002] 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, when comparing the expression levels of nectin-2 in various normal and cancerous tissues, it is found to be highly expressed 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.

[0003] Nectin-2 is present in various cells and induces different responses in these cells. The hetero-trans-interaction between nectin-2 and nectin-3 is important for accelerating lymphocyte extravasation through endothelial cell junctions, and nectin-2 regulates the migration and proliferation of outgrowth endothelial cells (OECs), endothelial progenitor cells that promote angiogenesis and improve vascular formation. Furthermore, nectin-2 is located at the Sertoli-sperm junction in the testis, where it has been identified as a site of canal constriction for neural tube formation in the Xenopus laevis model, and nectin-2 regulates neuronal-astrocytic synapse formation in the brain. Nectin-2 is overexpressed in poorly differentiated gastric and colon cancer tissues, and may be associated with poor prognosis in pancreatic cancer. Nectin-2 deficiency leads to structural disruptions, including dysfunction of intercalated discs and cardiac myofibrils, male infertility, and degeneration of astrocytes and neurons.

[0004] Nectin-2 can act as a receptor or ligand mediating signal transduction. Nectin-2 functions as a receptor for glycoprotein D (gD) for entry of porcine pseudorabies virus (PRV), herpes simplex virus (HSV)-2, and variant forms of HSV-1, but not HSV-1. Met89 of nectin-2 is known to be important for viral entry, as replacement of Met89 with Phe89 significantly reduces viral entry activity.

[0005] Therefore, as described above, Nectin-2 functions as a ligand or receptor, and thus can be a surface marker of cells highly expressed in certain diseases, and has the potential to be developed as a treatment for cancer and autoimmune diseases.

[0006] An object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to Nectin-2.

[0007] Another object of the present invention is to provide a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid molecule, and an isolated cell transformed with the recombinant expression vector.

[0008] Another object of the present invention is to provide a composition for detecting Nectin-2 antigen, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0009] Another object of the present invention is to provide a composition for diagnosing cancer in which Nectin-2 is overexpressed, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0010] 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.

[0011] The present invention also provides a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.

[0012] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0013] In addition, the present invention provides an isolated cell transformed with the recombinant expression vector.

[0014] In addition, the present invention provides a composition for detecting Nectin-2 antigen, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0015] In addition, the present invention provides a composition for diagnosing cancer in which Nectin-2 is overexpressed, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0016] The present invention relates to an antibody that specifically binds to nectin-2 and a use thereof. A mouse anti-nectin-2 antibody (m12G1 clone) capable of specifically targeting nectin-2 was discovered and characterized, and a chimeric anti-nectin-2 antibody (chimeric 12G1: c12G1) was produced. In addition, to verify the specificity of the mouse 12G1 antibody, siRNA against nectin-2 was produced and transduced into HEK-293 cells. The expression level of nectin-2 was confirmed through reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, and fluorescence-activated cell sorting techniques. In addition, epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization analysis were performed to characterize the chimeric 12G1 antibody. Therefore, since the antibody produced in the present invention has specific reactivity to Nectin-2, it suggests the possibility of being used as an antibody therapeutic agent for cancer and autoimmune diseases.

[0017] Figure 1 shows the results of the confirmation of mouse monoclonal Nectin-2 antibody.

[0018] Figure 2 shows the results of multiple production and purification of mouse monoclonal Nectin-2 antibodies.

[0019] Figure 3 shows the results of binding affinity measurement.

[0020] Figure 4 is V H and V L Shows the amplification results of the area.

[0021] Figure 5 shows V in 12G1 antibody H and V L It shows the results of amino acid and nucleic acid sequence analysis.

[0022] Figure 6 shows the optimization results for FACS analysis.

[0023] Figure 7 shows the results of immunoprecipitation analysis.

[0024] Figure 8 shows the expression level of Nectin-2 in HEK-293 cells.

[0025] Figure 9 shows the results that the 12G1 antibody specifically recognizes Nectin-2 on the cell surface.

[0026] Figure 10 shows the results of FACS analysis of Nectin-2 expression in various cancer cell lines.

[0027] Figure 11 shows the results of qRT-PCR analysis of Nectin-2 expression in various cancer cell lines.

[0028] Figure 12 shows the results of characterization of chimeric 12G1 (c12G1) antibody.

[0029] Figure 13 shows the results of confirming the Nectin-2 binding domain of c12G1.

[0030] 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.

[0031] 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.

[0032] Preferably, the antibody may be a mouse antibody or a chimeric antibody, but is not limited thereto.

[0033] Preferably, the antibody may comprise a kappa chain or a constant region derived from human IgG1, but is not limited thereto.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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, as long as they 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.

[0040] The present invention also provides a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.

[0041] As used herein, the term "nucleic acid molecule" has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified. The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified, and the modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0042] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0043] In the present invention, "vector" means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).

[0044] In the present invention, an "expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked to a specific host organism. The expression vector may preferably include one or more selectable markers. The markers are nucleic acid sequences having characteristics that can be selected, typically by chemical methods, and include all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin, and chloramphenicol, and can be appropriately selected by those skilled in the art.

[0045] Any of a wide variety of expression control sequences may be used in the vector to express the DNA sequence of the present invention. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the promoter and enhancer of CMV, the LTR of retroviruses, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd-encoded protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of such phosphatases, e.g., Pho5, the promoter of the yeast alpha-mating system, and any other sequence known to regulate the expression of genes of prokaryotes or eukaryotes or their viruses, and various combinations thereof.

[0046] The vector expressing the antibody of the present invention can be either a vector system in which the light chain and the heavy chain are simultaneously expressed from a single vector, or a system in which the light chain and the heavy chain are each expressed from separate vectors. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation. Co-transformation is a method in which vector DNA encoding the light chain and the heavy chain are simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected. Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing a heavy chain (or light chain), thereby finally selecting cells expressing both the light chain and the heavy chain.

[0047] In addition, the present invention provides an isolated cell transformed with a recombinant expression vector.

[0048] The cell capable of stably and continuously cloning and expressing the vector of the present invention may be any host cell known in the art, including, but not limited to, prokaryotic host cells such as strains of the genus Bacillus such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0049] In the method for producing the above antibody or antigen-binding fragment thereof, the culture of transformed cells can be performed according to appropriate media and culture conditions known in the relevant technical field. Such culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Cell culture is classified into suspension culture and attachment culture depending on the cell growth method, and batch, fed-batch, and continuous culture methods depending on the culture method. The culture medium used must appropriately satisfy the requirements of the specific strain.

[0050] In addition, the present invention provides a composition for detecting Nectin-2 antigen, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0051] In addition, the present invention provides a composition for diagnosing cancer in which Nectin-2 is overexpressed, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

[0052] 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.

[0053] 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.

[0054]

[0055] <Experimental Example>

[0056] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0057]

[0058] 1. Cell culture and cell lines

[0059] 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 DMEM (Dulbecco's high glucose modified Eagle's medium) (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.

[0060] 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

[0061]

[0062] 2. 항체 제작

[0063] 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 Balb / c mice with pristane (Sigma-Aldrich, St. Louis, USA) 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).

[0064] 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).

[0065]

[0066] 3. Indirect ELISA

[0067] 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).

[0068]

[0069] 4. RNA extraction and cDNA synthesis

[0070] 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).

[0071]

[0072] 5. Cloning of variable regions in monoclonal antibodies

[0073] 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 min, followed by denaturation at 94°C for 1 min, 5 cycles per temperature from 61°C to 58°C with 1 min of binding per cycle (exceptionally, MHV7 or 11 primers were bound from 57°C to 53°C), 25 cycles of 1 min of extension at 72°C, a final extension at 72°C for 7 min, and storage at 4°C until use. In addition, the amplification conditions of the light chain in the antibody had an association temperature of 12 cycles per temperature from 51°C to 52°C (for 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.

[0074] 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)

[0075]

[0076] 6. Surface plasmon resonance analysis

[0077] 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).

[0078]

[0079] 7. FACS analysis

[0080] 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).

[0081]

[0082] 8. Immunoprecipitation analysis and Western blot

[0083] 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.

[0084] 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).

[0085] 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).

[0086]

[0087] 9. RNA interference

[0088] 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.

[0089]

[0090] 10. RT-PCR and qRT-PCR

[0091] 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.

[0092]

[0093] 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)

[0094]

[0095] 11. ELISA

[0096] 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).

[0097] 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.

[0098]

[0099] 12. Effector Function Analysis

[0100] 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).

[0101] 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.

[0102]

[0103] 13. Statistical Analysis

[0104] Data were expressed as mean ± standard error of the mean (SEM) or ± standard deviation (SD) and analyzed using one-way ANOVA and unpaired Student's test to determine statistical significance. p < 0.05 was considered statistically significant.

[0105] <Example 1> Production of monoclonal Nectin-2 antibody

[0106] 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.

[0107] Mouse sera were harvested before and after immunization with nectin-2 protein and assessed for the presence of nonspecific (Fig. 1A) and specific antibodies (Fig. 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 (Fig. 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 (Fig. 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, 12G1, a hybridoma clone that 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. Cell culture supernatants obtained from the expanded cells of the clone were verified by indirect ELISA and Western blotting (Figures 1C and 1D). The OD value of each cell culture supernatant was ≥1.0 (Figure 1C). These results indicate the presence of high titer antibodies. To confirm that the 12G1 clone 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).

[0108] 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).

[0109] 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).

[0110] 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).

[0111] CloneO.D. 450nmIgG1IgG2aIgG2bIgG3IgAIgMKappaLambda12G10.5830.050.0430.0440.0440.0420.1440.048

[0112] OD values ​​were measured at a wavelength of 450 nm.

[0113]

[0114] 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

[0115]

[0116] <Example 2> Characterization of Nectin-2 mouse monoclonal antibody

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121]

[0122] <Example 3> Expression of Nectin-2 in cell lines

[0123] 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).

[0124] 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.

[0125] 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.

[0126] 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).

[0127]

[0128] <Example 4> Characterization of chimeric 12G1 (c12G1) antibody

[0129] 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).

[0130] 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).

[0131] 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.

[0132]

[0133] 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

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. An antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, characterized in that in claim 1, 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. An antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, characterized in that the antibody in claim 1 is a mouse antibody or a chimeric antibody. In claim 3, an antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, characterized in that the antibody comprises a kappa chain or a constant region derived from human IgG1. A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. A recombinant expression vector comprising the nucleic acid molecule of claim 5. A cell isolated by transformation with the recombinant expression vector of Article 6. A composition for detecting a Nectin-2 antigen comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 as an active ingredient. A composition for diagnosing cancer in which Nectin-2 is overexpressed, comprising an antibody or an antigen-binding fragment thereof of any one of claims 1 to 4 as an active ingredient. A composition for diagnosing cancer, characterized in that in claim 9, the cancer is breast cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, esophageal cancer, gallbladder cancer, or acute myeloid leukemia.

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