Anti-claudin 18.2 antibodies and uses thereof

Abstract

The present invention relates to novel antibodies and antibody fragments that specifically bind to Claudin 18.2 and compositions containing the antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding the antibodies or antibody fragments thereof and host cells comprising the same, and related uses. Furthermore, the present invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

[0001] This application is based on and claims priority to CN application number 202010570517.X, filed on June 19, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This invention relates to novel antibodies and antibody fragments that specifically bind to Claudin18.2, as well as compositions containing said antibodies or antibody fragments. Furthermore, this invention relates to nucleic acids encoding said antibodies or antibody fragments thereof, host cells containing them, and related uses. Additionally, this invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments. Background of the Invention

[0004] Claudins are a family of proteins that are essential components of tight junctions in cells. They establish intercellular barriers that control the flow of molecules between cells. Claudins have four transmembrane domains, with their N-terminus and C-terminus both located in the cytoplasm. Different Claudins are expressed in different tissues, and alterations in their function are associated with cancer development in various tissues. For example, Claudin-1 is expressed in colon cancer and has prognostic value, Claudin-18 is expressed in gastric cancer, and Claudin-10 is expressed in hepatocellular carcinoma. As cell membrane surface proteins, Claudins are useful targets for various therapeutic strategies.

[0005] Claudin-18 isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker, whose expression in normal tissues is strictly limited to epithelial cells differentiated from the gastric mucosa, but not in gastric stem cell regions. CLDN18.2 is expressed in a significant proportion of primary gastric cancers and retains its expression level in metastatic gastric cancer tissues. In addition to gastric cancer, CLDN18.2 expression has also been found in pancreatic cancer, making it an ideal target molecule for the treatment of these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017). https: / / doi.org / 10.1186 / s13045-017-0473-4 ).

[0006] Given the significant unmet clinical needs in oncology treatment, the development of drugs targeting Claudin18.2 is essential. While existing technologies have led to the development of several monoclonal antibodies against Claudin18.2, most of these antibodies are chimeric, carrying a potentially high risk of immunogenicity, and exhibit low affinity, poor specificity, and limited ADCC activity.

[0007] Therefore, it is necessary to develop new antibodies against Claudin18.2 that have lower immunogenicity, stronger binding affinity to Claudin18.2, better specificity, stronger ADCC and CDC activity, and better antitumor activity. Invention Overview

[0009] In some aspects, the present invention relates to antibodies or antigen-binding fragments thereof that bind to CLDN18.2, comprising the three heavy chain variable regions (CDRs) and three light chain variable regions (CDRs) described in this invention.

[0010] In some aspects, the antibody or antigen-binding fragment of the present invention that binds to CLDN18.2 includes the heavy chain variable region and / or light chain variable region described in the present invention.

[0011] In some aspects, the antibody or antigen-binding fragment of the present invention that binds to CLDN18.2 further comprises the heavy chain constant region and / or light chain constant region described in the present invention.

[0012] In some aspects, the present invention also relates to the following embodiments:

[0013] 1. The antibody or antigen-binding fragment of the present invention that binds to CLDN18.2, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1, IgG2, IgG3 or IgG4, preferably an antibody or antigen-binding fragment in the form of IgG1.

[0014] 2. The antibody or antigen-binding fragment thereof that binds to CLDN18.2 of the present invention, wherein the antibody is a monoclonal antibody.

[0015] 3. The antibody or antigen-binding fragment of the present invention that binds to CLDN18.2, wherein the antibody is a humanized antibody, a human antibody, or a chimeric antibody.

[0016] 4. The antibody or antigen-binding fragment of the present invention that binds to CLDN18.2, wherein the antigen-binding fragment is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), or linear antibody.

[0017] 5. The antibody or antigen-binding fragment thereof binding to CLDN18.2 of the present invention, wherein the antibody or antigen-binding fragment thereof has one or more of the following properties:

[0018] (i) binds to CLDN18.2 (e.g., human CLDN18.2) with high affinity, but not to CLDN18.1 (e.g., human CLDN18.1);

[0019] (ii) with the following equilibrium dissociation constant (K) D ) combined with human CLDN18.2, the K D Less than about 15 nM, preferably less than or equal to about 10 nM, 9.5 nM, 9 nM, 8.5 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM;

[0020] (iii) It binds to CLDN18.2 on the cell surface, but not to CLDN18.1 on the cell surface;

[0021] (iv) Possesses ADCC or CDC activity, such as ADCC or CDC activity comparable to that of a known antibody (e.g., Zmab), or higher ADCC or CDC activity;

[0022] (v) Suppress tumor cells, such as tumor cells expressing CLDN18.2;

[0023] (vi) It can effectively inhibit tumor growth, with a tumor inhibition rate greater than or equal to approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0024] 6. The isolated nucleic acid encoding the light chain variable region or heavy chain variable region, or light chain or heavy chain, of the antibody or antigen-binding fragment of the present invention that binds to CLDN18.2.

[0025] 7. A vector containing the nucleic acid of the present invention, preferably an expression vector.

[0026] 8. A host cell containing the nucleic acid or vector of the present invention, preferably a prokaryotic or eukaryotic cell, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, such as CHO-S cells or HEK293 cells) or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0027] 9. A method for preparing an antibody or antigen-binding fragment thereof that binds to CLDN18.2, the method comprising culturing host cells of the present invention under conditions suitable for expressing nucleic acids encoding the antibody or antigen-binding fragment thereof that binds to CLDN18.2 of the present invention, optionally isolating the antibody or antigen-binding fragment thereof, and optionally further comprising recovering the antibody or antigen-binding fragment thereof that binds to CLDN18.2 from the host cells.

[0028] 10. An immunoconjugate comprising the antibody of the present invention binding to CLDN18.2 or its antigen-binding fragment and other substances, such as cytotoxic agents.

[0029] 11. A pharmaceutical composition comprising the antibody of the present invention binding to CLDN18.2 or an antigen-binding fragment thereof or an immunoconjugate of the present invention, and optionally one or more other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators, and optionally pharmaceutical excipients.

[0030] 12. A drug combination comprising the antibody of the present invention binding to CLDN18.2 or its antigen-binding fragment or the immunoconjugate of the present invention, and one or more other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

[0031] 13. A method for preventing or treating tumors in a subject, the method comprising administering to the subject an effective amount of the antibody of the present invention binding to CLDN18.2 or an antigen-binding fragment thereof, or an immunoconjugate, pharmaceutical composition or combination thereof of the present invention.

[0032] 14. A method for inducing ADCC and / or CDC in a subject, the method comprising administering to the subject an effective amount of the antibody of the present invention binding to CLDN18.2 or an antigen-binding fragment thereof, or an immunoconjugate, pharmaceutical composition or combination thereof of the present invention.

[0033] 15. Use of the antibody or immune conjugate of the present invention in the preparation of a medicament or combination of medicaments for the treatment or prevention of tumors and / or the induction of ADCC and / or CDC.

[0034] 16. The method or use of the present invention, wherein the tumor is cancer, preferably, the cancer has elevated levels (e.g., nucleic acid or protein levels) of CLDN18.2.

[0035] 17. The method of the present invention, wherein the method further comprises administering one or more therapies to a patient, such as treatment modalities and / or other therapeutic agents, preferably, the treatment modalities include surgical treatment and / or radiotherapy, and other therapeutic agents are selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

[0036] 18. Use of the present invention, wherein the drug or combination of drugs can be administered to a subject in combination with one or more therapies, such as treatments and / or other therapeutic agents, preferably, the treatments include surgical treatment and / or radiotherapy, and the other therapeutic agents are selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.

[0037] 19. A method for detecting CLDN18.2 in a sample, the method comprising:

[0038] (a) Contacting the sample with the antibody or antigen-binding fragment of the present invention; and

[0039] (b) Detection of the formation of a complex between the antibody or its antigen-binding fragment and CLDN18.2: Optionally, the antibody is detectably labeled. Attached image description:

[0040] Figure 1 The antibody against CLDN18.2 was shown to specifically bind to CLDN18.2 on the cell surface.

[0041] Figure 2 It was shown that the anti-CLDN18.2 antibody does not bind to CLDN18.1 on the cell surface.

[0042] Figure 3 The reporter gene-based ADCC activity of the anti-CLDN18.2 antibody against CHO-hCLDN18.2 was demonstrated.

[0043] Figure 4 The reporter gene-based ADCC activity of the humanized anti-CLDN18.2 antibody against CHO-hCLDN18.2 was demonstrated.

[0044] Figure 5 The binding of the anti-CLDN18.2 antibody to the gastric cancer cell lines NUGC-4, KATO III-hCLDN18.2, and DAN-G-hCLDN18.2 was demonstrated.

[0045] Figure 6 The CDC activity assay for the anti-CLDN18.2 antibody is shown.

[0046] Figure 7 The ADCC activity assay of the anti-CLDN18.2 antibody is shown.

[0047] Figure 8 The anti-CLDN18.2 antibody demonstrated its anti-tumor effect in a mouse model of pancreatic cancer.

[0048] Figure 9 The anti-CLDN18.2 antibody demonstrated its anti-tumor effect in a mouse model of gastric cancer. Invention Details

[0050] I. Definition

[0051] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0053] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0054] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0055] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0056] The term "CLAUDIN" or "CLDN" used in this article refers to the most important skeletal protein determining the structure of tight junctions between cells. Claudin proteins participate in adhesion and play a crucial role in tumor cell metastasis and invasion. Claudin proteins are widely distributed in mammalian epithelial and endothelial cells, primarily on the lateral surfaces of epithelial cells and the plasma membrane of basal cells. Different Claudin proteins exhibit specific expression in different tissues. The Claudin18 (CLDN18) gene, located at 3q22.3, has a molecular weight of 24 kDa, contains 261 amino acid residues, and belongs to the Claudins superfamily. Its protein structure includes two extracellular loops and four transmembrane regions. The two isoforms of human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1) and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2). In their primary structural sequences, they differ only in certain amino acid residues at positions from the N-terminal signal peptide to the extracellular loop 1 structure, particularly at the extracellular loop 1, where CLDN18.1 and CLDN18.2 differ by only 8 amino acids. The two isoforms of CLDN18 also exhibit very high interspecies sequence homology. The extracellular loop 1 of CLDN18.2 is completely sequence-identical in different species, including humans, mice, and macaques, with 84% homology between human and mouse CLDN18.2 proteins, indicating extreme conservation of the CLDN18.2 protein sequence (O. Tureci. et al., Gene 481:83-92, 2011). CLDN18.2 or any variants and isotypes thereof can be isolated from cells or tissues that naturally express them, or recombinantly generated using techniques well known in the art and / or those described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0057] As used herein, the terms "anti-CLDN18.2 antibody," "anti-CLDN18.2," "CLDN18.2 antibody," or "CLDN18.2-binding antibody" refer to antibodies that bind to (human) CLDN18.2 with sufficient affinity such that the antibody can be used as a therapeutic agent targeting (human) CLDN18.2. In one embodiment, the (human) CLDN18.2 antibody binds to (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, the (human) CLDN18.2 antibody does not bind to CLDN18.1. In one embodiment, the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), thin-layer biofilm interferometry (BLI), MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0058] The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0059] "Antigen-binding fragment" refers to a molecule that is distinct from the intact antibody, contains a portion of the intact antibody, and binds the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; bivalent antibodies or fragments thereof; or camelid antibodies.

[0060] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0061] "Antibody that binds to the same or overlapping epitopes as the reference antibody" means an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay; conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay.

[0062] An antibody that competes with a reference antibody for binding to its antigen is one that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another; these assays include, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), and sandwich competitive assays.

[0063] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0064] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90%, or 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for measuring binding affinity and / or specificity.

[0065] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al., (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0066] For example, depending on the different CDR determination schemes, the residues of each CDR are as follows.

[0067]

[0068] CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0069] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0070] Unless otherwise stated, in this invention, when referring to the position of a residue in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0071] In one embodiment, the heavy chain variable region (CDR) of the antibody of the present invention is determined according to the following rules.

[0072] VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule.

[0073] In one embodiment, the light chain variable region (CDR) of the antibody of the present invention is determined according to the Kabat rule.

[0074] In one embodiment, the heavy chain variable region CDR of the antibody of the present invention is determined according to the following rules: VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule; and the light chain variable region CDR is determined according to the Kabat rule.

[0075] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined by the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0076] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs (within the same assignment system). However, although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0077] The term "Fc region" is used herein to define the constant regions of CH2 and CH3 of the immunoglobulin heavy chain, including both native and variant Fc regions. Fc regions can bind to different Fc receptors on the surface of immune cells, thereby eliciting CDC / ADCC / ADCP effector functions. Such effector functions generally require the Fc region to be coupled with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays, such as those disclosed herein.

[0078] "IgG form antibody" refers to the IgG form to which the antibody's heavy chain constant region belongs. All antibodies of the same type have the same heavy chain constant region, while antibodies of different types have different heavy chain constant regions. For example, an IgG4 form antibody means its heavy chain constant region comes from IgG4, or an IgG1 form antibody means its heavy chain constant region comes from IgG1.

[0079] A "humanized" antibody is an antibody comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of nonhuman antibodies, and all or substantially all of the FRs correspond to those of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized.

[0080] The terms "human antibody," "fully human antibody," or "fully human-derived antibody" are used interchangeably to refer to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody generated by a human or human cell or derived from a non-human source, utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0081] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0082] "Immune conjugates" are antibodies conjugated to one or more other substances (including but not limited to cytotoxic agents or markers).

[0083] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants).

[0084] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0085] "Chemotherapy agents" include chemical compounds that are useful in treating diseases of the immune system.

[0086] The term "small molecule drug" refers to low-molecular-weight organic compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0087] As used in this article, the term "immunomodulator" refers to natural or synthetic active agents or drugs that inhibit or modulate immune responses. Immune responses can be humoral or cellular. Immunomodulators include immunosuppressants.

[0088] The terms "immunosuppressant," "immunosuppressive drug," or "immunosuppressant" as used in this article refer to therapeutic agents used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

[0089] The term "effective amount" refers to such an amount or dose of the antibody, fragment, conjugate, composition, or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention.

[0090] "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or antibody fragment or its conjugate or composition or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, the "therapeutic effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 20%, more preferably at least about 40%, and even more preferably at least about 50%, 60%, or 70%.

[0091] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0092] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in their nucleic acid content and may contain mutations. This document includes mutant progeny with the same function or biological activity screened or selected from the initially transformed cells.

[0093] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0094] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0095] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some embodiments, the antibody is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0096] "Isolated nucleic acid encoding anti-CLDN18.2 antibody or a fragment thereof" refers to one or more nucleic acid molecules that encode the antibody heavy or light chain (or a fragment thereof, such as a heavy chain variable region or a light chain variable region), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0097] The following is a calculation of sequence identity between sequences.

[0098] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0099] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. A particularly preferred set of parameters (and, unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix employing a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the percentage of identity between two amino acid or nucleotide sequences can be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Additionally or alternatively, the nucleic acid and protein sequences described herein can be further used as “query sequences” to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0100] As used herein, the term “hybridization under stringent conditions (e.g., low-stringency, medium-stringency, high-stringency, or very high-stringency conditions)” describes the hybridization and washing conditions. Instructions for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which are incorporated herein by reference. Aqueous and non-aqueous methods are described in the references, and either method may be used. The preferred hybridization conditions mentioned herein are as follows: 1) Low-toughness hybridization conditions are in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes at at least 50°C (for low-toughness conditions, the washing temperature can be increased to 55°C) in 0.2X SSC, 0.1% SDS; 2) Medium-toughness hybridization conditions are in 6X SSC at about 45°C, followed by one or more washes at 60°C in 0.2X SSC, 0.1% SDS; 3) High-toughness hybridization conditions are in 6X SSC at about 45°C, followed by one or more washes at 65°C in 0.2X SSC, 0.1% SDS; and preferably 4) Very high-toughness hybridization conditions are in 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 65°C in 0.2X SSC, 0.1% SDS. Very high-toughness condition (4) is the preferred condition and, unless otherwise stated, should be used.

[0101] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0102] The terms “tumor” and “cancer” are used interchangeably in this article to cover both solid tumors and liquid tumors.

[0103] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the present invention include gastric or pancreatic cancer, including metastatic forms of those cancers.

[0104] The term "tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when used in this article.

[0105] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0106] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0107] The term "drug combination" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) an anti-CLDN18.2 antibody or a fragment thereof, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active agents. In some embodiments, the anti-CLDN18.2 antibody or a fragment thereof and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may be in separate formulations, and their formulations may be the same or different.

[0108] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0109] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0110] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0111] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0112] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0113] II. Antibodies

[0114] In some embodiments, the anti-CLDN18.2 antibody of the present invention, or its antigen-binding fragment, binds to CLDN18.2 (e.g., human CLDN18.2) with high affinity. In some embodiments, the anti-CLDN18.2 antibody of the present invention specifically binds to CLDN18.2 (e.g., human CLDN18.2) but not to CLDN18.1 (e.g., human CLDN18.1). In some embodiments, the antibody of the present invention, or its antigen-binding fragment, has a higher binding affinity to human CLDN18.2 than known CLDN18.2 antibodies, such as Zolbetuximab (Zmab) antibody. In some embodiments, the antibody affinity is determined by thin-layer interferometry or surface plasmon resonance assay.

[0115] In some embodiments, the anti-CLDN18.2 antibody of the present invention has the following equilibrium dissociation constant (K). D ) combined with human CLDN18.2, the K D Less than approximately 15 nM, preferably less than or equal to approximately 10 nM, 9.5 nM, 9 nM, 8.5 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, in some embodiments, the K D Between the above values ​​(including the endpoints).

[0116] In some embodiments, the antibody or antigen-binding fragment of the present invention binds to CLDN18.2 on the cell surface. In some embodiments, the antibody or antigen-binding fragment of the present invention does not bind to CLDN18.1 on the cell surface. In some embodiments, CLDN18.2 is expressed or overexpressed on the cell surface. In some embodiments, the cells are CHO cells or 293 cells expressing CLDN18.2, such as CHO-S cells or HEK293 cells. In some embodiments, the cells are cancer cells expressing CLDN18.2, such as naturally expressed CLDN18.2 or cells artificially transfected with CLDN18.2 or cells artificially transfected with increased expression levels of CLDN18.2, such as gastric cancer cells or pancreatic cancer cell lines expressing CLDN18.2, such as cell lines NUGC-4, KATO III, and DAN-G, such as KATO III and DAN-G cell lines overexpressing CLDN18.2.

[0117] In some embodiments, the binding is detected using flow cytometry. In some embodiments, the antibody of the present invention binds to CLDN18.2 overexpressed on CHO cells at EC50 values ​​less than or equal to about 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, and 5.5 nM. In some embodiments, the antibody of the present invention binds to cancer cells expressing CLDN18.2, such as gastric cancer cell lines or pancreatic cancer cell lines expressing CLDN18.2, such as cell lines NUGC-4, KATO III, and DAN-G, such as KATO III and DAN-G cell lines overexpressing CLDN18.2, at EC50 values ​​less than or equal to about 4 nM, 3.7 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, and 0.6 nM. In some embodiments, the EC50 of the antibody of the present invention binding to cells expressing CLDN18.2 is comparable to, or less than, the EC50 of a known antibody such as Zmab, or less than 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% of the EC50 of a known antibody such as Zmab.

[0118] In some embodiments, the antibody or antigen-binding fragment of the present invention has ADCC or CDC activity, such as ADCC or CDC activity comparable to that of a known antibody (e.g., Zmab), or higher ADCC or CDC activity.

[0119] In some embodiments, the antibody or antigen-binding fragment of the present invention can inhibit tumor cells, such as tumor cells expressing CLDN18.2.

[0120] In some embodiments, the antibodies or antigen-binding fragments of the present invention can be used to treat cancer. In some embodiments, the antibodies or antigen-binding fragments of the present invention can effectively inhibit tumor growth, with a tumor inhibition rate greater than or equal to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0121] In some embodiments, the anti-CLDN18.2 antibody of the present invention or its antigen-binding fragment comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, namely HCDR1, HCDR2 and HCDR3.

[0122] In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3.

[0123] In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region.

[0124] In some aspects, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region (VH). In some aspects, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention comprises both a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0125] In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region (HC). In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region (LC). In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region (HC) and a light chain constant region (LC).

[0126] In some embodiments, the heavy chain variable region of the present invention

[0127] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80, or 85; or

[0128] (ii) Containing or consisting of an amino acid sequence selected from or composed of said amino acid sequence, SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80, or 85; or

[0129] (iii) An amino acid sequence comprising having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80, or 85, preferably not occurring in the CDR region.

[0130] In some embodiments, the light chain variable region of the present invention

[0131] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83, or 86; or

[0132] (ii) Containing or consisting of an amino acid sequence selected from or composed of said amino acid sequence, SEQ ID NO: 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83, or 86; or

[0133] (iii) An amino acid sequence comprising having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83, or 86, preferably not occurring in the CDR region.

[0134] In some embodiments, the three complementary determinant regions (HCDRs) from the heavy chain variable region of the present invention, HCDR1, HCDR2, and HCDR3, are selected from...

[0135] (i) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 4

[0136] (ii) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 15 or 21

[0137] (iii) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 26 or 27

[0138] (iv) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 36 or 37

[0139] (v) The three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 45 or 48.

[0140] (vi) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 53 or 56

[0141] (vii) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 61 or 66

[0142] (viii) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 72 or 73,

[0143] (ix) The three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 80 or 85, or

[0144] (x) relative to any one of (i)-(ix) contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions.

[0145] In some embodiments, the three complementary determinant regions (LCDRs) from the light chain variable region of the present invention, LCDR1, LCDR2, and LCDR3, are selected from...

[0146] (i) The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 9,

[0147] (ii) The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 19 or 22,

[0148] (iii) The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 31 or 32,

[0149] (iv) The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 40 or 41,

[0150] (v) The three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 47 or 49,

[0151] (vi) The three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 55 or 57

[0152] (vii) The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 65 or 68,

[0153] (viii) The three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 75 or 76,

[0154] (ix) The three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 83 or 86, or

[0155] (x) relative to any one of (i)-(ix) contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions.

[0156] In some embodiments, HCDR1 comprises, or is composed of, the amino acid sequence of SEQ ID NO: 1, 12, 23, 33, 42, 50, 58, 69 or 77, or HCDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1, 12, 23, 33, 42, 50, 58, 69 or 77.

[0157] In some embodiments, HCDR2 comprises, or is composed of, the amino acid sequence of SEQ ID NO: 2, 13, 20, 24, 34, 43, 51, 59, 70, 78, 84, 93, or 94, or HCDR2 comprises, or is composed of, an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2, 13, 20, 24, 34, 43, 51, 59, 70, 78, 84, 93, or 94, wherein

[0158] SEQ ID NO: 93 is YIAPFXGDSRYNQKFKG, where X is any amino acid, preferably N or Q or a conserved amino acid thereof; or

[0159] SEQ ID NO: 94 is VIWGDXSTNYHSVLIS, where X is any amino acid, preferably G or V or a conserved amino acid thereof.

[0160] In some embodiments, HCDR3 comprises, or is composed of, the amino acid sequence of SEQ ID NO: 3, 14, 25, 35, 44, 52, 60, 71 or 79, or HCDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3, 14, 25, 35, 44, 52, 60, 71 or 79.

[0161] In some embodiments, LCDR1 comprises, or is composed of, the amino acid sequence of SEQ ID NO: 6, 16, 28, 38, 62, 67 or 96, or the amino acid sequence thereof, or LCDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6, 16, 28, 38, 62, 67 or 11.

[0162] Wherein SEQ ID NO: 11 is KSSQSLLXGGNQKNYLT, where X is any amino acid, preferably N or Q or its conserved amino acid substitution.

[0163] In some embodiments, LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO: 7, 17, 29, 63 or 81, or the amino acid sequence thereof, or LCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 7, 17, 29, 63 or 81.

[0164] In some embodiments, LCDR3 comprises, or is composed of, the amino acid sequence of SEQ ID NO: 8, 18, 30, 39, 46, 54, 64, 74 or 82, or the amino acid sequence thereof, or LCDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 8, 18, 30, 39, 46, 54, 64, 74 or 82.

[0165] In some embodiments, the antibody heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. In some embodiments, the antibody light chain constant region LC of the present invention is a lambda or Kappa light chain constant region, preferably the Kappa light chain constant region.

[0166] In some preferred embodiments, the antibody heavy chain constant region HC of the present invention

[0167] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 5;

[0168] (ii) Contains or is composed of an amino acid sequence selected from SEQ ID NO: 5; or

[0169] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 5.

[0170] In some embodiments, the amino acid change occurs in the Fc region. In one embodiment, the amino acid change in the Fc region increases the antibody's CDC or ADCC activity.

[0171] In some embodiments, the antibody light chain constant region LC of the present invention

[0172] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 10;

[0173] (ii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence; or

[0174] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 10.

[0175] In some specific embodiments of the present invention, the anti-CLDN18.2 antibody or its antigen-binding fragment comprises:

[0176] (i) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 4, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 9;

[0177] (ii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 15 or 21, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 19 or 22.

[0178] (iii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 26 or 27, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 31 or 32.

[0179] (iv) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 36 or 37, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 40 or 41.

[0180] (v) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 45 or 48, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 47 or 49.

[0181] (vi) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 53 or 56, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 55 or 57.

[0182] (vii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 61 or 66, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 65 or 68.

[0183] (viii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 72 or 73, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 75 or 76.

[0184] (ix) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 80 or 85, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 83 or 86.

[0185] In some specific embodiments of the present invention, the anti-CLDN18.2 antibody or its antigen-binding fragment comprises:

[0186] (i) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7 and 8, respectively;

[0187] (ii) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 12, 13 and 14, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 16, 17 and 18, respectively;

[0188] (iii) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 12, 20 and 14, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 16, 17 and 18, respectively;

[0189] (iv) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 12, 93 and 14, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 16, 17 and 18, respectively;

[0190] (v) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 23, 24 and 25, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 28, 29 and 30, respectively;

[0191] (vi) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 33, 34 and 35, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 38, 17 and 39, respectively;

[0192] (vii) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 42, 43 and 44, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 38, 17 and 46, respectively;

[0193] (viii) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 50, 51 and 52, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 28, 17 and 54, respectively;

[0194] (ix) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 58, 59 and 60, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 62, 63 and 64, respectively;

[0195] (x) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 58, 59 and 60, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 67, 63 and 64, respectively;

[0196] (xi) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 58, 59 and 60, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 11, 63 and 64, respectively;

[0197] (xii) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 69, 70 and 71, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 38, 17 and 74, respectively;

[0198] (xiii) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 77, 78 and 79, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 28, 81 and 82, respectively;

[0199] (xiv) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 77, 84 and 79, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 28, 81 and 82, respectively;

[0200] (xv) HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 77, 94 and 79, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 28, 81 and 82, respectively.

[0201] In some specific embodiments of the present invention, the anti-CLDN18.2 antibody or its antigen-binding fragment comprises:

[0202] (i) VH comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0203] (ii) VH comprising the amino acid sequence shown in SEQ ID NO: 15 or 21 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 19 or 22 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0204] (iii) VH comprising the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0205] (iv) VH comprising the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0206] (v) VH comprising the amino acid sequence shown in SEQ ID NO: 26 or 27 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 31 or 32 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0207] (vi) VH comprising the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0208] (vii) VH comprising the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 32 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0209] (viii) VH comprising the amino acid sequence shown in SEQ ID NO: 36 or 37 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 40 or 41 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0210] (ix) VH comprising the amino acid sequence shown in SEQ ID NO: 36 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0211] (x) VH comprising the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0212] (xi) VH comprising the amino acid sequence shown in SEQ ID NO: 45 or 48 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 47 or 49 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0213] (xii) VH comprising the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0214] (xiii) VH comprising the amino acid sequence shown in SEQ ID NO: 48 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 49 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0215] (xiv) VH comprising the amino acid sequence shown in SEQ ID NO: 53 or 56 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 55 or 57 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0216] (xv) VH comprising the amino acid sequence shown in SEQ ID NO: 53 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 55 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0217] (xvi) VH comprising the amino acid sequence shown in SEQ ID NO: 56 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0218] (xvii) VH comprising the amino acid sequence shown in SEQ ID NO: 61 or 66 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 65 or 68 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0219] (xviii) VH comprising the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0220] (xix) VH comprising the amino acid sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0221] (xx) VH comprising the amino acid sequence shown in SEQ ID NO: 72 or 73 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 75 or 76 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0222] (xxi) VH comprising the amino acid sequence shown in SEQ ID NO: 72 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 75 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0223] (xxii) VH comprising the amino acid sequence shown in SEQ ID NO: 73 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 76 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0224] (xxiii) VH comprising the amino acid sequence shown in SEQ ID NO: 80 or 85 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 83 or 86 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0225] (xxiv) VH comprising the amino acid sequence shown in SEQ ID NO: 80 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence, and VL comprising the amino acid sequence shown in SEQ ID NO: 83 or an amino acid sequence having at least 90% identity with it or composed of said amino acid sequence;

[0226] (xxv) contains the amino acid sequence shown in SEQ ID NO: 85 or an amino acid sequence having at least 90% identity with it or a VH composed of said amino acid sequence, and contains the amino acid sequence shown in SEQ ID NO: 86 or an amino acid sequence having at least 90% identity with it or a VL composed of said amino acid sequence.

[0227] In one embodiment of the invention, the amino acid alteration described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid alteration described herein is an amino acid substitution, and more preferably a conservative substitution.

[0228] In a preferred embodiment, the amino acid alteration described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described in this invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid alteration described in this invention occurs in the Fc region of the antibody heavy chain constant region, and in a preferred embodiment, the amino acid alteration in the Fc region increases the ADCC and / or CDC activity of the antibody.

[0229] In some implementations, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the table below:

[0230]

[0231]

[0232] In some embodiments, the substitution occurs in the CDR region of the antibody. Typically, the resulting variant has modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity) and / or will have some biological properties that are substantially retained by the parent antibody. An exemplary substitution variant is an affinity-matured antibody.

[0233] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the antibody contains an Fc region, the sugars attached to it can be altered. In some applications, modifications to remove unwanted glycosylation sites can be useful, for example, removing the fucosylation motif to enhance antibody-dependent cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be performed to modify complement-dependent cytotoxicity (CDC).

[0234] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to create Fc region variants that alter one or more functional properties of the antibody, such as serum half-life, complement binding, complement-dependent cytotoxicity, Fc receptor binding, and / or antibody-dependent cytotoxicity. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) containing amino acid alterations (e.g., substitutions) at one or more amino acid positions.

[0235] In one embodiment of the present invention, the antibody described herein introduces alterations in the Fc region to enhance the antibody's ADCC or CDC activity.

[0236] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0237] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0238] In some embodiments, the anti-CLDN18.2 antibody or its antigen-binding fragment of the present invention has one or more of the following characteristics:

[0239] (i) exhibits the same or similar binding affinity and / or specificity to CLDN18.2 as the antibody of the present invention;

[0240] (ii) Inhibit (e.g., competitively inhibit) the binding of the antibody of the present invention to CLDN18.2;

[0241] (iii) Epitopes that bind to the same or overlapping with the antibodies of the present invention;

[0242] (iv) Competing with the antibody of this invention to bind to CLDN18.2;

[0243] (v) Having one or more biological characteristics of the antibody of the present invention.

[0244] In some embodiments, the anti-CLDN18.2 antibody of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, preferably an antibody in the form of IgG1.

[0245] In some implementations, the anti-CLDN18.2 antibody is a monoclonal antibody.

[0246] In some implementations, the anti-CLDN18.2 antibody is humanized.

[0247] In some implementations, the anti-CLDN18.2 antibody is a human antibody.

[0248] In some implementations, the anti-CLDN18.2 antibody is a chimeric antibody.

[0249] In some implementations, at least a portion of the framework sequence of the anti-CLDN18.2 antibody is a human common framework sequence.

[0250] In one embodiment, the anti-CLDN18.2 antibody of the present invention also encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies.

[0251] III. The nucleic acid of the present invention and the host cell containing it.

[0252] In one aspect, the present invention provides a nucleic acid encoding any of the above-described anti-CLDN18.2 antibodies or fragments thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector, such as pcDNA3.1. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In another embodiment, the host cell is prokaryotic.

[0253] In one aspect, the present invention provides a nucleic acid encoding any anti-CLDN18.2 antibody or fragment thereof described herein. The nucleic acid may comprise an amino acid sequence encoding an amino acid sequence encoding the light chain variable region and / or the heavy chain variable region of the antibody, or a nucleic acid comprising an amino acid sequence encoding the light chain and / or the heavy chain of the antibody.

[0254] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80 or 85, or SEQ ID NO: 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83 or 86, or encoding an amino acid sequence selected from SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80 or 85, or SEQ ID NO: 85 ...85, 19, 22, 31, 32, 40, NO: Nucleic acid having an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity of the amino acid sequence shown in any one of NOs 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83, or 86.

[0255] The present invention also covers nucleic acids that hybridize under stringent conditions or that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions with the following nucleic acids: nucleic acids comprising a sequence encoding an amino acid sequence selected from SEQ ID NO: 4, 15, 21, 26, 27, 36, 37, 45, 48, 53, 56, 61, 66, 72, 73, 80, or 85, or SEQ ID NO: 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83, or 86; or nucleic acids ... The nucleic acid sequence of the amino acid sequence shown in any one of IDNO: 9, 19, 22, 31, 32, 40, 41, 47, 49, 55, 57, 65, 68, 75, 76, 83 or 86 has an amino acid sequence identity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0256] In one embodiment, one or more vectors containing the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, kinases, λ phages, or yeast artificial chromosomes (YAC). In one embodiment, the vector is pcDNA3.1.

[0257] In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing the vector encoding the antibody include prokaryotic or eukaryotic cells as described herein. For example, the antibody may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0258] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains whose glycosylation pathways have been “humanized” result in the production of antibodies with partial or complete human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F, or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in the art.

[0259] IV. Production and purification of the antibody molecules of the present invention

[0260] In one embodiment, the present invention provides a method for preparing an antibody molecule or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing a host cell under conditions suitable for expressing a nucleic acid encoding an antibody molecule or a fragment thereof (preferably an antigen-binding fragment), and optionally isolating the antibody or a fragment thereof (e.g., the antigen-binding fragment). In another embodiment, the method further comprises recovering the antibody molecule or a fragment thereof (e.g., the antigen-binding fragment) from the host cell.

[0261] In one embodiment, a method for preparing the antibody molecule of the present invention is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0262] To recombinantly generate the antibody molecules of the present invention, nucleic acids encoding antibodies (such as those described above, such as any one or more polypeptide chains) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0263] The antibody molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibody molecules of this invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0264] V. Determination Method

[0265] The anti-CLDN18.2 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the antibodies of the present invention is tested, for example by known methods such as ELISA, Western blotting, etc. Binding to CLDN18.2 can be determined using methods known in the art, exemplary methods of which are disclosed herein. In some embodiments, it is measured using radioimmunoassay (RIA), thin-layer interferometry, MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0266] On the other hand, competitive assays can be used to identify antibodies that compete with any anti-CLDN18.2 antibody disclosed herein for binding to CLDN18.2. In some embodiments, such competitive antibodies bind to epitopes (e.g., linear or conformational epitopes) that are the same as or overlap with the epitopes bound by any anti-CLDN18.2 antibody disclosed herein.

[0267] This invention also provides an assay for identifying biologically active anti-CLDN18.2 antibodies. Biological activity may include, for example, binding to CLDN18.2 (e.g., binding to human CLDN18.2), binding to cells expressing CLDN18.2, activity against cellular CDC or ADCC, inhibitory effect on tumor cells, etc. Antibodies exhibiting such biological activity in vivo and / or in vitro are also provided.

[0268] In some embodiments, the antibodies of the present invention are tested for such biological activity.

[0269] Cells used for any of the above in vitro assays include cell lines that naturally express CLDN18.2 or are engineered to express or overexpress CLDN18.2. Such cells also include cell lines transfected with CLDN18.2-encoding DNA that expresses CLDN18.2 and those that do not normally express CLDN18.2. In some embodiments, these cells are gastric cancer cells or pancreatic cancer cells. In some embodiments, these cells are CHO cells that express CLDN18.2. In some embodiments, these cells are cell lines NUGC-4, KATO III, and DAN-G, such as KATO III and DAN-G cell lines overexpressing CLDN18.2.

[0270] It is understood that the immunoconjugate of the present invention can be used to replace or supplement the anti-CLDN18.2 antibody for any of the above assays.

[0271] Understandably, any of the above assays can be performed using a combination of anti-CLDN18.2 antibody and other active agents.

[0272] VI. Immunoconjugates

[0273] In some embodiments, the present invention provides an immunoconjugate comprising any anti-CLDN18.2 antibody provided herein and other substances, such as therapeutic agents including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., anti-inflammatory agents or immunosuppressants). In one embodiment, the other substances, such as cytotoxic agents, include any agents that are harmful to cells.

[0274] In some implementations, the immune conjugate is used for the prevention or treatment of cancer.

[0275] VII. Pharmaceutical compositions and pharmaceutical preparations

[0276] In some embodiments, the present invention provides compositions comprising any anti-CLDN18.2 antibody described herein or a fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof, preferably pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example, a pharmaceutical composition, comprises an anti-CLDN18.2 antibody of the present invention or a fragment thereof or an immunoconjugate thereof, and a combination of one or more other therapeutic agents.

[0277] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an anti-CLDN18.2 antibody or an immunoconjugate thereof, or compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a polynucleotide encoding an anti-CLDN18.2 antibody. In some embodiments, the composition comprises one or more antibodies or fragments thereof that bind to CLDN18.2, or one or more polynucleotides encoding one or more antibodies or fragments thereof against CLDN18.2. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0278] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0279] For information on the use and applications of pharmaceutical excipients, see also “Handbook of Pharmaceutical Excipients”, 8th edition, R.C. Rowe, P.J. Seskey and S.C. Wen, Pharmaceutical Press, London, Chicago.

[0280] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0281] Pharmaceutical formulations containing the antibodies described herein can be prepared by mixing the antibodies of the present invention, having the desired purity, with one or more optional pharmaceutical excipients, preferably in the form of lyophilized formulations or aqueous solutions.

[0282] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use.

[0283] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0284] VIII. Drug Combinations and Medicine Boxes

[0285] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the anti-CLDN18.2 antibody of the present invention or a fragment thereof (preferably an antigen-binding fragment), or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators, etc.).

[0286] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0287] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0288] - A first container containing a pharmaceutical composition comprising an anti-CLDN18.2 antibody or a fragment thereof;

[0289] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0290] IX. Uses and Methods

[0291] In one aspect, the present invention provides a method for preventing or treating tumors (e.g., cancer) in a subject, comprising administering to the subject an effective amount of an antibody against CLDN18.2 of the present invention or a fragment thereof (preferably an antigen-binding fragment), an immunoconjugate, a pharmaceutical composition, a pharmaceutical combination, or a kit.

[0292] In some implementations, the tumor (e.g., cancer) patient has (e.g., elevated levels, such as nucleic acid or protein levels) CLDN18.2.

[0293] In some embodiments, the tumor, such as cancer, includes solid tumors, hematologic malignancies, and metastatic lesions. In one embodiment, examples of solid tumors include malignant tumors. Cancer can be in the early, intermediate, or late stage, or metastatic stage.

[0294] In some embodiments, the tumor treatment will benefit from the inhibition of nucleic acid or protein levels of CLDN18.2. In some embodiments, the tumor treatment will benefit from ADCC or CDC effects induced by the antibodies of the present invention.

[0295] In one specific embodiment, the anti-CLDN18.2 antibody of the present invention can inhibit the proliferation of tumor cells, such as tumor cells expressing CLDN18.2, such as gastric cancer cells or pancreatic cancer cells.

[0296] In one specific embodiment, the anti-CLDN18.2 antibody of the present invention has strong ADCC or CDC activity.

[0297] In some implementations, the tumor is a tumor immune escape.

[0298] In some implementations, the tumor is cancer, such as stomach cancer or pancreatic cancer.

[0299] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., an individual suffering from or at risk of suffering from the diseases described herein). In one embodiment, the subject suffers from or is at risk of suffering from the diseases described herein (e.g., cancer). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

[0300] In other respects, the present invention provides the use of antibody molecules or fragments thereof or their immunoconjugates or pharmaceutical compositions or combinations or kits in the manufacture or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0301] In some embodiments, the antibody molecule or fragment thereof or its immune conjugate or pharmaceutical composition or combination of drugs or cassette of the present invention may delay the onset of the disease and / or the symptoms associated with the disease.

[0302] In some embodiments, the antibody molecules or fragments thereof or their immunoconjugates or pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as treatments and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0303] In some implementation schemes, treatment methods include surgery; radiotherapy; local irradiation; or focused irradiation.

[0304] In some implementation schemes, the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, vaccines, other antibodies, small molecule drugs, or immunomodulators.

[0305] Exemplary immunomodulators include immunosuppressants or anti-inflammatory agents.

[0306] In some implementations, the antibody combinations described herein can be administered separately, for example, as individual antibodies.

[0307] Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the present invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceuticals.

[0308] The drug composition can be administered via known methods, such as oral, intravenous injection, intraperitoneal, intracerebral (internal parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; via a continuous release system or via an implantable device. In some embodiments, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0309] The composition can also be applied topically via an implantable membrane, a sponge, or another suitable material on which the desired molecules are absorbed or encapsulated. In some embodiments, when an implantable device is used, the device can be implanted into any suitable tissue or organ and can deliver the desired molecules via diffusion, timed release of a large pellet, or continuous administration.

[0310] X. Methods and compositions for diagnosis and detection

[0311] In some embodiments, any anti-CLDN18.2 antibody or fragment thereof (preferably an antigen-binding fragment) provided herein can be used to detect the presence of CLDN18.2 in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In some embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is derived from a lesion associated with hyperplastic or cancerous lesions.

[0312] In one embodiment, an anti-CLDN18.2 antibody or a fragment thereof is provided for diagnostic or detection methods. In another aspect, a method for detecting the presence of CLDN18.2 in a biological sample is provided. In some embodiments, the method comprises detecting the presence of the CLDN18.2 protein in a biological sample. In some embodiments, CLDN18.2 is human CLDN18.2. In some embodiments, the method comprises contacting a biological sample with an anti-CLDN18.2 antibody or a fragment thereof as described herein under conditions that allow the anti-CLDN18.2 antibody or a fragment thereof to bind to CLDN18.2, and detecting whether a complex is formed between the anti-CLDN18.2 antibody or a fragment thereof and CLDN18.2. The formation of a complex indicates the presence of CLDN18.2. This method may be an in vitro or in vivo method. In one embodiment, the anti-CLDN18.2 antibody or a fragment thereof is used to select subjects suitable for treatment utilizing the anti-CLDN18.2 antibody or a fragment thereof, for example, where CLDN18.2 is a biomarker for selecting said subjects.

[0313] In one embodiment, the antibodies of the present invention can be used to diagnose tumors, such as cancer, for example, to evaluate (e.g., monitor) the treatment or progression of the disease described herein in an individual, its diagnosis, and / or staging. In some embodiments, a labeled anti-CLDN18.2 antibody or a fragment thereof is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0314] In some embodiments provided herein, the sample is obtained prior to treatment with anti-CLDN18.2 antibody or a fragment thereof. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0315] In some embodiments, the sample is formalin-fixed and paraffin-coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical excision), or a fine-needle aspirate.

[0316] In some implementations, CLDN18.2 is detected before treatment, for example, before the start of treatment or before a treatment after a treatment interval.

[0317] In some embodiments, a method for treating the disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of CLDN18.2, thereby determining a CLDN18.2 value; comparing the CLDN18.2 value with a control value; and if the CLDN18.2 value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-CLDN18.2 antibody or a fragment thereof (e.g., the anti-CLDN18.2 antibody or a fragment thereof described herein) optionally in combination with one or more other therapies, thereby treating the disease.

[0318] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0319]

[0320] Example 1: Construction of a stable expression cell line

[0321] Preparation of human CLDN18.2 overexpression cell line

[0322] Use according to the manufacturer's instructions. The kit (Invitrogen, A1369601) was used to construct a cell line stably expressing human Claudin18.2 (hereinafter referred to as CLDN18.2). First, the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed into the vector pCHO1.0. The constructed plasmid was then transfected into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) using chemical transfection and electrotransfection methods, respectively. After two rounds of pressure selection, cell pools expressing CLDN18.2 were obtained. Then, the cells that highly expressed CLDN18.2 were sorted out using a flow cytometer (MoFlo XDP, Beckman Coulter), and the monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2 that stably expressed CLDN18.2 were obtained by dilution.

[0323] Preparation of human CLDN18.1 overexpressing cell lines

[0324] Use according to the manufacturer's instructions. A cell line stably expressing human Claudin18.1 (hereinafter referred to as CLDN18.1) was constructed using a kit (Invitrogen, A1369601). First, the full-length human CLDN18.1 (UniProt ID: P56856-1) gene was constructed into the vector pCHO1.0 (Invitrogen, A1369601). The constructed plasmid was then chemically transfected into CHO-S cells (Invitrogen, A1369601). After two rounds of pressure selection, cell pools expressing CLDN18.1 were obtained. Then, cells highly expressing CLDN18.1 were sorted using a flow cytometry system (MoFloXDP, Beckman Coulter), and a dilution method was used to obtain the stable CLDN18.1 monoclonal cell line CHO-hCLDN18.1.

[0325] Construction of tumor cell lines overexpressing CLDN18.2

[0326] The full-length human CLDN18.2 gene (UniProt ID: P56856-2) was constructed into the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence. This vector, along with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), was co-transfected into HEK293T (ATCC, CRL-3216) cells for viral packaging. Culture supernatants were collected after 48 and 72 hours of culture, and the lentivirus was concentrated using PEG8000. The concentrated virus was then used to transfect pancreatic cancer DAN-G cells and gastric cancer KATO III cells. Cells expressing CLDN18.2 were then sorted using a flow cytometry system (MoFlo XDP, Beckman Coulter) to obtain stably transfected CLDN18.2 tumor cell lines: DAN-G-CLDN18.2 and KATO III-CLDN18.2.

[0327] Example 2: Hybridoma screening for monoclonal antibodies against hCLDN18.2

[0328] This invention employs hybridoma technology. Mice are immunized with cells (CHO-huClaudin18.2) obtained in Example 1 (methods and procedures are shown in Table 1). Then, mouse spleen cells are fused with myeloma cells to obtain hybridoma cells capable of secreting CLDN18.2 antibodies.

[0329] mouse immunization

[0330] Table 1: Laboratory Animals and Immunological Information

[0331]

[0332] Hybridoma fusion

[0333] The mice were euthanized, and the spleen was removed from the abdominal cavity aseptically. The surrounding connective tissue was removed. The spleen cells were fully released by squeezing with a needle to prepare a spleen cell suspension. The cell suspension was filtered through a 70 μM cell strainer, washed once with RPMI-1640 medium, and centrifuged at 1200 rpm for 6 min. The supernatant was removed, and the cells were resuspended in RBC lysis buffer (GIBCO). Red blood cells were lysed, centrifuged to remove the supernatant, and the cells were resuspended in RPMI-1640 medium and counted. SP2 / 0 and lysed red blood cells were mixed at a ratio of 1:2 to 1:1 and centrifuged at 1000 rpm for 6 min. The supernatant was removed, and the mixed cells were resuspended in fusion buffer. 15 ml of fusion buffer was added, and the mixture was centrifuged at 1000 rpm for 5 min, removing the supernatant. This process was repeated once, and an appropriate volume of fusion buffer was added to resuspend the cells, adjusting the mixed cell density to 1 × 10⁶ cells / mL. 7 Cells / ml. After fusion using an electrofusion apparatus, cells were incubated at room temperature for 5 min in an electrofusion dish. Cells were then transferred to centrifuge tubes and diluted to 1–2 × 10⁶ cells / ml. 4 Cells / ml. Add 100 μl of cell suspension to each well of a 96-well plate. Change the culture medium on day 5 after confluence. Collect the supernatant on day 10 (or longer, depending on cell growth status) for flow cytometry (FACS) analysis to screen for positive clones.

[0334] High-throughput screening of hybridoma cells

[0335] Hybridoma cells that specifically express anti-CLDN18.2 antibodies were screened using flow cytometry (FACS), and the secreted antibodies did not bind to CLDN18.1.

[0336] The cells to be tested (HEK293-hCLDN18.2) obtained in Example 1 were counted and diluted to 1×10⁻⁶. 6 Cells / ml, add 100 μl / well to a U-bottom 96-well plate. Centrifuge at 500g for 5 min to remove cell culture medium. Add the supernatant from the hybridoma 96-well plate to the U-bottom plate and resuspend the cells, adding 100 μl to each well, and incubate on ice for 30 min. Centrifuge at 500g for 5 min to remove the supernatant, and wash the cells once with PBS. Add 100 μl of FITC-labeled anti-mouse Fab secondary antibody (1:500 diluted in PBS) to each well, and add 100 μl of FITC-labeled anti-human Fab secondary antibody to the positive control antibody. Incubate on ice in the dark for 30 min. Centrifuge at 500g for 5 min to remove the supernatant, and wash the cells once with PBS. Resuspend the cells in 50 μl of 1×PBS and analyze using FACS.

[0337] The positive clones were then rescreened for CHO-hCLDN18.1 using the same method described above, resulting in nine hybridoma cell lines that bound to human CLDN18.2 but not to human CLDN18.1. Eight of these lines were derived from Bal b / c mice, and one line was derived from H2L2 fully human transgenic mice, as detailed in Table 2.

[0338] Table 2. Candidate antibody clones obtained by hybridoma high-throughput screening

[0339]

[0340]

[0341] Example 3: Obtaining the antibody gene

[0342] Using molecular biology techniques, the antibody light and heavy chain gene sequences were retrieved from nine hybridoma cell lines (3G3, 14A5, 25C7, 38F8, 69H9, 46F6, 59C1, 51H10, and HB37A6) obtained in Example 2. Total RNA was extracted from the nine hybridoma cells using an RNA extraction kit (Biomiga, R6311-02), and then cDNA was obtained by reverse transcription of the total RNA using a PrimeScript II 1st StrandcDNA Synthesis Kit (Takara, 6110A). The heavy and light chain variable region sequences of the antibody were amplified using degenerate primers, inserted into the pMD20-T vector (Takara, 6028), ligated, transformed, and then cloned for sequencing.

[0343] The antibody sequence is shown in Table A.

[0344] Example 4: Preparation of recombinant CLDN18.2 antibody

[0345] Nine antibodies (3G3, 14A5, 25C7, 38F8, 69H9, 46F6, 59C1, 51H10, and HB37A6) screened in Example 2, along with the control antibody zolbetuximab (Zmab, sequence derived from INN117), were expressed as full-length monoclonal antibodies in HEK293 cells (Invitrogen, A14527). First, expression vectors were constructed by placing the nine paired heavy chain variable regions and light chain variable regions at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 5) and light chain kappa constant region (SEQ ID NO: 10), respectively, into a pcDNA3.1 expression vector containing an N-terminal signal peptide, thus obtaining the light and heavy chain expression vectors. The obtained light and heavy chain expression vectors were transiently transfected into HEK293 cells via co-transfection with PEI (Polysciences Inc., 23966), and the culture supernatant was collected after 7 days of culture. The supernatant was purified using a Protein A column (Hitrap Mabselect Sure, GE11-0034-95), then ultrafiltered and the solution was changed to PBS (Gibco, 70011-044). The concentration was detected by the A280 method and the purity was determined by SEC-HPLC to obtain an antibody solution with a purity greater than 95%.

[0346] Example 5: Determination of the affinity between recombinant CLDN18.2 antibody and antigen using the BLI method.

[0347] The equilibrium dissociation constant (K0) of the antibody binding to human CLDN18.2 in this invention was determined using biolayer interferometry (BLI). D BLI affinity assay was performed according to existing methods (Estep, P et al., Highthroughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8). The AHC (18-5060, Fortebio) sensor was immersed in SD buffer (1x PBS, BSA 0.1%, Tween-20 0.05%). 100 μl of SD buffer, each antibody, and human Claudin18.2 protein (P50251802, GenScript) were added to 96-well black polystyrene half-volume microplates (Greiner, 675076). The assay was performed using Fortebio Octet Red96, and the K-values ​​were analyzed using ForteBio Octet analysis software. DValues. The affinity data of the antibodies are shown in Table 3: all 9 antibodies have high affinity, except for 3G3, which has an affinity comparable to the control antibody Zmab, the others are superior to the control antibody Zmab.

[0348] Table 3: Affinity constants of CLDN18.2 antibody

[0349]

[0350]

[0351] Example 6: Binding specificity of CLDN18.2 antibody to CLDN18 cells

[0352] The binding of the nine antibodies to the CHO-S cell lines stably transfected with human CLDN18.2 and human CLDN18.1 obtained in Example 1 (i.e., CHO-hCLDN18.2 and CHO-hCLDN18.1 prepared as described in the examples) was determined by flow cytometry (FACS). The experimental methods were the same as in Example 2, and the experimental data were analyzed using GraphPad Prism software. Figure 1 and Figure 2 .like Figure 1 and Figure 2 As shown, all nine antibodies specifically bind to human CLDN18.2 but not to human CLDN18.1.

[0353] Example 7: Detection of ADCC activity of CLDN18.2 antibody based on reporter gene

[0354] ADCC activity of the above nine antibodies was detected using ADCC effector cells (Promega, G7102) containing a luciferase reporter gene. Target cells (CHO-hCLDN18.2) and ADCC effector cells in logarithmic growth phase were counted separately and added in a 1:6 ratio (50 μL total) to a 96-well white plate, with 1.75 × 10⁶ cells per well. 5 Then, add the above-mentioned antibody to each well and incubate at 37°C and 5% CO2 for 8-10 hours. The concentrations of the antibodies are as follows: the initial concentration is 3 nM, followed by 3-fold dilution, resulting in a total of 9 concentration points, i.e., the concentrations used for each antibody are: 3 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, 0.123 nM, 0.0041 nM, 0.0014 nM, and 0.00046 nM. Use Bio-Glo according to the manufacturer's instructions. TM Fluorescence was detected using Luciferase reagent (Promega, G7940). Figure 3 The results showed that all nine antibodies had ADCC activity comparable to the control antibody Zmab.

[0355] Example 8: Humanization of mouse antibodies

[0356] Eight murine antibodies (3G3, 14A5, 25C7, 38F8, 69H9, 46F6, 59C1, and 51H10) from Example 3 were selected for humanization. The CDR regions of the antibodies were determined; the heavy chain CDR1 followed Abm nomenclature, and the remaining CDRs followed Kabat nomenclature. Through sequence similarity comparison, the human germline gene sequence with the highest sequence similarity to the variable region of the murine antibody was selected as the framework region template for the humanized antibody, and then the CDR regions were transplanted into each template. The three-dimensional structures of each antibody were constructed using Discovery Studio software, and the amino acids in the framework region that affected the CDR region were reverse-mutated to obtain the humanized antibody sequences. The sequences of the humanized antibodies Hz3G3, Hz14A5, Hz25C7, Hz38F8, Hz69H9, Hz46F6, Hz59C1, and Hz51H10 are shown in Table A.

[0357] The humanized recombinant antibodies were prepared and purified according to the method described in Example 4 to obtain an antibody solution dissolved in PBS with a purity greater than 95%.

[0358] Example 9: Determination of CLDN18.2 antibody affinity by SPR method

[0359] The equilibrium dissociation constants (K0) of eight humanized antibodies (Hz3G3, Hz14A5, Hz25C7, Hz38F8, Hz69H9, Hz46F6, Hz59C1, and Hz51H10) and one fully human antibody (HB37A6) bound to human CLDN18.2 were determined using surface plasmon resonance (SPR). DFollowing the manufacturer's instructions, human Claudin 18.2 (GenScrip, P50251802) was conjugated onto the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino-conjugation kit (GE Healthcare, BR-1006-33). After conjugation, 1M ethanolamine was injected to block the remaining activation sites. Affinity and kinetic constants were obtained by detecting the binding and dissociation of the chip surface antigen with the antibody in the mobile phase using a Biacore (GE Healthcare, T200) analyzer. Serially diluted antibodies (0-100 nM) were flowed sequentially from low to high concentration onto the chip surface, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). Data were analyzed using Biacore T200 software with a 1:1 binding model for kinetic analysis. As shown in Table 4, except for Hz25C7 and Hz3G3, the affinity of the other antibodies was superior to that of the control antibody Zmab.

[0360] Table 4. Affinity constants (equilibrium dissociation constants) for SPR assay of CLDN18.2 antibody

[0361]

[0362] Example 10: Detection of ADCC activity of humanized antibodies based on reporter genes

[0363] Based on SPR affinity data, Hz14A5, Hz38F8, Hz69H9, Hz46F6, Hz59C1, and Hz51H10 were selected for reporter gene-based ADCC activity detection. The experimental procedure was as described in Example 7. Figure 4 As shown, the humanized antibodies Hz14A5, Hz38F8, Hz69H9, Hz46F6, Hz59C1, and Hz51H10 all exhibited ADCC activity comparable to the control antibody.

[0364] Example 11: Binding of CLDN18.2 antibody to tumor cell lines

[0365] Based on the above results, one humanized antibody Hz69H9 and one fully human antibody HB37A6 were selected for further testing. Referring to Example 4, the binding of the two antibodies to the gastric cancer cell line NUGC-4, the gastric cancer cell line KATOIII-hCLDN18.2, and the pancreatic cancer cell line DAN-G-hCLDN18.2 was determined by FACS. Figure 5The results showed that both the humanized antibody Hz69H9 and the fully human antibody HB37A6 exhibited good tumor cell-specific binding, which was superior to the control antibody Zmab.

[0366] Example 12: Detection of CDC activity of CLDN18.2 antibody

[0367] The CDC activity of antibody molecules was measured using the KATO III-CLDN18.2 cell line. 1×10⁻⁶ cells were added to each well of a 96-well plate. 5 KATO III-hCLDN18.2 target cells were incubated. An appropriate concentration of antibody molecules was added, and the cells were incubated at 37°C for 30 minutes. Then, freshly prepared human serum complement (Sigma, S1764) was added. The antibody concentrations were as follows: serial dilutions, starting at 150 μg / ml, 3-fold dilution, for a total of 9 concentration points. The reaction system was incubated again at 37°C for 3 hours. Data were then read using a CCK-8 assay kit (Tongren Chemical, CK04) and a microplate reader (ThermoFisher, MULTISKAN FC). The results are shown below. Figure 6 As shown, Hz69H9 and HB37A6 have CDC activity comparable to the control antibody Zmab.

[0368] Example 13: Detection of ADCC activity of CLDN18.2 antibody

[0369] Human peripheral blood mononuclear cells (PBMCs, Allcells, or Saily) were resuspended in complete culture medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03), and the PBMCs were adjusted to 1×10⁶ cells / mL. 7 Cells / ml. NUCGC-4 or Claudin18.2-overexpressing DAN-G tumor target cells (DAN-G-CLDN18.2) were labeled with Far-Red (Invitrogen) for 10 min, washed twice, and resuspended in complete medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03) to adjust the cell concentration to 2 × 10⁻⁶ cells / ml. 5 PBMCs were mixed with anti-claudin18.2 monoclonal antibodies Hz69H9 and HB37A6 and control antibody Zmab (each antibody started at 30 nM, serially diluted 3-fold, for a total of 12 concentration points), incubated at 37°C for 30 min, and then 50 μL of tumor target cells (1 × 10⁻⁶ cells / ml) were added at an effector-to-target ratio of 50:1. 4(Number of cells) was added to 50 μl of PBMC effector cells. After incubation at 37°C for 8 hours, the cells were centrifuged and resuspended in propidium iodide (PI, Invitrogen) at a final concentration of 10 μg / ml. Cells positive for both Far-Red and PI were detected by flow cytometry (BD, FACSCELESTA). The tumor target cell killing ratio was calculated using FACSDiva software (BD, Celestsa).

[0370] Results of cell-mediated NUGC-4 tumor cytotoxicity dependent on anti-Claudin18.2 monoclonal antibody: Figure 7 As shown in A and B, Hz69H9, HB37A6, and the control antibody Zmab all dose-dependently mediated cytotoxic killing of NUGC-4 cells by NK cells. Hz69H9 and HB37A6 exhibited stronger cytotoxic effects on tumor cells than the control antibody Zmab. This indicates that Hz69H9 and HB37A6 possess stronger cell-mediated tumor cytotoxicity. The results of cell-mediated DN-G tumor cytotoxicity dependent on anti-Claudin18.2 monoclonal antibody are shown below. Figure 7 As shown in C and D, Hz69H9, HB37A6, and the control antibody Zmab can all induce dose-dependent cytotoxic killing of tumor cells by NK cells. Among them, HB37A6 and Hz69H9 mediated greater cytotoxicity in PBMCs from two different donors than the control antibody Zmab. These results indicate that, compared to the control antibody Zmab, Hz69H9 and HB37A6 have stronger cell-mediated tumor cytotoxicity.

[0371] Example 14: In vivo antitumor effect of CLDN18.2 antibody

[0372] 1. Antibody activity against the DAN-G-CLDN18.2 tumor-bearing mouse model

[0373] The antitumor effects of HZ69H9 and HB37A6 antibodies in NOD-SCID mice with human pancreatic cancer (60 female NOD-SCID mice (15-18g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were tested. The human pancreatic cancer cells DAN-G-CLDN18.2 constructed in Example 1 were routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) to obtain a cell suspension with a cell density of 12×10^5 cells / ml. The cell suspension was mixed with Matrigel gel at a 1:1 ratio to prepare a cell suspension with a cell concentration of 6×10^5 cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously injected into the right abdominal region of NOD-SCID mice to establish a DAN-G-CLDN18.2 tumor-bearing mouse model.

[0374] Five days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with a tumor volume of 43.36 mm were selected. 3 ~89.47mm 3 Mice within the range were grouped in a serpentine pattern according to tumor size (8 mice per group).

[0375] Each mouse was administered hIgG (Equitech-Bio, batch number 160308-02), Hz69H9, HB37A6, and the control antibody Zmab at a dose of 10 mg / kg per administration, on days 5, 9, 12, and 16 post-inoculation. Tumor volume was monitored 2-3 times per week. Tumor volume was measured using calipers to determine the maximum long axis (L) and maximum wide axis (W) of the tumor. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0376] 2. Antibody activity against NUCG-4 tumor-bearing mouse model

[0377] The antitumor effects of HZ69H9 and HB37A6 antibodies in NOG mice with human gastric cancer (100 female NOG mice (15-18g) purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were tested. PBMC cells (Allcells) were resuscitated, collected by centrifugation, and dispersed in PBS (1×) at a cell density of 2.5×10^6 cells / ml. On day 0, 0.2ml of the cell suspension was injected intravenously into the eye vein of NOG mice to establish a humanized NOG mouse model.

[0378] NUGC-4 cells were routinely resuscitated and passaged for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1×) at a density of 12×10^6 cells / ml. The PBS was then mixed with Matrigel gel at a 1:1 ratio to prepare a cell suspension with a concentration of 6×10^6 cells / ml. On day 5, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOG humanized mice to establish a NUGC-4 tumor-bearing mouse model.

[0379] On day 1 after tumor cell inoculation, mice were randomly divided into groups of 7 mice each. Each mouse was administered hIgG (Equitech-Bio, batch number 160308-02), Hz69H9, HB37A6, and the control antibody Zmab at a dose of 10 mg / kg per administration. Administration was performed on days 1, 5, 8, and 12 post-inoculation. Tumor volume and body weight were monitored 2-3 times per week. Tumor volume was measured using calipers to determine the maximum long axis (L) and maximum wide axis (W) of the tumor. The tumor volume was calculated using the following formula: V = L × W 2 / 2. Weight was measured using an electronic balance.

[0380] 3. Results

[0381] The results are as follows Figure 8 As shown, Hz69H9, HB37A6, and the control antibody Zmab all inhibited tumor growth in a human pancreatic cancer DANN-G-hCLDN18.2 mouse model. Figure 9 As shown, Hz69H9 and HB37A6 exhibited better anti-tumor effects than the control antibody Zmab in the NUGC-4 human gastric cancer mouse model.

[0382] The relative tumor inhibition rate (TGI%) was calculated on day 26 after vaccination, using the following formula:

[0383] TGI% = 100% * (Tumor volume in control group - Tumor volume in treatment group) / (Tumor volume in control group - Tumor volume in control group before drug administration).

[0384] In the DANS-G-CLDN18.2 tumor-bearing mouse model, the TGI of Hz69H9 and HB37A were 70% and 28%, respectively, while the TGI of Zmab was 24%.

[0385] In the NUGC-4 tumor-bearing mouse model, the TGI of Hz69H9 and HB37A were 46% and 31%, respectively, while the TGI of Zmab was 0%.

[0386] Sequence information:

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395] sequence list <110> Innovent Biologics (Suzhou) Co., Ltd. <120> Anti-Claudin18.2 antibody and its uses <130> PF 210417PCT <160> 94 <170> PatentIn version 3.3 <210> 1 <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 1 Gly Phe Thr Phe Ser Ser Tyr Val Met Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 2 Thr Ile Ser His Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 13 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 3 Asp Ala Pro Tyr Tyr Asp Ile Leu Thr Gly Tyr Arg Tyr 1 5 10 <210> 4 <211> 122 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 4 Glu Val Gln Leu Leu Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asn Trp Val 35 40 45 Ser Thr Ile Ser His Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ile Asp Ala Pro Tyr Tyr Asp Ile Leu Thr Gly Tyr Arg Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 6 <211> 11 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 6 Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1 5 10 <210> 7 <211> 7 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 7 Lys Ala Ser Ser Leu Glu Ser 1 5 <210> 8 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 8 Gln Gln Tyr Asn Ser Tyr Ser Tyr Thr 1 5 <210> 9 <211> 107 <212> PRT <213> artificial <220> <223> synthetic peptide <400> 9 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 10 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 11 <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <220> <221> misc_feature <222> (8)..(8) <223> Xaa can be any naturally occurring amino acid. <400> 11 Lys Ser Ser Gln Ser Leu Leu Xaa Gly Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 12 <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 12 Gly Tyr Ser Phe Ser Ser Tyr Asn Ile His 1 5 10 <210> 13 <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 13 Tyr Ile Ala Pro Phe Asn Gly Asp Ser Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 14 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 14 Leu Asn Arg Gly Asn Ser Leu Asp Tyr 1 5 <210> 15 <211> 118 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 15 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Ser Phe Ser Ser Tyr 20 25 30 Asn Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ala Pro Phe Asn Gly Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Val Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Arg Leu Asn Arg Gly Asn Ser Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Leu Thr Val Ser Ser 115 <210> 16 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 16 Lys Ser Ser Gln Ser Leu Phe Asn Ser Gly Asn Gln Arg Asn Tyr Leu 1 5 10 15 Thr <210> 17 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 17 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 18 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 18 Gln Asn Asn Tyr Ile Tyr Pro Leu Thr 1 5 <210> 19 <211> 113 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 19 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Phe Tyr Tyr Cys Gln Asn 85 90 95 Asn Tyr Ile Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 20 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 20 Tyr Ile Ala Pro Phe Gln Gly Asp Ser Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 21 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Ser Tyr 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Ala Pro Phe Gln Gly Asp Ser Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Asn Arg Gly Asn Ser Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 22 <211> 113 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 22 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asn Tyr Ile Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> twenty three <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> twenty three Gly Phe Ser Phe Ser Asp Tyr Gly Met His 1 5 10 <210> twenty four <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> twenty four Tyr Ile Asn Ser Gly Ser Arg Thr Leu Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 25 <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 25 Asn Ala Tyr Tyr Gly Asn Ala Met Asp Tyr 1 5 10 <210> 26 <211> 119 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile 35 40 45 Ala Tyr Ile Asn Ser Gly Ser Arg Thr Leu Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Glu Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Arg Asn Ala Tyr Tyr Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 27 <211> 119 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Asn Ser Gly Ser Arg Thr Leu Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Ala Tyr Tyr Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 28 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 28 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Arg Asn Tyr Leu 1 5 10 15 Thr <210> 29 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptides <400> 29 Trp Ser Ser Thr Arg Gly Ser 1 5 <210> 30 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 30 Gln Asn Val Tyr Tyr Tyr Pro Phe Thr 1 5 <210> 31 <211> 113 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 31 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr Arg Gly Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Asn 85 90 95 Val Tyr Tyr Tyr Pro Phe Thr Phe Gly Ser Gly Thr Arg Leu Glu Val 100 105 110 Lys <210> 32 <211> 113 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 32 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr Arg Gly Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Val Tyr Tyr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys <210> 33 <211> 11 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 33 Gly Tyr Ser Ile Thr Ser Gly Tyr Gly Trp Asn 1 5 10 <210> 34 <211> 16 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 34 Tyr Ile His Phe Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 35 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 35 Ser Gly Lys Gly Asn Ala Met Asp Tyr 1 5 <210> 36 <211> 118 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 36 Gln Val Gln Leu Lys Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Gly Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile His Phe Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Lys Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 37 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Gly Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Phe Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Lys Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 38 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 38 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 39 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 39 Gln Asn Asp Tyr Tyr Phe Pro Phe Thr 1 5 <210> 40 <211> 113 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Val Ile Asn Met Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Tyr Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 41 <211> 113 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 41 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Tyr Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 42 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 42 Gly Tyr Thr Phe Thr Lys Tyr Ile Ile Gln 1 5 10 <210> 43 <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 43 Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 44 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 44 Thr Tyr Tyr Gly Asn Ser Phe Pro Asn 1 5 <210> 45 <211> 118 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 45 Glu Val Gln Leu Gln Gln Ser Val Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Ile Ile Gln Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Tyr Tyr Gly Asn Ser Phe Pro Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 46 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 46 Gln Asn Asp Tyr Ser Phe Pro Phe Thr 1 5 <210> 47 <211> 113 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 47 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ser Glu Asp Leu Ala Val Tyr Phe Cys Gln Asn 85 90 95 Asp Tyr Ser Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 48 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Ile Ile Gln Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Tyr Tyr Gly Asn Ser Phe Pro Asn Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 49 <211> 113 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 49 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 50 <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 50 Gly Phe Ser Leu Thr Asp Tyr Gly Val Ser 1 5 10 <210> 51 <211> 16 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 51 Val Met Trp Gly Gly Gly Asn Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 <210> 52 <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 52 Gln Arg Tyr Gly Gly Asn Ala Met Asp Tyr 1 5 10 <210> 53 <211> 118 <212> PRT <213> artificial <220> <223> synthetic peptide <400> 53 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Gly Gly Gly Asn Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Gln Arg Tyr Gly Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 54 <211> 9 <212> PRT <213> Artificial <220> <223> synthetic peptide <400> 54 Gln Asn Ser Tyr Phe Tyr Pro Phe Thr 1 5 <210> 55 <211> 113 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 55 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Ile Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asn Arg Phe Thr Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ser Tyr Phe Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 56 <211> 118 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 56 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Gly Gly Gly Asn Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gln Arg Tyr Gly Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 57 <211> 113 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 57 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ser Tyr Phe Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 58 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 58 Gly Phe Thr Phe Ser Asp Tyr Gly Met Ala 1 5 10 <210> 59 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 59 Phe Ile Asn Asn Leu Ala Tyr Ser Ile Tyr Tyr Val Asp Thr Val Thr 1 5 10 15 Gly <210> 60 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 60 Phe Thr Thr Gly Asn Val Met Asp Tyr 1 5 <210> 61 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 61 Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ala Phe Ile Asn Asn Leu Ala Tyr Ser Ile Tyr Tyr Val Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asp Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Phe Thr Thr Gly Asn Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 62 <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 62 Lys Ser Ser Gln Ser Leu Leu Asn Gly Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 63 <211> 7 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 63 Trp Ser Ser Thr Arg Glu Ser 1 5 <210> 64 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 64 Gln Asn Ser Tyr Ser Tyr Pro Leu Thr 1 5 <210> 65 <211> 113 <212> PRT <213> artificial <220> <223> Synthetic peptide <400> 65 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Gly 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Thr Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ser Tyr Ser Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 66 <211> 118 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Asn Asn Leu Ala Tyr Ser Ile Tyr Tyr Val Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Thr Thr Gly Asn Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 67 <211> 17 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 67 Lys Ser Ser Gln Ser Leu Leu Gln Gly Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thursday <210> 68 <211> 113 <212> PRT <213> artificially <220> <223> Synthetic peptide <400> 68 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Gln Gly 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ser Tyr Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 69 <211> 10 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 69 Gly Tyr Ser Phe Thr Thr Tyr Trp Met His 1 5 10 <210> 70 <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 70 Leu Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 71 <211> 6 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 71 Asn Arg Trp Leu Leu Gly 1 5 <210> 72 <211> 115 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 72 Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Arg Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Arg Trp Leu Leu Gly Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 73 <211> 115 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 73 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asp Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Arg Trp Leu Leu Gly Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 74 <211> 9 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 74 Gln Asn Asp Tyr Ser Tyr Pro Leu Thr 1 5 <210> 75 <211> 113 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 75 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 76 <211> 113 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 76 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 77 <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 77 Gly Phe Ser Leu Asn Ser Tyr Gly Val Gly 1 5 10 <210> 78 <211> 16 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 78 Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Val Leu Ile Ser 1 5 10 15 <210> 79 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 79 Ile Thr Arg Gly Asn Ala Met Asp Tyr 1 5 <210> 80 <211> 117 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 80 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Ser Val Ser Gly Phe Ser Leu Asn Ser Tyr 20 25 30 Gly Val Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Val Leu Ile 50 55 60 Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Met Ile Thr Arg Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 81 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 81 Trp Ala Ser Thr Arg Lys Ser 1 5 <210> 82 <211> 9 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 82 Gln Asn Asn Tyr Leu Phe Pro Leu Thr 1 5 <210> 83 <211> 113 <212> PRT <213> artificially <220> <223> synthetic peptide <400> 83 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Ile Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Thr Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Ser Cys Gln Asn 85 90 95 Asn Tyr Leu Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 84 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 84 Val Ile Trp Gly Asp Val Ser Thr Asn Tyr His Ser Val Leu Ile Ser 1 5 10 15 <210> 85 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 85 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Ser Tyr 20 25 30 Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Asp Val Ser Thr Asn Tyr His Ser Val Leu Ile 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ile Thr Arg Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 86 <211> 113 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 86 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asn Tyr Leu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 87 <211> 10 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 87 Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn 1 5 10 <210> 88 <211> 16 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 88 Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 <210> 89 <211> 9 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 89 Ser Trp Arg Gly Asn Ser Phe Asp Tyr 1 5 <210> 90 <211> 118 <212> PRT <213> artificial <220> <223> Synthetic peptides <400> 90 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 91 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 91 Gln Asn Asp Tyr Ser Tyr Pro Phe Thr 1 5 <210> 92 <211> 113 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 92 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 93 <211> 17 <212> PRT <213> artificial <220> <223> Synthetic peptides <220> <221> misc_feature <222> (6)..(6) <223> Xaa can be any naturally occurring amino acid. <400> 93 Tyr Ile Ala Pro Phe Xaa Gly Asp Ser Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 94 <211> 16 <212> PRT <213> artificial <220> <223> Synthetic peptides <220> <221> misc_feature <222> (6)..(6) <223> Xaa can be any naturally occurring amino acid. <400> 94 Val Ile Trp Gly Asp Xaa Ser Thr Asn Tyr His Ser Val Leu Ile Ser 1 5 10 15

Claims

1. An anti-CLDN18.2 antibody or its antigen-binding fragment, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 are, in turn, the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:4, and wherein LCDR1, LCDR2, and LCDR3 are, in turn, the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:9; HCDR1 is defined according to AbM rules, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to Kabat rules.

2. An anti-CLDN18.2 antibody or its antigen-binding fragment, comprising: HCDR1, HCDR2, HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 6, 7 and 8, respectively.

3. The antibody or antigen-binding fragment of claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:

9.

4. The antibody or antigen-binding fragment of claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 4, and the amino acid change does not occur in the CDR region; and the light chain variable region comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 9, and the amino acid change does not occur in the CDR region.

5. The antibody of claim 1 or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:4 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

9.

6. The antibody or antigen-binding fragment of claim 1, 2 or 5, further comprising a heavy chain constant region and / or a light chain constant region.

7. The antibody of claim 6 or its antigen-binding fragment, wherein the heavy chain constant region comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 5; or comprises an amino acid sequence having one or no more than 20 amino acid substitutions compared to the amino acid sequence of SEQ ID NO:

5.

8. The antibody of claim 6 or its antigen-binding fragment, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

5.

9. The antibody of claim 6 or its antigen-binding fragment, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

5.

10. The antibody of claim 6 or its antigen-binding fragment, wherein the light chain constant region comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 10; or comprises an amino acid sequence having one or no more than 20 amino acid substitutions compared to the amino acid sequence of SEQ ID NO:

10.

11. The antibody of claim 6 or an antigen-binding fragment thereof, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

10.

12. The antibody of claim 6 or its antigen-binding fragment, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

10.

13. The antibody of claim 7 or its antigen-binding fragment, wherein the amino acid substitution occurs in the Fc region of the heavy chain constant region.

14. The antibody or antigen-binding fragment thereof of claim 1, 2 or 5, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment in the form of IgG1, IgG2, IgG3 or IgG4.

15. The antibody or antigen-binding fragment thereof of claim 1, 2 or 5, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment in the form of IgG1.

16. The antibody of claim 1, 2 or 5, or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody.

17. The antibody of claim 1, 2 or 5, or an antigen-binding fragment thereof, wherein the antibody is a human antibody.

18. The antibody of claim 1 or an antigen-binding fragment thereof, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, scFv, or (Fab')2.

19. The antibody or antigen-binding fragment thereof of claim 1, 2 or 5, wherein said antibody or antigen-binding fragment thereof has one or more of the following properties: (i) It binds to human CLDN18.2 with high affinity, but not to human CLDN18.1; (ii) with the following equilibrium dissociation constant (K) D ) combined with human CLDN18.2, the K D Less than 15 nM; (iii) It binds to CLDN18.2 on the cell surface, but not to CLDN18.1 on the cell surface; (iv) It has ADCC or CDC activity; (v) Inhibit tumor cells; (vi) It can effectively inhibit tumor growth, with a tumor inhibition rate of greater than or equal to 25%.

20. An isolated nucleic acid encoding a light chain variable region or a heavy chain variable region, or a light chain or heavy chain, of an antibody or antigen-binding fragment of any one of claims 1 to 19 that binds to CLDN18.

2.

21. A vector comprising the nucleic acid of claim 20.

22. The carrier of claim 21, wherein the carrier is an expression carrier.

23. A host cell comprising the nucleic acid of claim 20 or the vector of claim 21 or 22.

24. The host cell of claim 23, wherein the host cell is prokaryotic or eukaryotic.

25. The host cell of claim 23, wherein the host cell is selected from yeast cells or mammalian cells.

26. The host cell of claim 23, wherein the host cell is selected from 293 cells or CHO cells.

27. The host cell of claim 23, wherein the host cell is a CHO-S cell or a HEK293 cell.

28. A method for preparing an antibody or antigen-binding fragment thereof that binds to CLDN18.2, said method comprising culturing a host cell of any one of claims 24-27 under conditions suitable for expressing a nucleic acid encoding an antibody or antigen-binding fragment thereof of any one of claims 1 to 19.

29. The method of claim 28, wherein the method further comprises separating the antibody or its antigen-binding fragment.

30. The method of claim 28 or 29, wherein the method further comprises recovering the antibody or its antigen-binding fragment from the host cell.

31. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to CLDN18.2 according to any one of claims 1 to 19.

32. The pharmaceutical composition of claim 31, further comprising one or more other therapeutic agents.

33. The pharmaceutical composition of claim 32, wherein the other therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

34. The pharmaceutical composition according to any one of claims 31-33, further comprising pharmaceutical excipients.

35. A pharmaceutical combination product comprising an antibody or antigen-binding fragment thereof of any one of claims 1 to 19, and one or more other therapeutic agents.

36. The pharmaceutical combination product of claim 35, wherein the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

37. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 19, or the pharmaceutical composition of any one of claims 31 to 34, or the pharmaceutical combination product of claim 35 or 36 in the preparation of a medicament for the prevention or treatment of tumors, wherein the tumor is gastric cancer or pancreatic cancer.

38. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 19, or the pharmaceutical composition of any one of claims 31 to 34, or the pharmaceutical combination product of claim 35 or 36 in the preparation of a medicament for inducing ADCC and / or CDC in a subject, wherein the subject has a tumor, and the tumor is gastric cancer or pancreatic cancer.

39. The use of claim 37 or 38, wherein the tumor is CLDN18.2 having elevated nucleic acid or protein levels.

40. The use of claim 37 or 38, wherein the drug is further administered in combination with one or more therapies.

41. The use of claim 40, wherein the therapy is a treatment method and / or other therapeutic agent.

42. The use of claim 41, wherein the treatment method includes surgical treatment and / or radiotherapy.

43. The use of claim 41, wherein the other therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators.

44. A non-diagnostic or non-treatment method for detecting CLDN18.2 in a sample, the method comprising: (a) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 19; and (b) Detect the formation of a complex between the antibody or its antigen-binding fragment and CLDN18.

2.

45. The method of claim 44, wherein the antibody or its antigen-binding fragment is detectably labeled.

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