Anti-claudin-18.2 antibody and its antibody-drug conjugate

Abstract

The present invention provides antibodies and antibody-drug conjugates that target claudin-18.2, and their use in the treatment of cancer. The present invention also provides nucleotides encoding the claudin-18.2 antibodies, combinations of polynucleotides, expression vectors, and combinations of expression vectors, pharmaceutical compositions containing the claudin-18.2 antibodies or antibody-drug conjugates, and their use in the manufacture of medicaments for treating or preventing cancer.

Description

[Technical field]

[0001] The present invention relates to the field of biopharmaceuticals, in particular to an anti-claudin-18.2 antibody and its antibody-drug conjugate, and also to uses of the antibody and its antibody-drug conjugate. [Background technology]

[0002] In recent years, the overall incidence of malignant tumors worldwide has been on an increasingly upward trend, seriously threatening human health and survival. Currently, the clinical treatment of malignant tumors is centered on surgery, chemotherapy and radiotherapy, but it is difficult to achieve satisfactory therapeutic effects. Antibody-drug conjugates (ADCs) are a type of biological preparation in which a biologically active drug is linked to an antibody by a chemical linker. In recent years, some antibody-drug conjugates have achieved breakthroughs in the treatment of malignant tumors, becoming the third major new treatment after surgery, chemotherapy and radiotherapy. However, as of March 2021, only 11 types of antibody-drug conjugates have been approved worldwide (10 items approved by the US FDA and 1 item approved by the Japanese PMDA), and the types of approved indications are limited, so the clinical needs of patients with malignant tumors are still unmet.

[0003] [Table 1]

[0004] Cell-junction claudins (CLDNs) are widely distributed in various epithelial tissues and are important structural components of cell tight junctions. Research has shown that CLDNs proteins are closely related to epithelial cells maintaining osmotic pressure, barrier function, and cell polarity (Non-Patent Document 1), and are also involved in the process of immune defense against pathogens (Non-Patent Document 2). In addition, it has been confirmed that the expression pattern of CLDNs changes during the development and development of many tumors, and research on targeted therapy using the CLDNs lineage as a specific marker protein has attracted widespread attention. However, while most CLDNs are widely expressed, individual members among them, such as the CLDN18 protein, are often highly selectively expressed only in specific tissues such as the gastrointestinal tract. The CLDN18 gene is located on human chromosome 3 at 3q22.3, and the first exon of the gene has two options, which can form two different splice variants and express two protein isoforms, claudin-18.1 protein and CLDN18.2 protein, which have different amino acid sequences of 69 amino acids at the N-terminus (Non-Patent Document 3). Here, the claudin-18.1 protein is a specific antigen selectively expressed by alveolar epithelial cells, and is highly expressed only in normal alveolar tissues, but is not found in other normal tissues, including tissues such as the pancreatic duct (Non-Patent Document 4). The claudin-18.2 protein is also a highly selective marker protein, but is distributed in tissues completely different from the claudin-18.1 protein. The expression of the claudin-18.2 protein is highly restricted in healthy tissues, but it is abnormally activated and overexpressed in various primary malignant tumors such as gastric cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, and is particularly frequently expressed in malignant tumors of the digestive system, such as gastric cancer (70%), pancreatic cancer (50%), and esophageal cancer (30%) (Non-Patent Document 5). In addition, another study showed that CLDN18.2 protein is not limited to expression in primary tumors, but is also highly expressed in metastatic tumors, and may be involved in the processes of proliferation and chemotaxis of malignant tumor cells (Non-patent Documents 6, 7).Therefore, claudin-18.2 is a very ideal tumor patient marker and target for antitumor drug development, especially for gastric cancer, pancreatic cancer, esophageal cancer, lung cancer and ovarian cancer. However, due to the specificity of its target (compared with claudin-18.1, its structure is highly similar, and the first domain in the extracellular region of both is exactly the same), it is more difficult to develop therapeutic antibodies against claudin-18.2, which is also the main reason for limiting the development of claudin-18.2 targeted drugs. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Gunzel D. Claudins: vital partners in transcellular and paracellular transport coupling[J].Pflugers Arch,2017,469(1):35-44. [Non-Patent Document 2] Colpitts CC,Baumert TF.Claudins in viral infection:from entry to spread[J].Pflugers Arch,2017,469(1):27-34. [Non-Patent Document 3] Hayashi D, Tamura A, Tanaka H, ​​et al.Deficiency of claudin-18 causes paracellular H+leakage,up-regulation of interleukin-1β,and atrophic gastritis in mice[J].Gastroenterology,2012,142(2):292-304. [Non-Patent Document 4] Li G, Flodby P, Luo J, et al.Knockout mice reveal key roles for clau‐din18 in alveolar barrier properties and fluid homeostasis[J].Am J Respir Cell Mol Biol,2014,51(2):210-222. [Non-Patent Document 5] Kumar V, Soni P, Garg M, et al.Emerging Therapies in the Management of Advanced-Stage Gastric Cancer[J].Front Pharmacol,2018,9:404. [Non-Patent Document 6] Woll S, Schlitter AM, Dhaene K, et al. Claudin-18.2 is a target for IMAB362 antibody in pancreatic neoplasms[J].Int J Cancer,2014,134(3):731-739. [Non-Patent Document 7] Jiang H, Shi Z, Wang P, et al.Claudin-18.2-Specific Chimeric Antigen Receptor Engineered T Cells for the Treatment of Gastric Cancer[J].J Natl Cancer Inst,2018,111(4):1-10. Summary of the Invention [Problem to be solved by the invention]

[0006] Currently, all antibody-drug conjugates targeting claudin-18.2 are in the preclinical research stage, and none have yet progressed to the clinical stage. Therefore, it is urgent to develop antibody-drug conjugates targeting claudin-18.2 to provide more clinical options. [Means for solving the problem]

[0007] The present invention provides antibodies targeting claudin-18.2, antibody-drug conjugates, and their use in the treatment of cancer. The present invention also provides nucleotides encoding the claudin-18.2 antibodies, combinations of polynucleotides, expression vectors, and combinations of expression vectors, pharmaceutical compositions comprising the claudin-18.2 antibodies and antibody-drug conjugates, and their use in the manufacture of medicines for treating or preventing cancer.

[0008] Specifically, the present invention relates to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the CDR of the heavy chain variable region and / or the CDR of the light chain variable region have the same CDR sequence as an antibody defined by the following sequence, or are made by substituting one or two amino acids in the CDR of the antibody defined by the following sequence, and the antibody defined by the sequence is (1) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1, and / or (2) An anti-claudin-18.2 antibody or an antigen-binding fragment thereof is provided, the amino acid sequence of the light chain variable region of which is shown in SEQ ID NO:2.

[0009] In certain embodiments, the complementarity determining regions CDR1-3 of the corresponding heavy and light chain variable regions identified by different assay methods or systems are shown in Table 2.

[0010] [Table 2]

[0011] The invention also provides, in some specific embodiments, an anti-claudin-18.2 antibody or antigen-binding fragment comprising a heavy chain and a light chain variable region sequence: (1) the amino acid sequence of the heavy chain variable region CDR1 is represented by SEQ ID NO: 3, 4, 5, 6 or 7, or an amino acid sequence having one or two amino acid substitutions with respect to SEQ ID NO: 3, 4, 5, 6 or 7; the amino acid sequence of CDR2 is represented by SEQ ID NO: 8, 9, 10, 11 or 12, or an amino acid sequence having one or two amino acid substitutions with respect to SEQ ID NO: 8, 9, 10, 11 or 12; and the amino acid sequence of CDR3 is represented by SEQ ID NO: 13, 14, 15, 16 or 17, or an amino acid sequence having one or two amino acid substitutions with respect to SEQ ID NO: 13, 14, 15, 16 or 17; (2) The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 18, 19, 20, 21 or 22, or an amino acid sequence having one or two amino acid substitutions with respect to SEQ ID NO: 18, 19, 20, 21 or 22; the amino acid sequence of CDR2 is shown in SEQ ID NO: 23, 24, 25, 26 or 27, or an amino acid sequence having one or two amino acid substitutions with respect to SEQ ID NO: 23, 24, 25, 26 or 27; and the amino acid sequence of CDR3 is shown in SEQ ID NO: 28, 29, 30, 31 or 32, or an amino acid sequence having one or two amino acid substitutions with respect to SEQ ID NO: 28, 29, 30, 31 or 32.

[0012] In some specific embodiments, the anti-claudin-18.2 antibodies or antigen-binding fragments provided herein are as follows: (1) the amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NO: 3, 8, or 13, or amino acid sequences with one or two amino acid substitutions with respect to SEQ ID NO: 3, 8, or 13, and / or the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NO: 18, 23, or 28, or amino acid sequences with one or two amino acid substitutions with respect to SEQ ID NO: 18, 23, or 28, (2) the amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NO: 4, 9, or 14, or are amino acid sequences with one or two amino acid substitutions with respect to SEQ ID NO: 4, 9, or 14, and / or the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NO: 19, 24, or 29, or are amino acid sequences with one or two amino acid substitutions with respect to SEQ ID NO: 19, 24, or 29, (3) the amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NO: 5, 10, or 15, or are amino acid sequences having one or two amino acid substitutions with respect to SEQ ID NO: 5, 10, or 15, and / or the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NO: 20, 25, or 30, or are amino acid sequences having one or two amino acid substitutions with respect to SEQ ID NO: 20, 25, or 30, (4) the amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NO: 6, 11, or 16, or are amino acid sequences of SEQ ID NO: 6, 11, or 16 with one or two amino acid substitutions, and / or the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NO: 21, 26, or 31, or are amino acid sequences of SEQ ID NO: 21, 26, or 31 with one or two amino acid substitutions, (5) The amino acid sequences of the heavy chain variable region CDR1 to 3 are SEQ ID NO: 7, 12, or 17, or are amino acid sequences with one or two amino acid substitutions relative to SEQ ID NO: 7, 12, or 17, and / or the amino acid sequences of the light chain variable region CDR1 to 3 are SEQ ID NO: 22, 27, or 32, or are amino acid sequences with one or two amino acid substitutions relative to SEQ ID NO: 22, 27, or 32.

[0013] In some specific embodiments, the antibodies or antigen-binding fragments thereof provided herein are (1) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:1, or has the same CDRs 1 to 3 as SEQ ID NO:1 and has a sequence identity of more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% compared to SEQ ID NO:1; and / or (2) The amino acid sequence of the light chain variable region is set forth in SEQ ID NO:2, or comprises a variable region selected from the group consisting of combinations having the same CDRs 1 to 3 as SEQ ID NO:2 and having a sequence identity of more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% compared to SEQ ID NO:2.

[0014] In a specific embodiment, the amino acid sequence of the heavy chain variable region of the anti-claudin-18.2 antibody of the present invention is as follows (SEQ ID NO: 1): QVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWMGL INPGSGGTNY 60 NEKFKGRVTM TRDTSTSTVY MELSSLRSED TAVYYCARGG YYGNSFAYWG QGTLVTVSS 119

[0015] In a specific embodiment, the amino acid sequence of the light chain variable region of the anti-claudin-18.2 antibody of the present invention is as follows (SEQ ID NO: 2): DIVMTQSPLS LPVTPGEPAS ISCKSSQSLL NSGNQKNYLT WYLQKPGQSP QLLIYWASTR 60 ESGVPDRFSG SGSGTDFTLK ISRVEAEDVG VYYCQNAYYY PYTFGGGTKV EIK 113

[0016] In a specific embodiment, the anti-claudin-18.2 of the present invention has (1) an amino acid sequence of a heavy chain variable region set forth in SEQ ID NO: 1 and / or (2) an amino acid sequence of a light chain variable region set forth in SEQ ID NO: 2.

[0017] In some specific embodiments, the antibody or antigen-binding fragment thereof provided herein has (1) a heavy chain amino acid sequence set forth in SEQ ID NO: 33 and / or (2) a light chain amino acid sequence set forth in SEQ ID NO: 34.

[0018] The antibodies provided herein may be a monoclonal antibody, a Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv ("scFv"), a diabody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, or a fusion protein comprising the antigen-binding portion of an antibody, although preferably the antibody is a humanized monoclonal antibody.

[0019] The antibodies provided by the present invention further comprise a human or mouse constant region, preferably said constant region is selected from IgG1, IgG2, IgG3, IgG4.

[0020] The present invention also provides an antibody-drug conjugate comprising any of the above antibodies or antigen-binding fragments thereof.

[0021] Furthermore, the structure of the antibody-drug conjugate is shown in formula (I) below. Ab-LD (I) wherein Ab is any one of the antibodies or antigen-binding fragments thereof described above; D is an active drug unit; L is an optional linking group covalently attached to the antibody or antigen-binding fragment thereof Ab and to the active drug unit D, respectively; wherein said Ab links one or more active drug units D via one or more linking groups L.

[0022] Alternatively, L is covalently bound to an amino or sulfhydryl residue on the antibody Ab. Preferably, L is covalently bound to a sulfhydryl residue on the antibody Ab. More preferably, L is covalently bound to a sulfhydryl residue formed after cleavage of an interchain disulfide bond on the antibody Ab.

[0023] Furthermore, L is a cleavable linker and a non-cleavable linker.

[0024] Furthermore, the cleavable linker includes a peptide unit, the peptide unit including 2 to 20 amino acids, the peptide unit being preferably selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-arginine-(-Phe-Arg-) and combinations thereof.

[0025] Further, any of the above antibody-drug conjugates is characterized in that L contains the following existing linker structure (pages 7 to 10 of the Chinese invention patent specification with patent publication number CN110997010A):

[0026] [ka]

[0027] [ka]

[0028] [ka]

[0029] [ka]

[0030] The active drug unit D may be a cytotoxic molecule, a cell differentiation factor, a stem cell nutrition factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease. Preferably, the cytotoxic molecule includes, but is not limited to, a tubulin inhibitor or a DNA damaging agent. The tubulin inhibitor includes, but is not limited to, a dolastatin and auristatin cytotoxic molecule, and a maytansine cytotoxic molecule. The DNA damaging agent includes, but is not limited to, a calicheamicin, a duocarmycin, an anthramycin derivative PBD, camptothecins and camptothecin derivatives, and SN-38. More preferably, the auristatin cytotoxic molecule includes, but is not limited to, MMAE or MMAF or a derivative thereof. The maytansine cytotoxic molecule includes, but is not limited to, DM1, DM4, or a derivative thereof. Furthermore, the active drug unit D includes the existing active drug units used in the following ADCs (pages 12-14 of the Chinese invention patent specification with patent publication number CN110997010A):

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] In some preferred embodiments, the invention provides an antibody-drug conjugate having the following structure: Ab--Mc-Val-Cit-PAB-MMAE:

[0035] [ka]

[0036] (wherein p is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8.)

[0037] In another preferred embodiment, the present invention provides an antibody-drug conjugate having the following structure: Ab-D07-Val-Cit-PAB-MMAE:

[0038] [ka] (wherein q is an integer selected from 1, 2, 3, and 4.)

[0039] In another preferred embodiment, the present invention provides an antibody-drug conjugate having the following structure: Ab--PY-Val-Cit-MMAE:

[0040] [ka]

[0041] (wherein q is an integer selected from 1, 2, 3, and 4.)

[0042] The present invention also provides an isolated polynucleotide encoding any of the above antibodies or antigen-binding fragments thereof.

[0043] The present invention also provides an isolated polynucleotide combination comprising a polynucleotide encoding a heavy chain of any of the above-mentioned antibodies or antigen-binding fragments thereof, and a polynucleotide encoding a light chain of any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0044] The present invention also provides a nucleic acid construct comprising the above-mentioned polynucleotide.

[0045] Further, the nucleic acid construct is a vector.

[0046] The present invention also provides a host cell comprising the above-described nucleic acid construct or vector.

[0047] Furthermore, the host cell is a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an E. coli cell, or a CHO cell, an NS0 cell, an Sp2 / 0 cell, a BHK cell.

[0048] The present invention also provides a pharmaceutical composition comprising any of the above-mentioned antibodies or antigen-binding fragments thereof and / or antibody-drug conjugates, and a pharma- ceutically acceptable carrier.

[0049] The present invention also provides a method for producing an anti-claudin-18.2 antibody, comprising culturing the host cell described above under conditions suitable for expressing a vector encoding an anti-claudin-18.2 antibody or antigen-binding fragment, and recovering the antibody or fragment.

[0050] In the use of any of the above-mentioned antibodies or antigen-binding fragments thereof, antibody-drug conjugates, polynucleotides, combinations of polynucleotides, nucleic acid constructs, vectors, or pharmaceutical compositions in the manufacture of a medicament for treating or preventing cancer, the cancer is a solid tumor, and further, the solid tumor includes gastric cancer and pancreatic cancer. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 is a graph showing changes in tumor volume in animals after administration of anti-claudin-18.2 mouse antibodies CLN-03-3A7, CLN-03-4E5, and CLN-03-6H2. [Diagram 2] FIG. 2 is a graph showing changes in animal body weight following administration of anti-claudin-18.2 mouse antibodies CLN-03-3A7, CLN-03-4E5, and CLN-03-6H2. [Diagram 3] FIG. 3 is a comparative diagram of affinity detection of chimeric antibodies CLN-03-4E5-01, CLN-03-6H2-01 and IMAB362. [Figure 4A] FIG. 4A shows a comparison of the endocytosis efficiency of CLN-03-6H2-01 and IMAB362 in the gastric cancer model KATOIII. [Figure 4B] FIG. 4B shows a comparison of the endocytosis efficiency of CLN-03-6H2-01 and IMAB362 in the pancreatic cancer model AsPC-1. [Figure 4C] FIG. 4C shows a comparison of the endocytosis efficiency of CLN-03-6H2-01 and IMAB362 in the lung cancer model A549. [Diagram 5] Figure 5 is a comparative diagram of the endocytosis effects of RGCLN18.2, RGCLN18.2-PY-Val-Cit-PAB-MMAE, RGCLN18.2-MC-Val-Cit-PAB-MMAE, RGCLN18.2-D07-Val-Cit-PAB-MMAE, and IMAB362-MC-Val-Cit-MMAE detected by flow cytometry. [Figure 6] FIG. 6 is a graph showing the changes in animal body weight following administration of RGCLN18.2-MC-Val-Cit-PAB-MMAE, IMAB362-MC-Val-Cit-PAB-MMAE and PBS. [Figure 7] Figure 7 is a graph showing the change in tumor volume after administration of RGCLN18.2-MC-Val-Cit-PAB-MMAE, IMAB362-MC-Val-Cit-PAB-MMAE and PBS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] [Definition] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by those skilled in the art. For definitions and terms in this field, those skilled in the art may specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues refer to one of the standard three-letter and / or one-letter codes used in the art to represent the 20 commonly used L-amino acids.

[0053] In the present invention, methods for measuring or numbering the complementarity determining regions (CDRs) of an antibody variable region include the IMGT, Kabat, Chothia, AbM and Contact methods well known in the art.

[0054] In the present invention, "identity", "identity" or "similarity" between two nucleic acid or amino acid sequences refers to the percentage of common nucleotides or common amino acid residues that the two sequences compared have, obtained by performing an optimal alignment (preferred alignment), and this percentage is purely statistical, and the differences between the two sequences are randomly distributed and cover their entire length. Sequence comparison between two nucleic acid or amino acid sequences is usually performed by aligning the sequences in an optimal manner and then comparing the sequences, which can be performed by segments or by "comparison windows". Suitable alignments for comparing sequences can be performed manually or by the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], the local homology algorithm of Neddleman and Wunsch (1970) [J. MoI. Biol. 48:443], the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444], or computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or BLAST N or BLAST P comparison software).

[0055] As used herein, "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies). As used herein, "antigen-binding fragment" means that the antibody fragment consists of or comprises a subsequence of the heavy or light variable chain of the antibody from which it is derived, provided that the subsequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100, and more preferably at least 1 / 10, of the affinity of the antibody from which it is derived. Such functional fragments include those that contain at least 5 amino acids, preferably 10, 15, 25, 50 and 100 contiguous amino acids from the antibody sequence from which it is derived, such as (among others) Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, and contain at least one immunoglobulin fragment sufficient to bind a specific antigen to the polypeptide. The above fragments can be prepared synthetically, enzymatically, or by chemical cleavage of intact immunoglobulins, or genetically engineered by recombinant DNA technology. Methods for their production are well known in the art. A heavy chain includes a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region includes three domains: CH1, CH2, and CH3. A light chain includes a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region includes one domain: CL. The VH and VL regions can be subdivided into several hypervariable regions called complementarity determining regions (CDRs), interspersed with several more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains contain binding motifs that interact with an antigen.The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The antibodies of the present invention also include chimeric or humanized antibodies.

[0056] The term "humanized antibody" refers to an antibody that contains CDR regions derived from a non-human antibody and the other parts of the antibody molecule are derived from one (or more) human antibodies. Furthermore, some residues in the framework (called FR) segment may be modified to retain binding affinity (reference 8: Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332:323-327, 1988.). The humanized antibody or fragment thereof of the present invention can be prepared by techniques known to those skilled in the art (Reference 9: Singer et al., J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; or Bebbington et al., Bio / Technology, 10:169-175, 1992.).

[0057] The term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody. The chimeric antibody according to the present invention or a fragment thereof can be prepared using a gene recombination technique. For example, the chimeric antibody can be produced by cloning a recombinant DNA comprising a promoter, a sequence encoding the variable region of a non-human, particularly mouse, monoclonal antibody according to the present invention, and a sequence encoding the constant region of a human antibody. The chimeric antibody according to the present invention encoded by such a recombinant gene is, for example, a mouse-human chimera, whose specificity is determined by the variable region derived from the mouse DNA and whose isotype is determined by the constant region derived from the human DNA. For a method of producing a chimeric antibody, see, for example, Verhoeyn et al. (BioEssays, 8:74, 1988).

[0058] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition, which displays a single binding specificity and affinity for a particular epitope.

[0059] The term "isolated" nucleic acid molecule refers to a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is generally associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different from the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule present in natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express the antibody, where, for example, the nucleic acid molecule is located in a different chromosomal location than in natural cells.

[0060] In general, for the preparation of monoclonal antibodies or functional fragments thereof, in particular of murine origin, reference is made in particular to the techniques described in the manual "Antibodies" (Reference 10: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the technique for preparation from hybridoma cells described by Kohler and Milstein (Nature, 256: 495-497, 1975). EXAMPLES

[0061] Hereinafter, the embodiments of the present invention will be described with reference to examples. However, those skilled in the art will understand that the following examples are only used to explain the present invention and should not be considered as limiting the scope of the present invention.

[0062] Example 1 Anti-claudin-18.2 Mouse Antibody Screening and Affinity Detection

[0063] Claudin-18.2 knockout mice (from Biocytogen Pharmaceuticals (Beijing) Co., Ltd.) were immunized with cells containing a eukaryotic expression plasmid encoding the first extracellular domain protein of claudin-18.2 as an immunogen.

[0064] A total of 22 mouse antibodies were selected: CLN-40-3C8, CLN-07-4C3, CLN-39-8D11, CLN-39-1E1, CLN-03-6H2, CLN-03-4E5, CLN-03-1A8, CLN-03-4A11, CLN-07-5B10, CLN-07-5G11, CLN-03-3A7, CLN-03-4C11, CLN-38-4H3, CLN-03-4G7, CLN-39-1B6, CLN-39-3E7, CLN-03-1F5, CLN-38-8A1, CLN-39-7H7, CLN-03-6G10, CLN-40-6C9, and CLN-07-5D9.

[0065] The binding activity of the obtained 22 mouse antibodies to claudin-18.2 was detected by FACS. Samples diluted to final concentrations of 10ug / mL, 1ug / mL, 0.1ug / mL, 0.01ug / mL, and 0.001ug / mL were reacted with CHO-claudin-18.2 cells at 4℃ for 30 minutes, centrifuged to discard the supernatant, added 200μl of PBS to each well, washed twice at 2000rpm, 5min, and then added 50μL of 1:100 diluted anti-mouse IgGFc-FITC antibody (Abcam, catalog number ab97264) to each well and reacted at 4℃ for 30 minutes, then repeated the washing step, washed once, and finally added 200μL of PBS to each well, resuspended, and detected by machine. The detection results are shown in Table 3. The polyclonal antibody showed good binding activity.

[0066] [Table 3]

[0067] Example 2 Evaluation of the in vivo efficacy of anti-claudin-18.2 mouse antibodies We selected 11 high-affinity anti-claudin-18.2 mouse antibodies (CLN-40-3C8, CLN-07-4C3, CLN-39-8D11, CLN-39-1E1, CLN-03-6H2, CLN-03-4E5, CLN-03-1A8, CLN-03-4A11, CLN-07-5B10, CLN-07-5G11, CLN-03-3A7) and screened CLN-03-3A7, CLN-03-4E5, and CLN-03-6H2 for therapeutic potential in gastric cancer PDX models. We then performed the following in vivo efficacy evaluations on the above three antibodies.

[0068] Gastric cancer tumors were subcutaneously inoculated into B-NDG mice (this mouse model was provided by Biocytogen JiangSu Co., Ltd.) until the tumor volume reached 150 mm 3 When the mice reached the age of 18, they were grouped with 5 mice in each experimental group. After grouping, mouse antibodies with excellent endocytosis and binding ability, CLN-03-3A7, CLN-03-4E5, and CLN-03-6H2, were injected intraperitoneally into the mice at a dose of 10 mg / kg twice a week, respectively, and the tumor growth of the mice was monitored twice a week (tumor volume = 0.5 × major axis × minor axis 2). Specific results are shown in Figure 1 and Figure 2. The experimental results showed that claudin-18.2 mouse antibody had a significant inhibitory effect on tumors, the antibody was safe, the mouse body weight did not change significantly, and the tumor inhibitory activity of the three mouse antibodies was CLN-03-6H2>CLN-03-4E5>CLN-03-3A7.

[0069] Example 3: Preparation of chimeric antibodies and detection of affinity

[0070] The two mouse antibodies CLN-03-4E5 and CLN-03-6H were remodeled into chimeric antibodies to obtain CLN-03-4E5-01 and CLN-03-6H2-01 chimeric antibodies. The affinity of CLN-03-4E5-01, CLN-03-6H2-01 and IMAB362 (human-mouse chimeric monoclonal antibody, cladiximab) was compared. As can be seen from the results (shown in Table 4 and Figure 3), CLN-03-4E5-01, CLN-03-6H2-01 and IMAB362 all showed good affinity activity, and the two chimeric antibodies CLN-03-4E5-01 and CLN-03-6H2-01 provided by the present invention all had better binding activity than IMAB362.

[0071] [Table 4]

[0072] Example 4: Detection of endocytosis efficiency of chimeric antibodies The endocytosis efficiency of CLN-03-6H2-01 chimeric antibody and IMAB362 (human-mouse chimeric monoclonal antibody, cladiximab) was detected and compared. The purified chimeric antibody was labeled according to the specifications of Promega PHAb antibody dye labeling kit (Promega G9841), and the concentration before antibody labeling was 2 mg / mL. The labeled antibody was incubated with gastric cancer model KATOIII (ATCC), pancreatic cancer model AsPC-1 (ATCC), and lung cancer model A549 (ATCC) cells according to a certain concentration gradient at 37 °C. The time gradient was set to 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and the concentration gradient was set to 0.1 nM, 1 nM, and 10 nM. After the end of each sampling point, the fluorescence intensity was detected by flow cytometry. The experimental results (see Figures 4A, 4B, and 4C) showed that the endocytosis effect of the chimeric antibody CLN-03-6H2-01 in different tumor cell lines was superior to that of IMAB362. However, Figure 4A shows a comparison of the endocytosis efficiency of CLN-03-6H2-01 and IMAB362 in the gastric cancer model KATOIII, Figure 4B shows a comparison of the endocytosis efficiency of CLN-03-6H2-01 and IMAB362 in the pancreatic cancer model AsPC-1, and Figure 4C shows a comparison of the endocytosis efficiency of CLN-03-6H2-01 and IMAB362 in the lung cancer model A549.

[0073] Example 5 Humanization of chimeric antibody CLN-03-6H2-01 The chimeric antibody CLN-03-6H2-01 was humanized and engineered by grafting the light or heavy chain CDRs into the light or heavy chain framework regions of immunoglobulins. The light and heavy chain CDRs of the chimeric antibody CLN-03-6H2-01 antibody were determined using the Kabat system. The human IgG1 framework regions were determined by alignment against a database of antibody variable regions. Different humanized Clandin18.2 antibody light chain variable region sequences and different humanized Clandin18.2 antibody heavy chain variable region sequences were designed and synthesized. The humanized Clandin18.2 antibody light chain variable region and the human kappa constant region were fused by PCR to obtain the full-length humanized Clandin18.2 light chain, and the humanized Clandin18.2 heavy chain variable region and the IgG1 constant region were fused by PCR to obtain the full-length humanized Clandin18.2 heavy chain. Different combinations of light and heavy chains were expressed, and the affinities of the purified polyclonal humanized antibodies were detected by flow cytometry (FACS) (see Table 5), and the H-1 number antibody with the optimal affinity (named RGCLN18.2 antibody) was selected and sequenced.

[0074] [Table 5]

[0075] [Table 6]

[0076] Amino acid sequence of the heavy chain variable region of the RGCLN18.2 antibody (SEQ ID NO:1): QVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWMGL INPGSGGTNY 60 NEKFKGRVTM TRDTSTSTVY MELSSLRSED TAVYYCARGG YYGNSFAYWG QGTLVTVSS 119

[0077] Amino acid sequence of the light chain variable region of the RGCLN18.2 antibody (SEQ ID NO:2): DIVMTQSPLS LPVTPGEPAS ISCKSSQSLL NSGNQKNYLT WYLQKPGQSP QLLIYWASTR 60 ESGVPDRFSG SGSGTDFTLK ISRVEAEDVG VYYCQNAYYY PYTFGGGTKV EIK 113

[0078] Amino acid sequence of the heavy chain of the RGCLN18.2 antibody (SEQ ID NO:33): QVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWMGL INPGSGGTNY 60 NEKFKGRVTM TRDTSTSTVY MELSSLRSED TAVYYCARGG YYGNSFAYWG QGTLVTVSSA 120 STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 180 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP 240 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPSREEM 360 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 420 QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 449

[0079] Amino acid sequence of the light chain of the RGCLN18.2 antibody (SEQ ID NO:34): DIVMTQSPLS LPVTPGEPAS ISCKSSQSLL NSGNQKNYLT WYLQKPGQSP QLLIYWASTR 60 ESGVPDRFSG SGSGTDFTLK ISRVAEEDVG VYYCQNAYYY PYTFGGGTKV EIKRTVAAPS 120 VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS 180 LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC 220

[0080] Example 6 Preparation of Antibody Drug Conjugates (ADCs) The reducing agent and protecting agent were prepared with purified water as follows, and antibody-drug conjugates (ADCs) were prepared using a general conjugation method. 1-20 mM TCEP (Tris-2-carboxyethyl-phosphine) and 1-20 mM DTPA (Diethylene triamine pentacetate acid) were used as the mother solution, and a reducing agent was added in an amount within a certain concentration range depending on the desired conjugation rate. A certain concentration of monoclonal antibody (e.g., 5-30 mg / mL) was mixed at a certain volume ratio (1:1) so that the molar ratio of the final concentration of TCEP to antibody was 0.5-6.0:1, and the reaction was stirred at 25°C for 1 hour. The antibody reduced with TCEP was used as it was for conjugation.

[0081] A linker-active drug unit compound was prepared at a certain concentration (5 mM) and dissolved in 25% DMSO (dimethyl sulfoxide), and the drug was slowly added so that the molar ratio of drug to thiol group was 0.3-2.8:1, and the reaction was stirred at 25°C for 1-4 hours. After the reaction was completed, centrifugal ultrafiltration was performed three times with PBS buffer to remove residual unreacted drug and free small molecules such as DMSO by purification, and the degree of complexation was detected using SDS-PAGE electrophoresis and hydrophobic high performance liquid chromatography (HIC-HPLC) method.

[0082] The linker-active drug unit compounds used in this embodiment are MC-Val-Cit-PAB-MMAE, D07-Val-Cit-PAB-MMAE and Py-MAA-Val-Cit-PAB-MMAE, whose structural formulas are as follows (for synthesis methods, see patent applications CN108853514A (specification page 14), CN111433188A (specification page 53), and WO2019223579A1 (specification pages 25-27)).

[0083] [ka]

[0084] The following ADCs (wherein p is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8, q is an integer selected from 1, 2, 3, and 4, and Ab is the RGCLN18.2 antibody provided by the present invention) were prepared by the above method. The average DAR of these ADCs was 3.5 to 4.5.

[0085] [ka]

[0086] Example 7 Cellular endocytosis experiment of antibody-drug conjugate Human gastric cancer cell line NCI-N87 was added at approximately 1 × 10 cells per well. 5The cells were resuspended in 6-well plates at 100 μg / ml. RGCLN18.2, RGCLN18.2-PY-Val-Cit-PAB-MMAE, RGCLN18.2-MC-Val-Cit-PAB-MMAE, and RGCLN18.2-D07-Val-Cit-PAB-MMAE were each conjugated to pHAb amine-reactive dye and then diluted to 10 μg / ml in cell culture medium. 100 μl of the dye conjugates of RGCLN18.2 and ADC were added to the cells and incubated at 37°C for the indicated times (0 h, 1 h, 3 h, 5 h, 21 h, and 24 h). The endocytosis efficiency of RGCLN18.2 and ADC was measured by flow cytometry. The results are shown in Figure 5. The results of the endocytosis experiment showed that the endocytosis rate of RGCLN18.2-PY-Val-Cit-PAB-MMAE, RGCLN18.2-MC-Val-Cit-PAB-MMAE, and RGCLN18.2-D07-Val-Cit-PAB-MMAE ADCs was about 98% at 24 hours. The results showed that RGCLN18.2 ADCs have a very good endocytosis effect in human gastric cancer cells NCI-N87 cells.

[0087] Example 8 In Vitro Cellular Evaluation of Antibody Drug Conjugates Cell line suspensions of human gastric cancer cells NCI-N87 were added to 96-well plates at 100 μL / well and a density of 5000 cells / well, and incubated overnight in a water-saturated CO2 incubator at 37 °C. Serial dilutions of antibody-drug conjugates (RGCLN18.2-PY-Val-Cit-PAB-MMAE, RGCLN18.2-D07-Val-Cit-PAB-MMAE, RGCLN18.2-MC-Val-Cit-PAB-MMAE, IMAB362-MC-Val-Cit-MMAE) were added to the 96-well plates containing cells at 100 μL / well. The incubation was continued for 72 h in a 37 °C incubator. The OD value at 450 nm was read using a microplate reader. The formula for calculating the inhibition rate was IR% = (OD blank - OD drug) × 100 / OD blank. The IC50 values ​​were calculated by the curve fitting software Softmax Pro7.0.3 Gxp, and the results are shown in Table 7. The experimental data from the above in vitro efficacy test showed that the proliferation inhibitory effect of ADCs was superior to that of IMAB362-MC-Val-Cit-MMAE in RGCLN18.2-PY-Val-Cit-PAB-MMAE, RGCLN18.2-MC-Val-Cit-PAB-MMAE, and RGCLN18.2-D07-Val-Cit-PAB-MMAE.

[0088] [Table 7]

[0089] Example 9: Antibody-drug combination PDX (human tumor tissue derived transplant tumor model) experiment After washing, human pancreatic cancer tissue was cut into small pieces and inoculated into the right scapula of the back of nude mice with a trocar, so that the tumors were 100–300 mm 3 After the animals had grown to adulthood, they were randomly divided into groups, and the treatment status of the control and experimental groups is shown in Table 8.

[0090] [Table 8]

[0091] The experimental results are shown in Figures 6 and 7. However, Figure 6 shows the change curves of the body weight of the animals in the control group and the experimental group, and Figure 7 shows the change curves of the tumor-bearing volume of the animals in the control group and the experimental group, and the results show that the tumor inhibitory effect of RGCLN18.2-MC-Val-Cit-PAB-MMAE in the human pancreatic cancer PDX model is stronger than that of IMAB362-MC-Val-Cit-PAB-MMAE.

[0092] The above description is merely a preferred embodiment, serves only as an example, and does not limit the combination of features necessary to implement the present invention. The titles provided are not intended to limit the various embodiments of the present invention. Terms such as "includes", "including" and "comprises" are not intended to be limiting. Furthermore, unless otherwise specified, the absence of a numerical modification includes the plural form, and "or" means "and / or". Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0093] All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in terms of certain preferred embodiments, it should be understood that the invention for which protection is sought should not be unduly limited to these specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art are intended to be included within the scope of the appended claims.

Claims

1. An anti-claudin-18.2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, (1) The amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NOs: 3, 8, and 13, respectively, and the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NOs: 18, 23, and 28, respectively; (2) The amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NOs: 4, 9, and 14, respectively, and the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NOs: 19, 24, and 29, respectively; (3) The amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NOs: 5, 10, and 15, respectively, and the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NOs: 20, 25, and 30, respectively; (4) The amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NOs: 6, 11, and 16, respectively, and the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NOs: 21, 26, and 31, respectively; or (5) An antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the heavy chain variable region CDR1 to CDR3 are SEQ ID NOs: 7, 12, and 17, respectively, and the amino acid sequences of the light chain variable region CDR1 to CDR3 are SEQ ID NOs: 22, 27, and 32, respectively.

2. (1) The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1 or has an identity of more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% compared to SEQ ID NO: 1; and (2) The antibody or antigen-binding fragment thereof described in claim 1, characterized in that the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2 or has an identity of more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% compared to SEQ ID NO:

2.

3. (1) the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 33; and (2) The antibody or antigen-binding fragment thereof described in claim 2, wherein the amino acid sequence of the light chain is shown in SEQ ID NO:

34.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or functional binding fragment thereof includes a monoclonal antibody, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fv (scFv), bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, or a fusion protein comprising an antigen-binding portion of an antibody, preferably, the antibody is a humanized monoclonal antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, further comprising a human or mouse constant region, preferably the constant region being selected from IgG1, IgG2, IgG3, and IgG4.

6. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

7. The antibody-drug conjugate of claim 6, wherein the structure of the antibody-drug conjugate is shown in the following formula (I): Ab-L-D (I) (wherein Ab is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, D is an active drug unit; L is an optional linking group covalently attached to the antibody or antigen-binding fragment thereof Ab and to the active drug unit D, respectively; wherein Ab links one or more active drug units D via one or more linking groups L.

8. said L is covalently attached to an amino or sulfhydryl residue on said Ab; Preferably, said L is covalently attached to a sulfhydryl residue on said Ab; More preferably, the L is covalently attached to a sulfhydryl residue formed after cleavage of an interchain disulfide bond on the Ab.

9. The antibody-drug conjugate of claim 7 , wherein L includes a cleavable linker and a non-cleavable linker.

10. the cleavable linker comprises a peptide unit; 10. The antibody-drug conjugate of claim 9, wherein the peptide unit comprises 2 to 20 amino acids, and is preferably selected from -valine-citrulline-(-Val-Cit-), -glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-), -valine-alanine-(-Val-Ala-), -valine-lysine-(-Val-Lys-), -valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-(-Phe-Cit-), -phenylalanine-lysine-(-Phe-Lys-), -phenylalanine-arginine-(-Phe-Arg-) and combinations thereof.

11. The above L is: 【Chemistry 1】 【change】 【change】 The antibody-drug conjugate according to any one of claims 7 to 10, comprising a structure selected from the group consisting of:

12. the active drug unit D is a cytotoxic molecule, a cell differentiation factor, a stem cell nutrient factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; Preferably, the cytotoxic molecule includes, but is not limited to, a tubulin inhibitor or a DNA damaging agent; The tubulin inhibitors include, but are not limited to, dolastatin and auristatin cytotoxic molecules, maytansine cytotoxic molecules; The DNA damaging agent includes, but is not limited to, calicheamicin, duocarmycin, anthramycin derivative PBD (pyrrolobenzodiazepine), camptothecins and camptothecin derivatives, SN-38; More preferably, the auristatin-based cytoplasmic molecule includes, but is not limited to, MMAE or MMAF or a derivative thereof; 8. The antibody-drug conjugate of claim 7, wherein the maytansine-based cytotoxic molecule includes, but is not limited to, DM1, DM4, or derivatives thereof.

13. The active drug unit D is: 【Chemistry 2】 【change】 The antibody-drug conjugate of claim 7, having a structure selected from the group consisting of:

14. 4. The antibody-drug conjugate comprising: 【Chemistry 3】 (wherein p is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8, and q is an integer selected from 1, 2, 3, and 4.) The antibody-drug conjugate of claim 7, having a structure selected from the group consisting of:

15. An isolated polynucleotide or a combination thereof encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

16. A nucleic acid construct comprising the polynucleotide of claim 15.

17. The nucleic acid construct of claim 16 , wherein the nucleic acid construct is a vector.

18. A host cell comprising the nucleic acid construct of claim 16.

19. 19. The host cell of claim 18, wherein the cell is a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an E. coli cell, or a CHO cell, an NSO cell, an Sp2 / 0 cell, or a BHK cell.

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and / or the antibody-drug conjugate according to claim 6 or 13, and a pharma- ceutical acceptable carrier.

21. 20. A method for producing an anti-claudin-18.2 antibody, comprising culturing a host cell described in claim 18 containing a nucleic acid construct described in claim 17 under conditions suitable for expressing a vector encoding an anti-claudin-18.2 antibody or antigen-binding fragment, and recovering the antibody or fragment.

22. Use of the antibody or antigen-binding fragment thereof described in any one of claims 1 to 4 in the manufacture of a medicament for treating or preventing cancer that expresses claudin-18.

2.

23. The use of claim 22, wherein the cancer is a solid tumor, and further wherein the solid tumor includes gastric cancer and pancreatic cancer.

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