Monoclonal antibody that specifically binds to claudin 18.2 and its applications

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] This invention relates to a monoclonal antibody that specifically binds to claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2) and its applications. [Background technology]

[0002] Claudin 18 has a molecular weight of approximately 27 kDa and belongs to the family of endogenous membrane proteins located at the junction of the epithelium and endothelium. Its structure consists of two extracellular loops and four transmembrane (TM) domains. 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, while claudin 18.2 is expressed very restrictively in normal cells excluding gastric mucosa and is overexpressed in various cancers and their metastatic cancers, including gastric cancer, liver cancer, biliary tract cancer, breast cancer, kidney cancer, pancreatic cancer, non-small cell lung cancer, and mesothelioma (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 can play important roles in tumorigenesis, metastasis, and inflammation. Changes in claudin protein expression are known to lead to impaired tight junction function, affecting signaling pathways and promoting tumor growth.

[0003] Therefore, in order to develop superior anticancer drugs, there is a need to develop antibodies that specifically bind only to claudin 18.2, which exhibits tumor-specific expression, unlike claudin 18.1.

[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 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] Against this technological backdrop, the inventors used human claudin 18.2 protein as an antigen and screened monoclonal antibodies that specifically bind only to human claudin 18.2 using phage display technology composed of complete human antibody sequences. They confirmed that monoclonal antibodies in human antibody form, produced based on the variable regions of the heavy and light chains of the screened antibody clones, could selectively bind to human claudin 18.2 protein expressed on the cell surface and exhibited excellent internalization ability, thereby completing the present invention.

[0006] On the other hand, Korean Published Patent No. 2022-0136267 discloses "Antibody-drug conjugate containing an antibody against human CLDN18.2 and its uses," and Korean Published Patent No. 2022-0121873 discloses "Anti-claudin 18.2 antibody and its uses," but neither describes the "monoclonal antibody that specifically binds to claudin 18.2 and its uses" of the present invention. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Korean Published Patent No. 2022-0136267 [Overview of the Initiative] [Problems that the invention aims to solve]

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

[0009] Another object of the present invention is to provide polynucleotides that encode the antibody or its antigen-binding fragment.

[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] Another object of the present invention is to provide a cancer treatment composition comprising the antibody or its antigen-binding fragment. [Means for solving the problem]

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

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

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

[0015] Furthermore, the present invention provides a polynucleotide encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody or the antigen-binding fragment thereof.

[0016] Furthermore, the present invention provides an expression vector comprising the polynucleotide.

[0017] Furthermore, the present invention provides a transformant, excluding a human, transformed with the expression vector.

[0018] Furthermore, the present invention provides a method for producing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to Claudin 18.2, comprising culturing the transformant.

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

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

[0021] Furthermore, the present invention provides a composition for cancer diagnosis comprising the monoclonal antibody or the antigen-binding fragment thereof, and a kit for cancer diagnosis comprising the composition for cancer diagnosis.

[0022] Furthermore, the present invention provides an antibody-drug conjugate, wherein a drug is conjugated to the monoclonal antibody or the antigen-binding fragment thereof.

[0023] Furthermore, the present invention provides a chimeric antigen receptor (CAR) protein comprising i) the monoclonal antibody; ii) a transmembrane domain; and iii) an intracellular signaling domain characterized by inducing T cell activation when the antibody in i) binds to an antigen.

[0024] Furthermore, the present invention provides a multi-specific antibody comprising the monoclonal antibody or its antigen-binding fragment. [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 an intracellularly internalized fully human antibody sequence, exhibits specific antigen-binding affinity and diverse intracellular internalization capabilities, and has confirmed thermal stability. Therefore, it is expected to be useful in the treatment and / or prevention of cancer and related diseases that express claudin 18.2 in monoclonal antibody form or modified form (such as antibody-drug conjugate, chimeric antigen receptor, or multispecific antibody). [Brief explanation of the drawing]

[0026] [Figure 1] These are flow cytometry results using the HEK293E cell line, which does not express human claudin 18.2 (CLDN18.2). [Figure 2] This is the flow cytometry result using HEK293E cell lines (HEK293E / 18.2) that overexpress CLDN18.2. [Figure 3] This is the flow cytometry result using a CHO-K1 cell line (CHO-K1 / 18.2) that overexpresses CLDN18.2. [Figure 4] This is the result of ELISA analysis using human CLDN18.2 as the coated antigen. [Figure 5] This is the result of ELISA analysis using human CLDN18.1 as the coated antigen. [Figure 6] These are flow cytometry results using the BxPC3 cell line (a cell line that endogenously expresses human CLDN18.1). [Figure 7] Figure 6 shows the results of flow cytometry analysis of the CLDN18.1 expression level of the BxPC3 cell line used. [Figure 8] The intracellular internalization ability of monoclonal antibodies was analyzed using a live cell analysis instrument (Incucyte) in the HEK293E cell line (CLDN18.2 negative). [Figure 9] The intracellular relocation ability of monoclonal antibodies was analyzed using a live cell analysis instrument (Incucyte) in the HEK293E / 18.2 cell line (CLDN18.2 overexpression). [Figure 10] This study analyzed the intracellular relocation ability of monoclonal antibodies in the CHO-K1 / 18.2 cell line (CLDN18.2 overexpression) via cell immunofluorescence staining. [Figure 11] This study analyzed the intracellular relocation ability of monoclonal antibodies in the CHO-K1 / 18.2 cell line (CLDN18.2 overexpression) via cell immunofluorescence staining. [Figure 12] The intracellular integration and cytolytic activity of monoclonal antibodies were analyzed via the FabZAP assay in the HEK293E / 18.2 cell line (CLDN18.2 overexpression). [Figure 13] The intracellular integration and cytolytic activity of monoclonal antibodies were analyzed in the HEK293E cell line (CLDN18.2 negative) via the FabZAP assay. [Figure 14] This is the result of analyzing the thermal stability of a monoclonal antibody specific to CLDN18.2 using differential scanning fluorimetry. [Modes for carrying out the invention]

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

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

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

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

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

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

[0033] In this invention, the term "monoclonal antibody" means a protein molecule that is directed to a single antigenic site (single epitope) and specifically binds to it. The monoclonal antibody can be produced by various methods known in the art.

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

[0035] The monoclonal antibody of the present invention is selected from the group consisting of fully human antibody, humanized antibody, chimeric antibody, mouse antibody, and recombinant antibody, and is preferably a human antibody, but is not limited thereto. Since the entire structure of the human antibody is derived from humans, it has the advantage of having a lower probability of causing an immunoassay compared to existing humanized antibodies or mouse antibodies, and when administered to humans, it does not cause an undesirable immune response. Therefore, it can be used very effectively as a therapeutic antibody.

[0036] In this invention, the term "antigen-binding fragment" refers to a fragment that possesses antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv. Among the antibody fragments, Fab has a structure that has variable regions of the light chain and heavy chain, an invariant region of the light chain, and an initial invariant region (CH1) of the heavy chain, 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 antibody is produced with the cysteine ​​residues in the hinge region of Fab' forming disulfide bonds. Fv is the smallest antibody section that has only a heavy chain variable region and a light chain variable region, and the recombinant technology for producing Fv fragments is disclosed in international publication WO88 / 10649, etc. In double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by disulfide bonds, while in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by covalent bonds via a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (for example, restrictive cleavage of the whole antibody with papain yields Fab, and cleavage with pepsin yields F(ab')2 fragments), and preferably, they can be produced through genetic engineering techniques.

[0037] In this invention, the term "variable region" refers to a region that exhibits many variations in the sequence while performing the function of specifically binding to an antigen. The variable region contains complementarity-determining regions CDR1, CDR2, and CDR3. Framework regions (FR) exist between these complementarity-determining regions and play a role in supporting the ring of complementarity-determining regions.

[0038] In this invention, the term "complementarity determining region (CDR)" refers to a cyclic region involved in antigen recognition, where changes in the sequence of this region determine the specificity of the antibody to the antigen.

[0039] According to one embodiment of the present invention, the inventors screened scFv that specifically bind to claudin 18.2 from a native human single-chain Fv library, and then converted it to IgG form to produce a human antibody that specifically binds to claudin 18.2.

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

[0041] The monoclonal antibody or its antigen-binding fragment of the present invention may be glycated and / or PEGylated in order to increase its residence time in the body to which it is administered, but is not limited thereto.

[0042] In this invention, the term "glycosylation" refers to a processing method that transfers glycosyl groups to proteins. This glycation is carried out by glycosyltransmitters binding glycosyl groups to serine, threonine, asparagine, or hydroxylysine residues of target proteins. These glycated proteins are not only used as components of biological tissues, but also play an important role in cell recognition on the cell surface. Therefore, in this invention, the effectiveness of antibodies can be improved by glycating monoclonal antibodies or their antigen-binding fragments, or by altering the pattern of glycation.

[0043] In this invention, the term "PEGylation" 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, by PEGylating high molecular weight nanoparticles with polyethylene glycol, the surface aqueous properties of the nanoparticles increase, preventing rapid degradation in the body through a so-called stealth effect, which prevents recognition by immune functions, including macrophages that engulf and digest pathogenic bacteria, waste products, and externally infiltrated substances. Therefore, PEGylation can improve the blood residence time of the antibody. The PEGylation used in this invention is formed by a method in which an amide group is formed by bonding the carboxyl group of hyaluronic acid with the amine group of polyethylene glycol, but is not limited to this, and PEGylation can be performed by various methods. In this case, the polyethylene glycol used is not particularly limited, but it is preferable to use one that has a molecular weight of 100 to 1,000 and has a linear or branched structure.

[0044] The aforementioned glycation and / or pegulation can be modified in various ways by methods known in the art, as long as the function of the antibody of the present invention is preserved, and the antibody of the present invention includes either a mutant monoclonal antibody or its antigen-binding fragment in which a diverse glycation and / or pegulation pattern has been modified.

[0045] The term "affinity" refers to the ability of an antibody to specifically recognize and bind to a particular site on an antigen. Along with the specificity of an antibody to an antigen, a high degree of affinity is a crucial element in immune responses. Various methods known in the industry can be used to measure antigen affinity, with surface plasmon resonance technology being one example.

[0046] Furthermore, the present invention provides polynucleotides that code for the heavy chain variable region and the light chain variable region of the monoclonal antibody or its antigen-binding fragment.

[0047] Those skilled in the art will understand that the polynucleotides encoding the light and heavy chains of the monoclonal antibody or its antigen-binding fragment of the present invention are subject to various modifications in the coding region, within the limits that do not alter the amino acid sequence of the antibody light and heavy chains expressed from the coding region, either due to codon degeneracy or considering preferred codons in the organism intending to express the human antibody light and heavy chain or fragment. Furthermore, various modifications or alterations are also possible in the portion excluding the coding region, within the limits that do not affect gene expression, and such modified genes are also included within the scope of the present invention. In other words, the polynucleotides of the present invention are subject to mutations in one or more nucleic acid bases by substitution, deletion, insertion, or combination thereof, as long as they encode a protein with equivalent activity, and these are also included within the scope of the present invention. The sequences of such polynucleotides may be single-stranded or double-stranded, and may be DNA molecules or RNA (mRNA) molecules.

[0048] Furthermore, the present invention provides an expression vector comprising polynucleotides that encode the heavy chain and light chain variable regions of the monoclonal antibody or its antigen-binding fragment.

[0049] In the present invention, the term "expression vector" means a means for expressing a target gene in a host cell, and includes plasmid vectors; viral vectors such as cosmid vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and refers to a gene construct containing essential regulatory elements that are operably linked so as to express a gene insert. In the present invention, "operably linked" means that a nucleic acid expression regulatory sequence and a polynucleotide coding the target protein are functionally linked to perform a general function. Operational linking with an expression vector can be produced using genetic engineering techniques well known in the art, and site-specific DNA cleavage and linking can be easily performed using enzymes and the like that are well known in the art.

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

[0051] Expression vectors may include selective markers for selecting host cells containing the vector. These selective markers are used to sort cells transformed by the vector and may confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface proteins. Since only cells expressing the selective marker survive in an environment treated with the selective agent, transformed cells can be sorted. 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 host cells, such as viral (e.g., baculovirus) or phage vectors, and retroviral vectors, can also be used. Vectors that express whole antibodies or antibody fragments can be either vector systems in which the light and heavy chains are expressed simultaneously in a single vector, or systems in which the light and heavy chains are expressed in separate vectors. In the latter case, the two vectors are introduced into host cells through cotransformation and targeted transformation, cells transformed with the vector containing the light chain (or heavy chain) are sorted, and the sorted cells expressing the light chain are re-transformed with the vector containing the heavy chain (or light chain) to finally sort cells that express both the light and heavy chains.

[0052] To produce Fab-type antibodies, a vector is used in which genes encoding amino acids for the variable region (VL) and invariant region (CL) of the human light chain, and the variable region (VH) and initial invariant region domain (CH1) of the human heavy chain are inserted.

[0053] Furthermore, the present invention provides transformants excluding humans transformed with the expression vector of the present invention.

[0054] Suitable host cells for the aforementioned 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 cells from higher eukaryotes such as insects, as well as plant cells. Furthermore, the cells may also contain, but are not limited to, mammalian-derived 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 host cells" includes any method of introducing nucleic acids into an organism, cell, tissue, or organ, and can be carried out by selecting a standard technique suitable for the host cell, as known in the art. Such methods include, but are not limited to, electroporation, plasmofusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring with silicon carbide fibers, agrobacteria-mediated transformation, PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

[0056] The present invention also, (a) A step of culturing the transformed organism to produce a culture medium, The present invention provides a method for producing a monoclonal antibody or an antigen-binding fragment that specifically binds to claudin 18.2, comprising the step of (b) purifying the monoclonal antibody or an antigen-binding fragment of the present invention from the culture medium of step (a).

[0057] In the above-described manufacturing method, the culture of the transformants is carried out using a suitable culture medium and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art using selected bacterial strains or animal cells.

[0058] Antibodies obtained by culturing transformants are used in an unpurified state and can be further purified using various conventional methods, such as centrifugation or ultrafiltration to remove impurities, followed by dialysis, salt precipitation, or chromatography. These purified antibodies can then be used individually or in combination. Among these, affinity chromatography is the most commonly used, along with ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography. Antibodies produced by these methods have increased affinity for the antigen.

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

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

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

[0062] In this invention, the term "treatment of cancer" means all actions in which the symptoms of cancer improve or are alleviated by the administration of the composition.

[0063] In the pharmaceutical composition according to the present invention, the cancer may be selected from, but is not limited to, the group consisting of pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, gastric cancer, spleen cancer (e.g., colon cancer, rectal 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 osteomyeloma), pharyngeal cancer, melanoma, primary peritoneal cancer, salivary gland cancer, sarcoma, thyroid cancer, glioblastoma, and prostate cancer.

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

[0065] In this invention, the term "pharmaceutically acceptable carrier" means a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. A pharmaceutically acceptable carrier for a composition formulated into a liquid solution is one that is sterile and suitable for biological use, and can be used by mixing one or more components from saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and their components. Other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate the composition into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

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

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

[0068] The pharmaceutical composition of the present invention can be formulated as an active ingredient in parenteral dosage forms, such as injectable forms including subcutaneous, intravenous, or intramuscular injection, suppository infusion, or aerosol formulations for spray use that can be inhaled through the respiratory system. To formulate as an injectable dosage form, the composition of the present invention can be mixed with a stabilizer or buffer in water to produce a solution or suspension, which can then be formulated as a unit dose in ampoules or vials. For suppository infusion, it can 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 formulation such as an aerosol, propellants and other additives may be incorporated to disperse the water-dispersed concentrate or wet powder.

[0069] Furthermore, the pharmaceutical composition may be administered in a pharmaceutically effective amount.

[0070] In this invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined by factors including the individual's species and severity, age, sex, type of cancer, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The compositions of this invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. They may also be administered single or in multiple doses. It is important to administer the amount that provides the greatest effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

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

[0072] The aforementioned chemotherapeutic agent is one or more 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 target one or more of the following immune cells, but are not limited to them: 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] Furthermore, the present invention provides a method for treating cancer, comprising the step of administering the aforementioned cancer treatment pharmaceutical composition to an individual.

[0074] In the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by a suitable method. The administration routes of the compositions of the present invention are via a variety of oral or parenteral routes, insofar as they can reach the target tissue, and specifically, they can be administered in a conventional manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intra-arterial, transdermal, nasal, inhalation, intraocular, or intradermal routes.

[0075] The therapeutic method of the present invention involves administering the cancer therapeutic composition of the present invention in a pharmaceutically effective amount. It will be obvious to those skilled in the art that the appropriate total daily dose is determined by the treating physician within the correct range of medical judgment. The specific therapeutic effective dose for a particular patient is to be applied differently depending on a variety of factors, including the type and degree of response to be achieved, whether other formulations are used, the specific composition, the patient's age, weight, general health status, sex and diet, administration time, route of administration, and secretion rate of the composition, the duration of treatment, and any drugs used with or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical field. Accordingly, the effective dose of the cancer preventive or therapeutic composition suitable for the purposes of the present invention is to be determined taking the above matters into consideration.

[0076] Furthermore, the term "individual" refers to any animal in which diseases such as tumor development and neovascularization can occur due to excessive activity of claudin 18.2, and the term "individual" includes not only humans and primates, but also livestock such as cattle, 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 a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the 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 a monoclonal antibody or its antigen-binding fragment specific to claudin 18.2 of the present invention can be used to diagnose diseases related to the expression and degree of expression of claudin 18.2, such as cancer.

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

[0080] In the present invention, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, autopsy tissue samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems. These biological samples, either manipulated or unmanaged, can be reacted with the antibody 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" means a conjugate of a claudin 18.2 protein antigen in a sample and a monoclonal antibody according to the present invention that recognizes it or its antigen-binding fragment. The formation of such an antigen-antibody complex can be measured by conventional immunoassay methods, including, but is not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competitive analysis, sandwich analysis, 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 through the aforementioned immunoassay process. Specifically, 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 of a biological sample isolated from a normal individual, cancer can be diagnosed.

[0083] Furthermore, the kit according to the present invention includes an antibody against claudin 18.2 of the present invention, and cancer can be diagnosed by analyzing the signal shown by the reaction between the sample and the antibody. In this case, the signal is shown by an enzyme conjugated to the antibody (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, or cytochrome P450), and in this case, the substrate for the enzyme is, when alkaline phosphatase is used as the enzyme, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF (enhanced When a chromogenic reaction substrate such as chemifluorescence is used and 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), naphthol / pyronin, glucose oxidase, t-NBT, and m-PMS (phenzaine methosulfate) can be used, but are not limited to these.

[0084] Furthermore, the kit according to the present invention includes a label that generates a detectable signal, the label being a chemical substance (e.g., biotin), an enzyme (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P450), a radioactive substance (e.g., 14 C, 125 I, 32P and 35 S) may include, but is not limited to, fluorescent substances (e.g., fluoresin), luminescent substances, chemiluminescent substances, and FRET (fluorescence resonance energy transfer).

[0085] The measurement of enzyme activity or signaling used for cancer diagnosis is also carried out by a variety of methods known to the industry. Through these methods, 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 conjugated to a monoclonal antibody or its antigen-binding fragment that specifically binds to the claudin 18.2.

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

[0088] The drug used in the antibody-drug conjugate according to the present invention comprises any compound, part, or group having cytotoxic or cell proliferation inhibitory effects, and includes (i) chemotherapeutic agents that function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators; (ii) enzymatically functioning protein toxins; and (iii) radioisotopes (radionuclides), and one or more of the above compounds may be used.

[0089] Non-limiting examples of such drugs include various antitumor or anticancer agents, including but not limited to maytansinoids, auristatins, dolastatins, trichothecenes, CC1065 (Rachelmycin), calicheamicins 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, dactinomycin, mitomycins, bleomycins, esperamicins, 5-fluorouracil, melphalan, other nitrogen mustards and their stereoisomers, isosteres, homologs or derivatives, cis-platinum and cis-platinum homologs, other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, toxins (enzyme-active toxins or small molecule toxins of bacterial, fungal, plant or animal origin), cisplatin, CPT-11, doxorubicin, paclitaxel and docetaxel, and the like. In addition, the radioisotopes (radionuclides) include, 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 and the like, without being limited thereto, microRNA (miRNA), siRNA and shRNA capable of suppressing the expression of a specific oncogene can 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 characterized by inducing T cell activation when the antibody in i) binds to an antigen.

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

[0092] In the present invention, the term "CAR (chimeric antigen receptor)" means a non-naturally occurring receptor that can confer specificity to a specific antigen to an immunoeffector cell. Typically, the CAR refers to a receptor used to transfer the specificity of a monoclonal antibody to T cells. Typically, a CAR consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes an antigen recognition region, and in the present invention, the antigen recognition region is an antibody specific to claudin 18.2. The antibody specific to claudin 18.2 is as described above, and the antibody used as a CAR is preferably in the form of an antibody fragment, more preferably in the form of a Fab or scFv, but is not limited thereto.

[0093] Furthermore, the membrane-permeable domain of CAR is linked to the extracellular domain and can be of natural or synthetic origin. When derived from natural sources, it may originate from membrane-bound or membrane-permeable proteins, such as the membrane-permeable regions of various proteins, including the alpha, beta, or zeta forms of T cell receptors, such as CD28, CD3 epsilon, CD45, CD4, CD5, CDDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or CD8. The sequences of such membrane-permeable domains can be obtained from publicly known literature in the industry that provides information on the membrane-permeable regions of membrane-permeable proteins, but are not limited to this. Also, when the membrane-permeable domain is synthetic, it may mainly contain hydrophobic amino acid residues such as leucine and valine, and, for example, a triplet of phenylalanine, tryptophan, and valine may be present in the synthetic membrane-permeable domain, but is not limited to this.

[0094] In the CAR of the present invention, the intracellular domain is a part of the domain of the CAR that exists inside the cell and is linked to the membrane-permeable domain. The intracellular domain of the present invention may include an intracellular signaling domain that, when an antigen is bound to the antigen-binding site of the CAR, induces T cell activation, preferably T cell proliferation. The intracellular signaling domain is not particularly limited in type as long as it is a part that transmits a signal that leads to T cell activation when an antibody is bound to the antigen-binding site located outside the cell, and a variety of intracellular signaling domains can be used, for example, an immune receptor tyrosine-based activation motif (ITAM), and the ITAM may include, but is not limited to, those derived from CD3 zeta (ξ), 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 includes, but is not limited to, a costimultatory domain in addition to the intracellular signaling domain. The costimultatory domain is a portion of the CAR of the present invention that, in addition to the signal transmitted by the intracellular signaling domain, plays a role in transmitting signals to T cells, and means the intracellular portion of the CAR that includes the intracellular domain of a costimulatory molecule. The costimulatory molecule means a cell surface molecule that is necessary to elicit a sufficient response of lymphocytes to an antigen, and is, but is not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C, or B7-H3. The costimulatory domain is also the intracellular portion of a molecule selected from the group consisting of such costimulatory molecules and combinations thereof.

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

[0097] The present invention also provides a multispecific antibody comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the 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 according to the present invention is preferably in a form in which the anti-claudin 18.2 antibody according to the present invention is conjugated to an antibody or fragment thereof that has the ability to bind to an immunoassay cell-specific target molecule. The immunoassay cell-specific target molecule is preferably selected from, but is 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] Multiple-specific antibodies are antibodies that simultaneously recognize multiple (two or more) distinct epitopes for the same antigen or two or more other antigens. Antibodies belonging to the category of multiple-specific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. In the case of multiple-specific antibodies, for example, bispecific antibodies, two signals can be suppressed or amplified simultaneously, making them more effective than suppressing / amplifying a single signal. Compared to treating each signal with its own signal inhibitor, lower doses are possible, and two signals can be suppressed / amplified in the same time and space.

[0101] Methods for producing bispecific antibodies are widely known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two rabbit globulin heavy / light chain pairs under conditions in which the two heavy chains have different specificities.

[0102] In the case of bispecific antibodies based on scFv, hybrid scFvs are produced in a heterodimeric form by combining the light chain variable region (VL) and heavy chain variable region (VH) of different scFvs to create a diabody (Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444, 1993). Then, different scFvs are linked together to produce tendem scFvs, and the CH1 and CL of Fab are expressed at the ends of each scFv to produce 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 change it to a heterodimeric structure in the form of a "knob into hole," and these modified CH3 domains are expressed at the ends of different scFvs to produce heterodimeric antibodies. Minibodies in the scFv form can be manufactured (Merchant et al., Nat. Biotechnol., 16:677, 1998).

[0103] In the case of Fab-based bispecific antibodies, individual Fab's for specific antigens can be combined with each other using disulfide bonds or mediators to produce heterodimeric Fab forms. By expressing scFvs for different antigens at the ends of the heavy or light chains of specific Fabs, two antigen-binding valencies can be created, or by creating a hinge region between the Fab and scFvs, four antigen-binding valencies can be produced in a homodimeric form. Furthermore, methods for producing a bitarget bibody with three antigen-binding valencies by fusing scFvs for different antigens to the light and heavy chain ends of Fabs, a triple-target bibody with three antigen-binding valencies by fusing different scFvs to the light chain mardan and heavy chain ends of Fabs, and a simple form of triple-target antibody F(ab')3 obtained by chemically joining three different Fabs are known in the industry.

[0104] In the case of IgG-based bispecific antibodies, a method is known by TrionPharma to produce bispecific antibodies by crossbreeding mice and rat hybrids again to create hybrid hybrids, also known as quadromas. In addition, there is a method for producing bispecific antibodies in the so-called "Holes and Knob" form, in which the light chain is shared, but some amino acids in the CH3 homodimeric domain of Fc are modified to create a heterodimeric form for different heavy chains (Merchant et al., Nat. Biotechnol., 16:677, 1998). Besides heterodimeric bispecific antibodies, there is also a method in which two different scFvs are fused and expressed in the constant domains instead of the variable domains of the light and heavy chains of IgG, and then produced as homodimeric (scFv)4-IgG. Furthermore, ImClone has reported the creation of a bispecific antibody based on IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, by fusing only the single variable domain for mouse platelet-derived growth factor receptor-α to the light chain amino terminus of this antibody. Rossi et al. have also disclosed an antibody with multiple antigen-binding titers for CD20 through a so-called "dock and lock (DNL)" method using 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 the content of the present invention is not limited to the following examples. [Examples]

[0106] Example 1. Selection of claudin 18.2 (CLDN18.2) specific antibodies The CLDN18.2 (R&D, #RDC2149) gene was overexpressed in the HEK293E animal cell line to secure the cell line. CLDN18.2-VLPs antigen (Cusabio, #CSB-MP005498HU(A5)) was coated onto immunosorbent tubes, followed by blocking. Human scFv library phage (Y Biologics Co., Ltd.) was used to infect Bacillus malva, and the resulting Bacillus malva was cultured at 30°C for 16 hours. The culture medium was centrifuged, and the supernatant was concentrated with PEG (polyethylene glycol). This supernatant was then dissolved in PBS (phosphate-buffered saline) buffer to prepare the human antibody library phage.

[0107] The library phages were placed in the immunoadsorption tubes and reacted at room temperature for 2 hours. After washing with 1xPBST and 1xPBS, the samples were sequentially treated with 100 mM TAE and Tris-HCl (pH 7.5) solution 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 form and used in experiments.

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

[0109] [Table 1]

[0110] [Table 2]

[0111] Example 2. Characteristics of the selected CLDN18.2-specific monoclonal antibody 2-1. Nonspecificity of CLDN18.2 monoclonal antibody by flow cytometry Nonspecific binding affinity experiments were performed using the HEK293E cell line. Human-CLDN18.2 expression was confirmed using anti-CLDN18.2 (CUSABIO, #CSB-RA005498A1HU).

[0112] 0.5 x 10⁶ HEK293E cells that do not express human CLDN18.2 were collected per sample. 6 Cells were prepared, and the selection antibodies 0058-001, 0058-002, and 0058-003 were diluted to a concentration of 7.5 μg / ml and reacted with the prepared cells at 4°C for 30 minutes. Subsequently, the cells were washed three times with PBS (Welgene, #LB001-02) containing 2% fetal bovine serum, reacted with anti-human IgG antibody (Vectorlabs, #FI-3000) conjugated with the fluorescent substance (fluorescein isothiocyanate) at 4°C for 30 minutes, and then washed in the same manner as above. The cells were suspended in PBS containing 0.2 ml of 2% FBS (Thermo, #26140-079), and the binding strength was analyzed using a CytoFlex flow cytometer (Beckman Coulter, USA).

[0113] Analysis revealed that none of the screening 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 HEK293E / 18.2 cells and CHO-K1 / 18.2 cells overexpressing human CLDN18.2 were each measured at 0.5 x 10⁶ per sample. 6Cells were prepared, and the CLDN18.2 antibody was diluted to a specific multiple and reacted with the prepared cells at 4°C for 30 minutes. The cells were then washed three times with PBS containing 2% fetal bovine serum, reacted with FITC-conjugated anti-human IgG antibody at 4°C for 20 minutes, and washed again in the same manner. The cells were suspended in PBS containing 0.5 ml of 2% FBS, and the binding strength was analyzed using a CytoFlex flow cytometer.

[0115] Analysis revealed that the anti-CLDN18.2 screening antibodies 0058-001, 0058-002, and 0058-003 all bind in a concentration-dependent manner to HEK293E / 18.2 and CHO-K1 / 18.2 cell lines, which highly express CLDN18.2 (Figures 2 and 3).

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

[0117] 3-1. Analysis of binding activity to CLDN18 isoform via ELISA CLDN18.1 (EUPROTEIN, #EPY255141) and CLDN18.2 (Cusabio, #CSB-MP005498HU(A5)) proteins were prepared at 1 μg / ml using dilution solutions (4% skim milk / 0.05% tween-20 / PBS), and 100 μl of each was placed in each well of an immunoassay plate (Thermo, #439454) and incubated statically at 4°C for at least 16 hours. 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 each well, and the mixture was incubated at room temperature for 1 hour. After three washes, 100 μl of anti-CLDN18.2 antibody, diluted to a specific multiple, was added to each well, and the mixture was rocked at room temperature for 1 hour. After three washes, 100 μl of anti-human kappa-HRP (Sigma, #A7164), prepared at a dilution ratio of 1:3,000, was added to each well and reacted at room temperature for 1 hour. After three washes, 100 μl of TMB substrate (Sigma, #T0440) was added to each well and reacted at room temperature under light shielding. When color development was confirmed, 50 μl of reaction stop solution (1N H2SO4) was added to stop the reaction. Absorbance was measured using a spectrophotometer (Promega, GM3000).

[0118] Analysis revealed that the selective antibodies 0058-001, 0058-002, and 0058-003 bound to the CLDN18.2 antigen (Figure 4), but none bound to the CLDN18.1 antigen (Figure 5), confirming their selectivity for the CLDN18.2 antigen.

[0119] 3-2. Analysis of binding affinity to CLDN18 isoform via flow cytometry BxPC3 (Figure 7) endogenously expressing human CLDN18.1 was used at a rate of 0.5 x 10 per sample. 6Prepared cells were reacted with 50 nM CLDN18.1 antibody (Cusabio, #CSB-RA005498A2HU) and CLDN18.2 antibody according to the present invention, respectively, with prepared BxPC3 cells at 4°C for 30 minutes. Subsequently, the cells were washed three times with PBS containing 2% fetal bovine serum, reacted with FITC-conjugated anti-human IgG antibody at 4°C for 30 minutes, and then washed in the same manner as above. After suspending the cells in PBS containing 0.2 ml of 2% FBS, the binding strength 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, while none of the anti-CLDN18.2 screening antibodies bound to the BxPC3 cell line (Figure 6). Through these 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.

[0121] Example 4. Intracellular relocation ability of selected CLDN18.2-specific monoclonal antibodies. 4-1. Analysis of intracellular relocation ability using INCUCYTE The intracellular acquisition ability of the anti-CLDN18.2 control antibody SC0080 and the sorting antibodies 0058-001, 0058-002, and 0058-003 was monitored by conjugating each antibody with Incucyte® FabFluor human red fluorescent reagent (Essen Bioscience, USA), then treating HEK293E / 18.2 cells overexpressing CLDN18.2 and HEK293E cells not expressing CLDN18.2, and monitoring the amount of antibody flowing into the cells over time. The data was collected in 96-well plates (Nunc, USA) at a rate of 1 x 10⁶ per well. 4Individual cells were dispensed and allowed to adhere for 24 hours. Each cell was then treated with 4 μg / ml of antibody mixed with the same volume of Incucyte® FabFluor red antibody labeling reagent and allowed to react in the dark at 37°C for 15 minutes. The amount of antibody entering the cells was quantified by monitoring the amount of antibody accumulated in the cell lysosomes every 15 minutes over a total of 24 hours using the IncuCyte ZOOM HD / 2CLR System (Essen Biosciences, USA).

[0122] As a result, anti-CLDN18.2 sorting antibodies 0058-001, 0058-002, and 0058-003 bound to the cell surface did not show intracellular influx in HEK293E cells that do not express CLDN18.2 (Figure 8), while in HEK293E / 18.2 cells, 50% of the antibodies were confirmed to have entered the cells within 5 hours (Figure 9). In particular, sorting antibodies 0058-001 and 0058-002 showed superior intracellular infiltration ability compared to the control antibody (Ybiologics, #SC0080; IgG mAb zolbetuximab in-house). These results suggest that all anti-CLDN18.2 sorting antibodies can function as antibodies that deliver drugs intracellularly in a target-specific manner, and that they can be developed as antibody-drug conjugates.

[0123] 4-2. Analysis of intracellular relocation capacity using cell immunofluorescence To confirm whether the anti-CLDN18.2 antibody can enter the cell after binding to an antigen present on the cell surface, an endocytosis test was performed on CHO-K1 / 18.2 cells overexpressing CLDN18.2, and immunocytochemistry (ICC) was performed to confirm the intracellular location of the antibody. For reference, CHO-K1 / 18.2 cells are cells in which claudin 18.2 is overexpressed approximately 161 times compared to CHO-K1 cells, which was confirmed by the same method as in Example 2-2 (see bottom of Figure 10).

[0124] Place 12mm coverslips coated with PPL (SIGMA-ALDRICH, #P4707) into a 24-well plate and insert 5x10 cells per well. 4 Cells were seeded and treated with 10 μg / ml anti-CLDN18.2 antibodies 0058-001, 0058-002, and 0058-003, respectively, and allowed to react at 4°C for 1 hour to allow the antibodies to bind to antigens present on the cell surface. Subsequently, the cells were 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)] preheated to 37°C and 5% CO2 to induce antibody resorption into the cells. 2Cells were cultured for 24 hours under the specified conditions. After culturing, the culture medium was removed by washing the cells three times with PBS, and the cells were fixed with 4% paraformaldehyde. The fixed cells were washed three times with PBS and then reacted at room temperature for 1 hour with an endosome-labeled 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-labeled antibody. After labeling with the antibody, the cells were washed three times with PBS containing 10% FBS at 3-minute intervals. Then, anti-human IgG (Jackson ImmunoResearch, #709-545-149) conjugated with FITC, a secondary antibody against anti-CLDN18.2, and anti-rabbit IgG (Jackson ImmunoResearch, #711-165-152) conjugated with Cy3, a secondary antibody against anti-LAMP1, were diluted 1:200 in PBS containing 0.5% saponin and 10% FBS, and cultured in the dark at room temperature for 1 hour. The fluorescently stained cells were washed three times with PBS at 3-minute intervals, then mounted in a mounting solution containing DAPI (Vector Laboratories, #H1200) onto a slide, sealed with a coverslip to prevent air bubbles, and observed using a confocal laser scanning microscope (ZEISS, LSM9; Germany). The signal information associated with each fluorescent color is shown in Table 3 below.

[0125] [Table 3]

[0126] As a result, it was confirmed that the control antibody (SC0080) and the three CLDN18.2 monoclonal antibodies were influxed into cells after endocytosis induction, while SC0117 was not observed in cells at 0 and 24 hours in the isotype control group that does not bind to CLDN18.2 (Figures 10 and 11). On the other hand, the influxed anti-CLDN18.2 antibodies frequently colocalized with the endosomal / lysosome-labeled antibody anti-LAMP1, indicating that the anti-CLDN18.2 antibodies were transferred to lysosomes. This reaffirmed that the anti-CLDN18.2 selection antibodies 0058-001, 0058-002, and 0058-003 are all suitable antibodies for use as antibody-drug conjugates for delivering cytotoxic substances into cells.

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

[0128] On a sterile laboratory bench (Biosafety cabinet, #JSCB-1500SB, JSR), infuse 1x10 cells of HEK293E / CLDN18.2 overexpressing cell lines or HEK293E parental cell lines into 100 μl of DMEM (HyClone, #SH30243.01) culture medium. 3 Place each cell in a 96-well plate and add 5% CO2. 2The cells were incubated in a 37°C incubator for 16 hours. On the day of antibody treatment, saporin (ATSbio, #SAP-200), a ribosome inactivating protein from the Fab-ZAP Kit, was diluted tenfold in DMEM culture medium, starting from 10,000 nM (10 times the treatment concentration), down to a total of eight concentrations down to 0.001 nM. For diluting the test antibody, ZAP spike medium was prepared by adding Fab-ZAP Human (ATSbio, #IT-51-40) to DMEM to a concentration of 45 nM. Using this medium, the antibody was diluted tenfold in increments, starting from 100 nM (10 times the treatment concentration), down to a total of eight concentrations down to 0.00001 nM. Human IgG (Invitrogen, #31154) was used as a control for the test antibody. The control substance (Con-SAP) for the saporin, Fab IgG-SAP (ATSbio, #IT-67-40), is prepared in DMEM, the cell culture medium, at a concentration of 100 nM, which is 10 times the treatment concentration. The prepared saporin, test antibody, and Fab IgG-SAP are then added to each well of a 96-well plate using a multi-pyretto, after being cultured for 16 hours. The plate is then cooled in 5% CO2. 2 The cells were then incubated in a 37°C incubator for 72 hours.

[0129] On a sterile lab bench, thoroughly mix 5.5 ml of DPBS (HyClone, #SH30028.02) and 5.5 ml of XTT (ATSbio, #XTT) from the Fab ZAP Kit to prepare 11 ml of DPBS / XTT (1:1) reagent. Then, add 92 ml of PMS (ATSbio, #PMS-400) from the kit to prepare the XTT / PMS reagent. After culturing the plate for 72 hours, remove the plate and dispense 50 μl of the XTT / PMS reagent into each well that has been treated with antibody and saporin, etc. Then, cover the plate with 5% CO2. 2 The cells were then incubated in a 37°C incubator for 2 hours.

[0130] After 2 hours of incubation, the plates were removed and the absorbance was measured at a wavelength of 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 (9.4 version), with the value of the well not treated with the test antibody set to 100%. 50 The half-maximal inhibitory concentration (the amount of antibody needed to induce 50% cell death) was calculated.

[0131] As a result, as shown in Table 4, Figure 12 and Figure 13, no cytotoxicity of the test antibody was observed in the HEK293E parental cell line without antigen expression. However, in the HEK293E / 18.2 cell line overexpressing CLDN18.2, cytotoxic effects were observed at the highest concentration of test antibody treatment, 10 nM. 50 The values ​​in the 0.04–0.10 nM range indicated that the test antibodies could induce cytotoxicity after antigen-specific internalization, and that the effect varied among the test antibodies.

[0132] [Table 4]

[0133] Example 6. Thermal stability of selected CLDN18.2-specific monoclonal antibodies The thermal stability of the antibody was tested using differential scanning fluorimetry. The antibody protein was diluted with DPBS to prepare a 3 μM solution of 45 μl, which was mixed with 5 μl of 200x Sypro orange dye (Thermo, #S6650). 50 μl of this mixture was dispensed into qPCR tubes (Bio-Rad, #TLS0851-white), and the lids (Bio-Rad, #TCS0803) were closed. qPCR was performed using a Bio-Rad CFX96 real-time PCR instrument. The qPCR reaction was carried out at 25°C for 30 seconds, then increased by 0.5°C increments up to 99°C, with each temperature setting lasting 0.5 minutes. The final reaction was completed at 25°C for 10 seconds. The melting temperature (Tm) was used as the rate constant for the dissolution of the antibody structure.

[0134] [Table 5]

[0135] The analysis results, as shown in Table 5 above, confirmed that the control antibody and selected antibodies 0058-002 and 0058-003 had good thermal stability with Tm values ​​of 65°C or higher, while 0058-001 showed a slightly lower value of 64.5°C. The two Tm values ​​shown for antibodies 0058-002 and 0058-003 were thought to be due to differences in the dissolution rates between the domains (CH2, CH3, Fab) constituting the antibodies, or because the inactivated proteins that dissolved from the first melting temperature aggregated before dissolving in a secondary manner at the second melting temperature.

Claims

1. A monoclonal antibody or antigen-binding fragment that specifically binds to claudin-18.2 (claudin-18 spliced ​​variant 2, CLDN18.2), comprising a heavy chain variable region including heavy chain CDR1 described in SEQ ID NO: 1; heavy chain CDR2 described in SEQ ID NO: 2; and heavy chain CDR3 described in SEQ ID NO: 3; and a light chain variable region including light chain CDR1 described in SEQ ID NO: 5; light chain CDR2 consisting of an amino acid sequence DVS; and light chain CDR3 described in SEQ ID NO:

6.

2. The monoclonal antibody or its antigen-binding fragment is a monoclonal antibody that specifically binds to claudin 18.2, comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 4 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 7, as described in claim 1.

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

4. The antigen-binding fragments are 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 its antigen-binding fragment that specifically binds to claudin 18.2 according to claim 1 or 2, characterized by being selected from the group consisting of the following.

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

6. An expression vector comprising the polynucleotide described in claim 5.

7. A transformant, excluding a human being transformed with the expression vector described in claim 6.

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

9. A pharmaceutical composition for cancer treatment comprising a monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2 as an active ingredient.

10. The cancer treatment pharmaceutical composition according to claim 9, characterized in that the cancer is selected from the group consisting of pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, gastric cancer, intestinal 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, characterized in that the composition further comprises an anticancer agent.

12. The pharmaceutical composition for cancer treatment according to claim 11, characterized in that the anticancer agent is a chemotherapy agent or an immune checkpoint inhibitor.

13. A cancer diagnostic composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in claim 1 or 2.

14. A cancer diagnostic kit comprising the composition described in claim 13.

15. An antibody-drug conjugate comprising a monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, to which a drug is conjugated.

16. The antibody-drug conjugate according to claim 15, wherein the drug is selected from the group consisting of cytotoxic agents, immunomodulatory compounds, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, microRNA (miRNA), siRNA, shRNA, radioisotopes, and combinations thereof.

17. i) The antibody according to claim 1 or 2; ii) Transmembrane domain; and iii) A chimeric antigen receptor protein that includes an intracellular signaling domain, characterized in that when the antibody described in i) binds to a target antigen, it triggers T cell activation.

18. 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) as described in claim 1 or 2.

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