Humanized antibody specifically binding to nectin-2, and use thereof

Abstract

The present invention relates to a humanized antibody specifically binding to Nectin-2, and use thereof. The humanized anti-Nectin-2 antibody was prepared by humanizing a chimeric anti-Nectin-2 antibody capable of specifically targeting Nectin-2. The antigen binding equivalency of chimeric and humanized anti-Nectin-2 antibodies was identified using an enzyme-linked immunosorbent assay, and the affinity of the humanized anti-Nectin-2 antibody was measured. In addition, the Nectin-2 binding equivalency of the humanized anti-Nectin-2 antibody in live cells was identified, and the Nectin-2 binding equivalency expressed on the live cell surfaces of chimeric and humanized antibodies was also identified using flow cytometry. It was found that the humanized anti-Nectin-2 antibody of the present invention maintained freezing and thawing stability and also maintained pH-dependent Nectin-2 binding stability. Therefore, the humanized anti-Nectin-2 antibody developed in the present invention has specific reactivity to Nectin-2, and thus shows potential as an antibody therapeutic agent for cancer and autoimmune diseases.

Description

Humanized antibodies specifically binding to nectin-2 and uses thereof

[0001] The present invention relates to a humanized 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, 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.

[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 blood vessel 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] Antibodies for disease treatment are primarily produced using mice to produce monoclonal antibodies. However, non-human antibodies, such as mouse-derived monoclonal antibodies, are considered foreign antigens in the human body, triggering immune responses and having short half-lives, which limit their therapeutic efficacy. To address these issues, humanized antibodies have been developed, in which all but the antigen-binding portion of the antibody is replaced with a human antibody. The current method for replacing mouse antibodies with humanized antibodies involves selecting the most similar human antibody gene to the target antibody and then, using a process called CDR grafting, replacing only the mouse antibody CDR region with the human antibody CDR position. Because most of the genes in these humanized antibodies are humanized, they have the advantage of reducing immune responses in the body.

[0005] The purpose of the present invention is to provide a humanized antibody or an antigen-binding fragment thereof that specifically binds to Nectin-2.

[0006] Another object of the present invention is to provide a nucleic acid molecule encoding the humanized 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.

[0007] Another object of the present invention is to provide a composition for detecting Nectin-2 antigen, comprising the humanized antibody or antigen-binding fragment thereof as an active ingredient.

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

[0009] Another object of the present invention is to provide the humanized antibody or antigen-binding fragment thereof; and antibody drug conjugates (ADC) to which a drug is bound.

[0010] 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.

[0011] To achieve the above object, the present invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, comprising a heavy chain variable region comprising any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 5 to SEQ ID NO: 26; and a light chain variable region comprising any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 27 to SEQ ID NO: 48.

[0012] Additionally, the present invention provides a nucleic acid molecule encoding the humanized antibody or antigen-binding fragment thereof.

[0013] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0014] In addition, the present invention provides an isolated cell transformed with the recombinant expression vector.

[0015] In addition, the present invention provides a composition for detecting Nectin-2 antigen, comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.

[0016] In addition, the present invention provides a composition for diagnosing cancer in which Nectin-2 is overexpressed, comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.

[0017] In addition, the present invention provides an antibody-drug conjugate comprising the humanized antibody or an antigen-binding fragment thereof and a drug.

[0018] 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.

[0019] The present invention relates to a humanized antibody that specifically binds to nectin-2 and a use thereof. A chimeric anti-nectin-2 antibody capable of specifically targeting nectin-2 was humanized to produce a humanized anti-nectin-2 antibody. The antigen-binding equivalence of the chimeric and humanized anti-nectin-2 antibodies was confirmed using an enzyme-linked immunosorbent assay, and the affinity of the humanized anti-nectin-2 antibody was measured. In addition, the nectin-2 binding equivalence of the humanized anti-nectin-2 antibody in live cells was confirmed, and the nectin-2 binding equivalence of the chimeric and humanized antibodies on the surface of live cells was also confirmed using flow cytometry. Meanwhile, the humanized anti-nectin-2 antibody of the present invention was found to maintain freeze-thaw stability and pH-dependent nectin-2 binding stability. Therefore, the humanized anti-Nectin-2 antibody produced in the present invention has specific reactivity to Nectin-2, and thus presents the possibility of being used as an antibody therapeutic agent for cancer and autoimmune diseases.

[0020] Figure 1 shows the SPR results of chimeric and grafted anti-Nectin-2 antibodies.

[0021] Figure 2 shows the affinity analysis screening results of humanized anti-Nectin-2.

[0022] Figure 3 shows the SDS-PAGE results of purified humanized anti-Nectin-2 antibody.

[0023] Figure 4 shows the results of confirming the binding ability of chimeric and humanized anti-Nectin-2 antibodies using enzyme-linked immunosorbent assay.

[0024] Figure 5 shows the affinity analysis results of chimeric and humanized anti-Nectin-2 antibodies.

[0025] Figure 6 shows the FACS EC50 results of humanized anti-nectin-2 antibodies.

[0026] Figure 7 shows the results of comparisons between chimeric and humanized anti-Nectin-2 antibodies in ovarian cancer cell lines and Daudi. A) Flow cytometry results according to cell line and antibody type are shown. B) Flow cytometry results according to cell line and antibody treatment concentration are shown. For comparison with the chimeric antibody, the results for the chimeric antibody are shaded at each concentration.

[0027] Figure 8 shows the results of freeze-thaw stability experiments of chimeric and humanized anti-Nectin-2 antibodies.

[0028] Figure 9 shows the pH-dependent Nectin-2 binding stability results.

[0029] Figure 10 shows the internalization results of humanized anti-Nectin-2 antibodies.

[0030] Figure 11 shows the results of development of a DM1 drug conjugate (ADC) of a humanized anti-Nectin-2 antibody.

[0031] Figure 12 shows the results of confirming the Necitn-2 binding equivalence of a humanized anti-Nectin-2 antibody and an ADC using the same.

[0032] Figure 13 shows the results of drug response validation of a humanized anti-Nectin-2 antibody-DM1 conjugate (ADC).

[0033] The present invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, comprising a heavy chain variable region comprising any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 5 to SEQ ID NO: 26; and a light chain variable region comprising any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 27 to SEQ ID NO: 48.

[0034] Preferably, the humanized antibody or antigen-binding fragment thereof that specifically binds to Nectin-2 comprises a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 27, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 28, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 29, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 8; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 30, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 31, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 10; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 32, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 33, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 34, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 13; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 35, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 14; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 36, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15;And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 37, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 38, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 39, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 18; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 40, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 19; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 41, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 20; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 42, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 21; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 43, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 22; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 44, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 23; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 45, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 24; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 46, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 25; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 47, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 26;and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 48, but is not limited thereto.;

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In the present invention, the term "humanized antibody" refers to an antibody in which all or part of the CDR sequence of a mouse monoclonal antibody is grafted onto a human antibody. For example, the CDRs of a mouse monoclonal antibody can be recombined with FRs derived from a human antibody to produce a humanized variable region, which can then be recombined with the constant region of a desired human antibody to produce a humanized antibody. In other words, it refers to antibodies that are nonimmunogenic or have reduced immunogenicity in humans. A humanized antibody is an antibody with an altered amino acid sequence, and the amino acid sequence of the antibody can be reconstructed to suit the desired purpose. These possible changes are numerous and range from changing one or a few amino acids to completely reconstructing the variable and / or constant regions of the antibody. Generally, modifications in the variable region are performed to increase antigen binding ability and affinity, whereas modifications in the constant region are performed to increase intracellular functions such as fixation of complement, interaction with membranes, and other effector functions.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] Additionally, the present invention provides a nucleic acid molecule encoding the humanized antibody or antigen-binding fragment thereof.

[0043] 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.

[0044] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0045] In the present invention, "vector" means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In addition, the present invention provides an isolated cell transformed with a recombinant expression vector.

[0050] 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).

[0051] 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.

[0052] In addition, the present invention provides a composition for detecting Nectin-2 antigen, comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.

[0053] In addition, the present invention provides a composition for diagnosing cancer in which Nectin-2 is overexpressed, comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.

[0054] 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.

[0055] In addition, the present invention provides an antibody-drug conjugate comprising the humanized antibody or an antigen-binding fragment thereof and a drug.

[0056] 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.

[0057] 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).

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065]

[0066] <Example 1> Humanization of chimeric anti-Nectin-2 antibodies

[0067] The present inventors produced a chimeric anti-Nectin-2 antibody by grafting the variable domain of a mouse monoclonal antibody capable of specifically targeting Nectin-2 onto human IgG1.

[0068] The heavy chain variable domain (SEQ ID NO: 1) of the chimeric anti-Nectin-2 antibody was grafted onto the Germline: IGHV3-33*03 sequence (SEQ ID NO: 2), and the light chain variable domain (SEQ ID NO: 3) was grafted onto the Germline:IGKV1-39*01 sequence (SEQ ID NO: 4), and the binding affinity to Nectin-2 was confirmed by SPR (Table 1 and Fig. 1). The graft sequence was confirmed to have slightly lower affinity for Nectin-2 than the chimeric antibody. Using GeneScript's FASEBA (Fast Screening of Expression, Biophysical-properties, and Affinity) technology, random mutations were induced in the graft sequence by overlapping PCR, and the base sequences of the clones reacting with the antigen were analyzed to secure the final 22 non-overlapping amino acid sequences of the humanized anti-Nectin-2 antibody. To confirm the binding ability of 22 humanized anti-Nectin-2 antibodies (heavy chain variable region: SEQ ID NO: 5 to SEQ ID NO: 26; light chain variable region: SEQ ID NO: 27 to SEQ ID NO: 48) to Nectin-2, SPR (Surface Plasma Resonance) was performed. Anti-Histidine antibodies were immobilized on a CM5 chip (Cytiva, Cat#. BR-1005-30) using Biacore 8K (Cytiva, Sweden). After secondary immobilization of Nectin-2 (Sinobiological, China) at a concentration of 1 ug / ml, the culture medium expressing the chimeric and humanized anti-Nectin-2 antibodies was flowed, and the affinity KD value for Nectin-2 was analyzed using Biacore T200 evaluation software. The KD value is the value calculated by dividing the kd by the ka, and a lower value indicates a stronger binding ability to the target.When the affinity results of the humanized anti-Nectin-2 antibodies of 22 sequences were compared with those of the chimeric Nectin-2 antibody, the distribution of KD values ​​was equal to, equal to, or greater than, or less than equal to (Table 2, Table 3, and Figure 2). Three clones were selected based on the degree of back mutation in the sequence and the degree of RU max value and used for additional antibody characterization.

[0069] SPR results of chimeric and grafted anti-Nectin-2 antibodiesLigandAnalyteka (1 / Ms)kd (1 / s)KD (M)Rmax (RU)Chi 2 (RU 2 )Chimeric-1Nectin-21.57E+051.35E-038.62E-09107.30.305Chimeric-2Nectin-22.05E+051.47E-037.20E-09122.30.4 28Grafted-1Nectin-22.20E+053.39E-031.54E-0824.10.165Grafted-2Nectin-21.60E+053.07E-031.92E-0831.30.0432

[0070]

[0071] Affinity screening results of humanized anti-Nectin-2 Fab 1LigandAnalyteka (1 / Ms)kd (1 / s)KD (M)Rmax (RU)Chi 2 (RU 2 )AHF16887Nectin-21.98E+052.54E-031.28E-08460.0681AHF16892Nectin-21.90E+ 053.18E-031.67E-0812.60.0357AHF16894Nectin-21.65E+052.58E-031.56E-08350. 0689blankNectin-2NANANANANAChimericNectin-21.78E+051.89E-031.06E-0844.70.0659GraftedNectin-22.01E+054.35E-032.16E-08170.0297NCNectin-2NANANANANA

[0072]

[0073] Affinity screening results of humanized anti-Nectin-2 antibodies 2LigandAnalyteka (1 / Ms)kd (1 / s)KD (M)Rmax (RU)Chi 2 (RU 2AHF17272Nectin-22.71E+052.08E-037.69E-09225.33.05 AHF17273Nectin-22.68E+052.36E-038.81E-09240.13.78 AHF17275Nectin-22.81E+052.03E-037.24E-09204.74.17 AHF17276Nectin-22.73E+051.55E-035.69E-09259.55.76 AHF17280Nectin-22.77E+051.63E-035.86E-09223.54.49AHF 17281Nectin-22.40E+053.04E-031.27E-081283Nectin-22.44E+053.00E-031.23E-0847.30.487AHF17283Nectin-22.53E+052.10E-0 38.31E-09330.248AHF17284Nectin-22.79E+051.91E-036.86E-09220.34.4AHF17285Nectin-22.50E+052.50E-031.00E-0822.10.349AHF17286Nect in-22.46E+051.90E-037.72E-0988.30.882AHF17287Nectin-21.79E+054.04E-032.25E-0836.30.207AHF17288Nectin-22.71E+051.56E-035.76E- 092696.1AHF17289Nectin-22.76E+051.82E-036.60E-091803.3AHF17290Nectin-21.89E+054.25E-032.26E-0827.90.238AHF17292Nectin-22.14E+ 052.51E-031.17E-0835.60.31AHF17293Nectin-22.42E+052.87E-031.18 E-0828.90.371AHF17294Nectin-22.59E+051.87E-037.24E-092965.72AHF 17295Nectin-21.64E+054.16E-032.53E-0821.20.515U181-blankNectin-2NANANANANAU181-NCNectin-2NANANANANAChimericNectin-22.39E+051.56E-036.55E-09125.81.45GraftedNectin-22.24E+052.32E-031.04E-0855.70.514.

[0074]

[0075] For reference, the CDR sequences of the humanized antibodies produced in the present invention are listed in Table 4.

[0076] Sequence Information Amino Acid Sequence Heavy Chain CDR1 SEQ ID NO: 49 GFSLSRYGVHCDR2 SEQ ID NO: 50 VIWRGGSTDYNAAFMS CDR3 SEQ ID NO: 51 KRDNDGALDY Light Chain CDR1 SEQ ID NO: 52 RASENIIY SYLA CDR2 SEQ ID NO: 53 NAKTLAECDR3 SEQ ID NO: 54 QHHYGPPYT

[0077]

[0078] <Example 2> Production of humanized anti-Nectin-2 antibody

[0079] The DNA sequences of three humanized anti-Nectin-2 antibodies (AHF17275, AHF17284, and AHF17294) were cloned into the pcDNA3.4 vector. The heavy and light chains were cloned into separate plasmids, and two plasmids encoding the heavy and light chains, respectively, were transfected into Expi293 cells to produce humanized anti-Nectin-2 antibodies. The recombinant humanized IgG antibodies were secreted into the culture medium of transfected Expi293 cells and purified using protein A affinity chromatography (Table 5 and Fig. 3).

[0080] Humanized anti-Nectin-2 antibody purification results Sequence IDVLVHPurified Ab Con. (mg / mL)Purified Ab volume (mL)Purified Ab quantity (mg)Purified Ab quantity (SDS page)4548854849677179ChimericQVSLGLKF2.41312.413>90%AHF17275KVSVGFRF1.8011.01.801>90%AHF17284QVTVGFRF2.0421.02.042>90%AHF17294KITLGFRF3.2551.03.255>90%

[0081]

[0082] <Example 3> Confirmation of antigen binding equivalence of chimeric and humanized anti-Nectin-2 antibodies using enzyme-linked immunosorbent assay

[0083] To confirm and compare the nectin-2 binding of three humanized anti-nectin-2 antibodies and chimeric c12G1 antibody, an enzyme-linked immunosorbent assay was performed. Recombinant human nectin-2 protein (100 ng / well, Sinobiological, China) was coated on a 96-well plate overnight at 4 °C, and each well was reacted with 1 × DPBS containing 5% BSA for 1 hour and 30 minutes. Subsequently, suspensions diluted by antibody concentration in 1 × DPBS supplemented with 1% BSA and 0.1% Tween 20 were added to each well and reacted for 1 hour at room temperature. Each well was washed three times with 1× DPBS supplemented with 0.1% Tween-20, and 100 μl per well of HRP-conjugated goat anti-human secondary antibody (533 ng / ml, 1:1500, Invitrogen, IL, United States) diluted in 1× DPBS supplemented with 1% BSA and 0.1% Tween20 was added, and incubated for 1 hour at room temperature. Each well was washed three times with 1× DPBS supplemented with 0.1% Tween-20, and 100 μl per well of 1-StepTM Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, Waltham, United States) was added at RT, and the reaction was stopped after 3 minutes by adding 50 μl of 1 N H2SO4 solution. The results were analyzed by measuring absorbance at 450 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany). The results shown in Fig. 4 confirmed that the binding capacity of the three chimeric and humanized anti-Nectin-2 antibodies to Nectin-2 was equivalent.

[0084]

[0085] <Example 4> Affinity measurement of humanized anti-Nectin-2 antibody

[0086] To confirm the binding ability of purified chimeric and humanized anti-Nectin-2 antibodies to Nectin-2, SPR (Surface Plasma Resonance) was performed. Anti-Histidine antibody (20 ug / mL) was immobilized on a CM5 chip (Cytiva, Cat#. BR-1005-30) using a Biacore T200 (Cytiva, Sweden). After secondary immobilization of Nectin-2 (Sinobiological, China) at a concentration of 15 nM, chimeric and humanized anti-Nectin-2 antibodies were flowed at various concentrations, and the affinity KD values ​​for Nectin-2 were analyzed using Biacore T200 Control software (v.3.2) and Biacore T200 Evaluation software. The KD value is the kd divided by the kA, and a lower value indicates a stronger binding ability to the target. The results are shown in Fig. 5 and Table 6. The KD value of the chimeric anti-Nectin-2 antibody for human Nectin-2 was 2.136E-10M, indicating strong affinity, and the humanized anti-Necitn-2 antibody also showed a similar or equivalent level of affinity.

[0087] Affinity analysis results of chimeric and humanized anti-Nectin-2 antibodies. LigandAnalyteConc. (nM)ka(1 / Ms)kd(1 / s)KD(M)Rmax(RU)Chi 2 (RU 2)Nectin-2c12G1 (chimeric) 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 nM 4.586E+059.796E-052.136E-10211.610.217275 (humanized) 0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, 200 nM1.328E+058.986E-056.766E-10206.11.3117284(humanized)0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 nM2.778E+058.509E-053.063E-10212.12.4717294(humanized)0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 nM6.741E+051.239E-041.839E-10233.718.2

[0088]

[0089] <Example 5> Confirmation of nectin-2 binding equivalence and EC50 measurement of humanized anti-nectin-2 antibody in live cells

[0090] OV-90 cells were seeded at 1 × 10 per well in a 96-well plate. 550 μl of cell suspension containing cells was dispensed, and 50 μl of antibody dilution solution diluted to the indicated concentration was treated each, and incubated at 4 °C for 40 minutes. Afterwards, the cells were centrifuged at 300 g for 3 minutes, the supernatant was removed, and the cells were washed twice with 150 μl of pH 7.4 PBS. 100 μl of secondary antibody was treated each, and the cells were reacted at 4 °C for 3 minutes, centrifuged at 300 g for 3 minutes, the supernatant was removed, and the cells were washed twice with 150 μl of pH 7.4 PBS. 50 μl of PBS was dispensed into each well, and the fluorescence value of the samples was measured using a flow cytometer (BD Canto II, Unites States). The EC50 of the humanized anti-Nectin-2 antibody flow cytometry was 0.08702 to 0.1897 ug / ml, which showed an EC50 value at a similar level compared to the EC50 of the chimeric antibody, 0.1628 ug / ml (Fig. 6).

[0091]

[0092] <Example 6> Confirmation of binding equivalence of chimeric and humanized antibodies to Nectin-2 expressed on the surface of live cells using flow cytometry

[0093] Flow cytometry was performed to confirm the binding of humanized anti-Nectin-2 antibodies to cell surface nectin-2. Human IgG (HIgG) antibody was used as a negative control, and three humanized anti-Nectin-2 antibodies, including chimeric anti-Nectin-2 antibody (c12G1), were diluted in 1× DPBS supplemented with 2% BSA at three concentrations: 10 µg / ml, 1 µg / ml, and 0.1 µg / ml. The indicated cells (2.0 × 10 5) and incubated on ice for 1 hour. Then, the cells were transferred to a suspension of humanized anti-Nectin-2 antibody diluted to the concentrations indicated in 1 × DPBS containing 2% BSA and incubated on ice for 1 hour. After washing three times with 1 × DPBS containing 2% BSA, the cells were added to a suspension of FITC-conjugated anti-human IgG secondary antibody (Invitrogen, CA, USA) diluted to 0.3 μg / ml in 1 × DPBS containing 2% BSA and incubated on ice for 1 hour. After secondary antibody staining, the cells were washed three times with 1× DPBS containing 2% BSA, resuspended in 1× DPBS, and analyzed for fluorescence with CytoFLEX (Beckman Coulter, United States). It was confirmed that the binding affinity of the humanized anti-Nectin-2 antibody and the chimeric antibody to the nectin-2 expressed on the cell surface was equivalent in OV-90, SK-OV-3, and Caov-3 cell lines (Fig. 7). The binding affinity of the chimeric and humanized anti-Nectin-2 antibodies was tested in Daudi cells, known as a nectin-2-expressing negative control, and it was confirmed that when the concentration of the human IgG antibody used as a negative control increased, it bound to the antigen on the cell surface and showed a higher fluorescence value. These results showed that the humanized anti-Nectin-2 reacted with the Daudi cell line at the highest concentration (10 ug / ml), but the degree of reaction was small compared to the human IgG control antibody.

[0094]

[0095] <Example 7> Freezing and thawing stability

[0096] Chimeric and humanized anti-Nectin-2 antibodies were diluted to 40 ng / ml in 1× DPBS supplemented with 1% BSA and 0.1% Tween 20. For freeze / thaw stability testing, freeze and thaw cycles were defined as 3 min in liquid nitrogen and 5 min in a 37°C water bath. The diluted samples were exposed to freeze / thaw cycles once, twice, and three times, respectively, according to the conditions, and centrifuged at 4°C and 15,000 rpm for 10 min. The antibody stability according to the freeze and thaw cycles was compared using enzyme-linked immunosorbent assay. When the chimeric and humanized anti-Nectin-2 antibodies were diluted to 40 ng / ml, which is a non-saturating level in ELISA, and the freeze and thaw stability test was performed, it was confirmed that antibody stability was maintained over the tested cycles (Fig. 8).

[0097]

[0098] <Example 8> pH-dependent Nectin-2 binding stability

[0099] After preparing phosphate-buffered saline solutions of pH 5.5, pH 6.0, pH 6.5, and pH 7.2, chimeric and humanized anti-Nectin-2 antibodies were diluted to two concentrations (40 ng / ml, 1000 ng / ml). Enzyme-linked immunosorbent assay was performed to confirm the difference in nectin-2 binding ability according to the buffer solution pH. Recombinant human nectin-2 protein (100 ng / well, Sinobiological, China) was coated on a 96-well plate overnight at 4 °C, and then each well was treated with 1 × DPBS containing 5% BSA for 1 hour and 30 minutes. Antibodies diluted in phosphate-buffered saline of different pH were treated with 100 μl per condition in a 96-well plate and reacted for 1 hour at room temperature. Each well was washed three times with 1 × DPBS supplemented with 0.1% Tween-20, and 100 μl per well of HRP-conjugated goat anti-human secondary antibody (533 ng / ml, Invitrogen, IL, United States) diluted in 1 × DPBS supplemented with 1% BSA and 0.1% Tween-20 was added, and incubated at room temperature for 1 h. Each well was washed three times with 1 × DPBS supplemented with 0.1% Tween-20, and 100 μl per well of 1-StepTM Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, Waltham, United States) was added at RT. After 3 min, the reaction was stopped by adding 50 μl of 1 N H2SO4 solution. The final color development was analyzed at 450 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany). It was confirmed that the binding degree of chimeric and humanized anti-Nectin-2 antibodies to Nectin-2 was maintained regardless of pH, even under conditions other than normal physiological pH (Fig. 9).

[0100]

[0101] <Example 9> Confirmation of internalization of three types of humanized anti-Nectin-2 antibodies

[0102] To confirm the internalization of three types of humanized anti-Nectin-2 antibodies, flow cytometry was performed using ovarian cancer cell lines OV90, SKOV-3, and Caov-3. Each cell line was incubated for 25 minutes in a solution containing 1% BSA, 75 μg / mL cycloheximide, and Human Fc Block (BD Pharmingen™). Then, each antibody was diluted to a single concentration of 1 μg / mL and added to each cell line suspension and reacted for 1 hour at 4°C. Before the internalization reaction, the experimental groups that reacted with the antibodies for 1 hour at 4°C were washed three times and immediately stained with FITC-conjugated anti-human IgG secondary antibody (Invitrogen, CA, USA). This was indicated by the darkest shade on the rightmost side of each graph as a reference value for binding to cell surface nectin-2 (Fig. 10). After reacting the diluted antibodies with each cell line at 4℃ for 1 hour, the cells were incubated for 1 hour and 3 hours in an incubator at 37℃ and 5% CO2 for antibody internalization reaction, and further incubated for 1 hour and 3 hours at 4℃, followed by staining with FITC-conjugated anti-human IgG secondary antibody (Invitrogen, CA, USA). Other processes were the same as the flow cytometry method described above. The results of this internalization reaction are shown in Fig. 10. The 4℃-1 hour / 3 hour incubation condition, which is a negative control for the internalization reaction, is indicated by a solid line, and the 37℃-1 hour / 3 hour incubation condition is shaded. The non-fluorescent labeled condition and the condition treated with only the secondary antibody (FITC) are indicated by the black and red solid lines on the far right. In the internalization experiment, all three humanized anti-Nectin-2 antibodies were confirmed to exhibit an internalization response of 70-80% overall when incubated at 37°C for 3 hours.

[0103]

[0104] <Example 10> Development of humanized anti-Nectin-2 antibody conjugated with DM1 drug

[0105] Humanized anti-Nectin-2-ADCs (antibody-drug conjugates) containing N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine (DM1) conjugated with SMCC (N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) as a non-cleavable linker and a microtubule inhibitor were prepared. Specifically, two types of humanized anti-Nectin-2 antibodies (17284, 17294) were individually dialyzed against conjugation buffer (0.1 M sodium phosphate and 0.15 M NaCl, pH 7.2) and conjugated to SMCC-DM1 (MedChemExpress) at a molar ratio of 1:20 at RT for 1.5 h. To remove unbound SMCC-DM1 and aggregated humanized anti-Nectin-2-DM1, centrifugation (21,000 × g, 20 min, RT) was performed. After centrifugation, the supernatant was collected and the prepared humanized anti-Nectin-2 antibody-drug conjugate was dialyzed against formulation buffer (10 mM sodium succinate, 0.05% polysorbate 20, and 6% sucrose; pH 5.0). After collecting the dialyzed humanized anti-Nectin-2 antibody-drug conjugate, the presence of conjugation was measured by examining the optical density (OD) at 280 nm and 252 nm (DM1 absorbs UV at 252 nm) using a SPECTROstar Nano (BMG LABTECH), and the absorbance before and after drug conjugation was compared. The 252 nm absorbance of the humanized anti-Nectin-2 antibody conjugated with SMCC-DM1 was higher than that of the unconjugated antibody. The results are shown in Figure 11, and Figures 11A and 11B confirm that DM1 was potentially conjugated to the humanized anti-Nectin-2 antibody.The anti-humanized Nectin-2 antibodies before and after conjugation were mixed 1:1 with 2X sample buffer containing a reducing agent and 2X sample buffer without, reacted at 95℃ for 10 minutes, and loaded onto 10% and 6% SDS-PAGE gels, respectively, and electrophoresed. Afterwards, they were stained with Coomassie blue solution to confirm the structural changes of the drug-conjugated and unconjugated antibodies. As shown in Figure 11C, the antibody after drug conjugation showed a smeared shape and a slightly larger protein band position than the antibody before conjugation, indicating that the SMCC-DM1 drug existed in a bound state with the antibody and that the drug and antibody were bound at various ratios. The drug antibody ratios (DARs) of the two humanized anti-Nectin-2 antibodies produced, 17284-DM1 and 17294-DM1, were confirmed to be 7.81 and 6.81, respectively.

[0106]

[0107] <Example 11> Confirmation of Nectin-2 Binding Equivalence of Drug-Conjugated ADC of Humanized Anti-Nectin-2 Antibody Using Enzyme-Linked Immunosorbent Assay

[0108] To compare the nectin-2 binding ability of 17284-DM1 and 17294-DM1 developed as ADCs with that of naked antibodies 17284 and 17294, an enzyme-linked immunosorbent assay was performed. Recombinant human nectin-2 protein (100 ng / well, Sinobiological, China) was coated on a 96-well plate overnight at 4°C, and each well was reacted with 1 × DPBS containing 5% BSA for 1 hour and 30 minutes. Subsequently, suspensions diluted by antibody concentration in 1 × DPBS supplemented with 1% BSA and 0.1% Tween 20 were added to each well and reacted for 1 hour at room temperature. Each well was washed three times with 1 × DPBS supplemented with 0.1% Tween-20, and 100 μl per well of HRP-conjugated goat anti-human secondary antibody (533 ng / ml, 1:1500, Invitrogen, IL, United States) diluted in 1 × DPBS supplemented with 1% BSA and 0.1% Tween20 was added, and incubated at room temperature for 1 hour. Each well was washed three times with 1 × DPBS supplemented with 0.1% Tween-20, and 100 μl per well of 1-StepTM Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, Waltham, United States) was added at RT, and the reaction was stopped after 3 minutes by adding 50 μl of 1 N H2SO4 solution. The results were analyzed by measuring absorbance at 450 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, Germany). The results shown in Figure 12 confirmed that the binding affinity for nectin-2 of humanized anti-nectin-2 antibodies and ADCs (17284-DM1, 17294-DM1) conjugated to these antibodies with DM1 were equivalent.

[0109]

[0110] <Example 12> IC50 of humanized anti-Nectin-2 antibody-DM1 conjugate (ADC)

[0111] OV-90, SKOV-2, and Caov-3 cell lines were seeded at 1.25–2.5 × 10 in 96-well plates (Greiner Bio-One, Alphen aan den Rijn, Netherlands). 3 After seeding cells / well, the next day, four types of antibodies (17284, 17284-DM1, 17294, and 17294-DM1) were treated at concentrations of 400, 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512, and 0.001 ug / ml. Afterwards, the cells were cultured at 37°C and 5% CO2 for 3–5 days. For cell counting, the cells were stained with Hoechst 33342 (10 μM; Thermo Fisher Scientific, MA, USA) for 20 min at 37°C and analyzed by Celigo Imaging Cytometer (Nexcelom Bioscience, MA, USA) to verify the drug response. The results are shown in Fig. 13. IC 50 The values ​​are summarized in Table 7, and the IC for the previous chimeric antibody 50 It was confirmed to have cell cytotoxicity at a level similar to that of the value.

[0112] IC of humanized anti-Nectin-2-DM1 50 c12G1*17284-DM117294-DM1Cell lineIC50(μg / ml)IC50(μg / ml)R 2 IC50(μg / ml)R 2 OV-900.01590.020810.98970.016580.9922SK-OV-31.1170.51810.99840.91420.9972Caov-30.53220.45030.97281.1300.9847

[0113] *IC for c12G1 50 Please refer to the paper for the values

[0114]

[0115] 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. A humanized antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, comprising a heavy chain variable region comprising any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 5 to SEQ ID NO: 26; and a light chain variable region comprising any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NO: 27 to SEQ ID NO:

48.

2. In the first paragraph, the humanized antibody or antigen-binding fragment thereof that specifically binds to Nectin-2 comprises a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 27, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 28, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 29, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 8; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 30, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 9; and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 31, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO:

10. And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 32, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 33, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 12; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 34, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 13; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 35, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 14; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 36, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15;And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 37, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 16; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 38, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 17; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 39, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 18; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 40, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 19; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 41, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 20; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 42, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 21; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 43, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 22; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 44, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 23; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 45, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 24; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 46, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 25; And a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 47, or a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 26;A humanized antibody or antigen-binding fragment thereof that specifically binds to Nectin-2, characterized in that it comprises a light chain variable region comprising an amino acid sequence represented by SEQ ID NO:

48.

3. A nucleic acid molecule encoding a humanized antibody or an antigen-binding fragment thereof of claim 1 or 2.

4. A recombinant expression vector comprising the nucleic acid molecule of paragraph 3.

5. Cells isolated by transformation with the recombinant expression vector of Article 4.

6. A composition for detecting Nectin-2 antigen, comprising the humanized antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.

7. A composition for diagnosing cancer in which Nectin-2 is overexpressed, comprising the humanized antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.

8. A composition for diagnosing cancer, characterized in that the cancer in paragraph 7 is breast cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, esophageal cancer, gallbladder cancer, or acute myeloid leukemia.

9. A humanized antibody or antigen-binding fragment thereof according to claim 1 or 2; and an antibody drug conjugate (ADC) to which a drug is bound.

10. An antibody drug conjugate according to claim 9, characterized in that the drug is an anticancer agent.

11. In paragraph 10, 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.

12. An antibody-drug conjugate according to claim 9, characterized in that the humanized antibody or antigen-binding fragment thereof is linked to a drug via a linker.

13. An antibody drug conjugate according to claim 12, 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).

14. A pharmaceutical composition for the prevention or treatment of cancer in which Nectin-2 is overexpressed, comprising the antibody-drug conjugate of Article 9 as an active ingredient.

15. A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer in claim 14 is breast cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, esophageal cancer, gallbladder cancer, or acute myeloid leukemia.

Images