Monoclonal antibody that specifically binds to claudin 18.2 and uses thereof

Abstract

The present invention relates to a monoclonal antibody that specifically binds to claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2) and uses thereof. The antibody of the present invention has high binding affinity to CLDN18.2 and excellent cellular internalization ability, and therefore may be useful in the development of cancer therapeutic agents as a monoclonal antibody or in modified forms (antibody-drug conjugates, chimeric antigen receptors, or multispecific antibodies).

Description

[Technical Field]

[0001] The present invention relates to a monoclonal antibody that specifically binds to claudin-18 spliced ​​variant 2 (CLDN18.2) and uses thereof. [Background technology]

[0002] Claudin 18, with a molecular weight of approximately 27 kDa, belongs to a family of integral membrane proteins present at epithelial-endothelial junctions. Its structure consists of two extracellular loops and four transmembrane (TM) domains, and there are two splice variants, claudin 18.1 (Genebank accession number NM_016369) and claudin 18.2 (Genebank accession number NM_001002026), which differ from each other by only eight amino acids. Claudin 18.1 is expressed in normal lung and gastric epithelium, whereas claudin 18.2 expression is highly restricted in normal cells except for gastric mucosa. It is overexpressed in various cancers, including gastric cancer, liver cancer, biliary tract cancer, breast cancer, kidney cancer, pancreatic cancer, non-small cell lung cancer, and mesothelioma, as well as their metastatic forms (Okugawa T, et al., Dig Dis Sci (2012) 57:1562-7; Rohde C, et al., Jpn J Clin Oncol (2019) 49:870-6). Claudin proteins play an important role in tumorigenesis, metastasis, and inflammation. Altered expression of claudin proteins is known to lead to impaired tight junction function, affecting signaling pathways and promoting tumor growth.

[0003] Therefore, to develop an effective anti-cancer therapeutic agent, it is necessary to develop an antibody that specifically binds only to claudin 18.2, which, unlike claudin 18.1, shows tumor-specific expression.

[0004] Recently, zolbetuximab, a potent chimeric IgG1 mAb (IgG1 monoclonal antibody) that binds to claudin 18.2 on the surface of tumor cells, has been developed and is currently undergoing clinical trials. In particular, zolbetuximab, used as a first-line treatment in a phase 3 trial (SPOTLIGHT trial, NCT03504397), improved progression-free survival (PFS) and overall survival (OS) in patients with claudin 18.2-expressing gastric cancer compared to standard chemotherapy.

[0005] Under these technical backgrounds, the present inventors used human claudin 18.2 protein as an antigen to screen for monoclonal antibodies that specifically bind only to human claudin 18.2 using phage display technology composed of fully human antibody sequences, and confirmed that human antibody-type monoclonal antibodies prepared based on the variable regions of the heavy and light chains of the screened antibody clones can selectively bind to human CLDN18.2 protein expressed on the cell surface and have excellent cellular internalization ability, thereby completing the present invention.

[0006] Meanwhile, Korean Patent Publication No. 2022-0136267 discloses "an antibody-drug conjugate containing an antibody against human CLDN18.2 and uses thereof," and Korean Patent Publication No. 2022-0121873 discloses "an anti-claudin 18.2 antibody and uses thereof," but does not describe the "monoclonal antibody that specifically binds to claudin 18.2 and uses thereof" of the present invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent Publication No. 2022-0136267 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a novel antibody or antigen-binding fragment thereof against claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2).

[0009] Another object of the present invention is to provide a polynucleotide encoding said antibody or antigen-binding fragment thereof.

[0010] Another object of the present invention is to provide a vector containing the polynucleotide, a transformant into which the vector has been introduced, and a method for producing the same.

[0011] It is yet another object of the present invention to provide a composition for treating cancer, comprising said antibody or antigen-binding fragment thereof. [Means for solving the problem]

[0012] To achieve the above object, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2), comprising a heavy chain variable region comprising a heavy chain CDR (complementarity-determining region) 1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 described in SEQ ID NO: 5; a light chain CDR2 consisting of the amino acid sequence DVS; and a light chain CDR3 described in SEQ ID NO: 6.

[0013] The present invention also provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2, comprising a heavy chain variable region comprising a heavy chain CDR1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 8, and a light chain variable region comprising a light chain CDR1 described in SEQ ID NO: 10; a light chain CDR2 consisting of the amino acid sequence AAS; and a light chain CDR3 described in SEQ ID NO: 11.

[0014] The present invention also provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2, comprising a heavy chain variable region including a heavy chain CDR1 set forth in SEQ ID NO: 1; a heavy chain CDR2 set forth in SEQ ID NO: 2; and a heavy chain CDR3 set forth in SEQ ID NO: 13, and a light chain variable region including a light chain CDR1 set forth in SEQ ID NO: 15; a light chain CDR2 consisting of the amino acid sequence GAF; and a light chain CDR3 set forth in SEQ ID NO: 11.

[0015] The present invention also provides polynucleotides encoding the heavy chain variable region and light chain variable region of the monoclonal antibody or antigen-binding fragment thereof.

[0016] The present invention also provides an expression vector comprising the polynucleotide.

[0017] The present invention also provides a non-human transformant transformed with the expression vector.

[0018] The present invention also provides a method for producing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to claudin 18.2 by culturing the transformant.

[0019] The present invention also provides a pharmaceutical composition for treating cancer, comprising the monoclonal antibody or its antigen-binding fragment as an active ingredient.

[0020] The present invention also provides a method for treating cancer, comprising administering the pharmaceutical composition for treating cancer to an individual.

[0021] The present invention also provides a cancer diagnostic composition comprising the monoclonal antibody or its antigen-binding fragment, and a cancer diagnostic kit comprising the cancer diagnostic composition.

[0022] The present invention also provides an antibody-drug conjugate in which a drug is bound to the monoclonal antibody or its antigen-binding fragment.

[0023] The present invention also provides a chimeric antigen receptor (CAR) protein comprising: i) the monoclonal antibody; ii) a transmembrane domain; and iii) an intracellular signaling domain that induces T cell activation when the antibody of i) binds to an antigen.

[0024] The present invention also provides a multi-specific antibody comprising the monoclonal antibody or an antigen-binding fragment thereof. [Effects of the Invention]

[0025] The novel monoclonal antibody that binds to claudin 18.2 (CLDN18.2) according to the present invention is composed of a fully human antibody sequence that is internalized within cells, and exhibits specific antigen binding ability and diverse cellular internalization capabilities, and has been confirmed to have thermal stability. Therefore, it is expected that the monoclonal antibody or modified forms (such as antibody-drug conjugates, chimeric antigen receptors, or multispecific antibodies) can be usefully used for the treatment and / or prevention of cancer diseases and related diseases that express claudin 18.2. [Brief explanation of the drawings]

[0026] [Figure 1] This shows the results of flow cytometry using a HEK293E cell line in which human claudin 18.2 (CLDN18.2) is not expressed. [Figure 2] This shows the results of flow cytometry using a HEK293E cell line (HEK293E / 18.2) in which CLDN18.2 was overexpressed. [Figure 3] This shows the results of flow cytometry using a CHO-K1 cell line (CHO-K1 / 18.2) in which CLDN18.2 is overexpressed. [Figure 4] This shows the results of ELISA analysis using human CLDN18.2 as the coating antigen. [Figure 5] This shows the results of ELISA analysis using human CLDN18.1 as the coating antigen. [Figure 6] These are the results of flow cytometry using the BxPC3 cell line (a cell line that endogenously expresses human CLDN18.1). [Figure 7] FIG. 6 shows the results of flow cytometry to confirm the CLDN18.1 expression level in the BxPC3 cell line used. [Figure 8] The internalization ability of monoclonal antibodies in HEK293E cell lines (CLDN18.2 negative) was analyzed using a live cell analysis device (Incucyte). [Figure 9] The cellular internalization ability of the monoclonal antibody was analyzed in HEK293E / 18.2 cell line (overexpressing CLDN18.2) using a live cell analysis device (Incucyte). [Figure 10] The cellular internalization ability of the monoclonal antibody was analyzed in CHO-K1 / 18.2 cell line (CLDN18.2 overexpression) by cell immunofluorescence staining. [Figure 11] The cellular internalization ability of the monoclonal antibody was analyzed in CHO-K1 / 18.2 cell line (CLDN18.2 overexpression) by cell immunofluorescence staining. [Figure 12] The cellular internalization and cytolytic activity of the monoclonal antibodies were analyzed in HEK293E / 18.2 cell line (overexpressing CLDN18.2) via FabZAP assay. [Figure 13] The cellular internalization and cytolytic activity of the monoclonal antibodies were analyzed in HEK293E cell line (CLDN18.2 negative) via FabZAP assay. [Figure 14] The thermal stability of a monoclonal antibody specific to CLDN18.2 was analyzed by differential scanning fluorimetry. DETAILED DESCRIPTION OF THE INVENTION

[0027] To achieve the objectives of the present invention, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2), comprising a heavy chain variable region comprising a heavy chain CDR (complementarity-determining region) 1 set forth in SEQ ID NO: 1; a heavy chain CDR2 set forth in SEQ ID NO: 2; and a heavy chain CDR3 set forth in SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 5; a light chain CDR2 consisting of the amino acid sequence DVS; and a light chain CDR3 set forth in SEQ ID NO: 6.

[0028] The present invention also provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2, comprising a heavy chain variable region comprising a heavy chain CDR1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 8, and a light chain variable region comprising a light chain CDR1 described in SEQ ID NO: 10; a light chain CDR2 consisting of the amino acid sequence AAS; and a light chain CDR3 described in SEQ ID NO: 11.

[0029] The present invention also provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2, comprising a heavy chain variable region including a heavy chain CDR1 set forth in SEQ ID NO: 1; a heavy chain CDR2 set forth in SEQ ID NO: 2; and a heavy chain CDR3 set forth in SEQ ID NO: 13, and a light chain variable region including a light chain CDR1 set forth in SEQ ID NO: 15; a light chain CDR2 consisting of the amino acid sequence GAF; and a light chain CDR3 set forth in SEQ ID NO: 11.

[0030] The monoclonal antibody of the present invention that specifically binds to claudin 18.2 comprises a heavy chain variable region comprising a heavy chain CDR1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 described in SEQ ID NO: 5; a light chain CDR2 consisting of the amino acid sequence DVS; and a light chain CDR3 described in SEQ ID NO: 6, more specifically, the monoclonal antibody also comprises a heavy chain variable region described in the amino acid sequence of SEQ ID NO: 4 and a light chain variable region described in the amino acid sequence of SEQ ID NO: 7,

[0031] A monoclonal antibody that specifically binds to claudin 18.2 comprises a heavy chain variable region comprising a heavy chain CDR1 set forth in SEQ ID NO: 1; a heavy chain CDR2 set forth in SEQ ID NO: 2; and a heavy chain CDR3 set forth in SEQ ID NO: 8, and a light chain variable region comprising a light chain CDR1 set forth in SEQ ID NO: 10; a light chain CDR2 consisting of the amino acid sequence AAS; and a light chain CDR3 set forth in SEQ ID NO: 11, more specifically, the monoclonal antibody also comprises a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 9 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 12,

[0032] A monoclonal antibody that specifically binds to claudin 18.2 comprises a heavy chain variable region comprising a heavy chain CDR1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 described in SEQ ID NO: 15; a light chain CDR2 consisting of the amino acid sequence GAF; and a light chain CDR3 described in SEQ ID NO: 11, and more specifically, also comprises a heavy chain variable region described in the amino acid sequence of SEQ ID NO: 14 and a light chain variable region described in the amino acid sequence of SEQ ID NO: 16.

[0033] As used herein, the term "monoclonal antibody" refers to a protein molecule that is directed against and specifically binds to a single antigenic site (single epitope). Monoclonal antibodies can be prepared by various methods known in the art.

[0034] In the present invention, the term "antibody" refers to a whole antibody, which has two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG subtypes include IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) subclasses. The light chain constant region has kappa (κ) and lambda (λ) types.

[0035] The monoclonal antibody of the present invention is selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, and a recombinant antibody, and is preferably a human antibody, but is not limited thereto. Since the entire structure of a human antibody is derived from a human, the human antibody has the advantage of being less likely to cause an immune reaction compared to existing humanized or mouse antibodies, and therefore does not induce an undesired immune reaction when administered to a human. Therefore, it can be very useful as a therapeutic antibody.

[0036] In the present invention, the term "antigen-binding fragment" refers to a fragment that retains antigen-binding function and includes Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure comprising light-chain and heavy-chain variable regions, a light-chain constant region, and a heavy-chain first constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy-chain CH1 domain. F(ab')2 antibodies are produced by disulfide bonding between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the heavy-chain variable region and the light-chain variable region, and recombinant techniques for producing Fv fragments are disclosed in International Patent Publication WO88 / 10649, etc. In double-chain Fvs (dsFvs), the heavy-chain variable region and the light-chain variable region are linked by a disulfide bond, while in single-chain Fvs (scFvs), the heavy-chain variable region and the light-chain variable region are covalently linked via a peptide linker. Such antibody fragments can be obtained using protease hydrolases (e.g., whole antibodies can be digested with papain to obtain Fab fragments, or with pepsin to obtain F(ab')2 fragments), or, preferably, can be produced by genetic engineering.

[0037] In the present invention, the term "variable region" refers to a region that exhibits many variations in sequence while performing the function of specifically binding to an antigen, and the variable region contains complementarity-determining regions (CDR1, CDR2, and CDR3). Between the complementarity-determining regions, there is a framework region (FR), which plays a role in supporting the complementarity-determining regions.

[0038] In the present invention, the term "complementarity determining region (CDR)" refers to a cyclic region involved in antigen recognition, and the specificity of an antibody to an antigen is determined by changes in the sequence of this region.

[0039] According to one embodiment of the present invention, the inventors screened a native human single chain Fv library for scFvs that specifically bind to claudin 18.2, and then converted them into an IgG form to produce human antibodies that specifically bind to claudin 18.2.

[0040] In the present invention, the term "biopanning" refers to the process of selecting only phages that express peptides on their surface that have the ability to bind to target molecules (antibodies, enzymes, cell surface receptors, etc.) from a phage library that displays peptides on the outer coat of the phages.

[0041] The monoclonal antibody or antigen-binding fragment thereof of the present invention may be, but is not limited to, glycosylated and / or pegylated to increase its residence time in the body after administration.

[0042] As used herein, the term "glycosylation" refers to a processing method for transferring glycosyl groups to proteins. Glycosylation occurs when glycosyl groups are attached to serine, threonine, asparagine, or hydroxylysine residues of target proteins by glycosyltransferases. These glycosylated proteins not only serve as components of biological tissues but also play an important role in cell recognition on the cell surface. Therefore, in the present invention, the glycosylation of a monoclonal antibody or its antigen-binding fragment, or the glycosylation pattern, can be altered to improve the efficacy of the antibody.

[0043] The term "PEGylation" as used herein refers to a processing method that improves the blood residence time of an antibody by introducing polyethylene glycol into the monoclonal antibody or its antigen-binding fragment. Specifically, PEGylation of polymeric nanoparticles with polyethylene glycol increases the surface hydrophilicity of the nanoparticles, preventing rapid degradation in the body through the so-called stealth effect, which prevents recognition by immune systems, including macrophages, which ingest and digest pathogens, waste products, and foreign substances. Therefore, PEGylation can improve the blood residence time of an antibody. The PEGylation used in the present invention is formed by forming an amide group by bonding the carboxyl group of hyaluronic acid with the amine group of polyethylene glycol, but various methods for PEGylation are possible. The polyethylene glycol used in this method is not particularly limited, but preferably has a molecular weight of 100 to 1,000 and a linear or branched structure.

[0044] The glycosylation and / or pegylation may be modified to various glycosylation and / or pegylation patterns by methods known in the art, as long as the function of the antibody of the present invention is maintained. The antibody of the present invention includes any mutant monoclonal antibody or antigen-binding fragment thereof having various modified glycosylation and / or pegylation patterns.

[0045] The term "affinity" refers to the ability to specifically recognize and bind to a specific site on an antigen. Along with the specificity of an antibody to an antigen, high affinity is an important factor in immune reactions. Various methods known in the art can be used to measure affinity for an antigen, such as surface plasmon resonance technology.

[0046] The present invention also provides polynucleotides encoding the heavy chain variable region and light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0047] Those skilled in the art will readily understand that polynucleotides encoding the light and heavy chains of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be modified in various ways within the coding region without altering the amino acid sequence of the antibody light and heavy chains expressed from the coding region, taking into account codon degeneracy or the codon preferences of the organism in which the human antibody light and heavy chains or fragments thereof are to be expressed. Various modifications or alterations may also be made to portions excluding the coding region without affecting gene expression, and such modified genes are also within the scope of the present invention. That is, the polynucleotides of the present invention may be modified by substitution, deletion, insertion, or a combination of one or more nucleic acid bases, as long as they encode proteins with equivalent activity. Such polynucleotide sequences may be single-stranded or double-stranded, and may be DNA or RNA (mRNA) molecules.

[0048] The present invention also provides an expression vector comprising a polynucleotide encoding the heavy and light chain variable regions of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0049] As used herein, the term "expression vector" refers to a means for expressing a gene of interest in a host cell, including plasmid vectors; viral vectors such as cosmid vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors; and refers to a genetic construct containing essential regulatory elements operably linked to allow a gene insert to be expressed. As used herein, "operably linked" refers to the functional linkage between a nucleic acid expression regulatory sequence and a polynucleotide encoding a protein of interest so that they can perform their general functions. Operable linkage with an expression vector can be achieved using recombinant DNA techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes commonly known in the art.

[0050] In addition to expression regulatory elements such as promoters, initiation codons, termination codons, polyadenylation signals, and enhancers, suitable expression vectors of the present invention may also contain signal sequences for membrane targeting or secretion. The initiation and termination codons are generally considered part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in an individual when the gene construct is administered, and must be located in frame with the coding sequence. General promoters can be constitutive or inducible. Examples of promoters for prokaryotic cells include, but are not limited to, Lac, tac, T3, and T7 promoters. In eukaryotic cells, examples of promoters include, but are not limited to, the simian virus 40 (SV40) promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter, the HIV long terminal repeat (LTR) promoter, Moloney virus promoter, cytomegalovirus (CMV) promoter, and Epstein-Barr virus (EBV) promoter, as well as promoters derived from β-actin, human hemoglobin, human muscle creatine, and human metallothionein.

[0051] An expression vector can contain a selectable marker for selecting host cells containing the vector. The selectable marker is used to select cells transformed with the vector, and can be a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. Transformed cells can be selected because only cells expressing the selectable marker survive in an environment treated with a selective agent. Furthermore, if the vector is a replicable expression vector, it contains a replication origin, which is a specific polynucleotide from which replication is initiated. Vectors that are inserted into the genome of a host cell, such as viral (e.g., baculovirus) or phage vectors, and retroviral vectors, can also be used. Vectors expressing whole antibodies or antibody fragments can be either vector systems in which the light chain and heavy chain are co-expressed in a single vector, or systems in which the light chain and heavy chain are expressed in separate vectors. In the latter case, the two vectors are introduced into host cells via cotransformation and targeted transformation, followed by selection of cells transformed with the vector containing the light chain (or heavy chain), selection of cells expressing the light chain, and then retransformation with the vector containing the heavy chain (or light chain), followed by final selection of cells expressing both the light and heavy chains.

[0052] To produce Fab-type antibodies, a vector is used to insert genes encoding the amino acids of the human light chain variable region (VL) and constant region (CL) and the human heavy chain variable region (VH) and first constant region domain (CH1).

[0053] The present invention also provides a non-human transformant transformed with the expression vector of the present invention.

[0054] Suitable host cells for the vectors include prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp., fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa, other lower eukaryotic cells, and higher eukaryotic cells such as cells from insects and plant cells. The cells may also include, but are not limited to, mammalian-derived cells such as COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, HepG2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080 cells.

[0055] In the present invention, "transformation into a host cell" includes any method for introducing nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique appropriate for the host cell as known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, agitation using silicon carbide fibers, Agrobacteria-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation / quenching-mediated transformation.

[0056] The present invention also provides (a) culturing the transformant to produce a culture medium; (b) purifying the monoclonal antibody or antigen-binding fragment thereof of the present invention from the culture medium of step (a).

[0057] In the above-mentioned preparation method, the transformant is cultured in an appropriate medium and under appropriate conditions known in the art, and those skilled in the art can easily adjust and use such a culture process depending on the selected bacterial strain or animal cell.

[0058] Antibodies obtained by culturing transformants may be used unpurified, or may be further purified using various conventional methods, such as centrifugation or ultrafiltration to remove impurities, followed by dialysis, salt precipitation, chromatography, etc., which may be used alone or in combination. Among these, affinity chromatography is the most commonly used, followed by ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. Antibodies prepared by these methods have increased affinity for their antigens.

[0059] The present invention also provides a pharmaceutical composition for cancer treatment, comprising, as an active ingredient, the monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2.

[0060] The monoclonal antibody or antigen-binding fragment thereof is as described above.

[0061] The pharmaceutical composition of the present invention inhibits tumor growth in a subject when the antibody of the present invention is administered to the subject.

[0062] The term "treatment of cancer" as used herein means any action that improves or alleviates the symptoms of cancer by administering the composition.

[0063] In the pharmaceutical composition according to the present invention, the cancer may be selected from the group consisting of pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, gastric cancer, colon cancer (e.g., colon cancer, rectum cancer), liver cancer, biliary tract cancer, gallbladder cancer, breast cancer, kidney cancer, mesothelioma, head and neck cancer, bladder cancer, cervical cancer, endometrial cancer, fallopian tube cancer, gastrointestinal cancer, hematological cancer (e.g., leukemia, lymphoma, or myeloma), pharyngeal cancer, melanoma, primary peritoneal cancer, salivary gland cancer, sarcoma, thyroid cancer, glioblastoma, and prostate cancer, but is not limited thereto.

[0064] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, which may include a non-naturally occurring carrier.

[0065] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Pharmaceutical carriers acceptable for compositions formulated into liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable dosage forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, and tablets.

[0066] The cancer therapeutic composition of the present invention, comprising the monoclonal antibody and a pharmaceutically acceptable carrier, can be applied to any dosage form containing the monoclonal antibody as an active ingredient, and can be prepared into oral or parenteral dosage forms. Pharmaceutical dosage forms of the present invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or forms suitable for administration by inhalation.

[0067] Oral dosage forms containing the pharmaceutical composition of the present invention as an active ingredient can be formulated, for example, as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. Formulations into dosage forms such as tablets and capsules may contain binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, and lubricants such as massnezium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In the case of capsules, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned substances.

[0068] Dosage forms for parenteral administration containing the pharmaceutical composition of the present invention as an active ingredient may be formulated as injections such as subcutaneous injections, intravenous injections, or intramuscular injections, as suppository injections, or as sprays such as aerosols that can be inhaled through the respiratory tract. To formulate as an injection, the composition of the present invention is mixed with water along with a stabilizer or buffer to prepare a solution or suspension, which is then formulated into a unit dose in an ampule or vial. To be injected as a suppository, the composition may be formulated as a rectal administration composition such as a suppository or enema containing a conventional suppository base such as cocoa butter or other glycerides. When formulated as a spray, such as an aerosol, a propellant or other additive may be added to disperse a water-dispersed concentrate or a wettable powder.

[0069] The pharmaceutical composition may be administered in a pharmaceutically effective amount.

[0070] As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level can be determined based on factors including the type and severity of the individual, age, sex, type of cancer, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The compositions of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, they can be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy at the minimum dose without adverse effects, and this can be easily determined by one skilled in the art.

[0071] The pharmaceutical composition of the present invention may further include one or more known active ingredients having anti-cancer effects, and the known active ingredients having anti-cancer effects are preferably, but not limited to, chemotherapeutic agents or immune checkpoint inhibitors.

[0072] The chemotherapeutic agent may be at least one selected from the group consisting of alkylating anticancer agents such as carboplatin and paclitaxel; antimetabolite anticancer agents such as gemcitabine; anthracycline anticancer agents such as doxorubicin; and proteasome inhibitor anticancer agents such as bortezomib, but is not limited thereto. Immune checkpoint inhibitors may also target one or more selected from the group consisting of, but not limited to, PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, Transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0073] The present invention also provides a method for treating cancer, comprising administering the pharmaceutical composition for treating cancer to an individual.

[0074] In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention into a patient by an appropriate method. The administration route of the composition of the present invention can be through various routes, such as oral or parenteral, as long as it can reach the target tissue. Specifically, it can be administered in a conventional manner through oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular, or intradermal routes.

[0075] The therapeutic method of the present invention involves administering a pharmaceutically effective amount of the cancer therapeutic composition of the present invention. It will be apparent to those skilled in the art that the appropriate total daily dose will be determined by the treating physician within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will vary depending on a variety of factors, including the type and degree of response to be achieved, whether or not other formulations are used, the specific composition, the patient's age, weight, general health, sex, and diet, the time and route of administration, the excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or concomitantly with the specific composition, as well as similar factors well known in the pharmaceutical arts. Therefore, the effective amount of the cancer therapeutic composition suitable for the purpose of the present invention should be determined taking into account the above factors.

[0076] Furthermore, the individual refers to any animal in which diseases such as tumor development and neovascularization may occur due to excessive activity of claudin 18.2, and the animal includes not only humans and primates but also livestock such as cows, pigs, sheep, horses, dogs, and cats, and preferably mammals other than humans, but is not limited thereto.

[0077] The present invention also provides a cancer diagnostic composition comprising the monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2, and a cancer diagnostic kit comprising the cancer diagnostic composition.

[0078] The monoclonal antibody or its antigen-binding fragment, and cancer are as described above. A diagnostic composition comprising the claudin 18.2-specific monoclonal antibody or its antigen-binding fragment of the present invention can be used to diagnose diseases related to the expression and level of claudin 18.2, such as cancer.

[0079] The method for diagnosing cancer involves reacting the monoclonal antibody specific to claudin 18.2 of the present invention with a biological sample isolated from an individual suspected of having cancer, and analyzing the formation of an antigen-antibody complex, thereby providing information for diagnosing cancer.

[0080] In the present invention, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. These biological samples, whether manipulated or unmanipulated, can be reacted with the antibodies of the present invention to confirm the presence of claudin-18.2 or the presence or absence of cancer.

[0081] In the present invention, the term "antigen-antibody complex" refers to a complex between a claudin 18.2 protein antigen in a sample and a monoclonal antibody or its antigen-binding fragment according to the present invention that recognizes the antigen. The formation of such an antigen-antibody complex can be measured by a conventional immunoassay method, including, but not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competitive assay, sandwich assay, flow cytometry, and fluorescence immunoassay using an antibody against claudin 18.2.

[0082] Cancer can be diagnosed by analyzing the intensity of the final signal obtained by the immunoassay process. That is, if the claudin 18.2 protein of the present invention is overexpressed in a biological sample isolated from a suspected individual, and the signal is stronger than that in a biological sample isolated from a normal individual, cancer is diagnosed.

[0083] Furthermore, the kit according to the present invention includes an antibody against claudin 18.2 of the present invention, and can diagnose cancer by analyzing a signal generated by the reaction of the antibody with a sample. In this case, the signal is generated by an enzyme (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, or cytochrome P450, etc.) bound to the antibody. In this case, when alkaline phosphatase is used as the enzyme, the substrate for the enzyme is bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, or enhanced erythrocyte colony fluoride (ECF). When a colorimetric reaction substrate such as chemifluorescence is used, and when horseradish peroxidase is used as the enzyme, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), reserpine benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (o-Phenylenediamine, OPD), and naphthol / pyronin, glucose oxidase with t-NBT, and m-PMS (phenzaine methosulfate) can be used, but are not limited to these.

[0084] The kit according to the present invention also includes a label that generates a detectable signal, and the label may be a chemical (e.g., biotin), an enzyme (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P450), a radioactive material (e.g., 14 C. 125 I, 32P and 35 S), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and FRET (fluorescence resonance energy transfer).

[0085] The activity or signal of an enzyme used for cancer diagnosis can be measured by various methods known in the art, through which the expression of claudin 18.2 can be analyzed qualitatively or quantitatively.

[0086] The present invention also provides an antibody-drug conjugate in which a drug is bound to the monoclonal antibody or its antigen-binding fragment that specifically binds to claudin 18.2.

[0087] In the present invention, the term "drug" means a compound that binds to an antibody or antigen-binding fragment thereof specific to claudin 18.2 of the present invention, is separated from the antibody or antigen-binding fragment thereof under acidic conditions, and exhibits a therapeutic effect on target cells.

[0088] Drugs used in antibody-drug conjugates according to the present invention include any compound, moiety, or group that has a cytotoxic or cytostatic effect, and include (i) chemotherapeutic agents that function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators; (ii) enzymatically functioning protein toxins; and (iii) radioactive isotopes (radionuclides), of which one or more may be used.

[0089] Non-limiting examples of such drugs include maytansinoids, auristatins, dolastatins, trichothecenes, CC1065 (Rachelmycin), calicheamicin and other enediyne antibiotics, taxanes, anthracyclines, methotrexate, adriamycin, vindesine, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, carminomycin, aminopterin, dactylate ... These include, but are not limited to, cyclopropyl ... 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, etc., but are not limited thereto, and microRNA (miRNA), siRNA, shRNA, etc., which can suppress the expression of a specific oncogene, may also be used.

[0090] The present invention also provides a chimeric antigen receptor (CAR) protein comprising: i) a monoclonal antibody that specifically binds to claudin 18.2; ii) a transmembrane domain; and iii) an intracellular signaling domain that induces T cell activation when the antibody of i) binds to an antigen.

[0091] In the present invention, the CAR protein can be specified as being composed of the monoclonal antibody of the present invention, a known transmembrane domain, and an intracellular signaling domain.

[0092] In the present invention, the term "CAR (chimeric antigen receptor)" refers to a receptor that does not exist in nature and can confer specificity for a particular antigen to an immune effector cell. Typically, the CAR refers to a receptor used to transfer the specificity of a monoclonal antibody to a T cell. A CAR typically consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains an antigen-binding site, and in the present invention, the antigen-binding site is an antibody specific to claudin 18.2. The claudin 18.2-specific antibody is as described above. The antibody used in the CAR is preferably in the form of an antibody fragment, more preferably in the form of Fab or scFv, but is not limited thereto.

[0093] The transmembrane domain of the CAR, linked to the extracellular domain, may be naturally or synthetically derived. When derived from a naturally occurring protein, it may be derived from a membrane-bound or transmembrane protein, such as the alpha, beta, or zeta forms of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or CD8. The sequence of such a transmembrane domain may be obtained from, but is not limited to, literature known in the art that describes the transmembrane domain portions of transmembrane proteins. When the transmembrane domain is synthetic, it may primarily contain hydrophobic amino acid residues such as leucine and valine. For example, a synthetic transmembrane domain may contain, but is not limited to, a triplet of phenylalanine, tryptophan, and valine.

[0094] In the CAR of the present invention, the intracellular domain is a part of the CAR domain present intracellularly and is linked to a transmembrane domain. The intracellular domain of the present invention may include an intracellular signaling domain that induces T cell activation, preferably T cell proliferation, when an antigen binds to the antigen-binding site of the CAR. The intracellular signaling domain is not particularly limited as long as it transmits a signal that induces T cell activation when an antibody binds to the extracellular antigen-binding site. Various types of intracellular signaling domains can be used, including, but not limited to, immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAMs may include, but are not limited to, those derived from CD3 zeta (ξ, zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d, or FcεRIγ.

[0095] Furthermore, the intracellular domain of the CAR of the present invention preferably further comprises a costimulatory domain in addition to the intracellular signaling domain, but is not limited thereto. The costimulatory domain is a portion of the CAR of the present invention that transmits a signal to T cells in addition to the signal transmitted by the intracellular signaling domain, and refers to the intracellular portion of the CAR including the intracellular domain of a costimulatory molecule. The costimulatory molecule refers to a cell surface molecule required to induce a sufficient lymphocyte response to an antigen, and examples thereof include, but are not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. The costimulatory domain can also be the intracellular portion of a molecule selected from the group consisting of such costimulatory molecules and combinations thereof.

[0096] Alternatively, a short oligopeptide or polypeptide linker can link the intracellular domain and transmembrane domain of the CAR, and the linker is not particularly limited in length as long as it is a linker that can induce T cell activation via the intracellular domain when an antigen binds to an extracellularly located antibody, even when included in the CAR of the present invention.

[0097] The present invention also provides a multispecific antibody comprising the monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2.

[0098] In the present invention, the multispecific antibody is preferably a bispecific antibody, but is not limited thereto.

[0099] The multispecific antibody of the present invention is preferably a form in which the anti-claudin 18.2 antibody of the present invention is conjugated to an antibody or fragment thereof capable of binding to an immune cell-specific target molecule, preferably selected from, but not limited to, PD-1, PD-L1, CTLA-4, TIM-3, TIGIT, BTLA, KIR, A2aR, VISTA, B7-H3, TCR / CD3, CD16 (FcγRIIIa), CD44, Cd56, CD69, CD64 (FcγRI), CD89, and CD11b / CD18 (CR3).

[0100] A multispecific antibody is an antibody that simultaneously recognizes multiple (dual or more) different epitopes on the same antigen or two or more antigens, and antibodies belonging to the multispecific antibody category can be divided into scFv-based antibodies, Fab-based antibodies, IgG-based antibodies, etc. Multispecific, for example, bispecific, antibodies can simultaneously suppress or amplify two signals, which is more effective than suppressing / amplifying a single signal. Compared to treating each signal with a separate signal suppressor, lower dosages are possible, and two signals can be suppressed / amplified at the same time and space.

[0101] Methods for producing bispecific antibodies are widely known. Traditionally, recombinant production of bispecific antibodies relies on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities.

[0102] In the case of scFv-based bispecific antibodies, hybrid scFvs are prepared in a heterodimeric form by combining the light chain variable regions (VL) and heavy chain variable regions (VH) of different scFvs with each other to form diabodies (Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444, 1993). Different scFvs are linked to each other to prepare tendon scFvs, and the CH1 and CL of Fab are expressed at the termini of each scFv to prepare heterodimeric miniantibodies (Muller et al., FEBS lett., 432:45, 1998). Finally, some amino acids in the CH3 domain, which is the homodimeric domain of Fc, are switched to form a "knob-into-hole" heterodimeric structure, and these altered CH3 domains are expressed at the termini of different scFvs to form heterodimeric miniantibodies. Minibodies in scFv format can be produced (Merchant et al., Nat. Biotechnol., 16:677, 1998).

[0103] In the case of Fab-based bispecific antibodies, heterodimeric Fabs can be prepared by combining individual Fab's against specific antigens using a disulfide bond or an intermediary. Bivalent Fabs can be prepared by expressing scFvs against different antigens at the heavy or light chain ends of a specific Fab, resulting in two antigen binding valencies, or by inserting a hinge region between the Fab and scFvs to prepare homodimeric Fabs with four antigen binding valencies. Additionally, methods are known in the art for preparing bivalent bibodies, which have three antigen binding valencies by fusing scFvs against different antigens to the light and heavy chain ends of Fab; trivalent bibodies, which have three antigen binding valencies by fusing different scFvs to the light and heavy chain ends of Fab; and a simple trivalent antibody, F(ab')3, obtained by chemically conjugating three different Fabs.

[0104] In the case of IgG-based bispecific antibodies, a method is known in which TrionPharma crossbred mouse and rat hybridomas to produce hybrid hybridomas, also known as quadromas, to produce bispecific antibodies. Another method is to produce a "holes and knob" bispecific antibody, in which the light chain is shared but the heavy chains are heterodimeric by modifying some amino acids in the Fc CH3 homodimeric domain (Merchant et al., Nat. Biotechnol., 16:677, 1998). In addition to heterodimeric bispecific antibodies, a method is also known in which two different scFvs are fused to the constant domains of IgG light and heavy chains, respectively, to produce a homodimeric (scFv)4-IgG. ImClone reported the creation of a bispecific antibody based on the chimeric monoclonal antibody IMC-1C11 against human VEGFR-2 by fusing only a single variable domain against mouse platelet-derived growth factor receptor-α to the amino terminus of the light chain of this antibody. Rossi et al. also reported the creation of a multivalent antibody against CD20 using the so-called "dock and lock" (DNL) method, which uses the dimerization and docking domain (DDD) of the protein kinase A (PKA) R subunit and the anchoring domain of PKA (Rossi et al., Proc. Natl. Acad. Sci. USA, 103:6841, 2006).

[0105] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. [Example]

[0106] Example 1. Selection of claudin 18.2 (CLDN18.2)-specific antibodies The CLDN18.2 (R&D, #RDC2149) gene was overexpressed in HEK293E cell lines to establish a cell line. CLDN18.2-VLPs antigen (Cusabio, #CSB-MP005498HU(A5)) was coated onto an immunosorbent tube, followed by blocking. Human scFv library phage (Y Biologics, Inc.) was infected into E. coli, which was then cultured at 30°C for 16 hours. The culture medium was centrifuged, and the supernatant was concentrated with PEG (polyethylene glycol), then dissolved in PBS (phosphate buffered saline) to prepare human antibody library phage.

[0107] The library phages were placed in the immunosorbent tube and incubated at room temperature for 2 hours. After washing with 1x PBST and 1x PBS, the tube was treated sequentially with 100 mM TAE and Tris-HCl (pH 7.5) to elute only the scFv phages that specifically bound to the antigen. Panning and ELISA were then performed to select three positive phage clones that specifically bound to CLDN18.2. The scFv clones were then converted to IgG and used in the experiments.

[0108] The sequences of the heavy and light chain CDRs and the heavy and light chain variable regions containing them of the selected monoclonal antibodies are shown in Tables 1 and 2 below.

[0109] [Table 1]

[0110] [Table 2]

[0111] Example 2. Properties of selected CLDN18.2-specific monoclonal antibodies 2-1. Non-specificity of CLDN18.2 monoclonal antibodies via flow cytometry Nonspecific binding experiments were performed with the HEK293E cell line. Human CLDN18.2 expression was confirmed using anti-CLDN18.2 (CUSABIO, #CSB-RA005498A1HU).

[0112] 0.5x10 HEK293E cells that do not express human CLDN18.2 per sample 6 The cells were then incubated with the selection antibodies 0058-001, 0058-002, and 0058-003 at a concentration of 7.5 μg / ml for 30 minutes at 4°C. The cells were then washed three times with 2% fetal bovine serum in PBS (Welgene, #LB001-02) and incubated with a fluorescently conjugated anti-human IgG antibody (Vectorlabs, #FI-3000) at 4°C for 30 minutes, followed by washing in the same manner as above. The cells were suspended in 0.2 ml of 2% FBS in PBS (Thermo, #26140-079) and analyzed for binding affinity using a CytoFlex flow cytometer (Beckman Coulter, USA).

[0113] The analysis showed that none of the selected antibodies 0058-001, 0058-002, and 0058-003 bound to the HEK293E cell line, which does not express CLDN18.2 (Figure 1).

[0114] 2-2. Specificity of CLDN18.2 monoclonal antibody via flow cytometry 0.5x10 HEK293E / 18.2 cells and CHO-K1 / 18.2 cells overexpressing human CLDN18.2 were used per sample. 6The cells were prepared and the CLDN18.2 antibody was diluted at a certain ratio and reacted with the prepared cells for 30 minutes at 4°C. The cells were then washed three times with PBS containing 2% fetal bovine serum, reacted with an anti-human IgG antibody conjugated with FITC fluorescent dye for 20 minutes at 4°C, and washed in the same manner as above. The cells were suspended in 0.5 ml of PBS containing 2% FBS, and the binding strength was analyzed using a CytoFlex flow cytometer.

[0115] As a result of the analysis, it was confirmed that the anti-CLDN18.2 selected antibodies 0058-001, 0058-002, and 0058-003 all bound to HEK293E / 18.2 and CHO-K1 / 18.2 cell lines, which express CLDN18.2 at high levels, in a concentration-dependent manner (Figures 2 and 3).

[0116] Example 3. Analysis of binding affinity of selected CLDN18.2-specific monoclonal antibodies to CLDN18 isoforms Claudin 18.1 is an antigen expressed in normal lung and gastric epithelium. The CLDN18.1 / 18.2 isoforms are created depending on which ATG in the first exon of the claudin 18 gene is used as the initiation codon, and the protein sequences are 92% identical. Therefore, to select antibodies that specifically bind only to claudin 18.2 and not to claudin 18.1, we analyzed their selective binding ability to the isoforms.

[0117] 3-1. Binding Ability Analysis of CLDN18 Isoforms via ELISA CLDN18.1 (EUPROTEIN, #EPY255141) and CLDN18.2 (Cusabio, #CSB-MP005498HU(A5)) proteins were diluted to 1 μg / ml using a diluent (4% skim milk / 0.05% Tween-20 / PBS) and 100 μl of each was added to each well of an immunoplate (Thermo, #439454) and incubated for at least 16 hours at 4°C. After washing three times with 300 μl of washing solution (0.05% Tween-20 / PBS), 200 μl of blocking solution (4% skim milk / 0.05% Tween-20 / PBS) was added to the well and incubated for 1 hour at room temperature. After three washes, 100 μl of anti-CLDN18.2 antibody diluted at a fixed fold was added to the well and rocked at room temperature for 1 hour. After washing three times, 100 μl of anti-human kappa-HRP (Sigma, #A7164) diluted 1:3,000 was added to each well and incubated at room temperature for 1 hour. After washing three times, 100 μl of TMB substrate (Sigma, #T0440) was added to each well and incubated at room temperature in the dark. When color development was observed, 50 μl of stop solution (1N H2SO4) was added to stop the reaction. The absorbance was measured using a spectrophotometer (Promega, GM3000).

[0118] As a result of the analysis, the selected antibodies 0058-001, 0058-002, and 0058-003 bound to the CLDN18.2 antigen (Figure 4), but none of them bound to the CLDN18.1 antigen (Figure 5), confirming their selectivity for the CLDN18.2 antigen.

[0119] 3-2. Analysis of binding ability to CLDN18 isoforms via flow cytometry BxPC3 cells (Figure 7), which endogenously express human CLDN18.1, were added to the sample at 0.5x10 cells per sample. 6The cells were then incubated with 50 nM of CLDN18.1 antibody (Cusabio, #CSB-RA005498A2HU) and CLDN18.2 antibody of the present invention at 4°C for 30 minutes. The cells were then washed three times with PBS containing 2% fetal bovine serum, incubated with an anti-human IgG antibody conjugated with FITC fluorescent dye at 4°C for 30 minutes, and washed in the same manner as above. The cells were suspended in 0.2 ml of PBS containing 2% FBS, and the binding affinity was analyzed using a CytoFlex flow cytometer.

[0120] As a result, it was confirmed that the anti-CLDN18.1 antibody bound to the BxPC3 cell line, but none of the anti-CLDN18.2 selected antibodies bound to the BxPC3 cell line (Figure 6). These results confirmed that the selected antibodies 0058-001, 0058-002, and 0058-003 did not bind to the CLDN18.1 isoform, but selectively bound to the CLDN18.2 antigen.

[0121] Example 4. Cellular internalization ability of selected CLDN18.2-specific monoclonal antibodies 4-1. Analysis of cellular internalization using INCUCYTE The cellular internalization ability of the anti-CLDN18.2 control antibody SC0080 and the selected antibodies 0058-001, 0058-002, and 0058-003 was evaluated by conjugating the antibodies with Incucyte® FabFluor human red fluorescent reagent (Essen Bioscience, USA) and then applying them to HEK293E / 18.2 cells overexpressing CLDN18.2 and HEK293E cells not expressing CLDN18.2. The amount of antibody that entered the cells over time was monitored. 1 x 10 per well of a 96-well plate (Nunc, USA) was used. 4Cells were aliquoted and allowed to adhere for 24 hours. 4 μg / ml of each antibody was mixed with an equal volume of Incucyte® FabFluor red antibody labeling reagent, incubated in the dark at 37°C for 15 minutes, and then applied to the cells. The amount of antibody cellular influx was quantified by monitoring the amount of antibody accumulated in the lysosomes over time using an Incucyte ZOOM HD / 2CLR System (Essen Biosciences, USA) every 15 minutes for a total of 24 hours.

[0122] As a result, cell surface-bound anti-CLDN18.2 selected antibodies 0058-001, 0058-002, and 0058-003 did not show intracellular uptake in HEK293E cells, which do not express CLDN18.2 (Figure 8). However, in HEK293E / 18.2 cells, 50% of the antibodies were internalized within 5 hours (Figure 9). In particular, selected antibodies 0058-001 and 0058-002 demonstrated superior cellular internalization compared to the control antibody (Ybiologics, #SC0080; IgG mAb zolbetuximab in-house). These results demonstrate that all anti-CLDN18.2 selected antibodies can function as target-specific intracellular drug delivery antibodies, suggesting their potential for development as antibody-drug conjugates.

[0123] 4-2. Analysis of cellular internalization using cell immunofluorescence To determine whether the anti-CLDN18.2 antibody can enter cells after binding to antigens present on the cell surface, an endocytosis assay was performed in CLDN18.2-overexpressing CHO-K1 / 18.2 cells, and immunocytochemistry (ICC) was performed to confirm the intracellular location of the antibody. For reference, CHO-K1 / 18.2 cells overexpress claudin 18.2 approximately 161-fold compared to CHO-K1 cells, as confirmed by the same method as in Example 2-2 (see bottom of Figure 10).

[0124] 12mm coverslips coated with PPL (Sigma-Aldrich, #P4707) were placed in a 24-well plate and 5x10 cells were plated per well. 4 The cells were seeded with 10 μg / ml of anti-CLDN18.2 antibodies 0058-001, 0058-002, and 0058-003, and incubated at 4°C for 1 hour to allow the antibodies to bind to antigens present on the cell surface. The cells were then washed with cold PBS and replaced with complete medium [RPMI-1640 (GIBCO, #A1049101), 1% ANTI-ANTI (Thermo Fisher, #15240062), 10% FBS (Thermo Fisher, #26140-079)] pre-warmed to 37°C to induce cellular internalization of the antibodies. The medium was then incubated at 37°C in 5% CO. 2The cells were cultured for 24 hours under these conditions. After incubation, the cells were washed three times with PBS to remove the medium and then fixed with 4% paraformaldehyde. The fixed cells were then washed three times with PBS and incubated for 1 hour at room temperature with an endosome-labeling antibody diluted in PBS containing 0.5% saponin (Sigma, #47036) and 10% FBS. For endosome labeling, anti-rabbit LAMP1 (Abcam, USA) was used as a late endosome / lysosome-labeling antibody. After antibody binding, cells were washed three times with 10% FBS-containing PBS for 3 minutes each. Secondary antibodies for the anti-CLDN18.2 antibody, FITC-conjugated anti-human IgG (Jackson ImmunoResearch, #709-545-149) and anti-LAMP1 antibody, Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch, #711-165-152), were diluted 1:200 in 0.5% saponin and 10% FBS-containing PBS and incubated for 1 hour in the dark. Fluorescently stained cells were washed three times with PBS for 3 minutes each. After applying DAPI-containing mounting solution (Vector Laboratories, #H1200) to the slide, the slide was covered with a cover glass to avoid air bubbles and then observed under a confocal laser scanning microscope (ZEISS, LSM9; ZEISS, LSM9, Germany). The signal information for each fluorescent color is as shown in Table 3 below.

[0125] [Table 3]

[0126] The control antibody (SC0080) and the three CLDN18.2 monoclonal antibodies were confirmed to enter cells after endocytosis induction. SC0117, an isotype control that does not bind to CLDN18.2, was not observed intracellularly at 0 and 24 hours (Figures 10 and 11). Meanwhile, the anti-CLDN18.2 antibodies frequently colocalized with anti-LAMP1, an endosome / lysosome-targeting antibody, indicating that the anti-CLDN18.2 antibodies were delivered to lysosomes. This reaffirms that the anti-CLDN18.2-selected antibodies 0058-001, 0058-002, and 0058-003 are all suitable antibodies for use in antibody-drug conjugates for the delivery of cytotoxic substances into cells.

[0127] Example 5. Efficacy test of selected CLDN18.2-specific monoclonal antibodies in CLDN18.2-expressing cancer cells The cellular internalization and cell cytotoxicity of three anti-CLDN18.2 antibodies produced by Y Biologics Inc. were examined using the Fab ZAP Human Antibody Internalization Kit (ATSbio, #KIT-51-Z4).

[0128] In a sterile laboratory bench (Biosafety cabinet, #JSCB-1500SB, JSR), HEK293E / CLDN18.2-overexpressing cell lines or HEK293E parental cell lines were cultured at 1x10 in 100 μl of DMEM (HyClone, #SH30243.01). 3 Place 100 cells into each well of a 96-well plate and incubate at 5% CO 2The cells were then cultured in a 37°C incubator for 16 hours. On the day of antibody treatment, Saporin (ATSbio, #SAP-200), a ribosome-inactivating protein included in the Fab-ZAP Kit, was diluted in 10-fold increments from 10,000 nM (10x the treatment concentration) to 0.001 nM, for a total of eight concentrations. To prepare the ZAP spike medium for diluting the test antibody, Fab-ZAP Human (ATSbio, #IT-51-40) was prepared in DMEM to a concentration of 45 nM. This was then used to prepare the antibody in 10-fold increments from 100 nM (10x the treatment concentration) to 0.00001 nM, for a total of eight concentrations. Human IgG (Invitrogen, #31154) was used as a control for the test antibody. Fab IgG-SAP (ATSbio, #IT-67-40), a control substance for saporin (Con-SAP), was prepared in DMEM cell culture medium at 100 nM, 10 times the treatment concentration. Using a multi-tube, 10 μl of the prepared saporin, test antibody, and Fab IgG-SAP were added to each well of a 96-well plate that had been cultured for 16 hours. The plate was then incubated in 5% CO 2 The cells were cultured in an incubator at 37°C for 72 hours.

[0129] On a sterile laboratory bench, mix 5.5 ml of DPBS (HyClone, #SH30028.02) with 5.5 ml of XTT (ATSbio, #XTT) from the Fab ZAP Kit to make 11 ml of DPBS / XTT (1:1) reagent, then add 92 μl of PMS (ATSbio, #PMS-400) from the kit to make the XTT / PMS reagent. After 72 hours of incubation, remove the plate and dispense 50 μl of XTT / PMS reagent into each well treated with antibody and saporin. Then, place the plate in a 5% CO atmosphere. 2 The cells were cultured in an incubator at 37°C for 2 hours.

[0130] After 2 hours of incubation, the plate was removed and absorbance was measured at 450 nm using a GloMax® Discover Microplate Reader (Promega, #GM3000). Cell viability (%) was calculated using a 4-parameter nonlinear function curve (log vs response-variable slope 4 parameters) in GraphPad Prism (version 9.4), with the value of the wells not treated with the test antibody set at 100%. 50 The half maximal inhibitory concentration (the amount of antibody required to induce 50% cell death) was calculated.

[0131] As a result, as shown in Table 4, Figures 12 and 13, no cytotoxicity of the test antibody was observed in the HEK293E parent cell line, which does not express the antigen, but in the HEK293E / 18.2 cell line, in which CLDN18.2 was overexpressed, a cytotoxic effect was observed at the highest concentration of the test antibody treatment, 10 nM, and the IC 50 The values ​​ranged from 0.04 to 0.10 nM, and the test antibodies were able to induce cytotoxicity after antigen-specific internalization, with the effects differing among the test antibodies.

[0132] [Table 4]

[0133] Example 6. Thermal stability of selected CLDN18.2-specific monoclonal antibodies The thermostability of the antibodies was tested using differential scanning fluorimetry. The antibody protein was diluted with DPBS to 3 μM (45 μl), mixed with 5 μl of 200x Sypro orange dye (Thermo, #S6650), and dispensed into qPCR tubes (Bio-Rad, #TLS0851-white) in 50 μl aliquots. The tubes were capped with caps (Bio-Rad, #TCS0803). qPCR was performed using a Bio-Rad CFX96 real-time PCR instrument. The temperature was increased by 0.5°C in increments of 0.5°C up to 99°C for 0.5 minutes, and then finished at 25°C for 10 seconds. The melting temperature (Tm) was used as the rate constant for antibody unfolding.

[0134] [Table 5]

[0135] As a result of the analysis, as shown in Table 5, the control antibody and the selected antibodies 0058-002 and 0058-003 were confirmed to have good thermal stability with Tm values ​​of 65°C or higher, while 0058-001 was shown to have a slightly lower Tm of 64.5°C. The reason why antibodies 0058-002 and 0058-003 showed two Tms is thought to be due to different unfolding rates between the domains (CH2, CH3, Fab) that make up the antibodies, or due to aggregation of inactivated proteins generated by unfolding at the first melting temperature, followed by secondary unfolding at the second melting temperature.

Claims

1. (a) a monoclonal antibody that specifically binds to claudin 18.2, comprising a heavy chain variable region including a heavy chain CDR (complementarity-determining region) 1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 3, and a light chain variable region including a light chain CDR1 described in SEQ ID NO: 5; a light chain CDR2 consisting of the amino acid sequence DVS; and a light chain CDR3 described in SEQ ID NO: 6; (b) a monoclonal antibody that specifically binds to claudin 18.2, comprising a heavy chain variable region including a heavy chain CDR (complementarity-determining region) 1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 8, and a light chain variable region including a light chain CDR1 described in SEQ ID NO: 10; a light chain CDR2 consisting of the amino acid sequence AAS; and a light chain CDR3 described in SEQ ID NO: 11; (c) A monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin-18.2, selected from the group consisting of monoclonal antibodies that specifically bind to claudin-18.2, comprising a heavy chain variable region comprising a heavy chain CDR (complementarity-determining region) 1 described in SEQ ID NO: 1; a heavy chain CDR2 described in SEQ ID NO: 2; and a heavy chain CDR3 described in SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 described in SEQ ID NO: 15; a light chain CDR2 consisting of the amino acid sequence GAF; and a light chain CDR3 described in SEQ ID NO:

11.

2. The monoclonal antibody or antigen-binding fragment thereof (a) a monoclonal antibody that specifically binds to claudin 18.2, comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 4 and a light chain variable region represented by the amino acid sequence of SEQ ID NO: 7; (b) a monoclonal antibody that specifically binds to claudin 18.2, the monoclonal antibody comprising a heavy chain variable region set forth in the amino acid sequence of SEQ ID NO: 9 and a light chain variable region set forth in the amino acid sequence of SEQ ID NO: 12; and (c) A monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2 described in claim 1, selected from the group consisting of monoclonal antibodies that specifically bind to claudin 18.2 comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 14 and a light chain variable region described by the amino acid sequence of SEQ ID NO:

16.

3. A monoclonal antibody or its antigen-binding fragment that specifically binds to claudin 18.2 described in claim 1 or 2, characterized in that the monoclonal antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, and a recombinant antibody.

4. The antigen-binding fragments include scFv (single chain variable fragment), dsFv (disulfide stabilized Fv), Fab, F(ab') and F(ab') that bind to claudin 18.

2. 2 A monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin 18.2 according to claim 1 or 2, characterized in that it is selected from the group consisting of:

5. A polynucleotide encoding the heavy chain variable region and light chain variable region of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

6. An expression vector comprising the polynucleotide of claim 5.

7. A transformant, excluding humans, transformed with the expression vector of claim 6.

8. (a) culturing a non-human transformant transformed with an expression vector comprising a polynucleotide encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2) according to claim 1 or 2, to produce a culture medium; (b) purifying the monoclonal antibody or its antigen-binding fragment from the culture medium of step (a).

9. A pharmaceutical composition for cancer treatment, comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 as an active ingredient.

10. 10. The pharmaceutical composition for cancer treatment according to claim 9, wherein the cancer is selected from the group consisting of pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, gastric cancer, colon cancer, liver cancer, biliary tract cancer, gallbladder cancer, breast cancer, kidney cancer, mesothelioma, head and neck cancer, bladder cancer, cervical cancer, endometrial cancer, fallopian tube cancer, gastrointestinal cancer, hematological cancer, pharyngeal cancer, melanoma, primary peritoneal cancer, salivary gland cancer, sarcoma, thyroid cancer, glioblastoma, and prostate cancer.

11. The pharmaceutical composition for cancer treatment according to claim 9, further comprising an anti-cancer agent.

12. The pharmaceutical composition for cancer therapy according to claim 11, wherein the anticancer agent is a chemotherapeutic agent or an immune checkpoint inhibitor.

13. A method for treating cancer, comprising administering to an individual the pharmaceutical composition for cancer treatment according to claim 9.

14. A composition for diagnosing cancer, comprising the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

15. A cancer diagnostic kit comprising the composition of claim 14.

16. An antibody-drug conjugate in which a drug is bound to the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

17. 17. The antibody-drug conjugate of claim 16, wherein the drug is selected from the group consisting of a cytotoxic agent, an immunomodulatory compound, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, a microRNA (miRNA), an siRNA, an shRNA, a radioisotope, and a combination thereof.

18. i) an antibody according to claim 1 or 2; ii) a transmembrane domain; and iii) A chimeric antigen receptor protein comprising an intracellular signaling domain that is characterized by inducing T cell activation when the antibody of i) binds to a target antigen.

19. A multi-specific antibody comprising a monoclonal antibody or its antigen-binding fragment that specifically binds to claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2) described in claim 1 or 2.

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