Monoclonal antibody specifically binding to Claudin-18 splicing variant 2 and application thereof

Abstract

The present invention relates to a monoclonal antibody that specifically binds to Claudin-18 splice variant 2 (CLDN18.2), and a use thereof, the antibody of the present invention having a high binding force to CLDN18.2 and an excellent internalization capability, and the antibody of the present invention can be used for preparing a monoclonal antibody that can specifically bind to Claudin-18 splice variant 2 (CLDN18.2), and the antibody of the present invention can be used for preparing a monoclonal antibody that can specifically bind to Claudin-18 splice variant 2 (CLDN18.2). The present invention is useful as a monoclonal antibody or a deformed form (antibody-drug conjugate, chimeric antigen receptor, or multispecific antibody) in the development of a cancer therapeutic agent.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody that specifically binds to Claudin-18 spliced variant 2 (CLDN18.2) and its use. Background Art

[0002] Claudin-18 has a molecular weight of approximately 27 kDa and belongs to the endogenous membrane protein family present at the junction of epithelia and endothelia. The structure consists of two extracellular loops and four transmembrane (TM) domains, and there are two splice variants that differ by only eight amino acids, namely, Claudin-18 spliced variant 1 (Genebank accession number: NM_016369) and Claudin-18 spliced variant 2 (Genebank accession number: NM_001002026). CLDN18.1 is expressed in normal lung and gastric epithelia, but the expression of CLDN18.2 in normal cells other than gastric mucosa is very limited, while it is overexpressed in various cancers such as gastric cancer, liver cancer, gallbladder cancer, breast cancer, kidney cancer, pancreatic cancer, non-small cell lung cancer, mesothelioma, etc. and their metastatic cancers (Okugawa T, etal., Dig Dis Sci (2012) 57:1562-7; Rohde C, et al., Jpn J Clin Oncol (2019) 49:870-6). Claudin proteins can play important roles in tumorigenesis, metastasis, and inflammation. It is known that changes in the expression of Claudin proteins cause a decrease in the function of tight junctions, affecting signal transduction pathways and thus promoting tumors.

[0003] Therefore, in order to develop excellent anti-cancer therapeutic agents, it is necessary to develop antibodies that specifically bind to CLDN18.2, which is specifically expressed only in tumors and is different from CLDN18.1.

[0004] Recently, zolbetuximab, a potent chimeric IgG1 mAb (IgG1 monoclonal antibody) that binds to CLDN18.2 on the surface of tumor cells, has been developed and clinical trials have been conducted. In particular, zolbetuximab used as a first-line therapeutic agent in 3 trials (SPOTLIGHT trial, NCT03504397) has improved the progression-free survival (PFS) and overall survival (OS) of gastric cancer patients compared to standard chemotherapy methods.

[0005] Under such a technical background, the present inventors used human CLDN18.2 protein as an antigen and screened monoclonal antibodies that specifically bind only to human CLDN18.2 using phage display technology consisting entirely of human antibody sequences, and confirmed that monoclonal antibodies in the form of human 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 internalization ability, thus completing the present invention.

[0006] On the other hand, Korean Patent Publication No. 2022-0136267 discloses "antibody-drug conjugates containing antibodies against human CLDN18.2 and their uses", and Korean Patent Publication No. 2022-0121873 discloses "anti-CLDN18.2 antibodies and their uses", but there is no description of the "monoclonal antibodies specifically binding to CLDN18.2 and their uses" of the present invention. Summary of the Invention

[0007] (I) Technical Problems to be Solved

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

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

[0010] Still another object of the present invention is to provide a vector containing the polynucleotide, a transformant into which the vector is introduced, and a method for preparing the same.

[0011] Another object of the present invention is to provide a composition for treating cancer containing the antibody or an antigen-binding fragment thereof.

[0012] (II) Technical Solutions

[0013] To solve the above problems, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a heavy-chain variable region comprising heavy-chain CDR (complementarity-determining region) 1 shown in SEQ ID NO: 1, heavy-chain CDR2 shown in SEQ ID NO: 2, and heavy-chain CDR3 shown in SEQ ID NO: 3, and a light-chain variable region comprising light-chain CDR1 shown in SEQ ID NO: 5, light-chain CDR2 consisting of the amino acid sequence DVS, and light-chain CDR3 shown in SEQ ID NO: 6.

[0014] Furthermore, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a heavy-chain variable region comprising heavy-chain CDR1 shown in SEQ ID NO: 1, heavy-chain CDR2 shown in SEQ ID NO: 2, and heavy-chain CDR3 shown in SEQ ID NO: 8, and a light-chain variable region comprising light-chain CDR1 shown in SEQ ID NO: 10, light-chain CDR2 consisting of the amino acid sequence AAS, and light-chain CDR3 shown in SEQ ID NO: 11.

[0015] Furthermore, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a heavy-chain variable region comprising heavy-chain CDR1 shown in SEQ ID NO: 1, heavy-chain CDR2 shown in SEQ ID NO: 2, and heavy-chain CDR3 shown in SEQ ID NO: 13, and a light-chain variable region comprising light-chain CDR1 shown in SEQ ID NO: 15, light-chain CDR2 consisting of the amino acid sequence GAF, and light-chain CDR3 shown in SEQ ID NO: 11.

[0016] Furthermore, the present invention provides a polynucleotide encoding the heavy-chain variable region and light-chain variable region of the monoclonal antibody or antigen-binding fragment thereof.

[0017] Furthermore, the present invention provides an expression vector comprising the polynucleotide.

[0018] Furthermore, the present invention provides a transformant other than human transformed with the expression vector.

[0019] Furthermore, the present invention provides a method for preparing a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 by culturing the transformant.

[0020] Furthermore, the present invention provides a pharmaceutical composition for treating cancer comprising the monoclonal antibody or antigen-binding fragment thereof as an active ingredient.

[0021] Furthermore, the present invention provides a method for treating cancer, comprising the step of administering to an individual the pharmaceutical composition for treating cancer.

[0022] Furthermore, the present invention provides a composition for diagnosing cancer comprising the monoclonal antibody or an antigen-binding fragment thereof, and a kit for diagnosing cancer comprising the composition for diagnosing cancer.

[0023] Furthermore, the present invention provides an antibody-drug conjugate formed by binding of the monoclonal antibody or an antigen-binding fragment thereof to a drug.

[0024] Furthermore, the present invention provides a chimeric antigen receptor (CAR) protein, comprising: i) the monoclonal antibody; ii) a transmembrane domain; and iii) an intracellular signal transduction domain, characterized by causing activation of T cells upon binding of the antibody in i) to a target antigen.

[0025] Furthermore, the present invention provides a multi-specific antibody of the monoclonal antibody or an antigen-binding fragment thereof.

[0026] (III) Beneficial effects

[0027] The novel monoclonal antibody that binds to CLDN18.2 of the present invention is composed of a fully human antibody sequence that is internalized by cells, exhibits specific antigen-binding ability and various intracellularization abilities, and has confirmed thermal stability. It is expected to be effectively utilized as a monoclonal antibody or a modified form (such as an antibody-drug conjugate, a chimeric antigen receptor, or a multi-specific antibody) in the treatment and / or prevention of cancer diseases and related diseases expressing CLDN18.2. Description of the drawings

[0028] Figure 1 Results of flow cytometry analysis using HEK293E cell line not expressing human CLDN18.2.

[0029] Figure 2 Results of flow cytometry analysis using HEK293E cell line overexpressing CLDN18.2 (HEK293E / 18.2).

[0030] Figure 3 Results of flow cytometry analysis using CHO-K1 cell line overexpressing CLDN18.2 (CHO-K1 / 18.2).

[0031] Figure 4 Results of enzyme-linked immunosorbent assay (ELISA) analysis using human CLDN18.2 as the coated antigen.

[0032] Figure 5 Results of ELISA analysis using human CLDN18.1 as the coated antigen.

[0033] Figure 6 Results of flow cytometry analysis using the BxPC3 cell line (a cell line that endogenously expresses human CLDN18.1).

[0034] Figure 7 Results for confirmation by flow cytometry Figure 6 of the CLDN18.1 expression level of the BxPC3 cell line used in

[0035] Figure 8 Results of analyzing the cellular internalization ability of monoclonal antibodies in HEK293E cell line (CLDN18.2 negative) by a live cell analyzer (Incucyte).

[0036] Figure 9 Results of analyzing the cellular internalization ability of monoclonal antibodies in HEK293E / 18.2 cell line (overexpressing CLDN18.2) by a live cell analyzer (Incucyte).

[0037] Figure 10 and Figure 11 Results of analyzing the cellular internalization ability of monoclonal antibodies in CHO-K1 / 18.2 cell line (overexpressing CLDN18.2) by cell immunofluorescence staining.

[0038] Figure 12 Results of analyzing the cellular internalization ability and cytolytic activity of monoclonal antibodies in HEK293E / 18.2 cell line (overexpressing CLDN18.2) by FabZAP assay.

[0039] Figure 13 Results of analyzing the cellular internalization ability and cytolytic activity of monoclonal antibodies in HEK293E cell line (CLDN18.2 negative) by FabZAP assay.

[0040] Figure 14 Results of analyzing the thermal stability of monoclonal antibodies that specifically bind to CLDN18.2 by Differential scanning fluorimetry. Detailed implementation

[0041] To achieve the object of the present invention, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a heavy-chain variable region comprising a heavy-chain CDR1 shown in SEQ ID NO: 1, a heavy-chain CDR2 shown in SEQ ID NO: 2, and a heavy-chain CDR3 shown in SEQ ID NO: 3, and a light-chain variable region comprising a light-chain CDR1 shown in SEQ ID NO: 5, a light-chain CDR2 consisting of the amino acid sequence DVS, and a light-chain CDR3 shown in SEQ ID NO: 6.

[0042] Furthermore, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a heavy-chain variable region comprising a heavy-chain CDR1 shown in SEQ ID NO: 1, a heavy-chain CDR2 shown in SEQ ID NO: 2, and a heavy-chain CDR3 shown in SEQ ID NO: 8, and a light-chain variable region comprising a light-chain CDR1 shown in SEQ ID NO: 10, a light-chain CDR2 consisting of the amino acid sequence AAS, and a light-chain CDR3 shown in SEQ ID NO: 11.

[0043] Furthermore, the invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to CLDN18.2, comprising: a heavy-chain variable region comprising a heavy-chain CDR1 shown in SEQ ID NO: 1, a heavy-chain CDR2 shown in SEQ ID NO: 2, and a heavy-chain CDR3 shown in SEQ ID NO: 13, and a light-chain variable region comprising a light-chain CDR1 shown in SEQ ID NO: 15, a light-chain CDR2 consisting of the amino acid sequence GAF, and a light-chain CDR3 shown in SEQ ID NO: 11.

[0044] More specifically, the monoclonal antibody that specifically binds to CLDN18.2 of the present invention, which comprises a heavy-chain variable region containing a heavy-chain CDR1 shown in SEQ ID NO: 1, a heavy-chain CDR2 shown in SEQ ID NO: 2, and a heavy-chain CDR3 shown in SEQ ID NO: 3, and a light-chain variable region containing a light-chain CDR1 shown in SEQ ID NO: 5, a light-chain CDR2 consisting of the amino acid sequence DVS, and a light-chain CDR3 shown in SEQ ID NO: 6, may further comprise a heavy-chain variable region shown in the amino acid sequence of SEQ ID NO: 4 and a light-chain variable region shown in the amino acid sequence of SEQ ID NO: 7.

[0045] More specifically, a monoclonal antibody that specifically binds to CLDN18.2 and comprises a heavy chain variable region containing a heavy chain CDR1 shown in SEQ ID NO: 1, a heavy chain CDR2 shown in SEQ ID NO: 2, and a heavy chain CDR3 shown in SEQ ID NO: 8, and a light chain variable region containing a light chain CDR1 shown in SEQ ID NO: 10, a light chain CDR2 consisting of the amino acid sequence AAS, and a light chain CDR3 shown in SEQ ID NO: 11 may further comprise a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 9 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 12.

[0046] More specifically, a monoclonal antibody that specifically binds to CLDN18.2 and comprises a heavy chain variable region containing a heavy chain CDR1 shown in SEQ ID NO: 1, a heavy chain CDR2 shown in SEQ ID NO: 2, and a heavy chain CDR3 shown in SEQ ID NO: 13, and a light chain variable region containing a light chain CDR1 shown in SEQ ID NO: 15, a light chain CDR2 consisting of the amino acid sequence GAF, and a light chain CDR3 shown in SEQ ID NO: 11 may further comprise a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 14 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 16.

[0047] The term "monoclonal antibody" in the present invention refers to a protein molecule that specifically binds thereto by indicating a single antigenic site (single epitope). The monoclonal antibody can be prepared by a variety of methods well known in the art.

[0048] In the present invention, an antibody refers to the form of a whole antibody. The whole antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The full-length antibody includes IgA (immunoglobulin A), IgD, IgE, IgM, and IgG. As subtypes of IgG, it includes IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) classes, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) classes.

[0049] The monoclonal antibody of the present invention may be selected from the group consisting of fully human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, and recombinant antibodies. Preferably, it may be a fully human antibody, but is not limited thereto. All structures of the fully human antibody are derived from humans. Therefore, compared with conventional humanized antibodies or mouse antibodies, the probability of generating an immune response is low, and thus when administered to humans, it has the advantage of not generating unwanted immune responses. Therefore, it can be effectively used as a therapeutic antibody.

[0050] The term "antigen-binding fragment" in the present invention refers to a fragment having antigen-binding ability, including Fab, F(ab'), F(ab')2, Fv, etc. Fab in the antibody fragment has a structure of the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and it has one antigen-binding site. The difference between Fab' and Fab is that Fab' has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated during the formation of a disulfide bond between the cysteine residues in the hinge region of Fab'. The recombinant technology for generating Fv fragments using the smallest antibody fragments having only the variable regions of the heavy and light chains is disclosed in International Publication Patent WO88 / 10649, etc. In double-stranded Fv (dsFv), the variable region of the heavy chain and the variable region of the light chain are connected by a disulfide bond. In single-chain Fv (scFv), the variable region of the heavy chain and the variable region of the light chain are covalently linked by a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by digesting whole antibody with papain, and F(ab')2 fragments can be obtained by digesting with pepsin). Preferably, they can be prepared by genetic recombination technology.

[0051] The term "variable region" in the present invention refers to the region that shows various variations in sequence while performing the function of specifically binding to an antigen. CDR1, CDR2, and CDR3, which are complementarity determining regions, exist in the variable region. A framework region (FR) part exists between the complementarity determining regions, thus playing a role in supporting the complementarity determining region loop (CDR loop).

[0052] The term "complementarity determining region (CDR)" in the present invention refers to the loop region that intervenes in the recognition of an antigen, and the specificity of the antigen of the antibody is determined according to the change in the sequence of this region.

[0053] According to an embodiment of the present invention, after the inventors screened for scFv that specifically binds to CLDN18.2 from a naive human single-chain Fv library, a human antibody that specifically binds to CLDN18.2 was prepared by converting it into an IgG form.

[0054] The term "biopanning" in the present invention refers to the process of selecting only phages that express peptides with the property of binding to a target molecule (antibody, enzyme, cell surface receptor, etc.) on the surface from a phage library that displays peptides on the outer coat of the phage.

[0055] Although the monoclonal antibody or its antigen-binding fragment of the present invention is not particularly limited, in order to increase the residence time in the organism to which it is administered, it can be glycosylated and / or PEGylated.

[0056] The term "glycosylation" in the present invention refers to a processing method of transferring a sugar group to a protein. The glycosylation is carried out by binding a sugar group to a serine, threonine, asparagine, or hydroxylysine residue of a target protein through a glycosyltransferase. The glycosylated protein can be used not only as a constituent of biological tissues but also plays an important role in cell recognition on the cell surface. Therefore, in the present invention, the effect of the antibody can be improved by changing the glycosylation or the pattern of the glycosylation of the monoclonal antibody or its antigen-binding fragment.

[0057] The term "PEGylation" in the present invention refers to a processing method of increasing the residence time of an antibody in the blood by introducing polyethylene glycol into the monoclonal antibody or its antigen-binding fragment. Specifically, polyethylene glycol is used to PEGylate high molecular nanoparticles to increase the hydrophilicity of the surface of the nanoparticles and prevent rapid decomposition in the body through the stealth effect. The so-called stealth effect refers to preventing recognition by the immune function of macrophages in the human body that prey on and digest pathogens, metabolites, and externally flowing substances. Therefore, the residence time of the antibody in the blood can be increased through the PEGylation. The PEGylation used in the present invention can be achieved by a method of forming an amide group by binding the carboxyl group of hyaluronic acid to the amino group of polyethylene glycol, but is not limited thereto, and various methods can be used for PEGylation. At this time, the polyethylene glycol used is not particularly limited thereto, but preferably has a molecular weight in the range of 100 to 1000, and preferably, polyethylene glycol with a linear or branched structure is used.

[0058] The glycosylation and / or PEGylation can deform various glycosylation and / or PEGylation patterns by methods well known in the art while maintaining the function of the antibody of the present invention. The antibody of the present invention comprises variant monoclonal antibodies or antigen-binding fragments thereof with various glycosylation and / or PEGylation pattern deformations.

[0059] The term "affinity" refers to the ability to specifically recognize and bind to a specific site of an antigen, and together with the specificity of the antibody for the antigen, is an important element in the immune response. Various methods well known in the technical field to which the present invention pertains can be used for measuring the affinity for an antigen. For example, surface plasmon resonance technology.

[0060] The present invention also provides polynucleotides encoding the heavy-chain variable region and the light-chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0061] Due to the degeneracy of codons or considering the preferred codons in organisms that need to express the light chain and heavy chain or fragments of the fully humanized antibody, the polynucleotides encoding the light chain and heavy chain of the monoclonal antibody or antigen-binding fragment thereof of the present invention can undergo various deformations within the range of not changing the amino acid sequences of the light chain and heavy chain of the antibody expressed from the coding region. Within the range of not affecting gene expression, various deformations or modifications can also occur in parts other than the coding region. Those of ordinary skill in the art can understand that such deformed genes are also included within the scope of the present invention. That is, when the polynucleotide of the present invention encodes a protein with equivalent activity, it can be mutated by substitution, deletion, insertion of one or more nucleic acid bases, or a combination thereof, and these are also included within the scope of the present invention. The sequence of such a polynucleotide. The sequence of such a polynucleotide can be single-stranded or double-stranded, a DNA molecule or an RNA (mRNA) molecule.

[0062] The present invention also provides an expression vector comprising a polynucleotide encoding the heavy-chain and light-chain variable regions of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0063] The term "expression vector" in the present invention refers to a gene construct containing the necessary regulatory elements operably linked to enable the expression of a gene insert, including plasmid vectors; viral vectors, such as cosmid vectors, Adenovirus vectors, Retrovirus vectors, adeno-associated virus vectors, etc., as means for expressing a target gene in a host cell. "Operably linked" in the present invention means a linkage that functionally links a nucleic acid expression regulatory sequence to a polynucleotide encoding a target protein for performing ordinary functions. The operable linkage with an expression vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily implemented using enzymes well known in the art.

[0064] Suitable expression vectors of the present invention may further include signal sequences for targeting or secretion in addition to expression regulatory elements such as a promoter, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer. The initiation codon and the termination codon are generally regarded as part of the nucleotide sequence encoding an immunogenic target protein, which must function in an object when a gene construct is administered and must be present in the coding sequence and in frame. The promoter can be a constitutive or inducible promoter. In prokaryotic cells, there are lac, tac, T3, and T7 promoters, but are not limited thereto. In eukaryotic cells, there are not only Simian virus 40 (SV40) promoter, Mouse mammary tumor virus (MMTV) promoter, Human immunodeficiency virus (HIV) promoter, the long terminal repeat (LTR) promoter of HIV, moloney virus promoter, Cytomegalovirus (CMV) promoter, Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, but also β-actin promoter, promoters derived from human hemoglobin, human muscle creatine, and human metallothionein, but are not limited thereto.

[0065] An expression vector may contain a selectable marker for selecting a host containing the vector. The selectable marker is used to screen cells transformed with the vector, and various markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins can be used. Only cells expressing the selectable marker survive in an environment treated with a selective agent, and thus, transformed cells can be screened.

[0066] Also, in the case where the vector is a replicable expression vector, it may contain an origin of replication as a specific polynucleotide for initiating replication. Viruses (e.g., baculoviruses) or phage vectors, as well as vectors such as retroviral vectors that can be inserted into the genome of a host, can also be used. Vectors expressing full antibodies or antibody fragments can be achieved in a vector system where the light and heavy chains are expressed simultaneously in one vector or in a system where the light and heavy chains are expressed in separate vectors. In the latter case, the two vectors are introduced into host cells by co-transformation and targeted transformation, cells transformed with the vector containing the light chain (or heavy chain) are screened, and the screened cells expressing the light chain are re-transformed with the vector containing the heavy chain (or light chain), thereby finally screening cells expressing both the light and heavy chains.

[0067] To prepare Fab-form antibodies, a vector into which genes encoding the variable region (VL) and constant region (CL) of the human light chain and the variable region (VH) of the human heavy chain and the first constant region domain (CH1) of the human heavy chain are inserted is used.

[0068] The present invention also provides a transformant transformed with the expression vector of the present invention.

[0069] Suitable host cells for the vector can be prokaryotic cells such as Escherichia coli ( Escherichia coli ), Bacillus subtilis ( Bacillus subtilis ), Streptomyces ( Streptomyces sp. ), Pseudomonas ( Pseudomonas sp. ), Proteus mirabilis ( Proteus mirabilis ), or Staphylococcus ( Staphylococcus sp. ). Also, suitable host cells for the vector can be fungi such as Aspergillus ( Aspergillus sp. ); yeasts such as Pichia pastoris ( Pichia pastoris ), Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Schizosaccharomyces ( Schizosaccharomyces sp. ), and Neurospora crassa ( Neurospora crassa), and yeast, etc. In addition, it can be cells of lower eukaryotes, cells of higher eukaryotes such as cells derived from insects, and plant cells. Moreover, suitable host cells for the vector can be derived from mammals. Preferably, it can be COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S or HT1080 derived from mammalian cells, but is not limited thereto.

[0070] In the present invention, "transformation of host cells" includes any method of introducing nucleic acid into an organism, cell, tissue or organ, and as described in the methods well-known in the art, appropriate standard techniques can be selected according to the host. Such methods include electroporation, cytoplasmic fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, cationic liposome (lipofectamine), and drying / inhibition-mediated transfer methods, etc., but are not limited thereto.

[0071] The present invention also provides a method for preparing a monoclonal antibody or its antigen-binding fragment that specifically binds to CLDN18.2, including:

[0072] Step (a), preparing a culture solution by culturing the transformant; and

[0073] Step (b), purifying the monoclonal antibody or its antigen-binding fragment of the present invention from the culture solution of step (a).

[0074] In the preparation method, the transformant can be cultured according to appropriate culture media and culture conditions well-known in the art. As long as it is an ordinary technician in the art, this culture process can be easily adjusted according to the selected strain or animal cell for use.

[0075] Moreover, the antibody obtained by culturing the transformant can be used in an unpurified state. For example, impurities can be removed by various conventional methods such as centrifugation or ultrafiltration, and the resultant can be dialyzed, salted out, chromatographed, etc., and these can be used alone or in combination. Among them, affinity chromatography is most commonly used, which includes ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc. The antibody prepared by the above method is an antibody with increased affinity for the antigen.

[0076] The present invention also provides a pharmaceutical composition for treating cancer comprising the monoclonal antibody specifically binding to CLDN18.2 or its antigen-binding fragment as an active ingredient.

[0077] The content of the monoclonal antibody or its antigen-binding fragment is as described above.

[0078] When the antibody of the present invention is administered to a subject, the pharmaceutical composition of the present invention inhibits tumor growth in the subject.

[0079] The term "treatment of cancer" in the present invention refers to all actions of improving or curing the symptoms of cancer by administering the composition.

[0080] In the pharmaceutical composition of the present invention, the cancer can be selected from the group consisting of pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, gastric cancer, colorectal cancer (e.g., colon cancer, rectal cancer), liver cancer, cholangiocarcinoma, gallbladder cancer, breast cancer, kidney cancer, mesothelioma, head and neck cancer, bladder cancer, cervical cancer, endometrial cancer, fallopian tube cancer, gastrointestinal cancer, blood cancer (e.g., leukemia, lymphoma or myeloma), throat cancer, melanoma, primary peritoneal cancer, salivary gland cancer, sarcoma, thyroid cancer, glioblastoma, and prostate cancer, but is not limited thereto.

[0081] The pharmaceutical composition of the present invention may further comprise an acceptable carrier, and the carrier may include a nonnaturally occurring carrier.

[0082] The term "pharmaceutically acceptable carrier" in the present invention refers to a carrier or diluent that does not stimulate the organism and does not hinder the biological activity and properties of the administered compound. The pharmaceutically acceptable carrier in a composition formulated as a liquid solution is a sterilized and organism-suitable carrier. Normal saline, sterile water, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerol, or one of them, or a mixture of one or more of these components can be used. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added. Furthermore, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate into injection dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0083] The composition for treating cancer containing the above-mentioned antibody of the present invention and a pharmaceutically acceptable carrier can be applicable to any dosage form containing it as an active ingredient and can be prepared into an oral or parenteral dosage form. The pharmaceutical dosage form of the present invention can include dosage forms suitable for oral, rectal, nasal, topical (including cheek and tongue), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration or dosage forms suitable for inhalation or insufflation administration.

[0084] As an oral dosage form containing the composition of the present invention as an active ingredient, for example, it can be formulated into tablets, lozenges, diamond-shaped lozenges, water-soluble or oil-based suspensions, powders or granules, emulsions, hard capsules or soft capsules, syrups, or bottled medications. For formulating into dosage forms such as tablets and capsules, binders such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin can be included; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In the case of capsule dosage forms, in addition to the substances mentioned above, liquid carriers such as fatty oils can also be contained.

[0085] As a parenteral dosage form containing the composition of the present invention as an active ingredient, it can be formulated into an injectable form such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method, or a jet form such as an aerosol that can be inhaled through the respiratory tract. To formulate an injectable dosage form, the composition of the present invention is mixed with a stabilizer or a buffer in water to prepare a solution or a suspension, and it can be formulated into a unit dosage amount in an ampoule or a vial. To inject as a suppository, it can be formulated into a rectal dosage composition such as a suppository or an enema containing a conventional suppository base such as cocoa butter or other glycerides. When formulated into a jet form such as an aerosol, a water-dispersible concentrate or a propellant for dispersing a wet powder can be combined with additives.

[0086] Furthermore, the pharmaceutical composition can be administered in a pharmaceutically effective amount.

[0087] The term "pharmaceutically effective amount" in the present invention refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective dose can be determined according to factors including the individual type, the severity of the disease, age, gender, the type of cancer, the activity of the drug, the sensitivity to the drug, the administration time, the administration route and the metabolic ratio, the treatment period, and the drugs used simultaneously, as well as other factors well-known in the medical field. The composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents. Moreover, it can be administered once or multiple times. The key point is to administer an amount that obtains the maximum effect with the minimum amount without side effects after considering all the above factors, which can be easily determined by relevant practitioners.

[0088] The pharmaceutical composition of the present invention may further contain one or more known active ingredients having an anti-cancer effect. Preferably, the known active ingredient having an anti-cancer effect may be a chemotherapeutic agent or an immune checkpoint inhibitor, but is not limited thereto.

[0089] The chemotherapeutic agent may be one or more selected from the group consisting of alkylating series anti-cancer agents such as Carboplatin and Paclitaxel, metabolic antagonist series anti-cancer agents such as Gemcitabin, anthracycline antibiotic series anti-cancer agents such as Doxorubicin, and proteasome inhibitor series anti-cancer agents such as Bortezomib, but is not limited thereto. The immune checkpoint inhibitor may target one or more selected from the group consisting of 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, MARCO, but is not limited thereto.

[0090] The present invention also provides a method for treating cancer, comprising the step of administering to a subject the pharmaceutical composition for treating cancer.

[0091] The term "administering" in the present invention refers to the act of introducing the pharmaceutical composition of the present invention to a patient by any suitable method. As long as it can reach the target tissue, the administration route of the composition of the present invention may be various routes such as oral or parenteral. Specifically, it can be administered orally, rectally, topically, intravenously, intraperitoneally, intramuscularly, intraarterially, transdermally, nasally, by inhalation, intravitreally or intradermally in a conventional manner.

[0092] The treatment method of the present invention comprises the step of administering a pharmaceutically effective amount of the composition for treating cancer of the present invention. For those of ordinary skill in the art, it is obvious that a physician can determine a suitable total daily dose within the scope of correct medical judgment. Preferably. The specific therapeutically effective amount for a particular patient varies according to the type and degree of the response to be achieved, whether or not to use a specific composition including other preparations according to the circumstances, the patient's age, weight, general health status, gender and diet, the administration time, the administration route and the secretion rate of the composition, the treatment period, and various factors such as the drugs used in combination with or simultaneously with the specific composition, and similar factors well known in the medical field. Therefore, preferably, the effective amount of the composition for preventing or treating cancer suitable for the purpose of the present invention is determined in consideration of the foregoing matters.

[0093] Furthermore, the above-mentioned subject refers to any animal that may develop diseases such as tumor development and angiogenesis due to the overactivity of CLDN18.2. The above-mentioned animals include not only humans and primates, but also domestic animals such as cattle, pigs, sheep, horses, dogs, and cats.

[0094] The present invention also provides a composition for diagnosing cancer comprising the monoclonal antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, and a kit for diagnosing cancer comprising the composition for diagnosing cancer.

[0095] The monoclonal antibody or antigen-binding fragment thereof and cancer are as described above. A composition for diagnosis comprising the monoclonal antibody or antigen-binding fragment thereof specific for CLDN18.2 of the present invention is used to diagnose diseases related to the expression or non-expression and expression level of CLDN18.2, for example, cancer.

[0096] In the above method for diagnosing cancer, detection is carried out by reacting the monoclonal antibody specific for CLDN18.2 of the present invention with a biological sample isolated from a subject suspected of having cancer and analyzing the formation of an antigen-antibody complex, thereby providing information for diagnosing cancer.

[0097] The term "biological sample" in the present invention may refer to tissues, cells, whole blood, serum, plasma, biopsy tissue samples (brain, skin, lymph nodes, spine, etc.), cell culture supernatants, disrupted eukaryotic cells, and bacterial expression systems, etc., but is not limited thereto. The presence or absence of CLDN18.2 or cancer is confirmed by reacting these biological samples, in an operated or unoperated state, with the antibody of the present invention.

[0098] The term "antigen-antibody complex" in the present invention refers to the conjugate of the CLDN18.2 protein antigen in the sample and the monoclonal antibody or antigen-binding fragment thereof of the present invention that recognizes it. The formation of such an antigen-antibody complex can be measured by conventional immunoassay methods, and can be measured by radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbent assay), capture ELISA, inhibition or competition assay, sandwich assay, flow cytometry, and fluorescence immunoassay using the antibody against CLDN18.2, etc., but is not limited thereto.

[0099] Cancer can be diagnosed by analyzing the intensity of the final signal based on the immunoassay process. That is, when the signal generated by the overexpression of the CLDN18.2 protein of the present invention in a biological sample isolated from a suspected subject is stronger than that in a biological sample isolated from a normal subject, it is diagnosed as cancer.

[0100] Moreover, the kit of the present invention contains the antibody against CLDN18.2 of the present invention. By reacting the sample with the antibody to analyze the generated signal, cancer can be diagnosed. In this case, the above signal can be manifested by an enzyme (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, or cytochrome P450, etc.) bound to the antibody. At this time, when alkaline phosphatase is used as the enzyme in the substrate for the enzyme, chromogenic reaction substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium, naphthol-AS-B1-phosphate, and enhanced chemifluorescence (ECF) are used as the substrate. When horseradish peroxidase is used as the enzyme, substrates such as chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), methylumbelliferyl benzyl ether, luminol, eosin red reagent (10-acetyl-3,7-dihydroxyphenazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), 2,2'-azino-di[3-ethylbenzthiazoline sulfonate] (ABTS), o-phenylenediamine (OPD), and naphthol / pyronin, glucose oxidase, t-NBT, and phenzaine methosulfate (m-PMS) can be used, but it is not limited thereto.

[0101] In addition, the kit of the present invention may include a label that generates a detectable signal, and the above label may include a chemical substance (e.g., biotin), an enzyme (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P450), a radioactive substance (e.g., 14C, 125I, 32P, and 35S), a fluorescent substance (e.g., fluorescein), a luminescent substance, a chemiluminescent substance, and fluorescence resonance energy transfer (FRET), but is not limited thereto.

[0102] The activity assay of the enzyme or the signal assay used for cancer diagnosis can be carried out by various methods well known in the art. Thus, the expression of CLDN18.2 can be analyzed normally or quantitatively.

[0103] The present invention also provides an antibody-drug conjugate formed by binding the monoclonal antibody or its antigen-binding fragment that specifically binds to CLDN18.2 with a drug.

[0104] The term "drug" in the present invention refers to a compound that can bind to an antibody or its antigen-binding fragment specific for CLDN18.2 of the present invention, can be separated from the above antibody or its antigen-binding fragment under acidic conditions, and has a therapeutic effect on target cells.

[0105] Drugs that can be used for the antibody-drug conjugate of the present invention include any compound, moiety, or group that has an inhibitory effect on cytotoxicity or cell proliferation, including (i) a tubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can have a DNA nucleic acid chelating function; (ii) a protein toxin that can have an enzyme function; and (iii) a radioactive isotope (radionuclide), etc. One or more of the above compounds can be used.

[0106] Non-limiting examples of such drugs include maytansine, auristatin, dolastatin, trichothecene, CC1065, calicheamicin and other enediyne antibiotics, taxane, anthracycline, methotrexate, doxorubicin, vindesine, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, carminomycin, aminopterin, actinomycin D, mitomycin C analogs, bleomycin analogs, esperamicin analogs, 5-fluorouracil, melphalan, other nitrogen mustards and their stereoisomers, bioisosteres, analogs or derivatives, cisplatin and cisplatin analogs, enzymes and their fragments as other intercalating agents, for example, nuclease, antibiotic, toxin (enzymatically active toxin or small molecule toxin derived from bacteria, fungi, plants or animals), cisplatin, CPT-11, doxorubicin, paclitaxel, docetaxel and other anti-tumor or anti-cancer agents, etc., but not limited thereto. And, radioactive isotopes (radionuclides) are 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, etc., but not limited thereto, and microRNA (miRNA), siRNA, shRNA, etc. that can inhibit the expression of specific oncogenes can also be used.

[0107] The present invention also provides a chimeric antigen receptor (CAR) protein, comprising: i) the monoclonal antibody that specifically binds to CLDN18.2; ii) a transmembrane domain; and iii) an intracellular signal transduction domain, characterized by causing T cell activation when the antibody in i) binds to an antigen.

[0108] In the present invention, the above chimeric antigen receptor protein can be specified by consisting of the monoclonal antibody of the present invention, a known transmembrane domain and an intracellular signal transduction domain.

[0109] The term "chimeric antigen receptor (CAR)" in the present invention refers to a non-natural receptor that can endow immune effector cells with specificity for a specific antigen. Generally, the above chimeric antigen receptor refers to a receptor used to transplant the specificity of a monoclonal antibody into T cells. CAR is generally composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The above extracellular domain contains an antigen-binding site, and in the present invention, the above antigen-binding site is an antibody specific for CLDN18.2. As described above, the antibody specific for CLDN18.2 is preferably in the form of an antibody fragment, and more preferably in the form of Fab or scFv, but is not limited thereto.

[0110] Moreover, the transmembrane domain of CAR is in a form connected to the extracellular domain and can be derived from a natural or synthetic form. In the case of being derived from a natural form, it can be from a membrane-bound or transmembrane protein and can be a part of the transmembrane region of various proteins such as the alpha, beta, zeta chains 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 can be obtained from publicly known documents in the art that describe the transmembrane region part of transmembrane proteins, but is not limited thereto. Moreover, in the case where the above transmembrane domain is in a synthetic form, it mainly contains hydrophobic amino acid residues such as leucine and valine. As an example, there may be a transmembrane domain synthesized with a triplet of phenylalanine, tryptophan, and valine, but is not limited thereto.

[0111] In the CAR of the present invention, the above intracellular domain is a part of the domain of the CAR present inside the cell and has a form connected to the transmembrane domain. The above intracellular domain of the present invention may include an intracellular signal transduction domain, and is characterized in that if an antigen binds to the antigen-binding site of the CAR, T cell activation is caused, and preferably, T cell proliferation is caused. As long as it is a part that transmits a signal capable of causing T cell activation by binding an antibody to the antigen-binding site present outside the cell, the type of the above intracellular signal transduction domain is not particularly limited, and various types of intracellular signal transduction domains can be used. As an example thereof, it may be an immunoreceptor tyrosine-based activation motif (ITAM). The above immunoreceptor tyrosine activation motif may include an immunoreceptor tyrosine activation motif derived from CD3 zeta (ξ, zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d or FcεRIγ, but is not limited thereto.

[0112] Furthermore, preferably, the intracellular domain of the chimeric antigen receptor of the present invention further includes a costimulatory domain, but is not limited thereto. The above costimulatory domain is a part that functions to transmit the signal based on the intracellular signal transduction domain included in the CAR of the present invention to the T cell, and refers to the intracellular part of the CAR including the intracellular domain of the costimulatory molecule. The above costimulatory molecule is a cell surface molecule and refers to a molecule required for a sufficient response of lymphocytes against an antigen. As an example thereof, it may be CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3, but is not limited thereto. The above costimulatory domain may be the intracellular part of a molecule selected from the group consisting of such costimulatory molecules and combinations thereof.

[0113] Furthermore, optionally, a short oligopeptide or polypeptide linker may connect the intracellular domain and the transmembrane domain of the chimeric antigen receptor. Even if the linker is included in the chimeric antigen receptor of the present invention, as long as it is a linker that can induce T cell activation through the intracellular domain when an antibody located outside the cell binds to an antigen, its length is not particularly limited.

[0114] The present invention also provides a multi-specific antibody comprising the monoclonal antibody or its antigen-binding fragment that specifically binds to CLDN18.2.

[0115] In the present invention, preferably, the multi-specific antibody may be a bispecific antibody, but is not limited thereto.

[0116] Preferably, the multi-specific antibody of the present invention has a form in which the anti-CLDN18.2 antibody of the present invention binds to an antibody or its fragment having the ability to bind to an immune effector cell-specific target molecule. Preferably, the immune effector cell-specific target molecule is selected from 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), but is not limited thereto.

[0117] A multi-specific antibody is an antibody that simultaneously recognizes different epitopes of the same antigen or two or more other antigens, and various antibodies belonging to multi-specific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, IgG-based antibodies, etc. In the case of a multi-specific antibody such as a bispecific antibody, two signals can be inhibited or amplified simultaneously. Therefore, it can be more effective than the case of inhibiting / amplifying one signal, and can be administered at a lower dose compared to the case of treating each signal with respective signal inhibitors, and can inhibit / amplify two signals in the same time and space.

[0118] Methods for preparing bispecific antibodies are well known. Generally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain / light chain pairs under the condition that the two heavy chains have different specificities.

[0119] In the case of scFv-based bispecific antibodies, the VL and VH of different scFvs are combined with each other to prepare hybrid scFvs in a heterodimeric form, thereby preparing diabodies (Holliger et al., Proc. Natl. Acad. Sci. U.S.A., (1993) 90:6444). Tandem ScFvs can be prepared by connecting different scFvs to each other. Heterodimeric miniantibodies can be prepared by expressing CH1 and CL of Fab at the ends of respective scFvs (Muller et al., FEBS lett., 432:45, 1998). Moreover, by substituting some amino acids in the CH3 domain, which is a homodimeric domain of Fc, into a "knob into hole" heterodimeric structure, and expressing these modified CH3 domains at the ends of different respective scFvs, minibodies in the form of heterodimeric scFvs can be prepared (Merchant et al., Nat. Biotechnol., (1998) 16:677).

[0120] In the case of Fab-based bispecific antibodies, individual Fab' against specific antigens are combined with each other using disulfide bonds or a medium to prepare heterodimeric Fab forms. By expressing scFvs against different antigens at the ends of the heavy or light chains of a specific Fab, the antigen valency becomes two, or by forming a hinge region between the Fab and the scFv, it has four antigen valencies in a heterodimeric form. Also, the preparation methods of bibodies with three antigen valencies by fusing scFvs against different antigens at the light-chain end and the heavy-chain end of the Fab, tribodies with three antigen valencies by separately fusing different scFvs at the light-chain end and the heavy-chain end of the Fab, and simple-form tritargeted antibodies F(ab')3 obtained by chemically conjugating different three Fabs are well-known in the art.

[0121] In the case of IgG-based bispecific antibodies, it is a well-known method to produce bispecific antibodies by re-hybridizing mouse and rat hybridomas by Trion Pharma to prepare hybrid-hybridomas, also known as quadromas. Also, a method for preparing a so-called 'Holes and Knob'-shaped bispecific antibody in a heterodimeric form by targeting partial amino acids of the CH3 heterodimeric domain of a different heavy-chain-deformed Fc while sharing the light-chain part (Merchant et al., Nat. Biotechnol., 16:677, 1998) is a well-known method. In addition to bispecific antibodies in heterodimeric form, a method for preparing (scFv)4-IgG in heterodimeric form by fusing and expressing two different scFvs in the constant domains of the variable structures of the light and heavy chains that are not IgG is a well-known method. Also, it has been reported that ImClone prepared a bispecific antibody by fusing only a single variable domain against mouse Platelet-derived Growth Factor Receptor-α to the amino terminus of the light chain of IMC-1C11, which is a chimeric monoclonal antibody against human VEGFR-2. And Rossi et al. revealed an antibody with multiple antigen-binding valences against CD20 by using the so-called 'dock and lock (DNL)' method of dimerization of the R subunit of protein kinase A (PKA) and the dimerization and docking domain (DDD) and the anchoring domain of PKA (Rossi et al., Proc. Natl. Acad. Sci. U.S.A., (2006) 103:6841).

[0122] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited to the following examples.

[0123] Example 1. Screening of CLDN18.2-specific antibodies

[0124] The gene of CLDN18.2 (R&D company, #RDC2149) was overexpressed in the HEK293E animal cell line to obtain the cell line. After coating the CLDN18.2-VLPs antigen (Cusabio company, #CSB-MP005498HU (A5)) on the immunosorb tube, blocking was performed. After transfecting the human scFv-display library phage ((strain) Ybiologics company) into Escherichia coli, the obtained Escherichia coli was cultured at a temperature of 30 °C for 16 hours. After centrifuging the culture solution and using the polyethylene glycol (PEG) supernatant, it was dissolved in a phosphate buffered saline (PBS) buffer solution to prepare the human antibody-display library phage.

[0125] The display library phage was placed in the immunosorb tube and reacted at room temperature for 2 hours. After washing with 1xPBST and 1x PBS, 100 mM of TAE and Tris-HCl (pH 7.5) solutions were sequentially treated to elute only the scFv-phage that specifically binds to the antigen. Then, 3 positive phage clones that specifically bind to CLDN18.2 were finally screened out by panning and ELISA. Then, the clones in the scFv form were converted to the IgG form and used in the experiment.

[0126] The sequences of the heavy chain and light chain CDRs of the screened monoclonal antibodies, as well as the heavy chain variable region and light chain variable region containing them, are shown in Table 1 and Table 2 below.

[0127] Table 1

[0128] Heavy chain and light chain CDR sequences of monoclonal antibodies 0058-001, 0058-002, and 0058-003

[0129]

[0130] Table 2

[0131] Heavy chain and light chain variable region sequences of monoclonal antibodies 0058-001, 0058-002, and 0058-003

[0132]

[0133] Example 2. Characteristics of the screened CLDN18.2-specific monoclonal antibodies

[0134] 2-1. Non-specificity of the CLDN18.2 monoclonal antibody by flow cytometry

[0135] The non-specific binding assay was performed in HEK293E cell line. The expression of human CLDN18.2 was confirmed using anti-CLDN18.2 (CUSABIO, #CSB-RA005498A1HU).

[0136] HEK293E cells that did not express human CLDN18.2 were prepared at a cell count of 0.5×10 per sample. Antibodies 0058-001, 0058-002, and 0058-003, which were used as screening antibodies, were diluted to a concentration of 7.5 μg / ml and reacted with the prepared cells at 4°C for 30 minutes. Then, the cells were washed 3 times with PBS (Welgene, #LB001-02) containing 2% fetal bovine serum. After reacting with an anti-human IgG antibody conjugated with fluorescein isothiocyanate (FITC, Vectorlabs, #FI-3000) at 4°C for 30 minutes, the cells were washed in the same manner as described above. After suspending the cells in 0.2 ml of PBS containing 2% FBS (Thermo, #26140-079), the binding was analyzed using a CytoFlex flow cytometer (Beckman coulter, USA). 6 As a result of the analysis, none of the three screening antibodies, 0058-001, 0058-002, and 0058-003, bound to the HEK293E cell line that did not express CLDN18.2 (

[0137] ). Figure 1 ).

[0138] 2-2. Specificity of CLDN18.2 monoclonal antibodies by flow cytometry

[0139] For each sample, 0.5×10 cells of HEK293E / 18.2 cells and CHO-K1 / 18.2 cells overexpressing human CLDN18.2 were prepared. The anti-CLDN18.2 antibodies were diluted at specified multiples and reacted with the prepared cells at 4°C for 30 minutes. Then, the cells were washed 3 times with PBS containing 2% fetal bovine serum. After reacting with an anti-human IgG antibody conjugated with FITC at 4°C for 20 minutes, the cells were washed in the same manner as described above. After suspending the cells in 0.5 ml of PBS containing 2% FBS, the binding was analyzed using a CytoFlex flow cytometer. 6

[0140] ​Analysis results confirmed that 0058-001, 0058-002, and 0058-003, which are anti-CLDN18.2 screening antibodies, bind to HEK293E / 18.2 and CHO-K1 / 18.2 cell lines with high expression of CLDN18.2 in a concentration-dependent manner ( Figure 2 and Figure 3 ).

[0141] Example 3. Analysis of the binding ability of the screened CLDN18.2-specific monoclonal antibody to CLDN18 isoforms

[0142] Claudin-18 splicing variant 1 is an antigen expressed in normal lung and gastric epithelia. Depending on which ATG in the first exon of the Claudin-18 gene is used as the start codon to form the CLDN18.1 / 18.2 isoforms, since the protein sequences are 92% identical, in order to screen for antibodies that do not bind to Claudin-18 splicing variant 1 but specifically bind to CLDN18.2, the selective binding ability to the isoforms was analyzed.

[0143] 3-1. Analysis of the binding ability to CLDN18 isoforms by ELISA

[0144] Prepare CLDN18.1 (EUPROTEIN, #EPY255141) and CLDN18.2 (CUSABIO, #CSB-MP005498HU (A5)) proteins at a concentration of 1 μg / ml using a dilution solution (4% skim milk / 0.05% tween-20 / PBS). Then, add 100 μl to each well of an immunization plate (Thermo, #439454) and incubate at 4°C for more than 16 hours. After washing 3 times with 300 μl of a washing solution (0.05% tween-20 / PBS), add 200 μl of a blocking solution (4% skim milk / 0.05% tween-20 / PBS) to each well and react at room temperature for 1 hour. After washing 3 times, add 100 μl of anti-CLDN18.2 antibody diluted to a specified multiple to each well and rock at room temperature for 1 hour. After washing 3 times, add anti-human kappa-HRP (Sigma, #A7164) prepared at a dilution factor of 1:3000 to each well and react at room temperature for 1 hour. After washing 3 times, add 100 μl of TMB substrate (Sigma, #T0440) to each well and react under light-shielded conditions at room temperature. When color development is confirmed, add 50 μl of a reaction termination solution (1N H2SO4) to stop the reaction. Measure the absorbance using a spectrophotometer (Promega, GM3000).

[0145] As a result of the analysis, it was screened that antibodies 0058-001, 0058-002, and 0058-003 bind to the CLDN18.2 antigen ( Figure 4 ), but do not bind to the CLDN18.1 antigen ( Figure 5 ), thus confirming selectivity for the CLDN18.2 antigen.

[0146] 3-2. Analyze the binding ability to CLDN18 isoforms by flow cytometry

[0147] Prepare BxPC3 that endogenously expresses human CLDN18.1 at a rate of 0.5×10 6 cells per sample ( Figure 7),(50 nM concentration of anti-CLDN18.1 antibody (Cusabio, #CSB-RA005498A2HU) and the anti-CLDN18.2 antibody of the present invention were reacted with BxPC3 cells at 4 °C for 30 minutes. Then, the cells were washed 3 times with PBS containing 2% fetal bovine serum, reacted with anti-human IgG antibody conjugated with FITC fluorescent substance at 4 °C for 30 minutes, and then washed in the above manner. After suspending the cells with 0.2 ml of PBS containing 2% FBS, the binding ability was analyzed using a flow cytometer CytoFlex flowcytometer.)

[0148] As a result, it was confirmed that the anti-CLDN18.1 antibody binds to the BxPC3 cell line, and none of the anti-CLDN18.2 screening antibodies bind to the BxPC3 cell line ( Figure 6 ). From the above results, it was confirmed that the screening antibodies 0058-001, 0058-002, and 0058-003 do not bind to the CLDN18.1 isoform, but selectively bind to the CLDN18.2 antigen.)

[0149] Example 4. Cellular internalization ability of the screened CLDN18.2-specific monoclonal antibody

[0150] 4-1. Analysis of cellular internalization ability using INCUCYTE

[0151] The cellular internalization abilities of the anti-CLDN18.2 control antibody SC0080 and the screening antibodies 0058-001, 0058-002, and 0058-003 were confirmed as follows: After conjugating each antibody with Incucyte ® FabFluor human red fluorescent reagent (Essen Bioscience, USA), the amount of the antibody flowing into the cells over time was detected after treating HEK293E / 18.2 cells overexpressing CLDN18.2 and HEK293E cells not expressing CLDN18.2, respectively. 1×10 was dispensed into each well of a 96-well plate (Nunc, USA) 4Cells were allowed to adhere for 24 hours. Each antibody at 4 μg / ml was mixed with an equal amount of Incucyte® FabFluor red antibody labeling reagent and incubated in the dark at 37°C for 15 minutes, then added to the cells. The amount of antibody accumulating in the lysosomes of the cells over time was confirmed every 15 minutes for a total of 24 hours using the IncuCyte ZOOM HD / 2CLR System (Essen Biosciences, USA), and quantification was performed to confirm the intracellular influx of the antibody.

[0152] As a result, no intracellular influx was confirmed for the anti-CLDN18.2 screening antibodies 0058-001, 0058-002, and 0058-003 bound to the cell surface in HEK293E cells that do not express CLDN18.2 ( Figure 8 ), while 50% of the antibody was confirmed to have entered the cells within 5 hours in HEK293E / 18.2 cells ( Figure 9 ). In particular, the screening antibodies 0058-001 and 0058-002 showed better intracellularization ability than the control antibodies (Ybiologics, #SC0080; in-house IgG mAb zolbetuximab). These results evaluate that all anti-CLDN18.2 screening antibodies can be used as antibodies that can specifically target the intracellular delivery of drugs, and it is considered that they can be developed into antibody-drug conjugates.

[0153] 4-2. Analysis of intracellularization ability using immunofluorescence

[0154] To confirm whether the anti-CLDN18.2 antibody can enter the cells after binding to the antigen present on the cell surface, an endocytosis assay was performed in CHO-K1 / 18.2 cells overexpressing CLDN18.2. To confirm the location of the antibody within the cells, immunocytochemistry (ICC) was performed. For reference, it was confirmed by the same method as in Example 2-2 that CHO-K1 / 18.2 cells are high-expressing cells that express Claudin-18.2 at approximately 161 times compared to CHO-K1 cells (refer to the lower end of Figure 10 ).

[0155] A 12-mm coverslip coated with PPL (SIGMA-ALDRICH, #P4707) was placed in a 24-well plate, and 5×10 4Seed cells in an amount, and after treating each with 10 μg / ml of anti-CLDN18.2 antibodies 0058-001, 0058-002, and 0058-003, incubate at 4°C for 1 hour to allow the antigen present on the cell surface to bind to the antibody. After washing the cells with cold PBS, to induce the internalization of the antibody, replace with complete medium (RPMI-1640 (GIBCO, #A1049101), 1% ANTI-ANTI (Thermo Fisher, #15240062), 10% FBS (Thermo Fisher, #26140-079)) pre-heated at 37°C, and then culture at 37°C under 5% CO2 for 24 hours. After culturing, wash the cells 3 times with PBS to remove the medium, and fix with 4% paraformaldehyde. After washing the fixed cells 3 times with PBS, react with an endosome-labeling antibody diluted in PBS containing 0.5% saponin (Sigma-Aldrich, #47036) and 10% FBS at room temperature for 1 hour. For endosome labeling, use anti-rabbit LAMP1 (Abcam, USA) as the late endosome / lysosome labeling antibody. After binding the labeling antibody, wash the cells 3 times with PBS containing 10% FBS at 3-minute intervals, and then dilute anti-human IgG conjugated with FITC (Jackson ImmunoResearch, #709-545-149), which is the secondary antibody against the anti-CLDN18.2 antibody, and anti-rabbit IgG conjugated with Cy3 (Jackson ImmunoResearch, #711-165-152), which is the secondary antibody against anti-LAMP1, in PBS containing 0.5% saponin and 10% FBS at a dilution of 1:200 respectively, and incubate at room temperature for 1 hour. After washing the fluorescence-stained cells 3 times with PBS at 3-minute intervals, drop a mounting solution containing DAPI (Vector laboratories, #H1200) on a glass slide, cover with a coverslip without generating bubbles, and then observe using a confocal laser scanning microscope (ZEISS, LSM9, Germany). The signal information for each fluorescence color is shown in Table 3 below.

[0156] Table 3

[0157] Signal information regarding fluorescence color

[0158]

[0159] As a result, it was confirmed that the control antibody (SC0080) and three CLDN18.2 monoclonal antibodies flowed into the cells after inducing endocytosis. SC0117, as an isotype control that did not bind to CLDN18.2, was not observed in the cells at 0 hours and 24 hours later ( Figure 10 and Figure 11 ). On the other hand, the frequency of colocalization of the internalized anti-CLDN18.2 antibody with the anti-LAMP1 antibody, which is an endosome / lysosome marker antibody, was high, indicating that the anti-CLDN18.2 antibody was delivered to the lysosome. Through this, it was reconfirmed that the anti-CLDN18.2 screening antibodies 0058-001, 0058-002, and 0058-003 are all suitable antibodies for antibody-drug conjugates used to deliver cytotoxic substances into cells.

[0160] Example 5. Efficacy test of the screened CLDN18.2-specific monoclonal antibodies in CLDN18.2-expressing cancer cells

[0161] The antibody internalization and cell cytotoxicity of three anti-CLDN18.2 antibodies produced by Ybiologics Co., Ltd. were confirmed using the Fab ZAP Human Antibody Internalization Kit (ATSbio, #KIT-51-Z4).

[0162] In a sterile workbench (biosafety cabinet, #JSCB-1500SB, JSR), HEK293E / CLDN 18.2 overexpressing cell line or HEK293E parental cell line was added to each well of a 96-well plate containing 100 μl of DMEM (HyClone, #SH30243.01) as the culture medium, so that each well contained 1×10 3Cells were cultured in a 5% CO2, 37 °C incubator for 16 hours. On the day of antibody treatment, the Fab-ZAP Kit (Kit) containing lysosome-inactivating protein saponin (ATSbio, #SAP-200) was serially diluted 10-fold from 10,000 nM, which is 10 times the treatment concentration, to 0.001 nM in DMEM as the culture medium, for a total of 8 concentrations. The ZAP spike medium for diluting the test antibody was prepared by adding Fab-ZAP Human (ATSbio, #IT-51-40) to DMEM to a concentration of 45 nM, and then the antibody was prepared at 8 concentrations from 100 nM, which is 10 times the treatment concentration, to 0.00001 nM using this medium. Human IgG (Invitrogen, #31154) was used as a control for the test antibody. Fab IgG-SAP (ATSbio, #IT-67-40), a control saponin (Con-SAP), was prepared at 100 nM, which is 10 times the treatment concentration, in DMEM as the cell culture medium. Using a multi-channel pipette, 10 μl of the prepared saponin, test antibody, and Fab IgG-SAP were added to each well of the 96-well plate that had been cultured for 16 hours. Then, the plate was cultured in a 5% CO2, 37 °C incubator for 72 hours.

[0163] In a sterile test bench, 5.5 ml of DPBS (HyClone, #SH30028.02) was thoroughly mixed with 5.5 ml of XTT from the Fab ZAP Kit (ATSbio, #XTT) to prepare 11 ml of DPBS / XTT (1:1) reagent, and then 92 μl of PMS from the kit (ATSbio, #PMS-400) was added to prepare the XTT / PMS reagent. The plate that had been cultured for 72 hours was taken out, and 50 μl of the XTT / PMS reagent was dispensed into each well where the antibody and saponin had been treated. Then, the plate was cultured in a 5% CO2, 37 °C incubator for 2 hours.

[0164] After the 2-hour culture ended and the plate was taken out, the absorbance was measured at a wavelength of 450 nm using a GloMax® Discover microplate reader (Promega, #GM3000). The cell viability (%) was calculated using the 4-parameter non-linear function curve (log vs response-variable slope 4 parameters) of GraphPad prism (9.4 version), with the value of the well without the test antibody treatment as the 100% reference for calculating the IC 50(the half maximal inhibitory concentration; the amount of antibody required to induce apoptosis in 50% of the cells)。

[0165] As a result, as shown in Table 4, Figure 12 and Figure 13 no cytotoxicity of the test antibody was observed in the parental HEK293E cell line without antigen expression. In the HEK293E / 18.2 cell line overexpressing CLDN18.2, cytotoxicity effects were observed at 10 nM, the highest concentration used as the treatment of the test antibody. The IC 50 value was in the range of 0.04 - 0.10 nM, indicating that the test antibody could induce cytotoxicity after antigen-specific internalization, and there were differences among the test antibodies in this effect.

[0166] Table 4

[0167] Cytotoxicity effects after cell internalization

[0168]

[0169] Example 6. Thermal stability of the selected CLDN18.2-specific monoclonal antibodies

[0170] The thermal stability of the antibody was tested using Differential Scanning Fluorimetry. Dilute the antibody protein to a concentration of 3 μM in DPBS to prepare 45 μl, mix it with 5 μl of 200x Sypro orange dye (Thermo, #S6650), and dispense 50 μl into a qPCR tube (Bio-Rad, #TLS0851-white), then cover the tube with a lid (Bio-Rad, #TCS0803). Perform qPCR using a Biorad CFX96 real-time PCR instrument. The qPCR reaction is carried out at 25°C for 30 seconds, then increased by 0.5°C each time to 99°C, reacting for 0.5 minutes at each temperature, and finally ending with a 10-second reaction at 25°C. Use Tm (melting temperature) as the rate constant for antibody structure disassembly.

[0171] Table 5

[0172] Thermodynamic stability of the antibody

[0173]

[0174] The analysis results, as shown in Table 5 described above, show that the Tm values of the control antibody and the screening antibodies 0058-002 and 0058-003 are above 65°C, confirming good thermal stability, while 0058-001 shows a slightly lower value of 64.5°C. Two Tm values are shown for antibodies 0058-002 and 0058-003, which is thought to be due to the different disassembly rates between the domains (CH2, CH3, Fab) that make up the antibody, or due to the aggregation of inactivated proteins generated by disassembly starting from the first melting temperature, followed by a second disassembly at the second melting temperature.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds to Claudin-18 splicing variant 2, characterized in that, Selected from the group consisting of the following (a), (b), and (c): (a) A monoclonal antibody that specifically binds to Claudin-18 splice variant 2, comprising a heavy chain variable region containing the heavy chain CDR1 shown in SEQ ID NO: 1, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 3, and a light chain variable region containing the light chain CDR1 shown in SEQ ID NO: 5, the light chain CDR2 consisting of the amino acid sequence DVS, and the light chain CDR3 shown in SEQ ID NO: 6; (b) A monoclonal antibody that specifically binds to Claudin-18 splice variant 2, comprising a heavy chain variable region containing the heavy chain CDR1 shown in SEQ ID NO: 1, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 8, and a light chain variable region containing the light chain CDR1 shown in SEQ ID NO: 10, the light chain CDR2 consisting of the amino acid sequence AAS, and the light chain CDR3 shown in SEQ ID NO: 11; and (c) A monoclonal antibody that specifically binds to Claudin-18 splice variant 2, comprising a heavy chain variable region containing the heavy chain CDR1 shown in SEQ ID NO: 1, the heavy chain CDR2 shown in SEQ ID NO: 2, and the heavy chain CDR3 shown in SEQ ID NO: 13, and a light chain variable region containing the light chain CDR1 shown in SEQ ID NO: 15, the light chain CDR2 consisting of the amino acid sequence GAF, and the light chain CDR3 shown in SEQ ID NO:

11.

2. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splice variant 2 according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding fragment is selected from the group consisting of the following (a), (b), and (c): (a) A monoclonal antibody that specifically binds to Claudin-18 splice variant 2, comprising a heavy chain variable region shown by the amino acid sequence of SEQ ID NO: 4 and a light chain variable region shown by the amino acid sequence of SEQ ID NO: 7; (b) A monoclonal antibody that specifically binds to Claudin-18 splice variant 2, comprising a heavy chain variable region shown by the amino acid sequence of SEQ ID NO: 9 and a light chain variable region shown by the amino acid sequence of SEQ ID NO: 12; and (c) A monoclonal antibody that specifically binds to Claudin-18 splice variant 2, comprising a heavy chain variable region shown by the amino acid sequence of SEQ ID NO: 14 and a light chain variable region shown by the amino acid sequence of SEQ ID NO:

16.

3. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splicing variant 2 according to claim 1 or 2, characterized in that, The monoclonal antibody is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, and recombinant antibodies.

4. The monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splicing variant 2 according to claim 1 or 2, characterized in that, The antigen-binding fragment is selected from the group consisting of scFv, dsFv, Fab, F(ab'), and F(ab')2 that bind to Claudin-18 splice variant 2.

5. A polynucleotide, characterized in that, The heavy chain variable region and the light chain variable region encoding the monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splice variant 2 as recited in claim 1 or 2.

6. An expression vector, characterized in that, Comprising the polynucleotide as recited in claim 5.

7. A transformant other than human transformed with the expression vector as recited in claim 6.

8. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to Claudin-18 splice variant 2, characterized in that, Comprising: Step (a) of preparing a culture solution by culturing a transformant other than human transformed with an expression vector, the 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 splice variant 2 as recited in claim 1 or 2; and Step (b) of purifying the monoclonal antibody or antigen-binding fragment thereof from the culture solution of step (a).

9. A pharmaceutical composition for treating cancer, characterized in that, Comprising the monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splice variant 2 as recited in claim 1 or 2 as an active ingredient.

10. The pharmaceutical composition for treating cancer 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, cholangiocarcinoma, gallbladder cancer, breast cancer, kidney cancer, mesothelioma, head and neck cancer, bladder cancer, cervical cancer, endometrial cancer, fallopian tube cancer, gastrointestinal cancer, hematological malignancy, throat cancer, melanoma, primary peritoneal cancer, salivary gland cancer, sarcoma, thyroid cancer, glioblastoma, and prostate cancer.

11. The pharmaceutical composition for treating cancer according to claim 9, wherein, The composition further comprises an anticancer agent.

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

13. A method for treating cancer, characterized in that, Comprising the step of administering to an individual the pharmaceutical composition for treating cancer as recited in claim 9.

14. A composition for diagnosing cancer, characterized in that, Comprising the monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splice variant 2 as recited in claim 1 or 2.

15. A kit for diagnosing cancer, characterized in that, Comprising the composition for diagnosing cancer as recited in claim 14.

16. An antibody-drug conjugate, characterized in that, Composed of the monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splice variant 2 as recited in claim 1 or 2 and a drug.

17. The antibody-drug conjugate according to claim 16, wherein The drug is selected from the group consisting of cytotoxic drugs, immunomodulatory compounds, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, microRNAs, siRNAs, shRNAs, radioisotopes, and combinations thereof.

18. A chimeric antigen receptor protein, characterized in that, Comprising: i) the antibody as recited in claim 1 or 2; ii) a transmembrane domain; and iii) an intracellular signal transduction domain, characterized by causing T cell activation when the antibody of i) binds to the target antigen.

19. A multispecific antibody, characterized in that, Comprising the monoclonal antibody or antigen-binding fragment thereof that specifically binds to Claudin-18 splice variant 2 as recited in claim 1 or 2.

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