Antibody and application thereof

Abstract

The invention relates to the field of disease treatment, in particular to an anti-CLDN18.2 antibody or an antigen binding fragment thereof, nucleic acid molecules encoding the anti-CLDN18.2 antibody or the antigen binding fragment, and a method for preparing the anti-CLDN18.2 antibody or the antigen binding fragment. The anti-CLDN18.2 antibody or the antigen binding fragment of the anti-CLDN18.2 antibody has high specificity and high affinity to CLDN18.2, can be effectively combined with CLDN18.2 and can be used for mediating and killing CLDN18.2 expression cells. Therefore, the invention further relates to a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof, and application of the antibody or the antigen-binding fragment thereof in preparation of a medicine, and the medicine is used for preventing and / or treating tumors.

Description

[0001] This application is a divisional application of the invention application with application number 201980076474.5, application date December 19, 2019, and invention name “An antibody and its use”. Technical Field

[0002] The present invention belongs to the field of therapeutic monoclonal antibodies, and more specifically, the present invention relates to an antibody against CLDN18.2; and also relates to the use of the antibody in treating diseases. Technical Background

[0003] Claudin 18.2 (CLDN18.2) is a member of the Claudin protein family of tight junction proteins. Tight junction proteins are a class of proteins that mediate tight junctions between cells. Different subtypes of Claudin proteins are expressed in different tissues and are associated with different types of cancer. Claudin 1 is highly expressed in colon cancer, and Claudin 7 is associated with the recurrence of liver cancer. The expression of Claudin 18.2 in normal tissues is limited to gastric mucosal cells and is not expressed in other normal tissues. At the same time, Claudin 18.2 is expressed in 70% of primary gastric adenocarcinomas and their metastases, and is also expressed in some other cancers such as pancreatic cancer (50%), esophageal cancer (30%) and non-small cell lung cancer (25%). Gastric cancer and pancreatic cancer have poor prognosis and high mortality, and there is currently a huge demand for drugs.

[0004] The Claudin 18.2 protein includes four transmembrane regions, two extracellular loops and one intracellular loop, with its N-terminus and C-terminus in the cytoplasm. The two extracellular loops make it an ideal antibody target. The Claudin 18.2 protein sequence is highly conserved among species. Its homologous family protein Claudin 18.1 is specifically expressed in lung tissue and is very similar to the sequence of Claudin 18.2. There are only 8 amino acid differences in the extracellular loop D1, which determines the antibody recognition epitope. The high similarity between Claudin 18.2 and Claudin 18.1 makes the discovery of Claudin 18.2-specific antibody drugs more challenging.

[0005] Astellas' Zolbetuximab (IMAB362) is a human-mouse chimeric IgG1 antibody that targets Claudin 18.2. It binds to Claudin18.2 on the surface of tumor cells and activates antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions. It can also induce tumor cell apoptosis and inhibit tumor cell proliferation.

[0006] However, there is no relevant drug on the market yet. Therefore, it is urgent and necessary to develop anti-targeted Claudin 18.2 antibodies with higher specificity, lower toxicity and side effects, and better clinical efficacy, which will provide more drug options for cancer patients. Summary of the invention

[0007] In the present application, the inventors first developed a mouse antibody with excellent properties, which can specifically recognize / bind to CLDN18.2 but not to CLDN18.1. On this basis, the inventors have devoted a lot of creative work to conduct in-depth research and transformation of the mouse antibody, thereby developing chimeric antibodies and humanized antibodies of the mouse antibody. The humanized antibody of the present invention not only has a very high degree of humanization, but also has substantially the same (or even better) biological functions as mouse antibodies and human-mouse chimeric antibodies (chimeric antibodies have the same heavy chain variable region and light chain variable region as mouse antibodies).

[0008] Therefore, the antibodies of the present invention (especially humanized antibodies) are extremely advantageous, as they not only retain the functions and properties of the parent mouse antibodies, such as binding to CLDN18.2 with very high affinity and specificity, and thus have the potential to be used for the prevention and treatment of tumors; the humanized antibodies of the present invention have a very high degree of humanization, and thus can be safely administered to human subjects without inducing an immunogenic response. Therefore, the antibodies of the present invention have great clinical value.

[0009] Antibodies of the present invention

[0010] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CLDN18.2.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0012] (a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and / or

[0013] CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:2;

[0014] or

[0015] (b) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO: 3, 39 or 40; and / or

[0016] CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:4 or 41;

[0017] or (c) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:44; and / or

[0018] CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:45;

[0019] or (d) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:46; and / or

[0020] CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:47; or

[0021] (e) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:48; and / or

[0022] CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:49; or

[0023] (f) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO: 50; and / or

[0024] CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:51; or

[0025] (g) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:52; and / or

[0026] CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:53; or

[0027] (h) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein, compared with the heavy chain variable region and / or light chain variable region of any one of (a) to (g), at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation, and the mutation is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution;

[0028] In certain preferred embodiments, the CDR is defined according to the Kabat, IMGT, Chothia or AbM numbering systems; preferably, the VH and / or VL of the antibody or its antigen-binding fragment comprises a framework region (FRs) from a human or mouse immunoglobulin, and preferably, the antibody or its antigen-binding fragment binds to human CLDN18.2.

[0029] In some embodiments, an antibody or an antigen-binding fragment thereof that can specifically bind to CLDN18.2 is provided, wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0030] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0031] (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 5, CDR-H2 with a sequence of SEQ ID NO: 6, and CDR-H3 with a sequence of SEQ ID NO: 7; and / or,

[0032] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8, CDR-L2 with a sequence of SEQ ID NO: 9, and CDR-L3 with a sequence of SEQ ID NO: 10;

[0033] or

[0034] (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11, CDR-H2 with a sequence of SEQ ID NO: 12 or 110, and CDR-H3 with a sequence of SEQ ID NO: 13; and / or,

[0035] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14, CDR-L2 with a sequence of SEQ ID NO: 15, and CDR-L3 with a sequence of SEQ ID NO: 16;

[0036] or

[0037] (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 81, CDR-H2 with a sequence of SEQ ID NO: 82, and CDR-H3 with a sequence of SEQ ID NO: 83; and / or,

[0038] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 96, and CDR-L3 with a sequence of SEQ ID NO: 97;

[0039] or

[0040] (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 81, CDR-H2 with a sequence of SEQ ID NO: 84, and CDR-H3 with a sequence of SEQ ID NO: 85; and / or,

[0041] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 96, and CDR-L3 with a sequence of SEQ ID NO: 97;

[0042] or

[0043] (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 86, CDR-H2 with a sequence of SEQ ID NO: 87, and CDR-H3 with a sequence of SEQ ID NO: 88; and / or,

[0044] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 98, and CDR-L3 with a sequence of SEQ ID NO: 99;

[0045] or

[0046] (f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 89, CDR-H2 with a sequence of SEQ ID NO: 90, and CDR-H3 with a sequence of SEQ ID NO: 91; and / or,

[0047] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 98, and CDR-L3 with a sequence of SEQ ID NO: 99;

[0048] or

[0049] (g) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 92, CDR-H2 with a sequence of SEQ ID NO: 93, and CDR-H3 with a sequence of SEQ ID NO: 94; and / or,

[0050] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:95, CDR-L2 with the sequence of SEQ ID NO:100, and CDR-L3 with the sequence of SEQ ID NO:101.

[0051] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0052] (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 17, CDR-H2 with a sequence of SEQ ID NO: 18, and CDR-H3 with a sequence of SEQ ID NO: 19; and / or,

[0053] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 20, CDR-L2 with a sequence of SEQ ID NO: 21, and CDR-L3 with a sequence of SEQ ID NO: 22;

[0054] or

[0055] (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 23, CDR-H2 with a sequence of SEQ ID NO: 24 or 111, and CDR-H3 with a sequence of SEQ ID NO: 25; and / or,

[0056] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 26, CDR-L2 with a sequence of SEQ ID NO: 27, and CDR-L3 with a sequence of SEQ ID NO: 28;

[0057] or

[0058] (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 58, CDR-H2 with a sequence of SEQ ID NO: 62, and CDR-H3 with a sequence of SEQ ID NO: 67; and / or,

[0059] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 74, and CDR-L3 with a sequence of SEQ ID NO: 78;

[0060] or

[0061] (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 58, CDR-H2 with a sequence of SEQ ID NO: 63, and CDR-H3 with a sequence of SEQ ID NO: 68; and / or,

[0062] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 74, and CDR-L3 with a sequence of SEQ ID NO: 78;

[0063] or

[0064] (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 59, CDR-H2 with a sequence of SEQ ID NO: 64, and CDR-H3 with a sequence of SEQ ID NO: 69; and / or,

[0065] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 75, and CDR-L3 with a sequence of SEQ ID NO: 79;

[0066] or

[0067] (f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 60, CDR-H2 with a sequence of SEQ ID NO: 65, and CDR-H3 with a sequence of SEQ ID NO: 70; and / or,

[0068] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 76, and CDR-L3 with a sequence of SEQ ID NO: 79;

[0069] or

[0070] (g) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 61, CDR-H2 with a sequence of SEQ ID NO: 66, and CDR-H3 with a sequence of SEQ ID NO: 71; and / or,

[0071] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO: 73, CDR-L2 with the sequence of SEQ ID NO: 77, and CDR-L3 with the sequence of SEQ ID NO: 80.

[0072] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation compared to the heavy chain variable region and / or light chain variable region described in any one of (a) to (g) under the aforementioned IMGT, Chothia, Kabat or AbM definitions, and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution.

[0073] In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) framework region (FR) derived from a murine immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) framework region (FR) derived from a murine immunoglobulin. Therefore, in certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is of murine origin.

[0074] In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) framework region (FR) derived from a human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) framework region (FR) derived from a human immunoglobulin. Therefore, in certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is humanized. In such embodiments, the heavy chain variable region FR and / or light chain variable region FR of the antibody or antigen-binding fragment thereof of the present invention may comprise one or more non-human (e.g., mouse) amino acid residues, for example, the heavy chain framework region FR and / or light chain framework region FR may comprise one or more amino acid back mutations, in which there are corresponding mouse amino acid residues.

[0075] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0076] (a) a heavy chain framework region of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4 or 5 conservative substitutions of amino acids) compared to the germline antibody gene sequence from which it is derived; and / or

[0077] (b) a light chain framework region of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the sequence of the germline antibody gene from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0078] In certain preferred embodiments, the degree of humanization of the antibodies or antigen-binding fragments thereof of the invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

[0079] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0080] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0081] (i) a sequence shown in any one of SEQ ID NOs: 1, 29, 30, 31, and 32;

[0082] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 1, 29, 30, 31, 32; or

[0083] (iii) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 1, 29, 30, 31, 32;

[0084] and / or

[0085] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0086] (iv) a sequence shown in any one of SEQ ID NOs: 2, 33, 34, 35, 36, 37, and 38;

[0087] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 2, 33, 34, 35, 36, 37, 38; or

[0088] (vi) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 2, 33, 34, 35, 36, 37, 38.

[0089] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0090] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0091] (i) a sequence shown in any one of SEQ ID NOs: 3, 39, and 40;

[0092] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 3, 39, 40; or

[0093] (iii) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 3, 39, 40;

[0094] and / or

[0095] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0096] (iv) a sequence shown in any one of SEQ ID NOs: 4 and 41;

[0097] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 4 and 41; or

[0098] (vi) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in any one of SEQ ID NOs: 4, 41.

[0099] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have the following heavy chain variable region (VH) and / or light chain variable region (VL): the VH sequence shown in any one of SEQ ID NOs: 1, 29, 30, 31, 32, and / or the VL sequence shown in any one of SEQ ID NOs: 2, 33, 34, 35, 36, 37, 38.

[0100] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have the following heavy chain variable region (VH) and / or light chain variable region (VL): the VH sequence shown in any one of SEQ ID NOs: 3, 39, and 40, and / or the VL sequence shown in any one of SEQ ID NOs: 4 and 41.

[0101] In certain embodiments, the antibody or antigen-binding fragment thereof of the invention has the VH sequence shown in SEQ ID NO:44, and / or the VL sequence shown in SEQ ID NO:45.

[0102] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the VH sequence shown in SEQ ID NO:44, and / or the VL sequence shown in SEQ ID NO:45.

[0103] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the VH sequence shown in SEQ ID NO: 44, and / or the VL sequence shown in SEQ ID NO: 45. In a preferred embodiment, the substitutions are conservative substitutions.

[0104] In certain embodiments, the antibody or antigen-binding fragment thereof of the invention has the VH sequence shown in SEQ ID NO:46, and / or the VL sequence shown in SEQ ID NO:47.

[0105] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the VH sequence shown in SEQ ID NO:46 and / or the VL sequence shown in SEQ ID NO:47.

[0106] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the VH sequence shown in SEQ ID NO: 46, and / or the VL sequence shown in SEQ ID NO: 47. In a preferred embodiment, the substitutions are conservative substitutions.

[0107] In certain embodiments, the antibody or antigen-binding fragment thereof of the invention has the VH sequence shown in SEQ ID NO:48, and / or the VL sequence shown in SEQ ID NO:49.

[0108] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the VH sequence shown in SEQ ID NO: 48, and / or the VL sequence shown in SEQ ID NO: 49.

[0109] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the VH sequence shown in SEQ ID NO: 48, and / or the VL sequence shown in SEQ ID NO: 49. In a preferred embodiment, the substitutions are conservative substitutions.

[0110] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention has the VH sequence shown in SEQ ID NO: 50, and / or the VL sequence shown in SEQ ID NO: 51;

[0111] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the VH sequence shown in SEQ ID NO: 50, and / or the VL sequence shown in SEQ ID NO: 51.

[0112] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the VH sequence shown in SEQ ID NO: 50, and / or the VL sequence shown in SEQ ID NO: 51. In a preferred embodiment, the substitutions are conservative substitutions.

[0113] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention has the VH sequence shown in SEQ ID NO: 52, and / or the VL sequence shown in SEQ ID NO: 53;

[0114] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the VH sequence shown in SEQ ID NO: 52, and / or the VL sequence shown in SEQ ID NO: 53.

[0115] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the VH sequence shown in SEQ ID NO: 52, and / or the VL sequence shown in SEQ ID NO: 53. In a preferred embodiment, the substitutions are conservative substitutions.

[0116] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0117] (a) a VH having the sequence shown in SEQ ID NO: 1 and a VL having the sequence shown in SEQ ID NO: 2;

[0118] (b) a VH having the sequence shown in SEQ ID NO: 29 and a VH having the sequence shown in SEQ ID NO: 33

[0119] VL of the sequence shown;

[0120] (c) a VH having the sequence shown in SEQ ID NO: 29 and a VH having the sequence shown in SEQ ID NO: 34

[0121] VL of the sequence shown;

[0122] (d) a VH having the sequence shown in SEQ ID NO: 29 and a VH having the sequence shown in SEQ ID NO: 35

[0123] VL of the sequence shown; or

[0124] (e) a VH having the sequence shown in SEQ ID NO: 29 and a VH having the sequence shown in SEQ ID NO: 36

[0125] VL of the sequence shown.

[0126] (f) a VH having the sequence shown in SEQ ID NO: 29 and a VH having the sequence shown in SEQ ID NO: 37

[0127] VL of the sequence shown.

[0128] (g) a VH having the sequence shown in SEQ ID NO: 29 and a VH having the sequence shown in SEQ ID NO: 38

[0129] VL of the sequence shown.

[0130] (h) a VH having the sequence shown in SEQ ID NO: 30 and a VH having the sequence shown in SEQ ID NO: 33

[0131] VL of the sequence shown.

[0132] (i) a VH having the sequence shown in SEQ ID NO: 31 and a VH having the sequence shown in SEQ ID NO: 33

[0133] VL of the sequence shown.

[0134] (j) a VH having the sequence shown in SEQ ID NO: 32 and a VH having the sequence shown in SEQ ID NO: 33

[0135] VL of the sequence shown.

[0136] (k) a VH having the sequence shown in SEQ ID NO: 3 and a VL having the sequence shown in SEQ ID NO: 4;

[0137] (l) a VH having the sequence shown in SEQ ID NO: 39 and a VH having the sequence shown in SEQ ID NO: 41

[0138] VL of the sequence shown;

[0139] (m) a VH having the sequence shown in SEQ ID NO: 40 and a VH having the sequence shown in SEQ ID NO: 41

[0140] VL of the sequence shown;

[0141] (n) a VH having the sequence shown in SEQ ID NO: 44 and a VH having the sequence shown in SEQ ID NO: 45

[0142] The VL sequence shown

[0143] (o) a VH having the sequence shown in SEQ ID NO: 46 and a VH having the sequence shown in SEQ ID NO: 47

[0144] The VL sequence shown

[0145] (p) a VH having the sequence shown in SEQ ID NO: 48 and a VH having the sequence shown in SEQ ID NO: 49

[0146] VL of the sequence shown;

[0147] (q) a VH having the sequence shown in SEQ ID NO: 50 and a VH having the sequence shown in SEQ ID NO: 51

[0148] VL of the sequence shown; or

[0149] (r) a VH having the sequence shown in SEQ ID NO: 52 and a VH having the sequence shown in SEQ ID NO: 53

[0150] VL of the sequence shown.

[0151] In certain preferred embodiments, the heavy chain variable region (VH) and the light chain variable region (VL) are compared with any one of the groups (a) to (r), the heavy chain variable region (VH) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and / or, the light chain variable region (VL) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0152] In certain preferred embodiments, the heavy chain variable region (VH) and light chain variable region (VL) are compared with any one of the groups (a) to (r), the heavy chain variable region (VH) has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); and / or, the light chain variable region (VL) has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

[0153] In any of the above aspects, the antibody or antigen-binding fragment thereof of the present invention may further comprise a constant region sequence derived from a mammalian (eg, mouse or human) immunoglobulin or a variant thereof. In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human or murine immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); and / or, the light chain of an antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human or murine immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0154] In certain preferred embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids); and / or, the light chain of the antibody or antigen-binding fragment thereof of the present invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0155] In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a murine immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids); and / or, the light chain of an antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a murine immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0156] In some embodiments, the constant region is altered, for example, mutated, to modify the properties of the anti-CLDN18.2 antibody molecule (e.g., to change one or more of the following properties: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue, for example, changing the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby changing the effector function (e.g., enhancing). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc. In some cases, these effector functions are required for therapeutic antibodies.

[0157] In certain embodiments, the anti-CLDN18.2 antibody molecule has a heavy chain constant region (Fc), which is selected from, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular, selected from, for example, the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, more particularly selected from the heavy chain constant region of IgG1 or IgG4 (for example, human IgG1 or IgG4). In some embodiments, the anti-CLDN18.2 antibody molecule has a light chain constant region, which is selected from, for example, the light chain constant region of κ or λ, preferably the κ light chain constant region (for example, human κ light chain).

[0158] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from:

[0159] (1) human IgG1 heavy chain constant region; or

[0160] (2) Human IgG4 heavy chain constant region.

[0161] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0162] (a) a heavy chain constant region (CH) comprising an amino acid sequence selected from the group consisting of:

[0163] (i) the sequence shown in SEQ ID NO:42;

[0164] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 42; or

[0165] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:42;

[0166] and / or

[0167] (b) a light chain constant region (CL) comprising an amino acid sequence selected from the group consisting of:

[0168] (iv) the sequence shown in SEQ ID NO:43;

[0169] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 43; or

[0170] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:43.

[0171] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0172] In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) as shown in SEQ ID NO:42 and a light chain constant region (CL) as shown in SEQ ID NO:43.

[0173] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 1 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 2 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0174] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH having a sequence as shown in SEQ ID NO: 29 and a heavy chain constant region (CH) as shown in SEQ ID NO: 42, and a light chain comprising a VL having a sequence as shown in SEQ ID NO: 33 and a light chain constant region (CL) as shown in SEQ ID NO: 43.

[0175] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 29 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 34 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0176] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 29 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 35 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0177] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 29 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 36 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0178] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 29 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 37 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0179] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 29 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 38 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0180] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 30 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 33 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0181] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 31 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 33 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0182] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 32 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 33 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0183] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 3 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 4 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0184] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 39 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 41 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0185] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO:40 and a heavy chain constant region (CH) shown in SEQ ID NO:42, and a light chain comprising a VL sequence shown in SEQ ID NO:41 and a light chain constant region (CL) shown in SEQ ID NO:43.

[0186] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO:44 and a heavy chain constant region (CH) shown in SEQ ID NO:42, and a light chain comprising a VL sequence shown in SEQ ID NO:45 and a light chain constant region (CL) shown in SEQ ID NO:43.

[0187] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH having a sequence as shown in SEQ ID NO:46 and a heavy chain constant region (CH) as shown in SEQ ID NO:42, and a light chain comprising a VL having a sequence as shown in SEQ ID NO:47 and a light chain constant region (CL) as shown in SEQ ID NO:43.

[0188] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO:48 and a heavy chain constant region (CH) shown in SEQ ID NO:42, and a light chain comprising a VL sequence shown in SEQ ID NO:49 and a light chain constant region (CL) shown in SEQ ID NO:43.

[0189] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH sequence shown in SEQ ID NO: 50 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 51 and a light chain constant region (CL) shown in SEQ ID NO: 43.

[0190] In certain preferred embodiments, the antibodies of the present invention comprise a heavy chain comprising a VH having a sequence as shown in SEQ ID NO:52 and a heavy chain constant region (CH) as shown in SEQ ID NO:42, and a light chain comprising a VL having a sequence as shown in SEQ ID NO:53 and a light chain constant region (CL) as shown in SEQ ID NO:43.

[0191] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0192] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0193] (i) the sequence shown in SEQ ID NO: 102;

[0194] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 102; or

[0195] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 102;

[0196] and

[0197] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0198] (iv) the sequence shown in SEQ ID NO: 103;

[0199] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 103; or

[0200] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:103.

[0201] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0202] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0203] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0204] (i) the sequence shown in SEQ ID NO: 106;

[0205] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 106; or

[0206] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 106;

[0207] and

[0208] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0209] (iv) the sequence shown in SEQ ID NO: 107;

[0210] (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 107; or

[0211] (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:107.

[0212] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0213] In certain exemplary embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0214] (1) a heavy chain having the sequence shown in SEQ ID NO: 102 and a light chain having the sequence shown in SEQ ID NO: 103; or

[0215] (2) A heavy chain having the sequence shown in SEQ ID NO: 106 and a light chain having the sequence shown in SEQ ID NO: 107.

[0216] In certain preferred embodiments, the antibodies of the present invention are chimeric antibodies or humanized antibodies. In certain preferred embodiments, the antibodies of the present invention or their antigen-binding fragments are selected from ScFv, Fab, Fab', (Fab') 2 , Fv fragments, disulfide-linked Fv (dsFv), diabodies, bispecific antibodies, and multispecific antibodies.

[0217] In certain embodiments, the antibody molecules or antigen-binding fragments thereof of the invention may exhibit at least one of the following properties:

[0218] (a) with a K of less than about 100 nM, such as less than about 10 nM, 1 nM, 0.1 nM or less D Binds to CLDN18.2 (eg, human CLDN18.2); eg, the K D Biofilm interferometry (BLI) (such as ForteBio ) measured;

[0219] (b) binds to CLDN18.2 (e.g., human CLDN18.2) with an EC50 of less than about 500 nM, e.g., less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less; e.g., the EC50 is measured by flow cytometry or cell competition ELISA;

[0220] (c) the antibody or antigen-binding fragment thereof does not bind to CLDN18.1 (such as human CLDN18.1).

[0221] In certain embodiments, the antibody or antigen-binding fragment thereof has ADCC activity.

[0222] In certain embodiments, the antibody or antigen-binding fragment thereof has CDC activity.

[0223] In certain preferred embodiments, the antibody or antigen-binding fragment thereof has ADCC and CDC activities.

[0224] In certain preferred embodiments, the antibody or antigen-binding fragment thereof has enhanced ADCC and / or CDC activity.

[0225] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention have at least one of the following biological functions:

[0226] (a) Inducing apoptosis of tumor cells;

[0227] (b) inhibiting tumor cell proliferation;

[0228] (c) increasing immune cell activity in vitro or in vivo in a subject (e.g., a human);

[0229] (d) enhancing an immune response in a subject (e.g., a human);

[0230] (e) treating a tumor in a subject (e.g., a human);

[0231] (f) inducing and / or increasing T cell infiltration;

[0232] (g) inducing and / or enhancing an immune response;

[0233] (h) inducing and / or increasing complement-dependent cytotoxic activity;

[0234] (i) inducing and / or increasing antibody-dependent cellular cytotoxicity activity;

[0235] (j) Increase NK cell activity;

[0236] (k) inhibiting the expression and activation of CLDN18.2; or

[0237] (l) Inhibit CLDN18.2-mediated signal transduction.

[0238] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention possess any combination of the above-mentioned biological functions.

[0239] Derivatized antibodies

[0240] The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). Generally, the derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to CLDN18.2 (particularly human CLDN18.2). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection agent, a pharmaceutical agent, and / or a protein or polypeptide (e.g., avidin or a polyhistidine tag) that is capable of mediating the binding of an antibody or antigen-binding fragment to another molecule.

[0241] One type of derivatized antibody (e.g., bispecific antibody) is produced by cross-linking 2 or more antibodies (of the same type or different types). Methods for obtaining bispecific antibodies are well known in the art, and examples thereof include, but are not limited to, chemical cross-linking, cell engineering (hybrid hybridoma) or genetic engineering.

[0242] Another type of derivatized antibody is a labeled antibody. For example, the antibody of the present invention or its antigen-binding fragment can be connected to a detectable label. The detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. Patents teaching the use of such labels include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). Detectable labels such as those described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of a colored label. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, detectable labels as described above can be linked to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

[0243] In addition, the antibodies or antigen-binding fragments thereof of the present invention may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups may be used to improve the biological properties of the antibodies, such as increasing serum half-life.

[0244] As one of the derivatives of the antibody, the present invention provides a conjugate, which includes the monoclonal antibody or its antigen-binding fragment of the present invention and a conjugated portion, wherein the conjugated portion is selected from: a detectable label, a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, an enzyme, polyethylene glycol (PEG), a nuclide, a nucleic acid, a small molecule toxin, and has the ability to bind to active polypeptides, proteins, receptors, ligands, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0245] As one of the derivatives of the antibody, the present invention provides a chimeric antigen receptor, which includes the monoclonal antibody or its antigen-binding fragment. In certain preferred embodiments, the chimeric antigen receptor includes a monoclonal antibody or its antigen-binding fragment (e.g., ScFv) of the present invention, a transmembrane domain, and one or more intracellular T cell signaling domains. The present invention also provides a host cell containing or expressing the chimeric antigen receptor, such as an immune cell (e.g., T lymphocyte, NK cell).

[0246] As one of the derivatives of antibodies, the present invention provides a multispecific antibody, which is formed by coupling a first antibody or a fragment thereof with other antibodies or fragments thereof or antibody analogs, each antibody or fragment thereof or antibody analog maintains the original binding specificity, and the first antibody or fragment thereof is the antibody or antigen-binding fragment thereof described in the present invention; preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0247] Antibody preparation

[0248] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present invention.

[0249] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be directly obtained from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 Fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab or F(ab') 2 Fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

[0250] Therefore, in another aspect, the invention provides a kind of isolated nucleic acid molecule, it comprises the nucleotide sequence encoding the antibody of the present invention or its antigen-binding fragment, or its heavy chain variable region and / or light chain variable region.According to the codon degeneracy of this area, in certain embodiments, the nucleotide sequence can be replaced according to the codon degeneracy.In certain embodiments, the nucleotide sequence is codon optimized.

[0251] In certain preferred embodiments, the isolated nucleic acid molecule comprises a first and a second nucleic acid encoding a heavy chain variable region and a light chain variable region of an antibody or antigen-binding fragment thereof of the present invention, respectively, or a first nucleic acid encoding a heavy chain variable region and a heavy chain constant region of an antibody or antigen-binding fragment thereof of the present invention, respectively, and a second nucleic acid encoding a light chain variable region and a light chain constant region, respectively, or a first and a second nucleic acid encoding a heavy chain and a light chain of an antibody or antigen-binding fragment thereof of the present invention, respectively, the antibody being selected from any one of the following groups: 1E9.2, 1 or a sequence substantially identical to the first and second nucleic acids. For example, the isolated nucleic acid molecule can comprise the nucleotide sequence shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:109 in the sequence listing, or a sequence substantially identical thereto (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity thereto or a sequence having one or more nucleotide substitutions, or a sequence that differs from the sequence shown in the sequence listing by no more than 3, 6, 15, 30 or 45 nucleotides).

[0252] In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region has a sequence selected from the following: (a). A nucleotide sequence as shown in SEQ ID NO: 54, or (b). A sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence of (a), or (c). A sequence that differs from the nucleotide sequence of (a) by no more than 3, 6, 15, 30 or 45 nucleotides; the nucleic acid molecule encoding the antibody light chain variable region has a sequence selected from the following: (d). A nucleotide sequence as shown in SEQ ID NO: 54. NO:55, or (e). A sequence substantially identical to the nucleotide sequence described in d) (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence described in d), or (f). A sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0253] In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region has a sequence selected from the following: (a). A nucleotide sequence as shown in SEQ ID NO: 56, or (b). A sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence of (a), or (c). A sequence that differs from the nucleotide sequence of (a) by no more than 3, 6, 15, 30 or 45 nucleotides; the nucleic acid molecule encoding the antibody light chain variable region has a sequence selected from the following: (d). A nucleotide sequence as shown in SEQ ID NO: 56. NO:57, or (b) a sequence substantially identical to the nucleotide sequence described in (d) (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence described in (a), or (f) a sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0254] In certain preferred embodiments, the nucleic acid molecule encoding the antibody heavy chain variable region has a nucleotide sequence as shown in SEQ ID NO: 54, and the nucleic acid molecule encoding the antibody light chain variable region has a nucleotide sequence as shown in SEQ ID NO: 55. In certain preferred embodiments, the isolated nucleic acid molecule of the present invention comprises a nucleic acid molecule encoding an antibody heavy chain variable region as shown in SEQ ID NO: 54, and / or a nucleic acid molecule encoding an antibody light chain variable region as shown in SEQ ID NO: 55.

[0255] In certain preferred embodiments, the nucleic acid molecule encoding the antibody heavy chain variable region has a nucleotide sequence as shown in SEQ ID NO: 56, and the nucleic acid molecule encoding the antibody light chain variable region has a nucleotide sequence as shown in SEQ ID NO: 57. In certain preferred embodiments, the isolated nucleic acid molecule of the present invention comprises a nucleic acid molecule encoding an antibody heavy chain variable region as shown in SEQ ID NO: 56, and / or a nucleic acid molecule encoding an antibody light chain variable region as shown in SEQ ID NO: 57.

[0256] In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule. It comprises a nucleic acid molecule encoding an antibody heavy chain, and / or a nucleic acid molecule encoding an antibody light chain, wherein the nucleic acid molecule encoding the antibody heavy chain has a sequence selected from the following: (a). A nucleotide sequence as shown in SEQ ID NO: 104 or 108, or (b). A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence described in (a), or (c). A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides, and / or, the nucleic acid molecule encoding the antibody light chain has a sequence selected from the following: (d). A nucleotide sequence as shown in SEQ ID NO: 104 or 108, or (b). A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence described in (a)), or (c). A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides, and / or, the nucleic acid molecule encoding the antibody light chain has a sequence selected from the following: (d). A nucleotide sequence as shown in SEQ ID NO:105 or 109, or (e). A sequence substantially identical to the nucleotide sequence described in (d) (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence described in (d), or (f). A sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0257] In certain preferred embodiments, the nucleic acid molecule encoding the antibody heavy chain has a nucleotide sequence as shown in SEQ ID NO: 104, and / or the nucleic acid molecule encoding the antibody light chain has a nucleotide sequence as shown in SEQ ID NO: 105;

[0258] In certain preferred embodiments, the nucleic acid molecule encoding the antibody heavy chain has a nucleotide sequence as shown in SEQ ID NO:108, and / or the nucleic acid molecule encoding the antibody light chain has a nucleotide sequence as shown in SEQ ID NO:109.

[0259] In another aspect of the present invention, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the present invention. In certain preferred embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain preferred embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a mammal, such as a human).

[0260] In certain embodiments, the antibodies or antigen binding fragments thereof of the present invention can be used to construct a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen binding domain (e.g., ScFv) that specifically binds to CLDN18.2, a transmembrane domain, and one or more intracellular T cell signaling domains. In such embodiments, the isolated nucleic acid molecules of the present invention may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antibody or antigen binding fragment thereof (e.g., ScFv) of the present invention. In certain embodiments, the isolated nucleic acid molecules of the present invention encode a chimeric antigen receptor comprising an antigen binding fragment (e.g., ScFv) of an antibody of the present invention.

[0261] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention can be used in immune cells modified with chimeric antigen receptors, wherein the chimeric antigen receptors can be used to construct chimeric antigen receptors (CARs), as well as immune cells (eg, T lymphocytes, NK cells).

[0262] In another aspect of the present invention, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., an intestinal bacteria). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain preferred embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0263] In certain embodiments, the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may include a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further includes a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (eg, ScFv). In certain embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of an antibody of the present invention.

[0264] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0265] Methods of treatment and pharmaceutical compositions

[0266] In another aspect of the present invention, a pharmaceutical composition is disclosed, which comprises the antibody or antigen-binding fragment thereof, a vector, a host cell, a conjugate, a chimeric antigen receptor, or a multispecific antibody of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0267] In certain preferred embodiments, the pharmaceutical composition of the present invention comprises the antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0268] In certain preferred embodiments, the pharmaceutical composition of the present invention comprises a vector or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody or its antigen-binding fragment (eg, ScFv) of the present invention; the host cell comprises an isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of an antibody of the present invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0269] In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent. In certain preferred embodiments, the additional pharmaceutically active agent is a drug for treating immune-related diseases. In certain preferred embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity. In certain preferred embodiments, the additional pharmaceutically active agent is an interferon, interleukin-2 or a chemotherapeutic agent.

[0270] In certain preferred embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent are provided as separate components or as components of the same composition. Therefore, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0271] In certain preferred embodiments, the pharmaceutical composition may further comprise another pharmaceutically active agent, which is selected from one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, albumin-bound paclitaxel, a combination of epirubicin + oxaliplatin + 5-fluorouracil, FOLFOX4, FOLFOX6, and mFOLFOX6 (comprising oxaliplatin, folinic acid, and 5-fluorouracil).

[0272] In another aspect, the antibody or antigen-binding fragment thereof, vector, host cell, conjugate, chimeric antigen receptor, or multispecific antibody in the pharmaceutical composition of the present invention is sufficient to:

[0273] (a) Inducing apoptosis of tumor cells;

[0274] (b) inhibiting tumor cell proliferation;

[0275] (c) induce and / or increase T cell infiltration;

[0276] (d) inducing and / or enhancing an immune response;

[0277] (e) inducing and / or increasing complement-dependent cytotoxic activity;

[0278] (f) inducing and / or increasing antibody-dependent cellular cytotoxicity activity;

[0279] (g) Increase NK cell activity;

[0280] (h) inhibiting the expression and activation of CLDN18.2;

[0281] (i) inhibiting CLDN18.2-mediated signaling; or

[0282] (j) Any combination of (a) to (i).

[0283] On the other hand, the pharmaceutical composition of the present invention further comprises a second antibody that specifically binds to a receptor or ligand selected from the following or a nucleic acid encoding the second antibody, wherein the receptor or ligand is selected from: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3 and any combination thereof.

[0284] In another aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, vector, host cell, conjugate, chimeric antigen receptor, or multispecific antibody of the present invention in the preparation of a medicament, wherein the medicament is used for:

[0285] (a) Inducing apoptosis of tumor cells;

[0286] (b) inhibiting tumor cell proliferation;

[0287] (c) increasing immune cell activity in vitro or in vivo in a subject (e.g., a human);

[0288] (d) enhancing an immune response in a subject (e.g., a human);

[0289] (e) treating a tumor in a subject (e.g., a human);

[0290] (f) inducing and / or increasing T cell infiltration;

[0291] (g) inducing and / or enhancing an immune response;

[0292] (h) inducing and / or increasing complement-dependent cytotoxic activity;

[0293] (i) inducing and / or increasing antibody-dependent cellular cytotoxicity activity;

[0294] (j) Increase NK cell activity;

[0295] (k) inhibiting the expression and activation of CLDN18.2;

[0296] (l) inhibiting CLDN18.2-mediated signal transduction; or

[0297] (m) Any combination of (a)-(l).

[0298] In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare a drug, the isolated nucleic acid molecule contained in the vector comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody or its antigen-binding fragment (eg, ScFv) of the present invention; the host cell comprises an isolated nucleic acid molecule or vector as described above. In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (eg, ScFv) of an antibody of the present invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0299] In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare a medicament, the medicament is used to treat a tumor in a subject (eg, a human).

[0300] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof, vector, host cell, conjugate, chimeric antigen receptor, or multispecific antibody of the present invention is selected from solid tumors, blood tumors (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma) and metastatic, refractory or recurrent lesions of cancer; for example, including but not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia.

[0301] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof described in the present invention is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, and lung cancer (e.g., non-small cell lung cancer).

[0302] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof described in the present invention is selected from gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, such as locally advanced unresectable or metastatic gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma.

[0303] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof described in the present invention is Claudin 18.2 positive, and further, the tumor is HER2 negative.

[0304] In certain preferred embodiments, the tumors targeted by the antibodies or antigen-binding fragments thereof described herein are HER2 negative.

[0305] In another aspect, the present invention provides a method for preventing and / or treating a tumor in a subject. In another aspect, the present invention provides a method for delaying tumor progression in a subject. In another aspect, the present invention provides a method for reducing or inhibiting tumor recurrence in a subject. The above method comprises administering an effective amount of the antibody or antigen-binding fragment thereof, vector, host cell, conjugate, chimeric antigen receptor, or multispecific antibody, pharmaceutical composition of the present invention to a subject in need thereof.

[0306] In certain embodiments, the invention provides a method for preventing and / or treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, vector, host cell, conjugate, chimeric antigen receptor, or multispecific antibody of the present invention, and a pharmaceutically acceptable carrier and / or excipient; separately, in combination, simultaneously, or sequentially with another pharmaceutically active agent;

[0307] In certain preferred embodiments, the additional pharmaceutically active agent is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, albumin-bound paclitaxel, a combination of epirubicin + oxaliplatin + 5-fluorouracil, FOLFOX4, FOLFOX6, and mFOLFOX6 (comprising oxaliplatin, folinic acid and 5-fluorouracil).

[0308] When the host cell of the present invention is used in the above method, the host cell expresses a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention. Therefore, in certain preferred embodiments, the isolated nucleic acid molecule contained in the host cell comprises a nucleotide sequence encoding a chimeric antigen receptor, and the nucleotide sequence encoding the chimeric antigen receptor further comprises a nucleotide sequence encoding an antibody of the present invention or its antigen-binding fragment (e.g., ScFv). In certain preferred embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention. In certain preferred embodiments, the host cell is a T cell. In certain preferred embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0309] In another aspect, the method described above further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0310] In certain embodiments, the second therapy can be used separately or in combination with the above-described method; or, the second therapy can be used separately or in combination with the above-described method, simultaneously or sequentially;

[0311] In certain preferred embodiments, the chemotherapy is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, albumin-bound paclitaxel, a combination of epirubicin + oxaliplatin + 5-fluorouracil, FOLFOX4, FOLFOX6, and mFOLFOX6 (comprising oxaliplatin, folinic acid and 5-fluorouracil).

[0312] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof, vector, host cell, conjugate, chimeric antigen receptor or modified immune cell, or multispecific antibody of the present invention is selected from solid tumors, blood tumors (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma) and metastatic, refractory or recurrent lesions of cancer; for example, including but not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia.

[0313] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof described in the present invention is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, and lung cancer (e.g., non-small cell lung cancer).

[0314] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof described in the present invention is selected from gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, such as locally advanced unresectable or metastatic gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma.

[0315] In certain preferred embodiments, the tumor involved in the antibody or antigen-binding fragment thereof described in the present invention is Claudin 18.2 positive, and further, the tumor is HER2 negative.

[0316] In certain preferred embodiments, the tumors targeted by the antibodies or antigen-binding fragments thereof described herein are HER2 negative.

[0317] In another aspect, the method described above further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.

[0318] The antibodies or antigen-binding fragments thereof of the present invention and the pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: incorporating the recombinant protein of the present invention in a suitable solvent at a required dose, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.

[0319] In addition, the antibodies or antigen-binding fragments thereof of the present invention may be present in a pharmaceutical composition in a unit dosage form for ease of administration.

[0320] The antibody or its Fab of the present invention, pharmaceutical composition can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The technician will understand that the route of administration and / or mode will change according to the intended purpose. In a preferred embodiment, the antibody or its Fab of the present invention, pharmaceutical composition is administered by intravenous infusion or injection.

[0321] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof of the present invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0322] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be administered once, multiple times over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0323] The typical non-limiting range of the therapeutic or prophylactic effective amount of the recombinant protein of the present invention is 0.02-100 mg / kg, such as 0.1-100 mg / kg, 0.1-50 mg / kg, or 1-50 mg / kg. It should be noted that the dosage may vary depending on the type and severity of the symptoms to be treated. In addition, those skilled in the art understand that for any particular patient, the specific dosage regimen should be adjusted over time according to the patient's needs and the doctor's professional evaluation; the dosage range given here is only for illustrative purposes and does not limit the use or scope of the pharmaceutical composition of the present invention.

[0324] In the present invention, the subject may be a mammal, such as a human.

[0325] Detection methods and kits

[0326] The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to CLDN18.2, and thus can be used to detect the presence or level of CLDN18.2 in a sample.

[0327] Therefore, in another aspect, the present invention provides a kind of test kit, it comprises the antibody of the present invention or its antigen-binding fragment.In certain preferred embodiments, the antibody of the present invention or its antigen-binding fragment carries a detectable label.In a preferred embodiment, the test kit also comprises a second antibody, which specifically recognizes the antibody of the present invention or its antigen-binding fragment.Preferably, the second antibody also comprises a detectable label.

[0328] In the present invention, the detectable label can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. It is particularly preferred that such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art, including but not limited to enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. Patents teaching the use of such labels include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). The labels encompassed in the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of a colored label. In certain embodiments, the detectable labels described above can be linked to the recombinant proteins of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0329] In another aspect, the present invention provides a method for detecting the presence or level of CLDN18.2 in a sample, comprising the step of using an antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention also carries a detectable label. In another preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment thereof of the present invention using a detectably labeled reagent. The method can be used for diagnostic purposes, or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0330] In another aspect, the present invention provides a method for detecting the presence or level of CLDN18.2 in a sample, the method comprising forming a complex between the antibody or antigen-binding fragment thereof and CLDN18.2, contacting the sample with the antibody or antigen-binding fragment thereof of the present invention, and detecting the formation of the complex.

[0331] In another aspect, the invention provides a use of the antibody or antigen-binding fragment thereof in the preparation of a kit for detecting the presence or level of CLDN18.2 in a sample. In another aspect, the invention provides a diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof, a vector, a host cell, a conjugate, a chimeric antigen receptor, or a multispecific antibody described in the invention, and instructions for use.

[0332] In view of the low or no expression of Claudin 18.2 in normal tissues, and the expression or high expression in some cancers, tumors and tumor metastasis can be diagnosed by detecting the presence or level of CLDN18.2 in the sample. For example, if CLDN18.2 is detected in lung tissue, it can be basically determined that the lung tissue has undergone canceration. In combination with other indicators, such as pathological morphology, it can be determined whether the cancer is gastric cancer metastasis or non-small cell lung cancer, thereby determining the subtype of the cancer. Therefore, the antibody or antigen-binding fragment thereof, conjugate, multispecific antibody, or kit of the present invention can be used to diagnose tumors and tumor metastasis. Therefore, in another aspect, the use of the antibody or antigen-binding fragment thereof, conjugate, multispecific antibody, or kit of the present invention in diagnosing tumors and tumor metastasis is provided.

[0333] The antibodies of the present invention have high binding affinity to CLDN18.2 and have extremely strong specificity. Therefore, the antibodies of the present invention have the potential to be used for the prevention and / or treatment of tumors. The humanized antibodies of the present invention retain the functions and properties of the parent mouse antibodies. Moreover, the humanized antibodies of the present invention have a high degree of humanization, so that they can be safely administered to human subjects without inducing immunogenic reactions. Therefore, the antibodies of the present invention (especially humanized antibodies) have great clinical value.

[0334] Abbreviations

[0335] CDR Complementarity determining region in the variable region of an immunoglobulin

[0336] FR Antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues

[0337] VH antibody heavy chain variable region

[0338] VL Antibody light chain variable region

[0339] IgG Immunoglobulin G

[0340] AbM The AbM CDR definition method is derived from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). This definition method integrates part of the definitions of Kabat and Chothia.

[0345] Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0349] Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0353] IMGT is based on the international ImMunoGeneTics information system initiated by Lefranc et al. For the numbering system of Immunoglobulin G (IMGT), see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0357] mAb monoclonal antibody

[0358] EC50 Concentration that produces 50% efficacy or binding

[0359] IC50 concentration that produces 50% inhibition

[0360] ELISA enzyme-linked immunosorbent assay

[0361] PCR polymerase chain reaction

[0362] HRP Horseradish Peroxidase

[0363] IL-2 Interleukin 2

[0364] K D Equilibrium dissociation constant

[0365] Ka binding rate constant

[0366] Kd dissociation rate constant

[0367] ADCC Antibody-dependent cellular cytotoxicity

[0368] CDC Complement-dependent cytotoxicity

[0369] FACS flow cytometry

[0370] CDR-H1 Complementarity determining region 1 in the variable region of the heavy chain of an immunoglobulin CDR-H2 Complementarity determining region 2 in the variable region of the heavy chain of an immunoglobulin CDR-H3 Complementarity determining region 3 in the variable region of the heavy chain of an immunoglobulin CDR-L1 Complementarity determining region 1 in the variable region of the light chain of an immunoglobulin CDR-L2 Complementarity determining region 2 in the variable region of the light chain of an immunoglobulin CDR-L3 Complementarity determining region 3 in the variable region of the light chain of an immunoglobulin

[0371] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operating procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0372] As used herein, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The distribution of amino acids in various regions or domains can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883, or related research by AbM, Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272).

[0373] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0374] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, 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), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), or, Martin's related work (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), which integrates part of the definitions of Kabat and Chothia, and was first used in the Oxford Molecular antibody modeling software (Martin AC R. Protein sequence and structure analysis of antibody variable domains [M] / / Antibody engineering. Springer, Berlin, Heidelberg, 2010: 33-51.). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to a person skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0375] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia, IMGT or AbM numbering systems.

[0376] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0377] "Germline antibody genes" are immunoglobulin sequences encoded by non-lymphocytes that have not undergone the genetic rearrangement and maturation process that leads to the expression of specific immunoglobulins. An advantage provided by various embodiments of the present invention comes from the recognition that germline antibody genes retain more of the important amino acid sequence structure characteristic of individual animal species than mature antibody genes. Therefore, when applied therapeutically to the species, they are less recognized as foreign substances by the species.

[0378] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0379] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab'), 2 , Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0380] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains". Wherein, "full-length heavy chain" or "heavy chain" refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N to C; and, when the full-length antibody is an IgE isotype, optionally also includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N to C. A "full-length light chain" or "light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N to C. Two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be from a single species, such as human; or may be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0381] As used herein, the term "Fd fragment" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341: 544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of VH and CH1 domains).

[0382] As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0383] As used herein, the term "Fc fragment" means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond. The Fc fragment of an antibody has a variety of different functions, but does not participate in antigen binding.

[0384] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0385] As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0386] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0387] As used herein, "bispecific antibody" refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or an antibody analog through a coupling arm, and the coupling method includes but is not limited to chemical reaction, gene fusion, protein fusion, polypeptide fusion and enzymatic. "Multispecific antibody" includes, for example, trispecific antibody and tetraspecific antibody, the former is an antibody with three different antigen binding specificities, and the latter is an antibody with four different antigen binding specificities.

[0388] As used herein, "antibody mimetics" refers to antibodies that specifically bind to antigens like antibodies, but do not have antibody structures. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. For example, ankyrin repeat proteins (DARPins) and fynomers. Designed ankyrin repeat proteins (DARPins) are linked to IgG antibodies, scFv-Fc antibody fragments, or a combination thereof, such as CN104341529A. Anti-IL-17a fynomers are bound to anti-IL-6R antibodies, such as WO2015141862A1.

[0389] As used herein, "chimeric antigen receptor (CAR)" refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that can activate T cells. Commonly, the extracellular binding domain of CAR is derived from mouse or humanized or human monoclonal antibodies.

[0390] As used herein, "immunoglobulin" or "Ig" may refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called chimeric antigen receptors or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE, of which IgG is the most common circulating antibody. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody reactions, and is important in defending against bacteria and viruses.

[0391] Herein, the technology for obtaining antibodies can use conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) to obtain an antigen-binding fragment of the antibody (e.g., the above-mentioned antibody fragment) from a given antibody (e.g., an antibody provided by the present invention), and screen the antigen-binding fragment of the antibody for specificity in the same manner as for intact antibodies.

[0392] As used herein, the terms "monoclonal antibody", "single antibody", and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, relative to monoclonal antibodies, generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

[0393] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0394] For example, monoclonal antibodies can be prepared as follows. First, mice or other suitable host animals are immunized with immunogens (adjuvants are added when necessary). The injection method of immunogens or adjuvants is usually subcutaneous multi-point injection or intraperitoneal injection. The immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). The hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium preferably contains one or more substances that can inhibit the growth of unfused, parent myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin and thymidine (HAT culture medium) to the culture medium will inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have characteristics such as high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among them, myeloma cells are preferably mouse myeloma, such as MOP-21 or MC-11 mouse tumor derivatives (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md. USA). In addition, there are also studies reporting the use of human myeloma and human-mouse heteromyeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium of growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).For example, the affinity of monoclonal antibodies can be determined by the Scatchard analysis method described by Munson et al. in Anal. Biochem. 107: 220 (1980). After determining the specificity, affinity and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned by the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Suitable culture fluids can be DMEM or RPMI-1640, etc. In addition, hybridoma cells can also grow in animals in the form of ascites tumors. Using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by subcloned cells can be separated from cell culture fluid, ascites or serum.

[0395] Monoclonal antibodies can also be obtained through genetic engineering recombination technology. Using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody for PCR amplification, DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells. The obtained DNA molecules are inserted into expression vectors, and then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins), and cultured under appropriate conditions to obtain recombinantly expressed target antibodies.

[0396] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (target molecule recognized by the antibody) or its antigenic epitope can be immobilized on a column and the immunospecific antibody can be purified by immunoaffinity chromatography. The purification of immunoglobulins can be referred to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0397] As used herein, the term "mouse antibody" refers to the fusion of B cells from immunized mice with myeloma cells, followed by screening of mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, followed by screening, antibody preparation and antibody purification. Or, after antigens invade the mouse body, B cells differentiate and proliferate to form plasma cells, which can produce secretory antibodies. Produced by specific antigen stimulation, the production of antibodies is due to the interaction of various immune cells caused by antigens invading the human body, causing B cells in lymphocytes to differentiate and proliferate to form plasma cells, which can produce secretory antibodies.

[0398] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy chain and light chain variable regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0399] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0400] Humanized antibodies can retain the expected properties of non-human donor antibodies (e.g., mouse antibodies) and can effectively reduce the immunogenicity of non-human donor antibodies (e.g., mouse antibodies) in human subjects, and therefore are particularly advantageous. However, due to the matching problem between the CDR of the donor antibody and the FR of the acceptor antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response) are usually lower than those of non-human donor antibodies (e.g., mouse antibodies).

[0401] Therefore, although researchers in this field have conducted in-depth studies on antibody humanization and have made some progress (see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000)), the prior art does not provide detailed guidance on how to fully humanize a donor antibody so that the generated humanized antibody has the highest possible degree of humanization and can retain the expected properties of the donor antibody as much as possible. Technicians need to explore, investigate and modify specific donor antibodies and put in a lot of creative work to obtain humanized antibodies that have a high degree of humanization (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% humanization) and retain the expected properties of the specific donor antibodies.

[0402] In the present invention, in order to make the humanized antibody retain the properties of the donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, the ability to increase the activity of immune cells and / or the ability to enhance the immune response), the framework region (FR) in the humanized antibody of the present invention may contain both the amino acid residues of the human acceptor antibody and the amino acid residues of the corresponding non-human donor antibody.

[0403] Chimeric antibodies or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. DNA encoding heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques.

[0404] To prepare chimeric antibodies, methods known in the art can be used to link the murine immunoglobulin variable region to the human immunoglobulin constant region (see, e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). For example, the DNA encoding VH is operably linked to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, the DNA encoding VL is operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (as well as a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0405] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of not producing endogenous immunoglobulins after immunization and are capable of producing a complete human antibody repertoire. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies when challenged with antigen (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of the above transgenic animals include, HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); or "KM mice TM" carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996, Nature Biotech 14: 309).

[0406] As used herein, the term "humanization degree" is an indicator for evaluating the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted, for example, by using the IMGT website DomainGapAlign to predict the homology between the variable region sequence and the human V domain.

[0407] As used herein, "homologous antibody" refers to a variant of an antibody, the heavy and light chain variable regions of which comprise amino acid sequences homologous to the amino acid sequences of the antibodies or antigen-binding fragments thereof provided herein, and wherein the variant retains the desired functional properties of the anti-CLDN18.2 antibodies of the present invention.

[0408] Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith TF and Waterman MS, Adv. Appl. Math., 2:482, 1981; Higgins DG and Sharp PM, CABIOS 5:151, 1989. Altschul SF et al., Nature Genet., 6:119, 1994 provide detailed ideas for sequence alignment methods and homology calculations.

[0409] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50) of the interaction.

[0410] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, bioluminescence interferometry (e.g., ForteBio Octet method) can be used to measure the dissociation constant. In addition, surface plasmon resonance technology (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0411] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0412] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression regulatory sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0413] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, etc. In this article, host cells also include immune cells used to construct chimeric antigen receptor T cells (CAR-T), such as T lymphocytes, NK cells, etc.

[0414] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0415] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence, and the variant of the antibody obtained by conservatively substituting the amino acid retains the biological activity of its source sequence, such as specific binding to CLDN18.2. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0416] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0417] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0418] The term "treatment" as used herein refers to clinical intervention in an attempt to change the disease process caused by an individual or a cell, which can be either preventive or intervention in the clinical pathological process. The therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc.

[0419] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor, infection, or autoimmune disease) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival period (if not receiving treatment), extending the survival period.

[0420] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) suffers from a tumor, infection, or autoimmune disease, or has a risk of suffering from the above diseases.

[0421] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor, an infection, or an autoimmune disease) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a tumor, an infection, or an autoimmune disease); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0422] As used herein, the term "immune cell" includes cells that have a hematopoietic origin and play a role in the immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

[0423] As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen presenting cells, phagocytes, or granulocytes) and soluble macromolecules (including antibodies, cytokines, and complement) produced by immune cells or the liver, which results in selective damage, destruction, or removal from the human body of invasive pathogens, cells or tissues infected by pathogens, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation. In the present invention, the term "antigen-specific T cell response" refers to an immune response produced by T cells, which occurs when the T cells are stimulated by an antigen to which the T cells are specific. Non-limiting examples of responses produced by T cells upon antigen-specific stimulation include proliferation of T cells and production of cytokines (e.g., IL-2).

[0424] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance rate of antibodies and antigen-antibody complexes, and the like. Methods for altering the effector functions of antibodies are known in the art, for example, by introducing mutations in the Fc region.

[0425] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcR) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), causing these cytotoxic effector cells to specifically bind to target cells attached to the antigen, and then kill the target cells by secreting cytotoxins. Methods for detecting the ADCC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the test antibody and an Fc receptor (e.g., CD16a).

[0426] As used herein, the term "complement dependent cytotoxicity (CDC)" refers to a form of cytotoxicity that activates the complement cascade by binding complement component C1q to antibody Fc. Methods for detecting the CDC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the test antibody and an Fc receptor (e.g., C1q).

[0427] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade neighboring tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.

[0428] The term "hematological malignancies" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphomas. Hematological malignancies also include leukemias, such as secondary leukemias or acute lymphocytic leukemias. Hematological malignancies also include myeloma (e.g., multiple myeloma) and other hematological and / or B-cell or T-cell related cancers.

[0429] As used herein, the term "pharmaceutically acceptable" means that when the molecular entity, molecular fragment or composition is appropriately administered to an animal or a human, they do not produce adverse, allergic or other adverse reactions. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or their components include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0430] As used herein, combination therapy includes the use of an anti-CLDN18.2 antibody or antigen-binding fragment thereof encompassed by the invention in combination with one or more additional active therapeutic agents of a second therapy (eg, chemotherapeutic agents) or other prophylactic or therapeutic modality (eg, radiotherapy).

[0431] Exemplary anticancer agents of the second therapy may include chemotherapeutic agents (e.g., mitotic inhibitors), alkylating agents (e.g., nitrogen mustard), antimetabolites (e.g., folic acid analogs), natural products (e.g., vinca alkaloids), various agents (e.g., platinum coordination complexes), hormones and antagonists (e.g., adrenocortical steroids), immunomodulators (e.g., Bropirimine, Upjohn), etc. Other anticancer treatments include other antibodies that specifically target cancer cells.

[0432] In such combination therapies, various active agents often have different complementary mechanisms of action, and combination therapies may result in synergistic effects. Combination therapies include therapeutic agents that affect immune responses (e.g., enhance or activate responses) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells. Combination therapies can allow one or more of the reagents to be dosed to reduce or eliminate adverse effects associated with one or more of the reagents. Such combination therapies can have a synergistic therapeutic or preventive effect on underlying diseases, disorders, or conditions.

[0433] Herein, "combination" includes therapies that can be administered separately, such as separately formulated for separate administration (for example, they can be provided in a kit), and therapies that can be administered together in a single formulation (ie, a "co-formulation"). In certain embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present invention can be administered sequentially. In other embodiments, the anti-CLDN18.2 antibodies or antigen-binding fragments thereof can be administered simultaneously. The anti-CLDN18.2 antibodies or antigen-binding fragments thereof of the present invention can be used in combination with at least one other (active) agent in any manner.

[0434] In this article, Claudin 18.2 positivity was obtained by immunohistochemistry and staining intensity evaluation by professional clinical pathologists. HER2 negativity included IHC 1+, or IHC 2+ / FISH negative, and also included the range of IHC 0 to 1+ and IHC 1+ to 2+.

[0435] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0436] Figure 1A :HEK293T-Claudin 18.2 monoclonal stable cell line flow cytometry detection

[0437] Figure 1B :L929-Claudin 18.2 monoclonal stable cell line flow cytometry detection

[0438] Figure 1C :Flow cytometry detection of KATOIII-Claudin 18.2 monoclonal stable cell line

[0439] Figure 1D :NCI-N87-Claudin 18.2 monoclonal stable cell line flow cytometry detection

[0440] Figure 1E :HEK293T-Claudin 18.1 monoclonal stable cell line Western blot detection

[0441] Figure 2A Cellular affinity test of mouse antibody against human Claudin18.2 and Ba / F3-Claudin18.2

[0442] Figure 2B Cell affinity test of mouse antibody against human Claudin18.2 and Ba / F3-Claudin18.1

[0443] Figure 2C :2C6.9-hz21, IMAB362 antibody affinity detection (flow cytometry)

[0444] Figure 3A : 1E9.2-hz11, IMAB362 antibody ELISA affinity detection (L929-Claudin 18.2 cells)

[0445] Figure 3B :2C6.9-hz21, IMAB362 antibody affinity detection (L929-Claudin 18.2 cells) Figure 3C :1E9.2-hz11, 2C6.9-hz11, 2C6.9-hz21 antibody affinity detection (L929-Claudin18.2 cells)

[0446] Figure 3D :1E9.2-hz11, IMAB362 antibody positive rate detection on NUGC-4 cells

[0447] Figure 3E :1E9.2-hz11, IMAB362 antibody affinity detection (NUGC4 cells)

[0448] Figure 3F :Detection of the specificity of 1E9.2-hz11 antibody by flow cytometry

[0449] Figure 3G :Detection of the specificity of 2C6.9-hz21 antibody by flow cytometry

[0450] Figure 4A : Determination of CDC killing activity of 1E9.2-hz11 and IMAB362 on HEK293-Claudin 18.2 cells

[0451] Figure 4B:2C6.9-hz21 and IMAB362 CDC killing activity against HEK293-Claudin 18.2 cells

[0452] Figure 5A :1E9.2-hz11, IMAB362 antibody ADCC activity detection (HEK293T-Claudin18.2 cells)

[0453] Figure 5B :1E9.2-hz11, IMAB362 antibody ADCC activity detection (KATOIII-Claudin18.2 cells)

[0454] Figure 5C :1E9.2-hz11, IMAB362 antibody ADCC activity detection (L929-Claudin 18.2 cells)

[0455] Figure 5D :1E9.2-hz11, IMAB362 antibody ADCC activity detection (NUGC4 cells) Figure 5E :2C6.9-hz21, IMAB362 antibody ADCC activity detection (HEK293T-Claudin18.2 cells)

[0456] Fig. 6A :1E9.2-hz11 tumor suppression effect detection

[0457] Figure 6B :2C6.9-hz21 tumor inhibition effect detection

[0458] Figure 6C :Monitoring of body weight changes of 2C6.9-hz21 during tumor inhibition experiment

[0459] Sequence information

[0460] The information of the sequences involved in the present invention is described in the following table:

[0461]

[0462]

[0463]

[0464]

[0465]

[0466] DETAILED DESCRIPTION

[0467] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0468] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0469] Example 1 Construction and Identification of Human Claudin 18.2 and Human Claudin 18.1 Overexpressing Cell Lines 1.1 Construction of Human Claudin18.2 and Human Claudin18.1 Overexpressing Cell Lines

[0470] To verify the specificity and function of human Claudin 18.2 antibody, the complete coding sequence of human Claudin 18.2 (gene number: NM_001002026.2, synthesized by Nanjing GenScript Biotechnology Co., Ltd.) and the complete coding sequence of human Claudin 18.1 (gene number: NM_016369.3, synthesized by Nanjing GenScript Biotechnology Co., Ltd.) were cloned into the lentiviral vector pLVX-IRES-puro, and the virus was prepared by the lentiviral packaging system described in the literature (Mohammadi Z et al., Mol Biotechnol. 2015 Sep; 57(9): 793-800.). After the obtained viruses were obtained, HEK293T, L929, KATOIII and NCI-N87 cells were infected respectively, and monoclonal HEK293T-Claudin 18.1, HEK293T-Claudin 18.2, L929-Claudin were obtained by puromycin selection and monoclonal selection. 18.2, KATOIII-Claudin 18.2, NCI-N87-Claudin18.2 stable cell lines. Human Claudin18.2 and human Claudin 18.1 plasmids were transfected into BaF / 3 cells (DSMZ, Cat#ACC300) using the 4D-Nucleofector X transfection kit (Lonza, Cat#V4XC-3012). 1.25 mg / mL hygromycin (Thermo Fisher Sci. Cat#10687010) was added for screening 48 hours after transfection. After 12 days of screening, single clones were sorted to obtain monoclonal BaF / 3-Claud18.1 and BaF / 3-Claud18.2 cell lines.

[0471] 1.2 Detection of human Claudin18.2 and human Claudin18.1 overexpressing cell lines

[0472] HEK293T-Claudin 18.1 was identified by Western Blot (detection antibody: Proteintech, 66167-1-Ig), and other cell lines were identified by flow cytometry (flow cytometer: Beckman, CytoFlex; detection antibody IMAB362, sequence from patent: CN 101312989 B). Figures 1A-1DAs shown in the figure, the flow cytometry results showed that HEK293T-Claudin 18.2, L929-Claudin18.2, KATOIII-Claudin 18.2, and NCI-N87-Claudin 18.2 all obtained monoclonal cell lines with high positive rates (close to 100%) and good uniformity, which can be used for subsequent experiments. Western Blot results ( Figure 1E ) showed that the three HEK293T-Claudin 18.1 stable cell lines all overexpressed human Claudin 18.1, among which the HEK293T-Claudin 18.1-1C2 monoclonal clone had a higher expression level and could be used for subsequent experiments.

[0473] Example 2 Preparation of anti-human Claudin18.2 mouse monoclonal antibody

[0474] Wild-type mice were immunized by DNA immunization and cell immunization to obtain anti-human Claudin18.2 mouse monoclonal antibodies. Each Balb / c mouse was injected with 100 μg of plasmid containing the complete coding sequence of human Claudin18.2 by tail vein injection. After the fourth and sixth immunizations, the serum titer was detected by flow cytometry. Mice with higher titers were selected and boosted with BaF / 3-Claudin18.2 overexpressing cell lines 3-5 days before fusion. Mouse spleen cells and Sp2 / 0 (ATCC, Cat#CRL-1581) mouse myeloma cell lines were fused by PEG fusion using a standard fusion process, followed by HAT pressure screening and flow screening 10-14 days later.

[0475] 6000 hybridoma clone supernatants were screened by flow cytometry (purchased from Sartorius, model: iQue Screener Plus), and 43 positive hybridoma clones that could recognize the HEK293T-Claudin18.2 cell line were obtained for subcloning. HEK293T-Claudin18.2 and HEK293T-Claudin18.1 cell lines were selected and further screened by flow cytometry to obtain 14 positive clones that only bind to human Claudin18.2 but not human Claudin18.1. Finally, subclone sorting was performed by limiting dilution method to obtain single clones.

[0476] The human gastric cancer cell line NUGC4 (purchased from the JCRB cell bank in Japan, catalog number: JCRB0834) endogenously expresses Claudin18.2 protein and is widely used to detect the binding of antibodies to endogenous Claudin18.2 and the development of functional assays. The candidate clones were tested using NUGC4 cells, and 7 subclones were finally selected as candidate clones, which were numbered as 1E9.2, 2C6.9, 6B9.22, 9C8.1, 16A9.11, 19G10.14 and 19H11.6.

[0477] Example 3: Affinity detection of anti-human Claudin18.2 mouse antibody

[0478] The affinity of candidate molecules to Claudin18.2 was detected by flow cytometry. Hybridoma monoclonal 1E9.2, 2C6.9, 6B9.22, 9C8.1, 16A9.11, 19G10.14 and 19H11.6 were cultured without serum to obtain 100 mL of supernatant, which was then purified by protein A (MabSelect SuRe, GE) to obtain mouse antibodies. Ba / F3-Claudin18.1 and Ba / F3-Claudin18.2 cells were incubated on ice for 30 minutes with purified mouse antibodies of different concentration gradients, and then washed twice with flow buffer (PBS + 2% FBS) using mouse IgG as a negative control (Thermo Fisher, Cat: 31903), and then incubated on ice for 30 minutes with goat anti-mouse fluorescent secondary antibody (Jackson ImmunoResearch, Cat: 115-605-071), and finally detected on the machine.

[0479] Test results such as Figure 2A-2B As shown in the figure, the seven anti-human Claudin18.2 mouse antibodies can specifically recognize human Claudin18.2, but not bind to human Claudin18.1. At the same time, as shown in Table 1, the seven anti-human Claudin18.2 mouse antibodies showed high affinity to Ba / F3-Claudin18.2 cells, with EC50 ranging from 3 to 20 nM.

[0480] Table 1 Anti-human Claudin18.2 mouse antibody Ba / F3-Claudin18.2 cell affinity test

[0481] Clone number EC50(nM) <![CDATA[Max MFI*(×10 6 )]]> 1E9.2 8.506 5.085 19H11.6 10.94 6.008 16A9.11 10.2 5.815 9C8.1 9.399 4.969 6B9.22 3.64 3.624 19G10.14 16.27 3.501 2C6.9 10.57 6.111 Control IgG No combination No combination

[0482] *Max MFI is the maximum mean fluorescence intensity

[0483] Example 4: Identification of subtypes and variable region amplification of anti-Claudin18.2 mouse monoclonal antibodies

[0484] In order to detect the antibody subtype of the candidate hybridoma clones, the Pierce Rapid Isotyping Kit (Thermo Fisher Sci.Cat#26179) was used to identify the antibody subtypes of 7 candidate clones. The identification results showed that the heavy chains of all candidate molecules were IgG1 subtypes and the light chains were Kappa subtypes.

[0485] Hybridoma cells were cultured to about 8,000 cells, and the cells were lysed and the first-strand cDNA was synthesized using a cDNA reverse transcription kit (ThermoFisher Sci. Cat#18080-200). Primers were used to amplify VH and VK (VLKappa) genes from cDNA by PCR, and the PCR products were purified by a DNA purification kit (Qiagen, Cat#28104) and connected to a TOPO vector (Thermo Fisher Sci. Cat#K457540). About 12 clones were selected for sequencing for each ligation reaction. The sequence was analyzed by Vector NTI 11.5 (Thermo Fisher Sci.) and Sequencer 5.4.6 (Genecodes). The variable region sequence and CDR sequence of the anti-human Claudin18.2 mouse antibody were obtained as shown in Table 2 below.

[0486] Table 2 Variable region and CDR amino acid sequences of anti-human Claudin18.2 mouse antibody

[0487]

[0488]

[0489] Example 5: Anti-Claudin18.2 chimeric antibody affinity detection

[0490] The light chain variable region amino acid sequences of 1E9.2, 2C6.9, 6B9.22, 9C8.1, 16A9.11, 19G10.14 and 19H11.6 were connected to the light chain kappa constant region amino acid sequence (SEQ ID NO: 43), and the heavy chain variable region amino acid sequence was connected to the IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 42) to construct chimeric antibodies. The cDNA was synthesized by codon optimization and connected to the plasmid pcDNA3.4 (commissioned to Nanjing GenScript Biotechnology Co., Ltd.). The pcDNA3.4 corresponding to the heavy chain and light chain of each chimeric antibody was simultaneously transfected into Expi293F cells (purchased from Thermo Company), and the supernatant was purified using protein A (MabSelect SuRe, GE). The purified chimeric antibodies were named 1E9.2-hz00, 2C6.9-hz00, 6B9.22-hz00, 9C8.1-hz00, 16A9.11-hz00, 19G10.14-hz00 and 19H11.6-hz00, respectively.

[0491] Detect the affinity of anti-human Claudin18.2 chimeric antibody to Ba / F3-Claundin18.2 cells. The specific experimental steps are as follows: Take 60,000 Ba / F3-Claundin18.2 cells in 10μl buffer, incubate with gradient dilution of anti-human Claudin18.2 chimeric antibody and control antibody IMAB362 on ice for 30 minutes, wash twice with flow buffer (PBS+2% FBS), and then incubate with goat anti-human fluorescent secondary antibody (Thermo Fisher Sci., Cat: A-21445) on ice for 30 minutes, and finally detect on the machine.

[0492] The results are shown in Table 3. The affinity EC50 of 1E9.2-hz00, 16A9.11-hz00 and 19H11.6-hz00 is lower than that of the positive control antibody IMAB362, indicating that the above anti-human Claudin18.2 chimeric antibody has a stronger affinity than IMAB362. The maximum average fluorescence intensity of 1E9.2-hz00, 6B9.22-hz00, 9C8.1-hz00, 16A9.11-hz00, and 19H11.6-hz00 antibodies is stronger than that of the positive control antibody IMAB362, indicating that the number of antigens bound by the antibody of the present invention under saturation is higher than that of IMAB362, showing that the antibody of the present invention has a stronger antigen binding ability.

[0493] Table 3 Cell affinity detection of anti-human Claudin18.2 chimeric antibodies

[0494]

[0495] Example 6 Humanization of anti-human Claudin 18.2 mouse antibody

[0496] The mouse antibodies 1E9.2 and 2C6.9 were humanized by using the CDR transplantation antibody humanization method. In short, the humanization involves the following steps: compare the amino acid sequence of the mouse monoclonal antibody with the amino acid sequence of the human embryonic antibody to find the sequence with high homology and better physical and chemical properties as the human embryonic framework sequence; analyze and examine the HLA-DR affinity to select the human embryonic framework sequence with low affinity; and then transplant the six CDRs of the mouse antibody to the selected heavy chain and light chain framework sequences.

[0497] Specifically, the heavy chain and light chain CDR regions of the murine antibodies 1E9.2 and 2C6.9 were transplanted onto the FR frameworks of the corresponding humanized templates. The heavy chain humanized template of 1E9.2 was the human germline gene sequence IGHV3-21*04 (see IMGT Accession No. HM855688), and the light chain humanized template was the human germline gene sequence IGKV4-1*01 (see IMGT Accession No. Z00023) and IGKV6-21*02 (see IMGT Accession No. KM455568). The heavy chain and light chain humanized templates of 2C6.9 were the human germline gene sequences IGHV4-59*01 (see IMGT Accession No. AB019438) and IGKV4-1*01 (see IMGT Accession No. Z00023), respectively.

[0498] Computer simulation technology was further used to apply molecular docking to analyze the variable region and its surrounding framework amino acid sequence to examine its spatial three-dimensional binding mode. By calculating electrostatic force, van der Waals force, hydrophilicity and entropy value, the key amino acids in the amino acid sequence of the mouse antibody that can interact with Claudin18.2 and maintain the spatial framework were analyzed, and these mouse amino acids were retained in the transplanted antibody. In other words, a series of back mutations were performed on the amino acid residues in the FR region of the above humanized template so that the humanized antibody can retain the antigen binding ability of the mouse antibody as much as possible.

[0499] According to the above method, based on the CDR of the mouse antibody 1E9.2, a total of 9 humanized antibodies were constructed, named 1E9.2hz11, 1E9.2hz12, 1E9.2hz13, 1E9.2hz14, 1E9.2hz15, 1E9.2hz17, 1E9.2hz21, 1E9.2hz31, 1E9.2hz41; at the same time, based on the CDR of the mouse antibody 2C6.9, 2 humanized antibodies were constructed, named 2C6.9hz11 and 2C6.9hz21. The heavy chain constant region of each antibody is the human wild-type IgG1 heavy chain constant region (SEQ ID NO: 42), and the light chain constant region of the antibody is the human wild-type IgG1 light chain constant region (SEQ ID NO: 43).

[0500] The amino acid sequences of the variable and constant regions of the above humanized antibodies are shown in Table 4.

[0501] Table 4: Amino acid sequences of variable and constant regions of humanized antibodies

[0502]

[0503] Example 7: Affinity and ADCC cell activity detection of anti-human Claudin18.2 humanized candidate antibodies

[0504] To evaluate the humanized candidate antibodies against human Claudin 18.2, the heavy and light chains of the candidate humanized antibodies in Example 6 were synthesized by codon optimization and connected to the plasmid pcDNA3.4 (commissioned to Nanjing GenScript Biotechnology Co., Ltd.). The pcDNA3.4 corresponding to each chimeric antibody heavy chain and light chain was simultaneously transfected into Expi293F cells (purchased from Thermo Company), and the supernatant was purified by protein A (MabSelect SuRe, GE). Ba / F3-Claudin 18.2 cells expressing human Claudin18.2 were used to identify the binding of humanized antibodies to Claudin18.2. First, Ba / F3-Claudin 18.2 cells (60,000 in 10 μl buffer) were incubated with purified humanized antibodies on ice for 30 minutes, eluted twice with buffer (PBS + 2% FBS), and then sheep anti-human IgG Fc antibody conjugated with Alexa647 (1:500 dilution) was added, placed on ice for 30 minutes, and detected using IntelliCyt.

[0505] The test results in Ba / F3-Claudin 18.2 overexpressing human Claudin 18.2 are shown in Table 5. The maximum average fluorescence intensity shows that all 1E9.2 chimeric antibodies, humanized antibodies and control antibody IMAB362 can bind to Claudin18.2, among which the maximum fluorescence signal values ​​of the chimeric antibodies and humanized candidate antibodies are stronger than that of the control antibody IMAB362, indicating that the number of candidate antibodies binding to the antigen under saturation state is higher than that of IMAB362, that is, the antibodies of the present invention have a stronger binding ability with the antigen.

[0506] Table 5 Binding ability test of anti-human Claudin18.2 chimeric and humanized candidate antibodies to Ba / F3-Claudin18.2 cells

[0507] Candidate antibodies <![CDATA[Max MFI(×10 6 )]]> 1E9.2-hz00 11.17 1E9.2-hz11 11.34 1E9.2-hz21 14.56 1E9.2-hz31 10.64 1E9.2-hz41 11.28 1E9.2-hz12 6.71 1E9.2-hz13 6.02 1E9.2-hz14 5.36 1E9.2-hz15 8.99 1E9.2-hz17 5.90 IMAB362 4.99*

[0508] * indicates that the value is the average of two measurements.

[0509] Alternatively, HEK293T-Claudin 18.2 cells were used to detect the affinity of 2C6.9-hz21 to human Claudin 18.2 on the cell membrane. The specific steps are as follows: digest HEK293T-Claudin18.2 cells, resuspend by centrifugation, and wash twice with PBS; resuspend the cells with PBS (containing 1% BSA), and plate the cells in a 96-well pointed bottom plate at 300,000 cells / well, with a volume of 50 μl per well, and plate 20 wells; add 50 μL of 2C6.9-hz21 and IMAB362 antibodies to each well, respectively, with a final concentration starting at 1000 nM, 3-fold gradient dilution, and a total of 11 concentration points; use Human IgG as a negative control; mix well, react at 4°C in the dark for 1 hour; wash 3 times with PBS, add FITC-labeled anti-human Fc secondary antibody (brand: BioLegend, catalog number: 409322), and incubate at 4°C in the dark for 0.5 hour; wash 3 times with PBS, and detect on a flow cytometer (flow cytometer brand: Beckman, model: Cytoflex).

[0510] The experimental results are as follows Figure 2C As shown in Table 6, the EC50 value of the binding affinity between 2C6.9-hz21 and HEK293T-Claudin 18.2 detected by flow cytometry technology was smaller than that of IMAB362, and the maximum fluorescence signal value was greater than that of IMAB362, indicating that the affinity of 2C6.9-hz21 to Claudin 18.2 on the cell membrane is better than that of IMAB362.

[0511] Table 6 Flow cytometry affinity test of humanized antibody 2C6.9-hz21 binding to HEK293T-Claudin 18.2

[0512] Antibody Name EC50(nM) Max MFI 2C6.9-hz21 5.767 165866.3 IMAB362 8.217 156715.7

[0513] ADCC cell activity detection. Take 10,000 Ba / F3-Claudin 18.2 cells in 40 μl NK-92 growth medium, dilute the antibody concentration in 10 μl PBS, and then add 50 μl NK-92 growth medium containing 50,000 NK-92MI_mCD16 cells; incubate at 37°C for 6 hours, then add 33.3 μl CytoTox-Glo (Promega, G9290) detection reagent, and read the results with an enzyme reader. As shown in Table 7, the EC50 and maximum killing rate of the ADCC killing activity of 1E9.2 chimeric antibody and all 1E9.2 humanized antibodies against Ba / F3-Claudin 18.2 cells are stronger than IMAB362, indicating that the 1E9.2 chimeric antibody and humanized antibody have stronger ADCC activity.

[0514] Table 7 Anti-human Claudin18.2 chimeric and humanized antibody ADCC cell activity detection

[0515]

[0516] * indicates that the value is the average of two measurements.

[0517] Example 8 Affinity and specificity determination of anti-human Claudin 18.2 humanized antibody

[0518] Human Claudin 18.2 is a membrane protein that spans four times and has a relatively complex structure. Cell ELISA was used for detection to ensure the complete configuration of the Claudin 18.2 antigen molecule. The L929-Claudin18.2 stable cell line constructed in Example 1 was selected for detection. The specific experimental steps are as follows: The L929-Claudin 18.2 cell line grown on the wall was digested with 2mM EDTA and resuspended to 2×10 5cells / ml, 100 μL per well was plated in a 96-well plate and cultured at 37°C overnight; the next day, the medium was removed, the cells were washed once with PBS, 100 μL of 4% formaldehyde was added to each well for fixation at room temperature for 30 minutes, the formaldehyde was removed, the cells were washed twice with PBS, and 100 μL of PBS (containing 2% BSA) was added for blocking at 37°C for 2 hours; the blocking solution was removed, the antibody to be tested was diluted with PBS (containing 2% BSA), starting at 1 μM, 4-fold dilution, 11 concentration points, 100 μL / well, incubated at 37°C for 2 hours; 250 μL PBST was washed 5 times, each time for 2 minutes; horseradish peroxidase (HRP)-labeled anti-human IgG secondary antibody (HRP-anti-Human IgG, Jackson ImmunoResearch, 109-035-003) was diluted with PBS (containing 2% BSA), 1:10000, 100 μL / well, incubated at 37°C for 1 hour; 250 μL Wash with PBST for 6 times, let stand for 2 minutes each time; add 100 μL TMB colorimetric solution (Thermo, 34029) to the corresponding wells, and color at 37°C for 20 minutes; add 50 μL 2 mol / L H 2 SO 4 The results were terminated and read at 450 nm using a microplate reader (MD, SpectraMax M2). The results were imported into Graphpad Prism for curve fitting.

[0519] The results of multiple experiments are as follows Figure 3A , 3B As shown in Tables 3C, 8, 9 and 10, the humanized antibodies 1E9.2-hz11 and 2C6.9-hz21 have significantly better affinity for human Claudin18.2 than IMAB362.

[0520] Table 8 ELISA affinity test of humanized antibody 1E9.2-hz11 binding to L929-Claudin 18.2 cells

[0521]

[0522] Table 9 ELISA affinity test of humanized antibody 2C6.9-hz21 binding to L929-Claudin 18.2 cells

[0523]

[0524] Table 10 ELISA affinity test of humanized antibodies binding to L929-Claudin 18.2 cells

[0525]

[0526] In order to detect the affinity of the candidate antibodies to the cell line endogenously expressing human Claudin 18.2, the expression of human Claudin 18.2 in the cell line NUGC-4 cells (purchased from the JCRB cell bank in Japan, catalog number: JCRB0834) endogenously expressing human Claudin 18.2 was first detected by flow cytometry analysis. Figure 3D As shown, Human IgG1 is a negative control. The positive rate of Claudin 18.2 expression in NUGC-4 cells detected by 1E9.2-hz11 is about 80%, and that of IMAB362 is about 20%, indicating that 1E9.2-hz11 can recognize more endogenous human Claudin 18.2 than IMAB362. The affinity of the candidate antibody to NUGC-4 cells was then determined by the cell ELISA method. The experimental steps are as described above. The experimental results are shown in Figure 3E As shown in Table 11, the EC50 value and the maximum signal value (OD450) indicated that 1E9.2-hz11 had better affinity and binding ability.

[0527] Table 11 Affinity detection of humanized antibodies binding to NUGC-4 cells

[0528] Antibody Name EC50(nM) OD450 1E9.2-hz11 0.4129 0.5 IMAB362 ≈663.7 Not reached

[0529] The present invention also detects the specificity of the candidate antibody by flow cytometry. The specific steps are as follows: digest HEK293T, HEK293T-human Claudin 18.1, and HEK293T-human Claudin18.2 cells, resuspend by centrifugation, and wash twice with PBS; resuspend the cells with PBS (containing 1% BSA), take 300,000 cells of each cell, add the candidate antibody with a final concentration of 1000nM, mix well, and react for 1 hour at 4°C in the dark; wash 3 times with PBS, add FITC-labeled anti-human Fc secondary antibody (brand: BioLegend, item number: 409322), incubate at 4°C in the dark for 0.5 hour; wash 3 times with PBS, and detect by flow cytometer (flow cytometer brand: Beckman, model: Cytoflex).

[0530] The experimental results are as follows Figure 3F and 3G As shown, both 1E9.2-hz11 and 2C6.9-hz21 can specifically bind to human Claudin 18.2 but not to human Claudin 18.1.

[0531] Example 9 Complement Dependent Cytotoxicity (CDC) Detection of Anti-Human Claudin18.2 Humanized Antibody

[0532] 1E9.2-hz11 is an IgG1 subtype, which can effectively activate the classical complement pathway and exert complement dependent cytotoxicity (CDC) function. In order to determine the CDC effect of antibody 1E9.2-hz11, the present invention uses complement-rich guinea pig serum (purchased from Zhengzhou Baiji, item number: S0001) as the material for the experiment. The specific experimental steps are as follows: HEK293T-Claudin 18.2 cells were taken, and the cell density was adjusted after centrifugation to 5×10 4 / well plate overnight; the next day, DMEM + 20% guinea pig serum medium was prepared, and 1E9.2-hz11 and IMAB362 antibodies were diluted with this medium, starting at 20μg / mL, 2-fold dilution, 10 concentration points; the original medium for culturing HEK293T-Claudin18.2 cells was removed, and the diluted antibodies in the previous step were added to the corresponding wells, 100μL / well; a positive control group for complete killing was set up, that is, 10μl / well lysis buffer was added; 37℃, 5% CO 2 After 3 hours of static culture in the incubator, CCK8 (Rhinogen, QDY-003-D) was added at 20 μl / well and reacted for 2 hours. The microplate reader (MD, SpectraMax M2) read the results at 450 nm and imported them into Graphpad Prism for curve fitting.

[0533] The experimental results are as follows Figure 4A As shown, the CDC activity of 1E9.2-hz11 is significantly better than that of the control antibody IMAB362, which is 3.5 times higher; the maximum killing rate of 1E9.2-hz11 is about 82%, while that of IMAB362 is about 70%.

[0534] The CDC detection method of antibody 2C6.9-hz21 is similar to that of antibody 1E9.2-hz11. After adding culture medium containing guinea pig serum, the cells were incubated at 37°C and 5% CO 2 The cells were cultured in an incubator for 3 hours, and then CellTiter-Glo Luminescent (CTG, purchased from Promega, catalog number: G7573) dye solution was added for staining, 50 μl / well, mixed for 30 seconds, and placed at room temperature for 1 minute before measuring the fluorescence signal value with an enzyme reader (MD, SpectraMax M2). The results were imported into Graphpad Prism for curve fitting.

[0535] The experimental results are as follows Figure 4B As shown in Table 12, the CDC activity of 2C6.9-hz21 is superior to that of the control antibody IMAB362.

[0536] Table 12 CDC activity detection of anti-Claudin18.2 humanized antibody 2C6.9-hz21

[0537] Antibody Name EC50 value (ng / mL) 2C6.9-hz21 1363 IMAB362 3317

[0538] Example 10 Determination of Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity of Humanized Antibodies Against Human Claudin18.2

[0539] 1E9.2-hz11 is an IgG1 subtype and has strong antibody-dependent cell-mediated cytotoxicity (ADCC) activity. To determine the ADCC activity of 1E9.2-hz11, the present invention uses the NK cell killing method for detection. The specific experimental steps are as follows: HEK293T-Claudin18.2, L929-Claudin 18.2, KATOIII-Claudin 18.2, and NUGC-4 cells were taken, and the cell density was adjusted after centrifugation to 1×10 4 Cells / well were plated overnight; the next day, the medium in the plate was removed, NK92MI-CD16a cells (Huabo Bio) were centrifuged, and resuspended in empty MEMA medium to adjust the cell density to 1×10 6 / mL, 50μl / well added to the corresponding wells; dilute 1E9.2-hz11 and IMAB362 antibodies with empty MEMA medium, HEK293T-Claudin18.2, L929-Claudin18.2, KATOIII-Claudin18.2 cells 40μg / ml starting, 5-fold dilution, 10 concentration points; NUGC-4 cells 2mg / mL starting, 5-fold dilution, 11 concentration points; add 50μL / well of the diluted antibody to the corresponding wells, place at 37℃, 5% CO 2 After 5.5 hours of static culture in the cell culture incubator, lysis buffer was added to the positive control well. After another 0.5 hour of static culture, lactate dehydrogenase (LDH) detection reagent (Dongren Chemical, CK12) was added, 50 μl / well, and the microplate reader (MD, SpectraMax M2) was read at 490 nm every 10 minutes. The results were imported into Graphpad Prism for curve fitting.

[0540] The experimental results are as follows Figures 5A-5D As shown in Table 13, the ADCC cell killing activity of antibody 1E9.2-hz11 on HEK293T-Claudin 18.2, KATOIII-Claudin 18.2, and NUGC-4 was better than that of IMAB362.

[0541] Table 13 ADCC activity determination of anti-human Claudin18.2 humanized antibodies

[0542]

[0543] Antibody 2C6.9-hz21 is also of IgG1 subtype. The method for detecting its ADCC activity is the same as that for antibody 1E9.2-hz11. The cells used are HEK293T-Claudin18.2 cells. Figure 5E As shown in Table 14, the ADCC cell killing activity of antibody 2C6.9-hz21 on HEK293T-Claudin 18.2 is better than that of IMAB362.

[0544] Table 14 ADCC activity assay of anti-Claudin18.2 humanized antibody 2C6.9-hz21

[0545] Antibody Name EC50 value (ng / mL) Maximum killing rate (%) 2C6.9-hz21 67.67 30 IMAB362 57.66 20

[0546] Example 11 Determination of the pharmacokinetic (PK) of humanized anti-human Claudin18.2 antibody in mice

[0547] In order to detect the pharmacokinetic (PK) characteristics of 1E9.2-hz11 and IMAB362 in mice, SCID mice (Weitong Lihua) were selected and injected with 10 mg / kg 1E9.2-hz11 antibody or IMAB362 antibody by intravenous injection, 2 mice / group. Blood was collected 8 hours, 1 day, 3 days, 8 days, and 15 days after injection, and the blood drug concentration of the antibody in mice was detected by cell ELISA method. Specific detection steps refer to Example 8. After the reading is completed, the standard curve is drawn according to the 1E9.2-hz11 and IMAB362 data using Graphpad Prism software, and then the fitting curve is drawn according to the mouse serum reading to calculate the pharmacokinetic characteristics of the two in mice.

[0548] The experimental results are shown in Table 15. The half-life of 1E9.2-hz11 in mice is 42.5 hours, and the half-life of IMAB362 in mice is 35.77 hours. 1E9.2-hz11 is superior to IMAB362.

[0549] Table 15 PK determination of anti-human Claudin18.2 humanized antibody

[0550]

[0551] Example 12 In vivo efficacy experiment of humanized anti-human Claudin18.2 antibody

[0552] The in vivo efficacy was demonstrated by the anti-tumor effect of the humanized anti-human Claudin18.2 antibody on the subcutaneous human gastric cancer cell line NCI-N87-Claudin18.2 cell (NCI-N87 cells were modified to highly express human Claudin18.2) transplanted tumor model of Balb / c Nude mice. Specifically, the humanized anti-human Claudin18.2 antibody was injected into the subcutaneous NCI-N87-Claudin18.2 tumor-bearing mouse model through the tail vein, and the tumor volume and animal weight changes were measured regularly to calculate the efficacy (tumor inhibition effect) of the humanized anti-human Claudin18.2 antibody on tumor-bearing mice.

[0553] The specific steps are as follows: NCI-N87-Claudin18.2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration. 6 / mouse (suspended in 0.1ml PBS), subcutaneously inoculated into female Balb / cNude mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd., production license number: SCXK (Su) 2016-0004), to establish a subcutaneous transplant tumor model, and the day of inoculation was recorded as P0. The experiment set up three groups: intravenous human immunoglobulin (negative control, Chengdu Rongsheng Pharmaceutical Co., Ltd., referred to as Human IgG), epirubicin (Epirubicin) + oxaliplatin (Oxaliplatin) + 5-fluorouracil (5-fluorouracil) chemical drug combination (referred to as EOF chemical drug), 1E9.2-hz11 and EOF chemical drug combination group. On P4, 11, 18, and 25 days, EOF drugs, Epirubicin 1.25 mg / kg, Oxaliplatin 3.25 mg / kg, and 5-fluorouracil 56.25 mg / kg were intraperitoneally administered (once a week for 4 weeks). For the 1E9.2-hz11 drug combination group, in addition to EOF administration, 1E9.2-hz11 antibodies (10 mg / kg, twice a week for 4 weeks) were administered through the tail vein on P5, 8, 12, 15, 19, 22, 26, and 29 days. After administration, the tumor volume and body weight of the mice were observed and measured regularly.

[0554] The tumor diameter was measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day.

[0555] The tumor growth inhibition rate TGI (%) was calculated using the following formula for the tumor inhibition efficacy of the antibody: TGI (%) = [1-(V TEnd-V T Start) / (V C End-V C Start)]*100%

[0556] Where V T End: Mean tumor volume at the end of the treatment group experiment

[0557] V T Start: Mean tumor volume at the start of drug administration in the treatment group

[0558] V C End: Mean tumor volume of negative control group at the end of the experiment

[0559] V C Start: Mean tumor volume of negative control group at the beginning of drug administration

[0560] The relative tumor proliferation rate T / C (%) was calculated using the following formula to evaluate the tumor inhibition efficacy of the antibody: T / C (%) = (T t / T 0 ) / (C t / C 0 )×100%

[0561] T0: the average tumor volume of the treatment group at the initial time (i.e. P0)

[0562] T t : Mean tumor volume of treatment groups at each measurement

[0563] C 0 : The average tumor volume of the negative control group at the initial time (i.e. P0)

[0564] C t : The average tumor volume of the negative control group at each measurement.

[0565] The specific results are shown in Table 16 and Fig. 6A The experimental results show that the EOF chemical drug group has a certain inhibitory effect on the tumor growth of the NCI-N87-Claudin18.2 gastric cancer transplant tumor model. After the 1E9.2-hz11 antibody (10 mg / kg) is used in combination with the EOF chemical drug, the tumor inhibition rate is further improved, and the drug efficacy is significant.

[0566] Table 16 NCI-N87-Claudin18.2+Balb / c Nude Mouse Model

[0567]

[0568]

[0569] Note: TGI refers to tumor growth inhibition rate, T / C refers to relative tumor proliferation rate

[0570] The same method was used to establish a subcutaneous transplant tumor model of NCI-N87-Claudin18.2 cells in mice. When the average tumor volume was about 110 mm 3 At the same time, the mice were randomly divided into groups according to the size of the tumor, and were treated with anti-chicken lysozyme human IgG1 isotype control antibody (negative control, Sichuan Kelun Botai Biopharmaceutical Co., Ltd., referred to as IgG1WT) group, paclitaxel injection (albumin-bound, Hunan Kelun) single-use group, and 2C6.9-hz21 antibody combined with paclitaxel injection group. All samples were injected into the tail vein twice a week for a total of 3 weeks. After administration, the tumor volume and body weight of the mice were observed and measured regularly, and the data analysis method was as described above. The specific results are shown in Table 17 and Figure 6B The anti-tumor efficacy of the paclitaxel injection group (10 mg / kg) was significant, and the combination of 2C6.9-hz21 antibody and paclitaxel injection could further improve the tumor inhibition rate and show significant efficacy. No mice died or had significant weight loss during the observation period (such as Figure 6C There was no obvious drug toxicity, and the mice tolerated the 2C6.9-hz21 antibody well during the treatment period.

[0571] Table 17 NCI-N87-Claudin18.2+Balb / c Nude Mouse Model

[0572]

[0573] Note: TGI refers to tumor growth inhibition rate, T / C refers to relative tumor proliferation rate

[0574] In summary, humanized antibodies such as 1E9.2-hz11 and 2C6.9-hz21 can effectively inhibit tumor growth when used in combination with chemotherapeutic drugs (such as EOF or paclitaxel).

[0575] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2, wherein: The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3, 39 or 40; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:4 or 41; or (b) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO: 1; and / or CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:2; or (c) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:44; and / or CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:45; or (d) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:46; and / or CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:47; or (e) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:48; and / or CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:49; or (f) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO: 50; and / or CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:51; or (g) CDR-H1, CDR-H2 and CDR-H3 contained in VH shown in SEQ ID NO:52; and / or CDR-L1, CDR-L2 and CDR-L3 contained in VL shown in SEQ ID NO:53; or (h) the heavy chain variable region (VH) and / or light chain variable region (VL) described above, wherein, compared with the heavy chain variable region and / or light chain variable region described in any one of (a) to (g), at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation, and the mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution; Preferably, the CDRs are defined according to the Kabat, IMGT, Chothia or AbM numbering systems; Preferably, the VH and / or VL of the antibody or antigen-binding fragment thereof comprises framework regions (FRs) from human or mouse immunoglobulins. Preferably, the antibody or antigen-binding fragment thereof binds to human CLDN18.

2.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: (1) The following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 11, CDR-H2 with a sequence of SEQ ID NO: 12 or 110, and CDR-H3 with a sequence of SEQ ID NO: 13; and / or, A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 14, CDR-L2 with a sequence of SEQ ID NO: 15, and CDR-L3 with a sequence of SEQ ID NO: 16; or (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 5, CDR-H2 with a sequence of SEQ ID NO: 6, and CDR-H3 with a sequence of SEQ ID NO: 7; and / or, A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 8, CDR-L2 with a sequence of SEQ ID NO: 9, and CDR-L3 with a sequence of SEQ ID NO: 10; or (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 81, CDR-H2 with a sequence of SEQ ID NO: 82, and CDR-H3 with a sequence of SEQ ID NO: 83; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 96, and CDR-L3 with a sequence of SEQ ID NO: 97; or (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 81, CDR-H2 with a sequence of SEQ ID NO: 84, and CDR-H3 with a sequence of SEQ ID NO: 85; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 96, and CDR-L3 with a sequence of SEQ ID NO: 97; or (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 86, CDR-H2 with a sequence of SEQ ID NO: 87, and CDR-H3 with a sequence of SEQ ID NO: 88; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 98, and CDR-L3 with a sequence of SEQ ID NO: 99; or (f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 89, CDR-H2 with a sequence of SEQ ID NO: 90, and CDR-H3 with a sequence of SEQ ID NO: 91; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 95, CDR-L2 with a sequence of SEQ ID NO: 98, and CDR-L3 with a sequence of SEQ ID NO: 99; or (g) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 92, CDR-H2 with a sequence of SEQ ID NO: 93, and CDR-H3 with a sequence of SEQ ID NO: 94; and / or, a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 having a sequence of SEQ ID NO: 95, a CDR-L2 having a sequence of SEQ ID NO: 100, and a CDR-L3 having a sequence of SEQ ID NO: 101; or (2) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 23, CDR-H2 with a sequence of SEQ ID NO: 24 or 111, and CDR-H3 with a sequence of SEQ ID NO: 25; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 26, CDR-L2 with a sequence of SEQ ID NO: 27, and CDR-L3 with a sequence of SEQ ID NO: 28; or (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 17, CDR-H2 with a sequence of SEQ ID NO: 18, and CDR-H3 with a sequence of SEQ ID NO: 19; and / or, A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 20, CDR-L2 with a sequence of SEQ ID NO: 21, and CDR-L3 with a sequence of SEQ ID NO: 22; or (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 58, CDR-H2 with a sequence of SEQ ID NO: 62, and CDR-H3 with a sequence of SEQ ID NO: 67; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 74, and CDR-L3 with a sequence of SEQ ID NO: 78; or (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 58, CDR-H2 with a sequence of SEQ ID NO: 63, and CDR-H3 with a sequence of SEQ ID NO: 68; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 74, and CDR-L3 with a sequence of SEQ ID NO: 78; or (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 59, CDR-H2 with a sequence of SEQ ID NO: 64, and CDR-H3 with a sequence of SEQ ID NO: 69; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 75, and CDR-L3 with a sequence of SEQ ID NO: 79; or (f) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 60, CDR-H2 with a sequence of SEQ ID NO: 65, and CDR-H3 with a sequence of SEQ ID NO: 70; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 72, CDR-L2 with a sequence of SEQ ID NO: 76, and CDR-L3 with a sequence of SEQ ID NO: 79; or (g) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 61, CDR-H2 with a sequence of SEQ ID NO: 66, and CDR-H3 with a sequence of SEQ ID NO: 71; and / or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 73, CDR-L2 with a sequence of SEQ ID NO: 77, and CDR-L3 with a sequence of SEQ ID NO: 80; or (3) the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein, compared with the heavy chain variable region and / or light chain variable region described in any one of (a) to (g) in (1) or (2), at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation, and the mutation is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution; Preferably, the VH and / or VL of the antibody or antigen-binding fragment thereof comprises framework regions (FRs) from human or mouse immunoglobulins; Preferably, the antibody or antigen-binding fragment thereof binds to human CLDN18.

2.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) selected from any one of the following groups: (a) a VH sequence as shown in any one of SEQ ID NOs: 3, 39, and 40, and / or a VL sequence as shown in any one of SEQ ID NOs: 4 and 41; (b) a VH sequence as shown in any one of SEQ ID NOs: 1, 29, 30, 31, 32, and / or a VL sequence as shown in any one of SEQ ID NOs: 2, 33, 34, 35, 36, 37, 38; (c) the VH sequence shown in SEQ ID NO: 44, and / or the VL sequence shown in SEQ ID NO: 45; (d) the VH sequence shown in SEQ ID NO: 46, and / or the VL sequence shown in SEQ ID NO: 47; (e) the VH sequence shown in SEQ ID NO: 48, and / or the VL sequence shown in SEQ ID NO: 49; (f) the VH sequence shown in SEQ ID NO: 50, and / or the VL sequence shown in SEQ ID NO: 51; (g) the VH sequence shown in SEQ ID NO: 52, and / or the VL sequence shown in SEQ ID NO: 53; Optionally, the heavy chain variable region (VH) and / or the light chain variable region (VL) are compared with any one of the groups (a) to (g), the heavy chain variable region (VH) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and / or, the light chain variable region (VL) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; Optionally, the heavy chain variable region (VH) and / or light chain variable region (VL) have one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to any one of the groups (a) to (g); and / or the light chain variable region (VL) has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups: (a) VH of the sequence shown in SEQ ID NO: 40 and VL of the sequence shown in SEQ ID NO: 41; (b) VH of the sequence shown in SEQ ID NO: 39 and VL of the sequence shown in SEQ ID NO: 41; (c) VH of the sequence shown in SEQ ID NO: 3 and VL of the sequence shown in SEQ ID NO: 4; (d) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 2; (e) VH of the sequence shown in SEQ ID NO: 29 and VL of the sequence shown in SEQ ID NO: 33; (f) VH of the sequence shown in SEQ ID NO: 29 and VL of the sequence shown in SEQ ID NO: 34; (g) VH of the sequence shown in SEQ ID NO: 29 and VL of the sequence shown in SEQ ID NO: 35; (h) VH of the sequence shown in SEQ ID NO: 29 and VL of the sequence shown in SEQ ID NO: 36; (i) VH of the sequence shown in SEQ ID NO: 29 and VL of the sequence shown in SEQ ID NO: 37; (j) VH of the sequence shown in SEQ ID NO: 29 and VL of the sequence shown in SEQ ID NO: 38; (k) VH of the sequence shown in SEQ ID NO: 30 and VL of the sequence shown in SEQ ID NO: 33; (l) VH of the sequence shown in SEQ ID NO: 31 and VL of the sequence shown in SEQ ID NO: 33; (m) VH of the sequence shown in SEQ ID NO: 32 and VL of the sequence shown in SEQ ID NO: 33; (n) VH of the sequence shown in SEQ ID NO: 44 and VL of the sequence shown in SEQ ID NO: 45; (o) VH of the sequence shown in SEQ ID NO: 46 and VL of the sequence shown in SEQ ID NO: 47; (p) VH of the sequence shown in SEQ ID NO: 48 and VL of the sequence shown in SEQ ID NO: 49; (q) VH of the sequence shown in SEQ ID NO: 50 and VL of the sequence shown in SEQ ID NO: 51; (r) VH of the sequence shown in SEQ ID NO: 52 and VL of the sequence shown in SEQ ID NO: 53; Optionally, the heavy chain variable region (VH) and the light chain variable region (VL) are compared with any one of the groups (a) to (r), the heavy chain variable region (VH) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and / or, the light chain variable region (VL) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; Optionally, the heavy chain variable region (VH) and light chain variable region (VL) are compared with any one of the groups (a) to (r), the heavy chain variable region (VH) having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); and / or, the light chain variable region (VL) having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); preferably, the substitutions are conservative substitutions.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein: The antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody or a humanized antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the wild-type sequence from which it is derived; and (b) a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which has one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived; Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of: (1) Human IgG1 heavy chain constant region; (2) Human IgG4 heavy chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 42 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 42 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids); Preferably, the light chain constant region is a kappa light chain constant region; Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 43 or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 43 (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids); More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:42 and a light chain constant region (CL) as shown in SEQ ID NO:

43.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antibody is selected from any one of the following groups: (a) a heavy chain comprising a VH sequence of SEQ ID NO: 40 and a heavy chain constant region (CH) of SEQ ID NO: 42, and a light chain comprising a VL sequence of SEQ ID NO: 41 and a light chain constant region (CL) of SEQ ID NO: 43; (b) a heavy chain comprising a VH of SEQ ID NO: 39 and a heavy chain constant region (CH) of SEQ ID NO: 42, and a light chain comprising a VL of SEQ ID NO: 41 and a light chain constant region (CL) of SEQ ID NO: 43; (c) a heavy chain comprising a VH sequence set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) set forth in SEQ ID NO: 42, and a light chain comprising a VL sequence set forth in SEQ ID NO: 4 and a light chain constant region (CL) set forth in SEQ ID NO: 43; (d) a heavy chain comprising a VH sequence shown in SEQ ID NO: 1 and a heavy chain constant region (CH) shown in SEQ ID NO: 42, and a light chain comprising a VL sequence shown in SEQ ID NO: 2 and a light chain constant region (CL) shown in SEQ ID NO: 43; (e) a heavy chain comprising a VH of SEQ ID NO: 29 and a heavy chain constant region (CH) of SEQ ID NO: 42, and a light chain comprising a VL of SEQ ID NO: 33 and a light chain constant region (CL) of SEQ ID NO: 43; (f) a heavy chain comprising a VH of SEQ ID NO: 29 and a heavy chain constant region (CH) of SEQ ID NO: 42, and a light chain comprising a VL of SEQ ID NO: 34 and a light chain constant region (CL) of SEQ ID NO: 43; (g) a heavy chain comprising a VH of SEQ ID NO:29 and a heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising a VL of SEQ ID NO:35 and a light chain constant region (CL) of SEQ ID NO:43; (h) a heavy chain comprising a VH of SEQ ID NO:29 and a heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising a VL of SEQ ID NO:36 and a light chain constant region (CL) of SEQ ID NO:43; (i) a heavy chain comprising a VH of SEQ ID NO: 29 and a heavy chain constant region (CH) of SEQ ID NO: 42, and a light chain comprising a VL of SEQ ID NO: 37 and a light chain constant region (CL) of SEQ ID NO: 43; (j) a heavy chain comprising a VH of SEQ ID NO: 29 and a heavy chain constant region (CH) of SEQ ID NO: 42, and a light chain comprising a VL of SEQ ID NO: 38 and a light chain constant region (CL) of SEQ ID NO: 43; (k) a heavy chain comprising a VH of SEQ ID NO: 30 and a heavy chain constant region (CH) of SEQ ID NO: 42, and a light chain comprising a VL of SEQ ID NO: 33 and a light chain constant region (CL) of SEQ ID NO: 43; (l) a heavy chain comprising a VH sequence set forth in SEQ ID NO:31 and a heavy chain constant region (CH) set forth in SEQ ID NO:42, and a light chain comprising a VL sequence set forth in SEQ ID NO:33 and a light chain constant region (CL) set forth in SEQ ID NO:43; (m) a heavy chain comprising a VH of SEQ ID NO:32 and a heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising a VL of SEQ ID NO:33 and a light chain constant region (CL) of SEQ ID NO:43; (n) a heavy chain comprising a VH of SEQ ID NO:44 and a heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising a VL of SEQ ID NO:45 and a light chain constant region (CL) of SEQ ID NO:43; (o) a heavy chain comprising a VH of SEQ ID NO:46 and a heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising a VL of SEQ ID NO:47 and a light chain constant region (CL) of SEQ ID NO:43; (p) a heavy chain comprising a VH of SEQ ID NO:48 and a heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising a VL of SEQ ID NO:49 and a light chain constant region (CL) of SEQ ID NO:43; (q) a heavy chain comprising a VH of SEQ ID NO:50 and a heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising a VL of SEQ ID NO:51 and a light chain constant region (CL) of SEQ ID NO:43; or (r) a heavy chain comprising the VH of SEQ ID NO:52 and the heavy chain constant region (CH) of SEQ ID NO:42, and a light chain comprising the VL of SEQ ID NO:53 and the light chain constant region (CL) of SEQ ID NO:

43.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain comprising an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 106; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 106; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 106; and A light chain comprising an amino acid sequence selected from the group consisting of: (iv) the sequence shown in SEQ ID NO: 107; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 107; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 107; or (b) a heavy chain comprising an amino acid sequence selected from the group consisting of: (i) the sequence shown in SEQ ID NO: 102; (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 102; or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 102; and A light chain comprising an amino acid sequence selected from the group consisting of: (iv) the sequence shown in SEQ ID NO: 103; (v) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 103; or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 103; Preferably, the substitutions described in (ii) or (v) are conservative substitutions.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antibody or antigen-binding fragment thereof is selected from the group consisting of ScFv, Fab, Fab', (Fab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody and multispecific antibody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein The antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is detectably labeled, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance) or biotin.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof has: With a K of less than about 100 nM, such as less than about 50 nM, 40 nM, 40 nM, 20 nM, 10 nM, 1 nM, 0.1 nM or less D Binds to CLDN18.2 (eg, human CLDN18.2); preferably, the K D Measured by bio-film interferometry (BLI); and / or Binds to CLDN18.2 (e.g., human CLDN18.2) with an EC50 of less than about 500 nM, such as less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM or less; preferably, the EC50 is measured by flow cytometry or cell competition ELISA; Preferably, the antibody or antigen-binding fragment thereof does not bind to CLDN18.1 (eg, human CLDN18.1); Preferably, the antibody or antigen-binding fragment thereof has ADCC and / or CDC activity; More preferably, the antibody or antigen-binding fragment thereof has enhanced ADCC and / or CDC activity.

12. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, the heavy chain and / or light chain thereof, or the heavy chain variable region and / or light chain variable region thereof according to any one of claims 1 to 11, Preferably, the encoded antibody or antigen-binding fragment thereof, its heavy chain and / or light chain, or its heavy chain variable region and / or light chain variable region comprises a heavy chain variable region and / or a light chain variable region as defined in any one of claims 1 to 4, More preferably, the encoded antibody or antigen-binding fragment thereof, or its heavy chain and / or light chain comprises the antibody or antigen-binding fragment thereof according to claim 6, More preferably, the encoded antibody or antigen-binding fragment thereof, or the heavy chain and / or light chain thereof comprises the heavy chain and light chain as defined in any one of claims 7-8.

13. The isolated nucleic acid molecule of claim 12, comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein: The nucleic acid molecule encoding the antibody heavy chain variable region has a sequence selected from the following: (a) the nucleotide sequence shown in SEQ ID NO: 56 or 54, or (b) a sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence described in (a)), or (c) a sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; and / or, The nucleic acid molecule encoding the antibody light chain variable region has a sequence selected from the following: (d) the nucleotide sequence shown in SEQ ID NO: 57 or 55, or (e) a sequence substantially identical to the nucleotide sequence described in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence described in (d)), or (f) a sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides; Preferably, the nucleic acid molecule encoding the antibody heavy chain variable region has the nucleotide sequence shown in SEQ ID NO: 56, and / or the nucleic acid molecule encoding the antibody light chain variable region has the nucleotide sequence shown in SEQ ID NO: 57; Preferably, the nucleic acid molecule encoding the antibody heavy chain variable region has a nucleotide sequence as shown in SEQ ID NO:54, and / or the nucleic acid molecule encoding the antibody light chain variable region has a nucleotide sequence as shown in SEQ ID NO:

55.

14. The isolated nucleic acid molecule of claim 12 or 13, comprising a nucleic acid molecule encoding an antibody heavy chain, and / or a nucleic acid molecule encoding an antibody light chain, wherein: The nucleic acid molecule encoding the antibody heavy chain has a sequence selected from the following: (a) the nucleotide sequence shown in SEQ ID NO: 108 or 104, or (b) a sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence described in (a)), or (c) a sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; and / or, The nucleic acid molecule encoding the antibody light chain has a sequence selected from the following: (d) the nucleotide sequence shown in SEQ ID NO: 109 or 105, or (e) a sequence substantially identical to the nucleotide sequence described in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence described in (d)), or (f) a sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides; Preferably, the nucleic acid molecule encoding the antibody heavy chain has a nucleotide sequence as shown in SEQ ID NO: 108, and / or the nucleic acid molecule encoding the antibody light chain has a nucleotide sequence as shown in SEQ ID NO: 109; Preferably, the nucleic acid molecule encoding the antibody heavy chain has a nucleotide sequence as shown in SEQ ID NO:104, and / or the nucleic acid molecule encoding the antibody light chain has a nucleotide sequence as shown in SEQ ID NO:

105.

15. A vector comprising the nucleic acid molecule according to any one of claims 12 to 14; preferably, the vector is a cloning vector or an expression vector.

16. A host cell comprising the nucleic acid molecule according to any one of claims 12 to 14 or the vector according to claim 15.

17. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising culturing the host cell according to claim 16 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

18. A conjugate comprising an antibody or an antigen-binding fragment thereof and a conjugated moiety, wherein: The antibody is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, and the coupling portion is selected from: a detectable label, a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, an enzyme, polyethylene glycol (PEG), a nuclide, a nucleic acid, a small molecule toxin, a polypeptide with binding activity, a protein, a receptor, a ligand, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

19. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof (eg, ScFv) of any one of claims 1 to 11, a transmembrane domain, and one or more intracellular T cell signaling domains.

20. A multispecific antibody, wherein the antibody is formed by coupling a first antibody or a fragment thereof with other antibodies or fragments thereof or antibody analogs, each antibody or fragment thereof or antibody analog retains the original binding specificity, and the first antibody or fragment thereof is the antibody or antigen-binding fragment thereof described in any one of claims 1 to 11; preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the vector according to claim 15, or the host cell according to claim 16, or the conjugate according to claim 18, or the chimeric antigen receptor according to claim 19, or the multispecific antibody according to claim 20, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; More preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; More preferably, the additional pharmaceutically active agent is an interferon, interleukin-2 or a chemotherapeutic drug; More preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as components of the same composition; More preferably, the additional pharmaceutically active agent is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, albumin-bound paclitaxel, a combination of epirubicin + oxaliplatin + 5-fluorouracil, FOLFOX4, FOLFOX6, and m FOLFOX6 (comprising oxaliplatin, folinic acid and 5-fluorouracil).

22. The pharmaceutical composition of claim 21, wherein the antibody or antigen-binding fragment thereof in the pharmaceutical composition is used in a subject: (a) Inducing apoptosis of tumor cells; (b) inhibiting tumor cell proliferation; (c) induce and / or increase T cell infiltration; (d) inducing and / or enhancing an immune response; (e) inducing and / or increasing complement-dependent cytotoxic activity; (f) inducing and / or increasing antibody-dependent cellular cytotoxicity activity; (g) Increase NK cell activity; (h) inhibiting the expression and activation of CLDN18.2; (i) inhibiting CLDN18.2-mediated signaling; or (j) Any combination of (a) to (i).

23. The pharmaceutical composition according to claim 21 or 22, further comprising a second antibody or a nucleic acid encoding the second antibody that specifically binds to a receptor or ligand selected from the following: The receptor or ligand is selected from: PD-1, PD-L1, PD-L2, TIM-3, LAG-3, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, CD40, LAIR1, CD160, 2B4, TGF-R, KIR, ICOS, GITR, CD3, CD30, BAFFR, HVEM, CD7, LIGHT, SLAMF7, NKp80, B7-H3 and any combination thereof.

24. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the vector according to claim 15, or the host cell according to claim 16, or the conjugate according to claim 18, or the chimeric antigen receptor according to claim 19, or the multispecific antibody according to claim 20, or the pharmaceutical composition according to any one of claims 21 to 23, and optionally instructions for use.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the vector according to claim 15, or the host cell according to claim 16, or the conjugate according to claim 18, or the chimeric antigen receptor according to claim 19, or the multispecific antibody according to claim 20, or the pharmaceutical composition according to any one of claims 21 to 23 in the preparation of a drug for preventing and / or treating and / or adjuvanting the treatment of tumors; Preferably, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the vector according to claim 15, or the host cell according to claim 16, or the conjugate according to claim 18, or the chimeric antigen receptor according to claim 19, or the multispecific antibody according to claim 20 is administered separately, in combination, simultaneously or sequentially with another pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, albumin-bound paclitaxel, a combination of epirubicin + oxaliplatin + 5-fluorouracil, FOLFOX4, FOLFOX6, mFOLFOX6 (comprising oxaliplatin, folinic acid and 5-fluorouracil).

26. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the vector according to claim 15, or the host cell according to claim 16, or the conjugate according to claim 18, or the chimeric antigen receptor according to claim 19, the multispecific antibody according to claim 20, or the pharmaceutical composition according to any one of claims 21 to 23 in the preparation of a drug, wherein the drug is used for: (a) Inducing apoptosis of tumor cells; (b) inhibiting tumor cell proliferation; (c) increasing immune cell activity in vitro or in vivo in a subject (e.g., a human); (d) enhancing an immune response in a subject (e.g., a human); (e) treating a tumor in a subject (e.g., a human); (f) inducing and / or increasing T cell infiltration; (g) inducing and / or enhancing an immune response; (h) inducing and / or increasing complement-dependent cytotoxic activity; (i) inducing and / or increasing antibody-dependent cellular cytotoxicity activity; (j) Increase NK cell activity; (k) inhibiting the expression and activation of CLDN18.2; (l) inhibiting CLDN18.2-mediated signal transduction; or (m) Any combination of (a)-(l).

27. The use according to claim 25 or 26, wherein the tumor is selected from solid tumors, blood tumors and metastatic, refractory or recurrent lesions of cancer; Preferably, the tumor or cancer is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia; Preferably, the tumor is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer (eg, non-small cell lung cancer); Preferably, the tumor is selected from gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, such as locally advanced unresectable or metastatic gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma; Preferably, the tumor is CLDN18.2 positive, more preferably, the tumor is HER2 negative.

28. A method for preventing and / or treating a tumor, and / or delaying tumor progression, and / or reducing or inhibiting tumor recurrence in a subject, the method comprising administering an effective amount of the pharmaceutical composition of any one of claims 21-23 to a subject in need thereof.

29. The method of claim 28, further comprising administering to the subject a second therapy selected from surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and any combination thereof; Optionally, the second therapy can be applied separately or in combination with the method of claim 28; the second therapy can be applied separately or in combination with the method of claim 28, simultaneously or sequentially; Preferably, the chemotherapy is selected from: one or more of epirubicin, oxaliplatin, capecitabine, 5-fluorouracil, folinic acid, paclitaxel, albumin-bound paclitaxel, a combination of epirubicin + oxaliplatin + 5-fluorouracil, FOLFOX4, FOLFOX6, mFOLFOX6 (comprising oxaliplatin, folinic acid and 5-fluorouracil).

30. The method according to claim 28 or 29, wherein the tumor is selected from solid tumors, hematological tumors, and metastatic, refractory or recurrent lesions of cancer; Preferably, the tumor or cancer is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymic cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia; Preferably, the tumor is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer (eg, non-small cell lung cancer); Preferably, the tumor is selected from gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma, such as locally advanced unresectable or metastatic gastric cancer or gastroesophageal junction (GEJ) adenocarcinoma; Preferably, the tumor is CLDN18.2 positive, more preferably, the tumor is HER2 negative.

31. A method for detecting the presence or level of CLDN18.2 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and CLDN18.2, and detecting the formation of the complex.

32. Use of the antibody or antigen-binding fragment of any one of claims 1-11, the conjugate of claim 18, or the multispecific antibody of claim 20 in the preparation of a diagnostic kit for diagnosing or differentially diagnosing a tumor or tumor metastasis, wherein the tumor is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, and lung cancer (e.g., non-small cell lung cancer).

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